Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MEDICAL DEVICE
Technical field
The present invention relates to a medical device suitable for implantation
into a human or
animal, such as an implantable prosthetic device, combined with a nucleic acid
component,
which codes for a gene product that will asist in reducing restenosis,
increasing
endothelialisation and reducing inflammatory reaction, and in reducing
connective tissue
formation. The invention additionally relates to a method of reducing
connective tissue and
inflammatory reaction formation and to a method for increasing
endothelialisation with the
purpose of improving a human or animal body's acceptance of a medical device,
comprising at
least one synthetic surface. Also related to is a method of producing a
medical device according
to the invention.
Background of the invention
Diseased and damaged parts of the body can either be repaired or replaced with
several
methods. These procedures induce reactive changes in the tissues where the
intervention is
performed or a device implanted. These reactive changes in tissues are
difficult to control and
cause complications. Tissue reactive changes occur both in connection to all
traumatic handling
of tissues, transplantation of biological material or implantation of
synthetic material.
To repair tissues either endovascular, endoscopic or surgical methods are
performed. All these
procedures suffer from reaction to trauma caused by the intervention with
following scar tissue
formation or fibrotic reaction. In addition to repairing the body part it can
also be replaced.
Then the donor tissues are generally procured elsewhere: either from the
recipient's own body
(autograft); from a second donor (allograft); or, in some cases, from a donor
of another species
(xenograft). Replacement of the body part with native structures is usually
preferred method
but suffers from tissue reaction in the connection site. Tissue
transplantation is costly, and
suffers from significant failure rates because of acute inflammatory and long-
term fibrotic
reactions. Use of artificial or synthetic medical implant devices has been
subject of considerable
attention, but also this technology suffers from foreign body reaction against
the implants with
following increase in connective tissue or fibrosis.
Although implant devices can be used in some instances as an alternative to
donor-based
transplants, they too often produce unsatisfactory results because of the
tissue response to
trauma, implant's incompatibility with the body, induction of foreign body
inflammatory reaction
and induction of connective tissue formation with following geometrical
changes. Also, lack of
cell lining of the cardiovascular device synthetic surface sets up conditions,
which increases the
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is either restriction in the flow or occlusion when the device is implanted
intravascularly or
fibrotic capsule encasing the implant when implanted in the tissues. The final
consequence is
dysfunction and following other clinical medical complications.
One specific area where the growth factors, genes and implants are used is in
the
cardiovascular field. Cardiovascular diseases affect a large segment of the
human population,
and are a cause for significant morbidity, costs and mortality in the society.
About 60 million
adults in the USA have a cardiovascular disease, which is the major cause of
death in the USA.
There are one million acute myocardial infarctions or heart attacks per year
with 200000 deaths
a year. Claudicatio intermittens cause significant morbidity and yearly 150000
lower limb
amputations are required for ischemic disease with significant perioperative
mortality. Cerebral
vascular disease, strokes and bleedings also cause significant morbidity,
costs and mortality.
There are one million dialysis patients, and yearly 200000 arteriovenous
fistula operations are
required to surgically create access for dialysis.
Coronary and peripheral vascular diseases are characterised by blockages in
the blood vessels
providing blood flow and nutrition to the organs. Native blood vessels used as
grafts suffer from
increased connective tissue formation and accelerated atherosclerosis. This
causes subsequently
narrowing of the vessel lumen. Other significant disease groups are aneurysms,
i.e, local
dilatation of the vessels, pseudoaneurysm, and dissection of the vessel wall.
There are pharmacological, surgical and percutaneous strategies to treat these
diseases. In
pharmacological treatment of ischemic heart disease the goal is to make blood
less coagulable,
inhibit cholesterol accumulation to the vessel wall and to increase blood flow
by vessel dilation
or to reduce oxygen consumption.
Alternatively the vessel can be treated with percutaneous transluminal
angioplasty (balloon
angioplasty), laser angioplasty, atherectomy, roto-ablation, invasive surgery,
thrombolysis or a
combination of these treatments. The intent of percutaneous methods is to
maintain patency
after an occluded vessel has been re-opened. Angioplasty suffers from two
major problems-
abrupt closure and restenosis. Abrupt closure refers to occlusion of a vessel
immediately after or
within initial hours of dilation procedure. Restenosis refers to re-narrowing
of an artery after an
initially successful angioplasty. It occurs in 20-40 % of patients within the
first few months after
a successful intervention and is thought to happen because of injury the blood
vessels during
the balloon inflation. When vessel then heals the smooth muscle cells
proliferate faster than
endothelial cells narrowing the lumen of the blood vessel (Ip et al. 7. Am.
College of Cardiol.
1990; 15:1667-1687, Faxonj et al.. Am. J. of Cardiology: 1987;60:5B-9B). The
percentage of
patients that develop early restenosis after balloon angioplasty can be
reduced with stent
implantation, in which an intraluminal implant such as adjustable stent
structural supports,
tubular grafts or a combination of them after doing the angioplasty. However,
stents actually
increase the amount of late luminal narrowing due to intimal hyperplasia, and
the overall rate of
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devices suffer from both thrombosis until they are covered with endothelial
cells and bleeding
complications postoperatively. Because of the risk of thrombosis,
anticoagulant therapy is used
until the endothelial cell coverage has developed in the stent surface.
Endothelial surface does
not develop on tubular grafts in humans. Stents and tubular endovascular
grafts can also be
used to exclude a local vascular dilatation or dissection.
The surgical treatment for cardiovascular disease is to bypass, substitute or
reconstruct a
diseased vessel with a native or synthetic vascular graft or patch.
All these endovascular and surgical methods are complicated by same problems -
trauma to the
blood vessel endothelium, formation of excessive connective tissue and
inflammatory reaction
with following problems with occlusion because of thrombosis or restenosis.
In coronary artery surgery the obstructed vessel is bypassed with an
autologous vascular graft.
The operation is called CABG, which means coronary artery bypass grafting. In
peripheral artery
surgery a native or synthetic graft is usually implanted to bypass an
obstruction, for example
from the groin to the thigh. In some cases arterial segment may alternatively
be replaced with a
native or synthetic vascular graft. In access surgery for dialysis there is a
need for creating an
access to clean the blood with the dialysis machine. Usually a connection
called fistula is
constructed between the upper extremity artery and vein to create a high blood
flow required
for dialysis. Intracardiae patches are used to repair holes in the cardiac
septa or wall. Prosthetic
vascular patches are used in vascular surgery in several operations, which
requires an incision
in the wall of the blood vessel, such as thrombectomies, endarterectomies,
aneurysmal repairs
and vessel reconstructions. In percutaneous revascularisation catheters with
balloons, stents or
stent grafts are used to reduce the narrowing or exclude the dilatation or
dissection in different
anatomical locations such as cerebral, coronary, renal, other peripheral
arteries and veins, aorta
and in vascular grafts. Balloon dilatations, stents and stent grafts may also
be employed in
other sites, such as biliary tree, esophagus, bowels, tracheo-bronchial tree
and urinary tract.
All endovascular and surgical devices are complicated by same problems - lack
of endothelial
surfaces on the synthetic surface, formation of excessive connective tissue
and inflammatory
reaction.
Several strategies have been suggested to improve the patency after vascular
interventions and
implantation of synthetic vascular implants. About 1 600 000 angioplasties are
performed yearly
worldwide and stent is inserted vast in majority of these procedures (8th
International drug
delivery meeting and cardiovascular course on radiation and molecular
strategies, Geneva,
Switzerland, Feb 1, 2002). The problem with angioplasty or angioplasty with
following stenting
is the process of restenosis. Because of the trauma to the vessel an excessive
connective tissue
formation develops leading to narrowing of the vessel lumen in 20-30 percent
of cases after 6
months (Bittl JA: Advances in coronary angioplasty N. Engl. J. Med. 1996;
335:1290-1302,
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1998; 97:1298-1305). The problem with restenosis has been well described in
the art and
several approaches have been described both scientific literature and patents.
Currently there is
no strategy in the market to reduce restenosis after simple angioplasty
without a device
inserted. When using stent devices after angioplasty procedure
pharmaceutically coated stents
have not been evaluated for long term effects and there is no verified method
in humans which
would securely reduce long term restenosis rate in stents or stent grafts. The
main strategy has
been use of various pharmaceutical substances with stents to reduce
hyperplasia following the
trauma to the tissue such as rapamycin, sirolimus, paclitaxel, tacrolimus,
dexamethasone,
cytochalasine D and Actinomycin C. One drawback with the current
pharmacologically coated
devices is the possible disappearance of the effect after the substance has
been released from
the device surface. Furthermore, the nature of compounds eluting at high local
concentration
into the vessel wall and downstream vasculature or tissue is an issue of
concern. Another
drawback is that none of these substances is naturally occurring in the body
and thereafter fail
to promote natural healing of the foreign body surface. For example are
paclitaxel and
Actinomycin D cytotoxic to the cells.
In native vessel graft the main problem has been intimal hyperplasia both in
the site for
connection of the graft and the vessel in the specific body location and the
intimal hyperplasia in
the graft vessel lumen. The same problem of anastomotic hyperplasia exists
when connecting
the native vessel with a synthetic vascular graft. More than 350000 synthetic
vascular grafts are
implanted each year and numerous synthetic biomaterials have been developed as
vascular
substitutes. As a foreign material, grafts are targets for foreign body
reaction and because of
thrombogenicity prone to clot in a higher degree than autologous material. To
overcome
thrombogenicity, most approaches have concentrated on creating a surface that
is
thromboresistant, with the majority of these efforts being directed toward an
improved polymer
surface. Studies have demonstrated that selected materials, for example Dacron
and ePTFE
(expanded polytetrafluorethylene), successfully can be incorporated in both
large and small
caliber arteries in animal models (Zdrahala, J. Biomater. Appl. 1996; 10:309-
29). In humans,
Dacron and ePTFE vascular prostheses have met certain clinical success in
large and middle-
sized arterial reconstructions, but are yet not ideal. However, the success is
limited for vessel
substitutes smaller than 6 mm in diameter, due to anastomotic hyperplasia
i.e., the propensity
to develop excessive connective tissue growth in the area where either two
native blood vessels
or an synthetic artificial blood vessel and an autologous vessel are connected
or due to
thrombosis (i.e. propensity to develop clots) in the open thrombogenic surface
(Nojiri, Artif.
Organs 1995 Jan;l9 (1):32-8). The autologous vein grafts suffer from
development of stenosis
when implanted in arterial position. Gene therapy has been used in prior art
to reduce
development of restenosis as described in patents and scientific literature.
Sleeves impregnated
with genes have been described and used as devices around vascular anastomosis
to inhibit
hyperplasia (WO 98/20027, WO 99/55315). The major drawback in these systems is
the
cumbersome use of the sleeve and the used substances are growth factors or
encoding for a
growth factor. Furthermore, studies with thrombogenicity of the implant have
been reduced by
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The substance mostly used has been heparin, which either is bound to the
graft, or is given with
a local drug delivery device.
In humans flow surface of foreign implants remains uncovered with endothelial
cells except for
some case reprots (Wu, J. Vasc. Surg. 1995 May; 21(5):862-7, Guidon,
Biomaterials 1993 Jul;
14(9):678-93). In animals, complete endothelialisation of the vascular graft
has been shown to
occur in 2-4 weeks depending on species. This period without endothelial
surface may result in
undesired effects and problems due to e.g. thrombogenicity of the surface. The
lack of
endothelial surface has led to inferior performance of synthetic grafts
compared to autologous
grafts (Nojiri, Artif. Organs 1995 Jan;l9 (1):32-8). Berger, Ann. of Surg.
1972;175 (1):118-27,
Sauvage). Autologous grafts, on the other hand, comprise a step for the
harvesting thereof,
which leads to longer operation times and also possible complications in the
harvesting area.
Transposition of omentum with uncompromised vasculature around a porous
carotid artery PTFE
graft has been demonstrated to increase endothelial cell coverage in the graft
lumen in dogs
(Hazama, 7. of Surg. Res. 1999; 81; 174-180), however, entailing problems with
a cumbersome
and complex procedure, such as discussed above. Further, grafts have been
seeded with
endothelial cells, and sodded with endothelial cells or bone marrow (Noishiki,
Artif. Organs 1998
Jan; 22(1): 50-62, Williams & Jarrel, Nat. Medicine 1996;2: 32-34). In cell
seeding, endothelial
cells are mixed with blood or plasma after harvesting and then added to the
graft surface during
the preclotting period. The endothelial cells used in these methods may be
derived from
microvascular (fat), macrovascular (for example from harvested veins), or
mesothelial sources,
whereby the graft later on is implanted. More specifically, these methods
comprise several
steps, including harvesting of the tissue with endothelial cells, separation
of endothelial cells, in
some cases a culture of endothelial cells, seeding of endothelial cells on the
graft materials and
finally implanting the graft. Accordingly, a substantial drawback with these
methods is that they
are time consuming and cumbersome in practice, and they also require a
specific expertise in
the area as well as the suitable equipment. Furthermore, such seeded
endothelial cells have
been genetically engineered, with various results: transduction of the cells
with tissue
plasminogen activator (tPA) decreases endothelial cell adhesion to the graft
surface, and
transfection with retrovirus reduces endothelialisation. In order to improve
cell seeding, vascular
endothelial growth factor (VEGF) transfected endothelial cells or fat cells
have been used. In
addition to the drawbacks discussed above, this method is even more cumbersome
and
therefore costly to be useful in practice. A method to transduce endothelial
progenitor cells and
then re-administer them has been described. However, the problems are still as
mentioned
above. In order to improve the technology for endothelial cell growth on a
surface, ligand
treatment of graft surfaces has been suggested. In cell sodding, endothelial
cells are
administered directly on the polymeric graft surface after harvesting, whereby
the graft is
implanted, but this technique also includes several steps as mentioned above,
which makes it
cumbersome as well. Also, tissue engineering, which is also a complex and
therefore costly
procedure, has been used in order to construct vascular tissues for
implantation. Gene
technological platform with angiogenetic factors has been suggested to induce
endothelial
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of growth factors on cells could cause uncontrollably connective tissue
growth. Arterial
homografts have been described, but they give rise to problems regarding
arterial preservation
and antigenicity.
Further, worldwide 1 600 000 stenting procedures are performed in the yearly
with in average
1,7 stents per patient. Stents, i.e. relatively simple devices of fine network
structures, are well
known in the art. Stenting for vessel obstruction is usually combined with
opening of the artery
by dilatation, ablation, atherectomy or laser treatment. These interventions
cause trauma and
tissue injury to the vessel wall with disruption of the endothelial cell
lining. Usually, stents are
composed of network of some material, usually stainless steel, which is
entered to the diseased
area usually percutaneously with a catheter. Stents are of different designs
for example, self-
deployable/ pressure expandable, tubular/ conical/ bifurcated, permanent/
temporary,
nondegradable/ biodegradable, metal/ polymeric material, with or without
pharmaceutical
compound. They are implanted in a blood vessel in different anatomical
locations such as
cerebral, coronary, renal, other peripheral arteries and veins, and aorta.
Stents may also be
used in other locations such as biliary tree, esophagus, bowels, tracheo-
bronchial tree and
genitourinary tract. Stents may be used for example to treat stenoses,
strictures or aneurysms.
Stents characteristically have an open mesh construction, or otherwise are
formed with multiple
openings to facilitate the radial enlargements and reductions and to allow
tissue ingrowth of the
device structure. After the vessel dilatation stents have been associated with
subacute
thrombosis and neointimal thickening leading to obstruction. Before the stent
era balloon
dilatations alone were used to relieve vessel narrowing. A balloon with
hydrogel for delivery of
naked DNA coding for VEGF has been described (Riessen, Human Gene Therapy
1993, 4:749-
758). U.S. patent 5,830,879, and van Belle J. Am. Coll. of Cardiol. 1997;29:
1371-9) describes
also VEGF plasmid being attached to the balloon with simultaneous deployment
of endovascular
stent to induce vessel healing and reduce restenosis. Also, a balloon with
hydrogel and gene for
drug delivery (5,674,192, Sahatjian et al.) has been described. Catheters have
been used to
deliver angiogenic peptides, liposomes and viruses with encoding gene to the
vascular wall (WO
95/25807, U.S. 5,833,651 as above). Catheters have also been used to deliver
VEGF protein in
order to provide a faster endothelialization of stents (van Belle, Circ.
1997:95 438-448).
Further, stent for gene delivery (U.S. 5,843,089, Klugherz BD et al.. Nat
biotechnology
2000;18: 1181-84) and a stent for viral gene delivery (Rajasubramanian, ASAIO
J 1994; 40:
M584-89, U.S. 5,833,651) have been described. Endothelial cell seeding on the
stent has been
used as a method to deliver recombinant protein to the vascular wall, in order
to overcome
thrombosis, but as mentioned above, this technology is cumbersome and
therefore costly.
Stent grafts, also referred to as covered stents, are well known in the art.
Such stents are a
combination of two parts, namely a stent portion and a graft portion. In a
stent graft, a
compliant graft is coupled to a radially expandable stent. Stent grafts are
considered to be
usable, by forming a complete barrier between the stent and the blood flow
through the vessel.
The graft may serve as a biologically compatible inner covering, by preventing
turbulent blood
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preventing thrombotic or immunologic reactions to the metal or to other
materials of which the
stent is made, and by forming a barrier to separate a diseased or damaged
segment of the
blood vessel from the blood-flow passing there. In humans, the main problem
with stent grafts
is the formation of neointimal thickening and lack of complete
endothelialisation leading to
occlusion, as discussed above in relation to grafts. Stent grafts may be used
in aorta, cerebral,
coronary, renal, other peripheral arteries and veins, and aorta. Experimental
studies have
shown that vascular injuries, that arises when the endovascular device is
delivered, induces
inflammation, local expression and release of mitogens and chemotactic
factors, which mediates
neointimal lesion formation. Stent grafts may also be used in other locations
such as biliary
tree, esophagus, bowels, tracheobronchial tree and genitourinary tract.
Thus, at the moment, there is a great need and interest in inhibiting the
intimal hyperplasia in
the site for the tissue trauma, in the area of device implantation, in the
vascular connection site
or in a native graft. Also, at the moment, there is a great need and interest
to improve the
endothelialisation and graft healing in clinical practice. However, hitherto,
no such method that
works in practice has yet been developed.
Yearly, about 100000 heart valve replacement operations are performed. Heart
valve
prosthesises are well known in the art. There are of four types of grafts:
synthetic grafts,
xenografts, allografts and autografts. Xenografts are usually preserved
pericardial and porcine
valves e.g. Carpentier-Edwards, Ionescu-Shiley, Hancock, Pericarbon or
stentless valves.
Biological degeneration is a major concern in bioprosthetic valves.
Degeneration is characterised
by disruption of endothelial cell barrier and lack of endothelialisation,
increased permeability
leading to eased diffusion of circulating host plasma proteins into valve
tissue, and increased
activity of infiltration processes e.g. calcification and lipid accumulation,
and biodegradation of
the collagen framework. Also a mild to moderate infiltration of inflammatory
cells has been
described and studies have shown either no (Isomura J. Cardiovasc. Surg. 1986,
27:307-15) or
scarce growth of endothelium on bioprosthetic valve surface (Ishihara, Am. J.
Card. 1981:48,
443-454) after one year. Changing the method of preservation, neutralisation
of glutaraldehyde
preservative and pre-endothelialisation of bioprosthetic valves has been
suggested to improve
valve performance. Some studies have been made on endothelial seeding in this
context, but it
is clinically cumbersome due to the many steps required, as described above.
As mentioned above, implantable devices are also used in other fields than the
cardiovascular.
Various implantable devices have been described, such as for structural
support, functional
support, drug delivery, gene therapy, and cell encapsulation purposes. A
variety of devices,
which protect tissues or cells producing a selected product from the immune
system have been
explored for implant in a body, such as extravascular diffusion chambers,
intravascular diffusion
chambers, intravascular ultrafiltration chambers, and microencapsulated cells.
However, when
foreign biomaterials are implanted, an inflammatory foreign body reaction
starts, which in the
end encapsulates the device, and inhibits diffusion of nutritive substances to
the cells inside the
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diffusion of substances. It decreases long-term viability of the encapsulated
endocrine tissue,
and it also makes vascular implants more susceptible to infections. The
fibrotic capsule without
vascularity can also limit the device performance. In U.S. 5,882,354, a
chamber holding living
cells comprises two zones that by an unknown mechanism prevents the invasion
of connective
tissue and increases the close vascularisation of the implant.
Some other materials used in the procedure of implanted devices also encounter
similar
problems as the ones discussed above. For example, suture materials can be
mentioned, which
materials are used for repair, fixation and/or approximation of body tissues
during surgical
procedures. Strict requirements exist for sutures for attachment of prosthetic
devices or
implants regarding strength, biocompatibility and biodegradability.
To summarise, the major drawbacks in this field is the development of
excessive connective
tissue after endovascular and surgical procedures. For example, after balloon
dilatation
procedure the result is excessive connective tissue growth with following
complications. Also, in
the autologous vascular grafts excessive connective growth causes narrowing.
In vascular
surgical procedures with or without implanted devices the excessive connective
tissue formation
in the anastomotic areas leads to narrowing of the vessel connection with
limitation to the flow
and following dysfunction in the organ supplied by that vessel. In vascular
implants, when
synthetic materials are used, problems also arise due to open thrombotic
surfaces where the
implant is performed, which in turn generate blood clotting and inferior
performance. In
synthetic tissue implants, the consequence is a non-vascularised fibrotic non-
nutritive zone,
which leads to dysfunction of the implant. This together with the inflammatory
reaction causes
that biocompatibility of the mammalian body, especially the human body, with
implanted
medical devices cannot be achieved in any satisfactory degree using the prior
art methods.
Patent EP1016726 describes use of angiogenetic proteins and genes, such as
growth factors
(e.g. VEGF) or other genes (e.g. NOS) to create endothelial surfaces after
vessel damage and
when inserting a stent.
Patent EP1153129 describes use of oligosense nucleotides to inhibit
restenosis.
Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidative
enzyme, which is
widely expressed throughout the body and is the major SOD isoenzyme in plasma.
Vessel walls,
lung, kidney, thyroid gland and epidymis are shown to be the primary
expression sites for EC-
SOD.
Patent ES2004687 describes the sequence of EC-SOD. Articles by Li et al.
describe use of EC-
SOD in myocardial protection (Gene therapy with extracellular superoxide
dismutase attenuates
myocardial stunning in conscious rabbits. Circulation 1998;98:1438-1448, and
Gene therapy
with extracellular superoxide dismutase protects conscious rabbits against
myocardial infarction.
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Summary of the invention
The object of the present invention is to provide a solution to the
aforementioned problems.
More specifically, one object of the invention is to provide a medical device,
which solves the
problems of traumatised vascular tissue reaction resulting in connective
tissue hyperplasia.
Another object of the present invention is to provide a medical device, which
solves the problem
of thrombogenic medical implant surfaces resulting in occlusion and other
problems. Another
object of the present invention is to provide a medical device, which is less
cumbersome to use
in practice than prior art methods for reducing restenosis or otherwise
improving the
biocompatibility between foreign materials and the recipient or host thereof.
Another object of
the invention is to provide a medical device useful in vascular interventions,
which entails less
risk of being narrowed because of tissue hyperplasia or occluded and
reoccluded than hitherto
known devices. Yet another object of the invention is to provide a device
useful in measurement
and control of metabolic functions that is better accepted and maintained in
the human or
animal body than prior art devices.
The given objects above are achieved by providing a medical device with
improved biological
properties for an at least partial contact with blood, bodily fluids and/or
tissues when introduced
in a mammalian body. Said device comprises a core and a nucleic acid present
in a biologically
compatible medium and is characterised in that said nucleic acid encodes a
translation or
transcription product leading to production of extracellular superoxide
dismutase (EC-SOD)
protein capable of reducing connective tissue formation and promoting
endothelialisation in vivo
at least partially on a synthetic surface of said core.
In most prefered embodiments, the polypeptide is EC-SOD.
In another embodiments, the nucleic acid encodes EC-SOD protein or
polypeptide.
The nucleic acid is present in the biologically compatible medium in naked
form, in a viral
vector, such as retrovirus, Sendai virus, lenti virus, adeno associated virus,
and adenovirus, or
in a liposome or is an artificial chromosome.
In one embodiment the nucleic acid is administered locally in the vessel wall.
In another embodiment the nucleic acid is administered locally in the tissue
surrounding a
device.
In yet another embodiment, the biologically compatible medium is a biostable
polymer, a
bioabsorbable polymer, a biomolecule, a hydrogel polymer or fibrin.
In one advantageous embodiment, the nucleic acid is present in a reservoir
separate from said
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In an alternative embodiment, the nucleic acid has been attached to the core
by ionic or
covalent bonding.
5 The synthetic surface is either non-porous or porous, in which case it
allows capillary and
endothelial cell growth through pores. Preferably, the porosity is from about
0 mm to about
2000 mm.
The present gene transfer product can be used in several different contexts to
reduce
10 connective tissue formation e.g. in connection to interventional
procedures, implanting a native
graft or implanting a medical implant.
The present device is useful in a wide variety of contexts, and may e.g. be a
cardiovascular
implant, such as an artificial part of a blood vessel, or an endovascular
implant. In general
terms, the present device may be used as an implant used for replacement of
part of a
mammalian body, where said implant is adapted for an at least partial contact
with blood, bodily
fluids and/or tissues. Further, the present device is useful as a tissue
implant or a biosensor.
Preferably, the device is selected from the group consisting of vascular
grafts, stents, covered
stents, graft connectors and biosensors.
The present invention also relates to a method of producing a medical device
according to the
invention.
The invention also relates to the method of introducing nucleic acid to a
synthetic graft in a
biologically compatible medium, said administration of nucleic acid being
performed before,
simultaneously as or after the introduction of the device in the body. The
invention also relates
to the method of introducing a device comprising a an autologous, allogenic
and xenogenic
synthetic surface in the body with an at least partial contact with blood,
bodily fluids and/or
tissues and administering a nucleic acid present in a biologically compatible
medium to the
surroundings thereof. The method is characterised in that the nucleic acid
encodes or increases
expression of a translation or transcription product of EC-SOD capable of
reducing connective
tissue growth and inflammatory reaction and promoting endothelialisation and
biocompatibility
in vivo at least partially on said synthetic surface, said administration of
nucleic acid being
performed before, simultaneously as or after the introduction of the device in
the body.
40
According to a further aspect of the invention, a use of EC-SOD gene/cDNA or
EC-SOD protein is
provided for the manufacture of a medicament for the treating of conditions
caused by damages
due to vascular manipulations, such as restenosis or blood vessel thickening,
e.g. by inhibiting
inflammation.
Further details regarding the method of treatment are disclosed below and in
the appended
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claims. The method may include administering of the nucleic acid at least
once, depending on
the case in question.
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Figurelegends
Figure 1.
Comparison of neointima formation in EC-SOD treated (left) versus control
animals (right) in
normal vessel segments (upper panels) and in segments with pre-existing
atherosclerotic
lesions (lower panels). H&E (upper panels) and Masson trichrome (lower
panels), original
magnification 20x in upper and 10x in lower panels. Devices implanted to EC-
SOD treated
animals demonstrated improved biocompatibility. The unwanted host responses to
the implant
were reduced, and the recovery of protective endothelial cell layer,
indicating better healing,
was accelerated.
Aortic sections immunostained with PECAM for endothelial cells showed 90.1 ~
11.5 % recovery
of the endothelium in the EC-SOD group at 4 weeks, whereas in the LacZ control
group, the
endothelial recovery measured 35.6 t 9.4 % (P<0.05).
Figure 2.
Plasmidchart of AdBgIII with EC-SOD insert
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Definitions
Below, explanations are provided as to the meaning of some of the terms used
in the present
specification. Terms that are not specifically defined herein are to be
interpreted by the general
understanding thereof within the relevant technical field.
It must be noted that as used in this specification and the appended claims,
the singular forms
"a", "an", and "the" include plural referents unless the context clearly
dictates otherwise
A "restenosis" is here referred to as growth of connective tissue after
performing a dilating
procedure with or without an implant leading to connective tissue growth in
the tubular
structure with following narrowing of the tubular structure. The connective
tissue growth may
occur at any site in the body leading to narrowing of the tubular structure.
The connective tissue
growth comprises of either increase in some cell type in the area or increase
in the volume or
the constitutients of extracellular matrix,
A °fibrosis" is here referred to as growth of connective tissue and
formation of an acellular or
avascular layer either in an allogenic, autologous or xenogenic biological
implant or around a
synthetic implant.
25
"Hyperplastic connective tissue reaction" here defines the reaction leading to
an increase of
number of connective tissue cells and/or an increase in the volume of
extracellular matrix in the
tissue, excluding tumour formation, whereby the bulk of the connective tissue
may be
increased.
"Restenosis" and "fibrosis" may be used interchangeably if not specified in
another way.
A "medical implant" is here referred to as an implant, a device, scaffold or
prosthesis, and is
understood as an object that is fabricated for being implanted at least partly
in a mammalian.
It is intended to be in contact with bodily tissues and fluids providing at
least one contacting
surface towards the bodily tissues or fluids. A cardiovascular implant is here
referred to an
implant in a circulatory system, or an implant being connected with the blood-
flow, if not
specified in any other way. A tissue implant is here referred to as an implant
implanted in other
bodily tissues or fluids, if not specified in any other way. For example, a
medical implant may be
an implantable prosthetic device, and more particularly a cardiovascular
implant or a tissue
implant, as well as a blood-contacting medical implant, a tissue-contacting
medical implant, a
bodily fluid-contacting medical implant, an implantable medical device, an
extracorporeal
medical device, an artificial heart, a cardiac assist device, an
endoprosthesis medical device, a
vascular graft, a stent graft, a heart valve, a cardiovascular patch, a
temporary intravascular
implant, an annuloplasty ring, a catheter, a pacemaker lead, a biosensor, a
chamber for holding
_..n.. ..... ..r~,on nmnl~nt nr n hin~rhifirial nYnan.
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A medical implant is in the present context not meant to be a device that is
introduced to act as
a mere barrier for preventing undesirable formation of connective tissue
growth between soft
tissues, such as e.g. described in WO 96/29987.
An "attached transferable nucleic acid segment" referred to here, represent
the wide variety of
genetic material, which can be transferred to the tissues surrounding the
medical implant. For
example, a nucleic acid segment may be a double or single stranded DNA, or it
may also be
RNA, such as mRNA, tRNA or rRNA, encoding a protein or polypeptide. Optionally
the nucleic
acid may be in the form of anti-sense. Suitable nucleic acid segments may be
in any form, such
as naked DNA or RNA, including linear nucleic acid molecules and plasmids, or
as a functional
insert within the genomes of various recombinant viruses, such as DNA viruses
or retroviruses.
The nucleic acid segment may also be incorporated in other carriers, such as
salts, polymers,
liposomes or other viral structures. The attached transferable nucleic acid
segment is attached
to the medical implant in such a way, that it can be delivered to and taken up
by the
surrounding tissues.
The term "attached" refers to adsorption, such as physisorption,
chemisorption, ligand/receptor
interaction, covalent bonding, hydrogen bonding, or ionic bonding of the
chemical substance or
biomolecule, such as a polymeric substance, fibrin or nucleic acid to the
implant.
A "surrounding tissue" here refers to any or all cells, which have the
capacity to form or
contribute to the formation of hyperplastic connective tissue or fibrotic
reaction of the implant
surface. Surrounding tissue also refers to any or all cells that have the
capacity to form or
contribute to the formation of endothelialised surfaces either on biological
or synthetic surfaces.
This includes various tissues, such as fat, omentum, pleura, pericardium,
peritoneum muscle,
vessel wall, and fibrous tissue, but the particular type of surrounding tissue
is not important as
long as the cells are activated in a way that ultimately gives rise to the
formation of hyperplastic
connective tissue in the of the implant. "Surrounding tissue" is also used to
refer to those cells
that are located within (excluding cells in tissue chambers), are in contact
with, or migrate
towards the implant. Also, cells that upon stimulation further attract
hyperplastic connective
tissue cells or endothelial cells are considered to be surrounding tissue, as
well as cells or
tissues that arrive to the active site of cardiovascular implant connective
tissue hyperplasia,
tissue implant fibrosis or endothelialisation. "Surrounding tissue" is also
used to refer to
inflammatory cells that are either present at the implant area or arrive at
the perigraft arear
after implantation of the implant.
An °endothelium" is a single layer of flattened endothelial cells,
which are joined edge-to-edge
forming a membrane covering the inner surface of blood vessels, heart and
lymphatics.
"Endothelialisation" is here referred to the growth of endothelial cells on
all mammalian tissue or
Fi..:,a ..,...r.....~.:.... .......p~...~~ ~,f a hinmat'arial that- is mcarl
tn firm a porous or nonporous implant.
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Endothelialisation of surfaces can occur via longitudinal growth, ingrowth of
capillaries and/or
capillary endothelial cells through the pores in the implants, or seeding of
circulating endothelial
cells or endothelial precursor cells. In this disclosure, it will be used
interchangeably with the
phrase "capillary endothelialisation", to refer to the growth of endothelial
cells on substantially
5 all tissue contacting surfaces of a biomaterial, that is used to form a
porous or nonporous
implant, unless otherwise specified.
The terms "capillarisation" and "vascularisation" are here understood as the
formation of
capillaries and microcirculation on the implant surface, and they will be used
interchangeably
10 with endothelialisation, unless otherwise specified.
"Angiogenesis" and reflections thereof, such as "angiogenic", are here
referred to formation and
growth of endothelial cells in the existing mammalian tissue, such as in the
surrounding tissue.
15 A translational or a transcriptional product having "the potential to
prevent restenosis and
increase endothelialisation" of the medical implant, is here understood as a
chemical substance
or biomolecule, preferably a hormone, a receptor or a protein, which, as a
result of its activity,
can reduce formation of excessive connective tissue and induce
endothelialisation or
capillarisation of the medical implant.
"Porosity" and reflections thereof, such as "pores" and "porous", are here
referred, if not
otherwise specified, to a biomaterial having small channels or passages, which
start at a first
surface and extend substantially through to a second surface of the
biomaterial.
~5 "Surface" refers to the interface between the biomaterial and its
environment. It is intended to
include the use of the word in both its macroscopic sense (e.g. two major
faces of a sheet of
biomaterial), as well as in its microscopic sense (e.g. lining of pores
traversing the material).
The term compartment refers to any suitable compartment, such as for example a
vial or a
package.
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16
Detailed description of the invention
In a first aspect, the present invention relates to administration of nucleic
acid in a biologically
compatible medium to a synthetic graft, characterised in that said nucleic
acid encodes a
translational or transcriptional product leading to production of
extracellular superoxide
dismutase (EC-SOD) protein capable of reducing hyperplastic connective tissue
growth in vivo.
In another aspect, the present invention relates to administration of nucleic
acid in a biologically
compatible medium and an implant, characterised that said the nucleic acid
encodes a
translational or transcriptional product leading to production of
extracellular superoxide
dismutase (EC-SOD) protein capable of reducing hyperplastic connective tissue
growth in vivo in
the tissue surrounding the implant.
Extracellular superoxide dismutase (EC-SOD) is secreted antioxidative enzyme,
which is widely
expressed throughout the body and is the major SOD isoenzyme in plasma. Vessel
walls, lung,
kidney, thyroid gland and epidymis are shown to be the primary expression
sites for EC-SOD.
About 50 % of total SOD amount in human aorta is EC-SOD. In most tissues EC-
SOD represents
only a minor part of the total SOD activity, which suggests that EC-SOD has a
significant
physiological role in the redox balance of the vascular wall. Adenovirus
mediated EC-SOD
gene/cDNA transfer resulted in a significant inhibition of neointima formation
in rabbit aortas
after balloon denudation (see e.g. W002/087610). The therapeutic effect
affects the whole
abdominal aorta, suggesting a systemic effect. EC-SOD thus is shown to be an
efficient
therapeutic molecule to prevent restenosis.
In another aspect, the present invention relates to a medical device with
improved biological
properties for an at least partial contact with blood, bodily fluids and/or
tissues when introduced
in a mammalian body, which device comprises a core and a nucleic acid present
in a biologically
compatible medium, characterised in that said nucleic acid encodes a
translation or transcription
product capable of inhibiting inflammation in the surrounding tissue and/or
reducing
hyperplastic connective tissue reaction and promoting endothelialisation in
vivo at least partially
on a synthetic surface of said core. The nucleic acid is provided in a way
whereby transfer
thereof into cells of tissue surrounding the implant is allowed. In the
present specification, it is
to be understood that the term °'introduced in a mammalian body" is
used in a broad sense to
encompass both devices that are totally included in a body and devices which
are only in part
introduced, but wherein at least one surface made from a synthetic material is
in contact with
blood, bodily fluids and/or tissues of said body.
The reduction of hyperplastic connective tissue growth and induction of
endothelialisation
achieved according to the invention offers many of the advantages of a native
structures.
Connective tissue hyperplasia comprises both proliferation of cells in the
respective tissue and
....~,rlmr.~inn of ovtraralliilar mai-rix Fnr~ntnelium is a single layer of
flattened cells, which are
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17
joined edge to edge forming a membrane of cells covering the inner surface of
blood vessels,
heart and lymphatics. In theory, endothelialisation of the graft can occur
either via longitudinal
growth from the anastomosis area (transanastomotic), ingrowth of capillaries
and/or capillary
endothelial cells through the synthetic surface, such as a graft wall, and
into porosities
(transinterstitial), or seeding of circulating endothelial precursor cells. In
the transinterstitial
migration through the pores, the endothelial cells originate from capillaries
through attachment,
spreading, inward migration and proliferation.
Thus, even though efforts have been made in the prior art to avoid restenosis
and the resulting
narrowing of biological tubular structures and connections between polymeric
surfaces and
bodies own vessels, such efforts have not proved satisfactory with smaller
vessels, wherein
hyperplasia and thrombosis have caused substantial problems. Also, several
efforts have been
made in prior art to reduce thrombogenicity without result. Surprisingly, the
present invention
provides a gene transfer product, which reduces restenosis in biological
tissues such as blood
vessels after an interventional procedure, and the present invention also
provides a novel
device, which is protected from restenosis. The present invention provides a
versatile
technology useful with a large range of implants, and surprisingly also
efficient with small size
synthetic vessel sections and intravascular implants that have previously been
known to develop
connective tissue hyperplasia and occlude. The reduction in restenosis
achieved according to the
invention has not been observed to form in humans in long term studies
according to the prior
art. The present invention also provides a gene transfer product which
increases
endothelialisation on implant surfaces.
In one embodiment of the device according to the invention, the nucleic acid
is present in the
biologically compatible medium associated with adenovirus. In an alternative
embodiment, the
nucleic acid in introduced in any other viral vector selected from the group
consisting of
retrovirus, lentivirus, Sendai virus and adeno-associated virus. In another
embodiment the
nucleic acid is present as naked DNA. In yet another embodiment, the nucleic
acid is present in
a liposome.
The use of gene transfer has been postulated for the treatment or prevention
of diseases in
several publications. Gene therapy entails the use of genetic material as the
pharmacological
agent. While originally recognised as a means for treating hereditary
diseases, gene therapy is
now understood as a powerful tool for delivering therapeutic mRNA or proteins
for local and/or
systemic use. There are two approaches in gene therapy: ex vivo and in vivo.
In the ex vivo
approach, cells removed from the host are genetically modified in vitro before
they are returned
to the host, and in the in vivo approach the genetic information itself is
transferred directly to
the host without employing any cells as a vehicle for transfer. The gene can
be targeted
depending on where they are needed, either in stem cells or in situ. The
principle for gene
therapy is that the cell functions are regulated through the alteration of the
transcription of
genes and the production of a gene transcription product, such as a
polynucleotide or a
r~_ .~..i........i....~.:.1~. ...-1-1-,~ r,r,l~incntirla than intPrc7CtS Wlth
other cells to regulate the
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18
function of that cell. This transcription change is accomplished with gene
transfer. Losordo et al.
Circulation 1994, 89:785-792 have shown that gene products that are secreted
may have
profound biological effects even when the number of transduced cells remains
low in contrast to
genes that do not encode a secretory signal. For genes expressing an
intracellular gene product
a much larger cell population might be required for that intracellular gene
product to express its
biological effects and subsequently more efficient transfection may be
required (Isner et al.,
Circulation, 1995, 91:2687-2692). To illustrate the use of gene therapy this
far, genes have e.g.
been transferred to adipocytes having a particular utility with respect to
diseases or conditions
that can be treated directly by in vivo gene transfer to adipocytes. Transfer
of nucleic acids into
bone tissue has been shown in situ and the use of infected mesothelium either
in situ or after
isolation as therapeutic resource has also been described.
An extremely wide variety of genetic materials can be transferred to the
surrounding tissues
using the compositions and methods of invention. For example, the nucleic acid
may be DNA
(double or single stranded) or RNA (e.g. mRNA, tRNA, rRNA). It may also be a
coding nucleic
acid, i.e. one that encodes a protein or a polypeptide, or it may be an anti-
sense nucleic acid
molecule, such as anti-sense RNA or DNA, that may function to disrupt gene
expression.
Alternatively, it may be an artificial chromosome. Thus, the nucleic acids may
be genomic
sequences, including exons or introns alone, or exons and introns, or coding
DNA regions, or
any construct that one desires to transfer to the tissue surrounding the
prosthesis to reduce
restenosis, fibrosis and inflammation or promote endothelialisation. Suitable
nucleic acids may
also be virtually any form, such as naked DNA or RNA, including linear nucleic
acid molecules
and plasmids, or a functional insert within the genomes of various recombinant
viruses,
including viruses with DNA genomes, and retroviruses. The nucleic acid may
also be
incorporated in other carriers, such as liposomes and other viral structures.
The nucleic acid
backbone may also be altered or replaced in order to modify the properties
such as stability or
transfection efficacy.
Chemical, physical, and viral mediated mechanisms are used for gene transfer.
Several different
vehicles are employed in gene transfer. There are a number of viruses, live or
inactive, including
recombinant viruses, that can be used to deliver a nucleic acid to the
tissues, such as
retroviruses, lentivirus, adenoviruses (e.g. U.S. 5,882,887, U.S. 5,880,102)
and
hemagglutinating viruses of Japan (HVJ or Sendai virus) (U.S. 5,833,651).
Retroviruses have
several drawbacks in vivo which limit their usefulness. They provide a stable
gene transfer, but
current retroviruses are unable to transduce nonreplicating cells. The
potential hazards of
transgene incorporation into the host DNA are not warranted if short-term gene
transfer is
sufficient. Replication deficit adenoviruses are highly efficient and are used
in a wide variety of
applications. The adenovirus enters the cell easily through receptor
interactions, which has been
used as a means for transporting macromolecules into the cell. Non-viral
nucleic acids can be
packaged within the adenovirus, either as a substitute for, or in addition to
normal adenoviral
components. Non-viral nucleic acids can also be either linked to the surface
of the adenovirus or
._ .. ~.....r.....,..1.... ........ecc rn-intcrn~licorl anrl taltaan alnnn ac
a Cr7rOO In the receptor-endosome
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complex. Adenovirus-based gene transfer does not result in integration of the
transgene into
the host genome, and is therefore not stable. It also transfects nonreplicable
cells, which makes
adenovirus as an effective vector. Other examples of used viral vectors are
adeno-associated
viruses (AAV), herpes viruses, vaccinia viruses, lentivirus, poliovirus, other
RNA viruses and
influenza virus (Mulligan, Science 1993; 260: 926-32; Rowland, Ann Thorac
Surgery 1995,
60:721-728). DNA can also be coupled to other types of ligands promoting its
uptake and
inhibiting its degradation (e.g. 5,972,900, 5,166,320, 5,354,844, 5,844,107,
5,972,707) or
directing it to nuclear localisation (Luo & Saltzman, Nat Biotech; 2000, 18:33-
37). It can also be
coupled to a so-called cre-lox system (Sauer & Henderson, Proc Natl Acad Sci.;
1988, 85:5166).
Naked DNA can also be given and the empirical experience is consistent with
that double
stranded DNA is minimally immunogenic and is unlikely elicit an immunologic
reaction.
Plasmid DNA may be administered either in a simple salt solution referred as
naked DNA or
complexed with a carrier or an adjuvant. In the latter case nucleic acids can
be complexed with
polycations, proteins or other polymers, dendrimers, incapsulated or
associated with liposomes,
or coated on colloidal particles. The traditional chemical gene transfer
methods are calcium
phosphate co-precipitation, carbohydrates (heparansulfate, chitosan),
poloxamers, PEI, DEAE-
dextran, polymers (U.S. 5,972,707), and liposome-mediated transfer (for
example U.S.
5,855,910, U.S. 5,830,430, U.S. 5,770,220), and the traditional physical
methods are
microinjection, electroporation (U.S. 5,304,120), iontophoresis, a combination
of iontophoresis
and electroporation (U.S. 5,968,006), ultrasound and pressure (U.S. 5,922,687)
(Luo &
Saltzman, Nat Biotech; 2000, 18:33-37, Rowland). Transfection efficiency may
be improved by
any of the known of pharmaceutical measures recognised by skilled in the art
The invention may be employed to promote expression of EC-SOD in tissues
surrounding an
implant, and to impart a certain phenotype, and thereby promote prosthesis
protection from
hyperplastic connective tissue growth or fibrosis. This expression could be
increased expression
of a gene that is normally expressed (i.e. over-expression), or it could be
the expression of a
gene that is not normally associated with tissues surrounding the prosthesis
in their natural
environment. Alternatively, the invention may be used to suppress the
expression of a gene
which normally inhibits a gene expression i.e. gene suppression may be a way
of expressing a
gene that encodes a protein that exerts a down-regulatory function.
Thus, the nucleic acid used with the device according to the present invention
encodes
transcription and/or translation products capable of inhibiting connective
tissue hyperplasia and
tissue fibrosis and/or promoting or stimulating endothelialisation in vivo,
i.e. it is also an
antirestenotic or angiogenic factor. Thus, in all embodiments, the nucleic
acid encodes an EC-
SOD protein or polypeptide.
In another embodiment EC-SOD protein may be used to instead of using EC-SOD
encoding
genes.
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In another embodiment, the biologically compatible medium is a biostable
polymer, a
bioabsorbable polymer, a biomolecule, a hydrogel polymer or fibrin. In a
specific embodiment,
the medium is a mucin composition.
5 The synthetic surface of the device according to the invention may be either
non-porous or
porous. Thus, porous, as well as nonporous, implant materials may be used to
produce the
device, depending on the implant embodiment. For example, graft porosity has
been shown to
be of importance in vascular graft endothelialisation in animals (Wesolowski,
Thorac Cardiovasc
Surgeon 1982;30:196-208, Hara, Am. J. Surg.;1967;113:766-69). In the context
of sutures,
10 porous sutures have been described to promote tissue ingrowth into the
sutures or promote
endothelialisation of the sutures (U.S. 4,905,367, U.S. 4,355,426). In porous
grafts, such as
vascular grafts, capillary and endothelial cell growth is allowed through
pores, and the porosity
thereof may be from 0 mm to 2000 mm.
15 In one embodiment, the nucleic acid has been attached to the core by ionic
or covalent bonding.
In one advantageous embodiment, the nucleic acid is present in a reservoir
separate from said
core enabling a successive delivery thereof to a mammalian body. The tissue
surrounding an
implanted device can e.g. be pleura, pericardium, peritoneum, fascia, tendon,
fat, omentum,
20 fibrous, muscle, skin, or any other tissue in which inhibition of
hyperplastic connective tissue
growth, restenosis and fibrosis are required.
A plausible theory for the underlying mechanism, giving rise to at least parts
of the advantages
of the present invention, is that genes expressing anti-restenotic EC-SOD are
attached to the
implant or administered in the tissue surrounding the device. The cells in the
surrounding tissue
become transfected and inhibit restenosis and result in reduction of
connective tissue growth in
the tissue, a process that results in less hyperplastic or fibrotic tissue
reaction with the earlier
described advantages of such a tissue.
The surface of the present device may be treated in a variety of ways, in all
or parts thereof,
e.g. by coating or adding other pharmaceutical substances, as is discussed in
more detail below
in the experimental section in the general disclosure of materials and
methods. The optimal
internodal distance for PTFE grafts has been approximately 60 um.
The present device is useful in a wide variety of contexts and depending on
the intended use, it
may be made from a biomaterial selected from the group of non-soluble
synthetic polymers,
metals and ceramics with or without modification of the prosthesis surfaces.
Thus, in one embodiment, the device is an implant made of a biocompatible
material selected
from the group consisting of metal, titanium, titanium alloys, tin-nickel
alloys, shape memory
alloys, aluminium oxide, platinum, platinum alloys, stainless steel, MP35N,
elgiloy, stellite,
_,.....i,.~.;.. ..~,.h~r, cih~ar ~~rhr,n nlaccv rarhnn nnlvmar nnlvafYllde.
oolvcarbonate, pOlyether,
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polyester, polyolefin, polyethylene, polypropylene, polystyrene, polyurethane,
polyvinyl
chloride, polyvinylpyrrolidone, silicone elastomer, fluoropolymer,
polyacrylate, polyisoprene,
polytetrafluoretylene, rubber, ceramic, hydroxyapatite, human protein, human
tissue, animal
protein, animal tissue, bone, skin, laminin, elastin, fibrin, wood, cellulose,
compressed carbon
and glass.
Thus, the device may be a medical implant selected from the group consisting
of a blood-
contacting medical implant, a tissue-contacting medical implant, a bodily
fluid-contacting
medical implant, an implantable medical device, an extracorporeal medical
device, an
endoprosthesis medical device, a vascular graft, an endovascular implant, a
pacemaker lead, a
heart valve, temporary, intravascular implant, a catheter, pacemaker lead,
biosensor or artificial
organ. In one specific embodiment, the device is a cardiovascular implant,
such as an artificial
part of a blood vessel, or an endovascular implant. In general terms, the
present device may be
used as an implant used for replacement of a part of a mammalian body, where
said implant is
adapted for an at least partial contact with blood, bodily fluids and/or
tissues. Further, the
present device is useful as a tissue implant or a biosensor. In alternative
embodiments, the
present device may be any other bioartificial implant that provides a
metabolic function to a
host, such as a pump for the delivery of insulin or a biosensor to sense the
glucose levels etc.
In fact, the present device may be virtually any one of a variety of devices,
which protect
tissues or cells producing a selected product from the immune system have been
explored for
implant in a body, such as extravascular diffusion chambers, intravascular
diffusion chambers,
intravascular ultrafiltration chambers, and microencapsulated cells. Cells can
be derived from
other species (xenografts), they can be from the same species but different
individuals
(allografts), sometimes they are previously isolated from the same individual
but are modified
(autografts) or are of embryonal origin. Bioartificial implants are designed
to provide a needed
metabolic function to a host, either by delivering biologically active
moieties, such as insulin in
diabetes mellitus, or removing harmful substances. Membranes can be
hydrophobic, such as
PTFE and polypropylene, or hydrophilic, such as PAN/PVC and cuprophane.
More specifically, implants encompassed by the invention include, but are not
limited to,
cardiovascular devices, such as artificial vascular prosthesis, cardiovascular
patches, stent
grafts, prosthetic valves, artificial hearts, cardiac assist devices,
anastomotic devices, graft
connectors, annuloplasty ring, indwelling vascular catheters, pacemaker wires,
anti-embolism
filters, stents and stent grafts for other indications, and tissue implants,
such as chambers
holding living cells for implantation, biosensors, surgical suture materials,
surgical nets,
pledgets and patches, tracheal cannulas, bioartificial organs, surgical
implants, plastic surgical
implants and orthopedic implants. It is anticipated that the herein described
procedures may
lead to the development of other artificial organs or devices.
In a second aspect, the invention provides a method for producing an
implantable medical
T'h~ rlo"i.-m~~n ho fnrmorl Ptf'H1PY nV f'H1P nradYea~'Ind Of a biomaterial
with genes, and
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22
then fabricating the device from the treated biomaterial, or by first
fabricating the device and
then treating the exposed surfaces of the device.
The methods described in the present invention generally comprise to contact
the tissue,
surrounding the vascular or tissue implant, with a composition comprising a
nucleic acid, in a
manner effective to transfer said nucleic acid into the tissue, and to inhibit
hyperplastic tissue
growth and promote endothelialisation of the vascular grafts, cardiovascular
patches, stent
grafts, heart valves, indwelling vascular catheters, cardiac assist devices
and artificial hearts, or
to promote vascularisation of tissue implant surfaces. The tissue may be
wrapped around the
vascular- or tissue implant - nucleic acid composition before implantation to
the body.
Alternatively, the nucleic acid sequence-prosthesis composition may be
implanted in the tissues,
or the nucleic acid may be applied to the implantation site before or after
the prosthesis
implantation, in order to effect, or promote, nuclear acid transfer into the
surrounding tissues in
vivo. In the transferring of nucleic acids into surrounding tissues, the
preferred method involves
to first add the genetic material to the tissue compatible medium, to
impregnate the prosthesis
with the nucleic acid-medium composition, and then to use the impregnated
prosthesis to
contact an appropriate tissue site. Alternatively, the tissue compatible
medium can first be
administered on the implant, and then the nucleic acid is added, whereafter
the nucleic acid-
prosthesis composition is applied to the implantation site. Alternatively
nucleic acid is
administered to the tissues surrounding the implant, whereafter the implant is
implanted, or the
implant is first implanted, whereafter the nucleic acid is administered on the
implant or to the
tissues surrounding the implant. Also, an impregnated implant can be used in
combination with
administration of nucleic acid in the tissues surrounding the implant before
or after
implantation. When surrounding tissue is scarce and have a low amount of
cells, the
impregnated prosthesis can be surgically wrapped in a tissue of higher cell
content before
implantation. Some of the cardiovascular implants, such as vascular
prosthesis, cardiovascular
patch and stent grafts, have a porosity that is high enough to allow growth of
endothelial cells
through the pores, and some other cardiovascular implants, such as heart
valves are non-
porous.
More specifically, the method according to the invention for inhibiting
restenosis of medical
implants by transferring a nucleic acid to the surrounding tissues may be
disclosed as a method
of improving a mammalian, e.g. a human, body's acceptance of a synthetic
surface, which
method comprises introducing a device comprising a synthetic surface in the
body with an at
least partial contact with blood, bodily fluids and/or tissues and
administering a nucleic acid
present in a biologically compatible medium to the surroundings thereof. The
method is
characterised in that the nucleic acid encodes a translation or transcription
product capable of
inhibiting de novo stenosis or restenosis in vivo, said administration of
nucleic acid being
performed before, simultaneously as or after the introduction of the device in
the body. As
q.p discussed above in relation to the device according to the invention, the
nucleic acid can e.g. be
administered in naked form, in a viral vector such as a retrovirus, a Sendai
virus, an adeno-
..........:..~-.,.I ~..,-..~ ,..- ~., ~rlor,nvirme nr in a linncnma
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23
Depending on the nature of the device, i.e., the condition of the patient who
is to receive the
implant, the nucleic acid may encode an EC-SOD protein or a polypeptide or a
protein inhibiting
the downregulation of EC-SOD production. Also, a substance which promotes EC-
SOD
production can be used. Also, EC-SOD protein may be administered instead of a
nucleic acid.
In one embodiment, the nucleic acid or protein is administered to the
surroundings of the
device, i.e. the tissue, before introduction thereof in a mammalian body.
Alternatively, the
nucleic acid or protein is administered to such surroundings after the
introduction thereof. As
the skilled in this field will realise, combinations of such administrations
are possible, such as a
first administration of a certain amount to the surroundings, the introduction
of the device, and
thereafter one or more additional administration, either according to a
predetermined scheme or
depending on the body's acceptance thereof and the rate of growth of the new
endothelial layer
on the synthetic surface.
In another embodiment, the nucleic acid or protein is administered or attached
to the device
before introduction thereof in a mammalian body. In a specific embodiment,
this is achieved by
attaching the nucleic acid or protein to the core by ionic or covalent
bonding. This embodiment
may if appropriate be combined with any one mentioned above, so as to provide
a method
wherein the device has been pretreated with protein or nucleic acid, while the
tissue
surrounding the device is later supplemented with further additions of nucleic
acid or protein
present in a suitable carrier. Also, the treatment with protein and nucleic
acid can be combined
in different variations. In one embodiment, which is advantageous due to its
simplicity, said
carrier is sterile water or a sterile aqueous solution. The proteins and
nucleic acids of the
invention may also additionally be systemically delivered in any suitable
pharmaceutical
formulations comprising a pharmaceutically acceptable carrier. Examples
include aqueous and
non-aqueous sterile injection solutions, which may contain antioxidants,
buffers, bacteriostats,
bactericidal antibiotics; and aqueous and non-aqueos sterile suspensions,
which may include
suspending agents and thickening agents. The formulation may be presented in
unit dose or
multi-dose containers, for example sealed ampoules and vials, and may be
stored in a frozen or
freeze dried (lyophilized) condition requiring only the addition of the
sterile liquid carrier, for
example water for injection, immediately prior to use.
EC-SOD protein or nucleic acid is administered with a view to preventing or
treating de novo
stenosis or preventing or treating restenosis. It can however also be used to
increase
endothelialisation.
In alternative embodiments of the present method, the biologically compatible
medium is a
biostable polymer, a bioabsorbable polymer, a biomolecule, a hydrogel polymer
or fibrin.
Thus, as mentioned above and as further detailed below, the device used in the
present method
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emrr,er~~ n rloviro ranlarinn a naY1' of fi~'1P 17~C~V_ SIJCh
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24
as a vessel, a device for introduction into a human body, such as an
endovascular implant, a
tissue implant, a biosensor or other similar equipment.
In summary, with respect to the transfer and expression of therapeutic
proteins or genes
according to the present invention, the ordinary skilled artisan is aware that
different genetic
signals and processing events control levels of nucleic acids and
proteins/peptides in a cell, such
as transcription, mRNA translation, and post-translational processing. These
steps are affected
by various other components also present in the cells, such as other proteins,
ribonucleotide
concentrations and the like.
Accordingly, in general terms, the present invention concerns anti-stenotic,
anti-restenotic and
anti-fibrotic treatments and devices, which devices may be generally
considered as molded or
designed vascular implant-gene compositions. The devices of the invention are
naturally a
tissue-compatible implant in which one or more anti-restenotic or anti-
fibrotic EC-SOD gene or
EC-SOD proteins are associated with the implant. The combination of EC-SOD
gene or protein
and implant components is decided by the skilled in this field in order to
render the device
capable of inhibiting stenosis, restenosis or fibrosis, or stimulating
angiogenesis when
implanted. Devices according to the invention may be of virtually any size or
shape, so that
their dimensions are adapted to fit the implantation site in the body.
The following section is provided to illustrate the present invention and
should not be
interpreted as limiting the invention in any way. References given below and
elsewhere in the
present application are hereby included by reference.
This section describes alternative materials and methods that may be utilised
in this context in
order to offer as many possibilities as possible within the scope of the
appended claims.
Thereafter, under the headline examples, specific disclosures of the
experiment carried out to
describe the effect of the invention and the advantages thereof will be
provided.
EC-SOD as implant endothelialisation increasing or restenosis reducing gene
As used herein, the term °restenosis or fibrosis inhibiting gene and
endothelialisation promoting
gene" is used to refer to a gene or a DNA coding region that encodes a
protein, a polypeptide or
a peptide, that is capable of promoting, or assisting in promotion of EC-SOD
mediated inhibition
of restenosis and fibrosis or EC-SOD mediated endothelialisation or
vascularisation, or that
decreases the rate of EC-SOD mediated inhibition of restenosis or fibrosis, or
increases the rate
of EC-SOD mediated endothelialisation or vascularisation, or EC-SOD mediated
inhibition of
macrophage infiltration. The terms inhibiting and reducing or promoting,
inducing and
stimulating are used interchangeably throughout this text, to refer to direct
or indirect
processes that ultimately result in less formation connective tissue to the
site of tissue trauma
or implantation of device or increase the formation of implant endothelium
and/or capillaries, or
... .... ...,......,....a ....~,. .,f o.,.~.,+~holialieal-inn anrl/nr
ranillarlSatlOn either with Or without
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implantation of a device. Thus, an implant restenosis or fibrosis inhibiting
gene or
endothelialisation promoting gene is a gene, which, when it is expressed,
causes the phenotype
of the cell to change, so that the cell either differentiates, stimulates
other cells to differentiate,
attracts restenosis inhibiting genes or implant endothelialisation promoting
cells, or otherwise
5 functions in a manner that ultimately gives rise to new implant endothelium
through an increase
in EC-SOD either locally or systemically.
In general terms, a restenosis inhibiting gene or vascular implant
endothelialisation promoting
gene may also be characterised as a gene capable of reducing formation of
connective tissue or
10 capable of stimulating the growth of endothelium in the tissues surrounding
vascular prosthesis
and thereby reducing restenosis and fibrosis or promoting the
endothelialisation or the
vascularisation of the traumatised tissue or of the implant through increase
in EC-SOD. Thus, in
certain embodiments the methods and compositions of the invention may be to
stimulate
growth of endothelium in vascular prosthesis itself and also in tissues
surrounding it.
A variety of anti-restenotic factors are now known, of which all are suitable
for use in connection
with the present invention. Anti-restenotic genes and their encoding proteins,
include, for
example, hormones, many different growth factors and cytokines, growth factor
receptor genes,
enzymes and polypeptides. Examples of suitable anti-restenotic factors include
those of the
VEGF and FGF-family, TGF-(3 Type II receptor, NOS and HGF.
The preferred anti-restenotic gene product is EC-SOD. There is a considerable
variation in the
terminology currently employed in the literature referring to genes and
polypeptides. It will be
understood by those skilled in the art, that all genes that cause increase in
an active EC-SOD
protein are considered for use in this invention, regardless of the differing
terminology that may
be employed.
The DNA sequences for several EC-SOD genes have been described both in
scientific articles
(Genomics 22; 162-171, 1994, Hjalmarsson et al., Proc. Natl. Acad. Sci. USA
84; 6340-
6344,1987. Laukkanen et al., Arteriosclerosis, Thrombosis and Vascular Biology
19; 2171-2178,
1999. Laukkanen et al., Gene, 254, 173-179, 2000), US Patent 5,788,961 and in
WO 87/01384.
As disclosed in the above patents, and known to those skilled in the art, the
original source of a
recombination gene or a DNA to be used in a therapeutic regimen need not be of
the same
species as the animal to be treated. In this regard, it is contemplated that
any recombinant
anti-restenotic or anti-fibrotic gene may be employed to reduce excessive
connective tissue
formation or promote vascular prosthesis endothelialisation in a human subject
or an animal,
such as e.g., horse. Particularly preferred genes are those from human, as
such genes are most
preferred for use in human treatment regiments. Recombinant proteins and
polypeptides
encoded by isolated DNA and genes are often referred to with the prefix r for
recombinant and
rh for recombinant human.
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26
To prepare an anti-restenotic or anti-fibrotic gene, gene segment or cDNA, one
may follow the
teachings disclosed herein and also teachings of any of the patents or
scientific documents
referred to in the reference list or in the scientific literature. For
example, one may obtain EC-
SOD segments by using molecular biological techniques, such as polymerase
chain reaction
(PCR), or by screening a cDNA or genomic library, using primers or probes with
sequences
based on the above nucleotide sequence. The practice of such a technique is a
routine matter
for those skilled in the art, as taught in various scientific articles, such
as Sambrook et al..,
incorporated herein by reference. The anti-restenotic or anti-fibrotic gene
and DNA segments
that are particularly preferred for use in the present compositions and
methods, is EC-SOD or
parts of its coding or non-coding sequence. It is also contemplated that one
may clone further
genes or cDNA that encode a protein or polypeptide that increases EC-SOD
expression and
protein production or decreases EC-SOD downregulation. The techniques for
cloning DNA, i.e.
obtaining a specific coding sequence from a DNA library that is distinct from
other portions of
DNA, are well known in the art. This can be achieved by, for example,
screening an appropriate
DNA library. The screening procedure may be based on the hybridisation of
oligonucleotide
probes, designed from a consideration of portions of the amino acid sequence
of known DNA
sequences encoding related anti-restenotic proteins. The operation of such
screening protocols
are well known to those skilled in the art and are described in detail in the
scientific literature,
for example Sambrook et al.. (Sambrook et al., Molecular Cloning: a Laboratory
Manual, 1989,
Cold Spring Lab Press; Inniste et al., PCR strategies, 1995, Academic Press,
New York).
EC-SOD genes, with sequences that vary from those described in the literature,
are also
encompassed by the invention, as long as the altered or modified gene still
encodes a protein
that functions to stimulate surrounding tissues of cardiovascular or tissue
implants, in any direct
or indirect manner. These sequences include those caused by point mutations,
those due to the
degeneracy of the genetic code or naturally occurring allelic variants, and
further modifications
that have been introduced by genetic engineering such as a hybrid gene, i.e.
by the hand of
man.
Techniques for introducing changes in nucleotide sequences that are designed
to alter the
functional properties of the encoded proteins or polypeptides are well known
in the art. Such
modifications include the deletion, insertion or substitution of bases, and
thus, changes in the
amino acid sequence. Changes may be made to increase the EC-SOD activity of a
protein, to
increase its biological stability or half-life, to decrease its degradation,
increase its secretion,
change its glycosylation pattern, and the like. All such modifications of the
nucleotide sequences
are encompassed by this invention.
Furthermore, the present invention is of course not strictly limited to the
use of products of the
EC-SOD gene, but also encompasses any recombinant or synthetic compound that
effectively
q.p mimics the biological effects of EC-SOD. Special focus is herein laid on
EC-SOD mimics that are
in contrast to mimicking any of the other SOD family members, display their
activity in the
__.i____m..~_- _.....,.... ,....-.-.,..r,.~linn tha tarnoi- rally
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It will also be understood that one, or more than one, anti-restenotic or anti-
fibrotic gene may
be used in the methods and compositions of the invention. The nucleic acid
delivery may thus
entail the administration of one, two, three, or more anti-restenotic or anti-
fibrotic genes or
proteins. The maximum number of genes or proteins that may be applied is
limited only by
practical considerations, such as the effort involved in simultaneously
preparing a large number
of gene constructs or even the possibility of eliciting an adverse cytotoxic
effect. The particular
combination of genes may be two or more anti-restenotic genes, or it may be
such that a
growth factor inhibiting gene is combined with a hormone gene. A hormone or
growth factor
gene may even be combined with a gene encoding a cell surface receptor capable
of interacting
with a polypeptide product of the first gene. Also, an EC-SOD gene can be
combined with genes
encoding antisense products intracellular aptamer molecules or ribozymes. In
using multiple
genes, the genes may be combined on a single genetic construct under control
of one or more
promoters, or they may be prepared as separate constructs of the same or
different types.
Thus, an almost endless combination of different genes and genetic constructs
may be
employed. Certain gene combinations may be designed to, or their use may
otherwise result in,
achieving synergistic effects on reducing excessive connective tissue
formation and fibrosis or
angiogenesis and endothelialisation. Any of all those combinations are
intended to fall within the
scope of the present invention. A person skilled in the art readily would be
able to identify likely
synergistic gene combinations or gene protein combinations. Another gene may
encode a
protein that inhibits the growth of neointimal cells, for example inducible
nitric oxide synthase
(iNOS) or endothelial cell nitric oxide synthase (ecNOS). Proteins or products
of enzyme
proteins that inhibit thrombosis, e.g. prostacyclin, tissue plasminogen
activator (tPA),
urokinase, and streptokinase, are also of interest for co-transfection. Also
EC-SOD may be
combined with other genes, which later inhibit the overexpression of EC-SOD or
modulate EC-
SOD expression at any level such as transcription or translation.
Administration may occur
before, simultaneously or after administration of the EC-SOD nucleic acid.
It will also be understood that the nucleic acid or gene could, if desired, be
administered in
combination with further agents, such as, e.g. proteins, polypeptides, aptamer
oligonucleotides,
ribozymes, transcription factor decoy oligonucleotides or various
pharmacologically active
agents, growth factors inhibiting restenosis formation, substances such as
heparin to inhibit
excessive connective tissue growth etc. Also, immunosuppressants, anti-
inflammatory and other
anti-restenosis substances may be used. As long as genetic material or protein
forms part of the
composition, there is virtually no limit for including other components, given
that the additional
agent does not cause a significant adverse effect upon contact with the target
cells or tissues.
The nucleic acids or protein may thus be delivered along with various other
agents. Also, nucleic
acid or protein may be delivered along with an implant giving radiation,
ultrasound, and electric
current or light energy to the surrounding tissue to excert a specific effect
along with anti-
fibrosis.
It will also be understood that the nucleic acid or gene can be administered
in combination with
.. ....."...i~....,~,...... ..~y ~oorlinn nr cnrirlinn nrnr~t~llrP. or
simultaneous administration of stem cells
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28
or stimulation of stem cell population at the site of implant. It can also be
combined with
simultaneous seeding or sodding with genetically modified cells.
Gene constructs and nucleic acid
As used herein, the terms gene and nucleic acid are both used to refer to a
DNA molecule that
has been isolated, and are free of total genomic DNA of a particular species.
Therefore, a gene
or a DNA encoding EC-SOD refers to a DNA that contains sequences encoding an
EC-SOD
protein, but it is in isolated from, or purified free from, total genomic DNA
of the species from
which the DNA is obtained. Included within the term DNA are DNA segments and
smaller
fragments of such segments aptamers, and also recombinant vectors, including
for example
plasmids, cosmids, artificial chromosomes, phages, lentivirus, retroviruses,
adenoviruses, and
the like.
The term gene is used for simplicity to refer to a functional protein- or
peptide-encoding unit. As
will be understood by those skilled in the art, this functional term includes
both genomic
sequences and cDNA sequences. Of course, this refers to the DNA segment as
originally
isolated, and does not exclude genes or coding regions, such as sequences
encoding leader
peptides or targeting sequences, later added to the segment by man.
This invention provides novel ways to utilise various EC-SOD protein and known
EC-SOD DNA
segments and recombinant vectors. Many such vectors are readily available.
However, there is
no requirement for a highly purified vector to be used, as long as the coding
segment employed
encodes an EC-SOD protein, and does not include any coding or regulatory
sequences that
would have an adverse effect on the tissues. Therefore, it will also be
understood that useful
nucleic acid sequences may include additional residues, such as additional non-
coding
sequences flanking either of the 5' or 3'portions of the coding region or may
include various
internal sequences, i.e. introns, which are known to occur within genes.
After the identification of an appropriate EC-SOD encoding gene or DNA
molecule, it may be
inserted into any one of the many vectors currently known in the art. In that
way it will direct
the expression and production of the EC-SOD when incorporated into a tissue
surrounding the
implant. In a recombinant expression vector, the coding portion of the DNA
segment is
positioned under the control of a promoter. The promoter may be in a form that
is naturally
associated with an EC-SOD gene. Coding DNA segments can also be positioned
under the
control of a recombinant, or heterologous, promoter. As used herein, a
recombinant or
heterologous promoter is intended to refer to a promoter that is not normally
associated with an
EC-SOD gene in its natural environment. Such promoters may include those
normally
associated with other anti-restenotic genes, and/or promoters isolated from
any other bacterial,
viral, eukaryotic, or mammalian cell. Naturally, it will be important to
employ a promoter that
effectively directs the expression of the DNA segment in tissues. The use of
recombinant
___.,.........,.,. ~.~ ".h~o"A r,rntPin PxnrPCCinn iS aeneraIIV known to those
skilled in the art of
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29
molecular biology (Sambrook et al.). The promoters used may be constitutive,
or inducible, and
can be used under the appropriate conditions to direct high level or regulated
expression of the
introduced DNA segment. The currently preferred constitutive promoters are for
example CMV,
RSV LTR, immunoglobulin promoter, SV40 promoter alone, and the SV40 promoter
in
combination with the SV40 enhancer, and regulatable promoters such as the
tetracyclin-
regulated promoter system, or the metalothionine promoter. The promoters may
or may not be
associated with enhancers, where the enhancers may be naturally associated
with the particular
promoter or associated with a different promoter. A termination region is
provided 3 ~ to the EC-
SOD coding region, where the termination region may be naturally associated
with the
cytoplasmic domain or may be derived from a different source. A wide variety
of termination
regions may be employed without adversely affecting expression. After various
manipulations,
the resulting construct may be cloned, the vector isolated, and the gene
screened or sequenced
to ensure the correctness of the construct. Screening can be done with
restriction analysis,
sequencing or alike.
EC-SOD gene and DNA segments may also be in the form of a DNA insert, which is
located
within the genome of a recombinant virus, such as, for example, recombinant
adenovirus,
adenoassociated virus (AAV) or retrovirus. To place the gene in contact with a
tissue
surrounding an implant, one would, in such embodiments, prepare the
recombinant viral
particles, the genome that includes the EC-SOD gene insert, and simply contact
the tissues
surrounding the implant with the virus, whereby the virus infects the cells
and transfers the
genetic material. In some embodiments of the invention, one would attach virus
in a
composition to an implant, such as a vascular prosthesis, stent, stent graft
or graft connector,
and then contact the tissue surrounding the implant with the implant in site.
The virus is
released from the composition, whereby cells grow into the implant, thereby
contacting the
virus and allowing viral infection, which results in that the cells take up
the desired gene or
cDNA and express the encoded protein, which in turn results in inhibition of
connective tissue
formation.
In a preferred embodiment, the methods of the invention involve to prepare a
composition in
which the EC-SOD gene is attached to or are impregnated on a vascular
prosthesis, stent, a
stent graft, a heart valve, a graft connector, or a tissue implant to form a
vascular prosthesis-,
a stent-, an endovascular graft-, a graft connector-, a heart valve- or a
tissue implant-gene
composition and then the vascular prosthesis-, stent-, stent graft-, graft
connector-, heart
valve-, tissue implant-gene composition is placed in contact with tissue
surrounding the said
cardiovascular or tissue implant. Vascular prosthesis-, cardiovascular patch-,
stent graft-, heart
valve-, graft connector-, tissue implant-gene compositions are all those in
which a gene is
adsorbed, absorbed, or otherwise maintained in contact with the said implant.
Nucleic acid transfer into cells of tissue surrounding an implanted device
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Once a suitable vascular implant-gene composition has been prepared or
obtained, all that is
required for delivering the EC-SOD protein or EC-SOD gene to the surrounding
tissue, is to
place the cardiovascular implant-gene or tissue implant-gene composition
surgically, or with the
help of a catheter, in contact with the desired site in the body, with or
without first wrapping it
5 with the surrounding tissue. The methods are well known to a person skilled
in the art. The EC-
SOD gene or protein can also be administered systemically into the circulation
or to the tissue
before, during or after implanting the cardiovascular or tissue implant to the
site. This could be
an arteriovenous fistula, arterial bypass graft or interposition graft, a
venous graft,
cardiovascular patch, artificial heart, stent graft, stent, heart valve,
cardiac asssist device,
10 anastomotic device, annuloplasty ring, vascular catheter, pacemaker wire,
tracheal cannula,
biomedical sensor, chamber for living cells, artificial organ, organ implant,
orthopedic implant,
suture material, surgical patch, clip or pledget, or any medical device, all
of which comprise at
least one synthetic surface.
15 In the present invention, one or more vectors are transferred to any
surrounding tissue, which
preferably is a mammalian tissue. Several publications have postulated the use
of gene transfer
for the treatment or prevention of diseases (Levine and Friedman, Curr. Opin.
in Biotech. 1991;
2: 840-44, Mulligan, Science 1993; 260: 926-32, Crystal, Science 1995; 270:404-
410,
Rowland, Ann. Thorac Surgery 1995; 60:721-728; Nabel et al., Science 1990;
249: 1285-88).
20 The eukaryotic host cell is optimally present in vivo. According to the
present invention, the
contacting of cells with the vectors of the present invention can be by any
means by which the
vectors will be introduced into the cell. Such introduction can be by any
suitable method.
Preferably, the vectors will be introduced by means of transfection, i.e.
using the natural
capability of the naked DNA to enter cells (e.g., the capability of the vector
to undergo receptor-
25 mediated endocytosis). However, the vectors can also be introduced by any
other suitable
means, e.g. by transduction, calcium phosphate-mediated transformation,
microinjection,
electroporation, osmotic shock, and the like.
The method can be employed with respect to various cells, differing both in
number of vector
30 receptors as well as in the affinity of the cell surface receptors for the
vector. According to the
invention, the types of cells to which gene delivery is contemplated in vivo
include all
mammalian cells, more preferably human cells. The vectors can be made into the
compositions
appropriate for contacting cells with appropriate (e.g. pharmaceutically
acceptable) excipients,
such as carriers, adjuvants, vehicles, or diluents. The means of making such a
composition, and
means of administration, have been described in the art. Where appropriate,
the vectors can be
formulated into preparations in solid, semisolid, liquid, or aerosol forms,
such as aerosol, spray,
paste, ointment, gel, glue, powders, granules, solutions, injections, creme
and drops, in the
usual ways for their respective route of administration without excluding any
other method. A
pharmaceutically acceptable form, that does not inefFectuate the compositions
of the present
invention should be employed. In pharmaceutical dosage forms, the compositions
can be used
alone or in an appropriate association, as well as in combination with other
pharmaceutically
......:.... ..".,-.r,r,~inrlc Fnr a~tamnla_ nucleic acids encoding for EC-SOD
can be administered
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31
together with nucleic acids encoding for inhibiting platelet deposition or
smooth muscle cell
proliferation. Accordingly, the pharmaceutical composition of the present
invention can be
delivered via various ways and to various sites in a mammalian to achieve a
particular effect. A
person skilled in the art will recognise that although more than one way can
be used for
administration, a particular way can provide a more immediate and more
effective reaction than
the other way. Local delivery can be accomplished by administration comprising
topical
application or instillation of the formulation on the implant, or
administration of the formulation
directly, to the tissues surrounding the implant in vivo, or any other topical
application method.
Administration of the drug this way, enables the drug to be site-specific, in
a way that release of
high concentrations and/or highly potent drugs may be limited to direct
application to the
targeted tissue. When delivering the nucleic acids either systemically or
locally they can be
delivered in solution in naked form in any biocompatible salt solutions or
complexed with any
biocompatible substances. Examples of the ways to complex nucleic acids is to
use polycations
(eg oligodendromer), proteins (eg transferrin) or other polymers (eg DEAE-
Dextran, polylysine).
Also derivatives and salts of the examples are included. Other example is to
encapsulate the
nucleic acid or associate with liposomes or coated on colloidal particles.
Preferred methods is to
deliver nucleic acids in an aqueous solution incorporated in fibrin, hydrogel,
glycosaminoglycans,
glycopolysaccharides, or any other biocompatible polymeric carrier matrix,
such as alginate,
collagen, mucin, hyaluronic acid, polyurethane, cellulose, polylactic acid,
poloxamer which
covers at least a portion of the implant (U.S. 5,833,651). Nucleic acids can
be added to the
polymer-coated implant, either at the time of implant manufacture or by the
physician prior to,
during or after implantation. The polymer may also be either a biostable or a
bioabsorbable
polymer, depending on the desired rate of release or the desired degree of
polymer stability. It
may be naturally occuring or synthetic compound, also derivatives and salts of
the compounds
are included. A bioabsorbable polymer is more desirable, as it is supposed to
cause no chronic
local response. Bioabsorbable polymers that may be used include, but are not
limited to, poly(L-
lactic acid), polycaprolactone, poly(lactide-coglycolide),
poly(hydroxybutyrate),
poly(hydroxybuturate-co-valerate), polydioxanone, polyorthoester,
polyanhydride, poly(glycolic
acid), poly(D,L-lactic acid), polylactic-polyglycolic acid, polyglactin,
polydioxone, polygluconate,
poly(glycolic acid-cotrimethylene carbonate), polyphosphoester,
polyphosphoester urethane,
poly(amino acids), cyanoacrylates, poly(trimethylene carbonate),
poly(iminocarbonate),
copoly(ether-esters)(e.g. PEO/PLA), polyalkylene oxalates, polyphosphazenes,
and
biomolecules, such as fibrin, fibrinogen, cellulose, starch, collagen, mucin,
fibronectin, and
hyaluronic acid. Also, biostable polymers with a relatively low chronic tissue
response, such as
polyurethanes, silicones, and polyesters could be used if they can be
dissolved or polymerised
on the implant, such as polyelolefins, polyisobutylene and ethylene-
alphaolefin copolymers;
acrylic polymers and copolymers, vinyl halide polymers and copolymers, such as
polyvinyl
chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidine
halides, such as
polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile,
polyvinyl ketones;
q.0 polyvinyl aromatics, such as polystyrene, polyvinyl esters, such as
polyvinyl acetate;
copolymers of vinyl monomers with each other and olefins, such as ethylene-
methyl
___~~_..,."m+o ...,~m"r"Ar~ arrvinnitrile-styrene cooolvmers, ABS resins,
ethylene-vinyl acetate
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32
copolymers: polyamides, such as Nylon 66 and polycaprolactam;alkyd resins;
polycarbonates;
polyoxymethylenes; polyimides; polyethers; epoxy resins; polyurethanes; rayon;
rayon-
triacetate; cellulose, cellulose acetate, cellulose butyrate; cellulose
acetate butyrate;
cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and
carboxymethylcellulose
(5,776,184). Also fibrin together with other biocompatible polymers, either
natural or synthetic
and their derivatives and salts, may be used. Of the polymers
glycopolysaccharides may be
advantageous. In one aspect there is a solid/solid solution of polymer and
drug. This means that
the drug and the polymer both are soluble in the same solvent and have been
intimately
admixed in the presence of that solvent. The drug and polymer can be applied
in various ways,
such as by simply immersing the implant into the solution or by spraying the
solution onto the
implant (U.S. 5,776,184). Various hydrogel polymers can be used, such as those
selected from
the group consisting of polycarboxylic acids, cellulosic polymers, gelatin,
alginate, poly 2-
hydroxyethylmethylacrylate (HEMA) polyvinylpyrrolidine, malefic anhydride
polymers, polyamids,
polyvinyl alcohols, polyethylene oxides, polyethylene glycol, polyacrylamide,
polyacids, e.g.
polyacrylic acids, polysaccharide, e.g. a mucopolysaccharide such as
hyaluronic acid (U.S.
5,674,192 and U.S. 5,843,089). The polymer can be porous or nonporous on the
implant.
Several layers of polymers can be utilised and several different polymers can
be combined on
the same implant. Different layers and different polymers can carry different
pharmacological
substances (5,833,651). Also, one or more surfaces of the implant can be
coated with one or
more additional coats of polymer that is the same or different from the second
polymer. The
adhesion of the coating and the rate at which the therapeutic compound is
delivered can be
controlled by selection of an appropriate bioab~sorbable or biostable polymer,
and by the ratio of
therapeutic compound to polymer in the solution (U.S. 5,776,184). The dosage
applied to the
tissue may also be controlled by regulating the time of presoaking therapeutic
compound into
the hydrogel coating to determine the amount of absorption of the therapeutic
compound
solution by the hydrogel coating. Other factors affecting the dosage are the
concentration of the
therapeutic compound in the solution applied to the coating, and the
drugreleasability of the
hydrogel coating, determined by, for example, the thickness of the hydrogel
coating, its
resiliency, porosity and the ability of the hydrogel coating to retain the
therapeutic compound,
e.g. electrostatic binding or pore size, or the ionic strength of the coating,
e.g. changed by
changing the pH. It may be advantageous to select a hydrogel coating for a
particular drug,
such that the therapeutic compound is not substantially released into body
fluids prior to
application to the site. The release of the solid/solid solution of polymer
and therapeutic
compound can further be controlled by varying the ratio of therapeutic
compound to polymer in
the multiple layers. Coating need not be solid/solid solution of polymeric and
therapeutic
compound, but may instead be provided from any combination of drug and polymer
applied to
implant. The ratio of therapeutic substance to polymer in the solution will
depend on the efficacy
of the polymer in securing the therapeutic substance onto the implant and the
rate at which the
coating is to release the therapeutic substance to the tissues. More polymer
may be needed if it
has a relatively poor efficacy in retaining the therapeutic substance on the
implant, and more
polymer may be needed in order to provide an elution matrix that limits the
elution of a very
. ,,_ aL......,......a-:~. ~.mhcf~nnro TharafnrP_ a wide therapeutic substance-
to-polymer rate could
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33
be appropriate, and it could range from about 10:1 to 1:100 (U.S. 5,776,184).
Binding of the
therapeutic compound may also be accomplished by electrostatic attraction of
the drug to the
coating or to a coating additive or a mechanical binding, for example by
employing a coating
having a pore size that inhibits inward flow of body fluids or outward flow of
the therapeutic
compound itself, which might tend to release the therapeutic compound.
Hydrogels are particularly advantageous in that the therapeutic compound is
held within the
hydrogen-bond matrix formed by the gel (U.S. 5,674,192). Examples of hydrogels
are for
example HYDROPLUS.RTM (U.S. 5,674,192), CARBOPOL.RTM (U.S. 5,843,089),
AQUAVENE.RTM
(U.S. 4,883,699), HYPAN.RTM (U.S. 4,480,642). In some cases, the hydrogel may
be
crosslinked prior to lining the implant, for example the hydrogel coating on a
vascular or
endovascular graft may be contacted with a primer dip before the hydrogel is
deposited on the
implant. If crosslinked it forms a relatively permanent lining on the implant
surface, and if left
uncrosslinked it forms a relatively degradable lining on the implant surface.
For example, the
longevity of a crosslinked form of a given hydrogel in the stent lining, has
been at least twice to
that of its uncrosslinked form (U.S. 5,843,089). Alternatively, the hydrogel
lining may be
contacted with a crosslinking agent in situ (U.S. 5,843,089). In general, when
dry, the hydrogel
coating is preferably on the order of about 1 to 10 microns thick, and
typically of 2 to 5 microns.
Very thin hydrogel coatings, e.g., of about 0,2-0,3 microns (dry) and much
thicker hydrogel
coatings, e.g., more than 10 microns (dry) are also possible. Typically, the
hydrogel coating
thickness may swell with a factor of about 6 to 10 or more, when hydrogel is
hydrated (U.S.
5,674,192). Usually, the polymeric carrier will be biodegradable or bioeluting
(taught for
example by U.S. 5,954,706, U.S. 5,914,182, U.S. 5,916,585, U.S. 5,928,916).
The carrier can
also be constructed to be a biodegradable substance filling the pores, and
release one or more
substances into the surrounding tissue by progressive dissolution of the
matrix. Subsequently
the pores will open. The delivered vectors may be nucleic acids encoding for
therapeutic protein,
e.g. a naked nucleic acid or a nucleic acid incorporated into a viral vector
or liposome. By a
naked nucleic acid is meant a single or double stranded DNA or RNA molecule
not incorporated
into a virus or liposome. Antisense oligonucleotides, which specifically bind
to complementary
mRNA molecules, and thereby reduce or inhibit protein expression, can also be
delivered to the
implant site via the hydrogel coating (U.S. 5,843,089). Generally, attachment
of the nucleic acid
to the implant can also be done in several other ways, such as by using
covalent or ionic
attachment techniques. Typically, covalent attachment techniques require the
use of coupling
agents, such as glutaraldehyde, cyanogen bromide, p-benzoquinone, succinic
anhydrides,
carbodiimides, diisocyanates, ethyl chloroformate, dipyridyl disulphide,
epichlorohydrin, azides,
among others, without excluding any other agent, but any method that uses the
described
methods of this invention can be used and will be recognised by a person
skilled in the art.
Covalent coupling of a biomolecule to a surface may create undesirable cross-
links between
biomolecules, and thereby destroying the biological properties of the
biomolecule. Also, they
may create bonds amongst surface functional sites and thereby inhibit
attachment. Covalent
coupling of a biomolecule to a surface may also destroy the biomolecules three-
dimensional
r+rmrtmra anri tharahv ral'~~ICina Or destrovina the biological properties
(U.S. 5,928,916). Ionic
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34
coupling techniques have the advantage of not altering the chemical
composition of the
attached biomolecule, and ionic coupling of biomolecules also has an advantage
of releasing the
biomolecule under appropriate conditions. One example is (U.S. 4,442,133). The
current
techniques for immobilisation of biomolecules by an ionic bond have been
achieved by
introducing positive charges on the biomaterial surface utilising quaternary
ammonium salts,
polymers containing tertiary and quaternary amine groups, such as TDMAC,
benzalconium
chloride, cetylpyrridinium chloride, benzyldimethylstearyammonium chloride,
benzylcetyidimethylammonium chloride, guanidine or biguanide moiety (U.S.
5,928,916). When
delivering the vascular implant percutaneously, a sheath member may be
included to inhibit
release of the drug into body fluids during placement of the catheter. For
example, it can be
carbowax, gelatin, polyvinyl alcohol, polyethylene oxide, polyethylene glycol,
or a biodegradable
or thermally degradable polymer, e.g. albumin or pluronic gel F-127 (U.S.
5,674,192). The
particular type of attachment method when practising the methods and
compositions of the
invention is not important, as long as the nucleic acids released from the
implant stimulates the
surrounding tissue in such a way that they are activated and, in the context
of in vivo
embodiments, ultimately give rise to endothelialisation of the cardiovascular
or tissue implant
without causing adverse reactions. The methods described herein are by no
means all inclusive,
and further methods to suit the specific application will be apparent to the
skilled person of the
art.
The composition of the present invention can be provided in unit dosage form,
wherein each
dosage unit, e.g. solution, gel, glue, drops and aerosol, contains a
predetermined amount of the
composition, alone or in appropriate combination with other active agents. The
term unit dosage
form, as used herein, refers to physically discrete units suitable as unitary
dosages for human
and animal subjects, whereby each unit contains a predetermined quantity of
the compositions
of the present invention, alone or in combination with other active agents,
calculated in an
amount sufficient to produce the desired effect, in association with a
pharmaceutically
acceptable diluent, carrier, or vehicle, where appropriate. The specifications
for the unit dosage
forms of the present invention depend on the particular effect to be achieved,
and the particular
pharmacodynamics associated with the pharmaceutical composition in the
particular host.
Accordingly, the present invention also provides a method of transferring a
therapeutic gene to
a host, which comprises administering the vector of the present invention
preferably as a part of
composition with the implant, using the aforementioned ways of administration
or alternative
ways known to those skilled in the art. The effective amount of the
composition is such as to
produce the desired effect in a host, which can be monitored using several end-
points known to
those skilled in the art. Effective gene transfer of a vector to a host cell,
in accordance with the
present invention, can be monitored in terms of a therapeutic effect (e.g.
formation of
capillaries and endothelialisation of surfaces), or further by evidence of the
transferred gene or
expression of the gene within the host (e.g, using the polymerase chain
reaction in conjunction
with sequencing, Northern or Southern hybridisations, or transcription assays
to detect the
r,mrlair acid in host cells. or using immunoblot analysis, antibody-mediated
detection, mRNA or
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protein half-life studies, or particularised assays to detect protein or
polypeptide encoded by the
transferred nucleic acid, or impacted in level or function due to such
transfer). One such
particularised assay described in the examples includes Western immunoassay
for detection of
proteins encoded by the EC-SOD-gene. These methods are by no means all-
inclusive, and
5 further methods to suit the specific application will be apparent to a
person skilled in the art.
Moreover, the effective amount of the compositions can be further approximated
through
analogy to compounds known to exert the desired effect (e.g., compounds
traditionally
employed to inhibit restenosis can provide guidance in terms of the amount of
a EC-SOD nucleic
acid to be administered to a host).
Furthermore, the preferred amounts of each active agent included in the
compositions according
to the invention, EC-SOD is preferably included from about 0.1 micrograms to
10000
micrograms (although any suitable amount can be utilised either above, i.e.
greater than about
10000 micrograms, or below, i.e. less than about 0.1 micrograms), provide
general guidance of
the range of each component to be utilised by the practitioner upon optimising
the methods of
the present invention for practice in vivo. Similarly, EC-SOD plasmids are
included from 0.1 to
10000 micrograms (although any suitable amount can be utilised either above,
i.e. greater than
about 10000 micrograms, or below, i.e. less than about 0.1 micrograms). The EC-
SOD vector
preferably has between 107 and 1013 viral particles, although any suitable
amount can be
utilised, either more than 1013 or less than 107. Moreover, such ranges by no
means preclude
use of a higher or lower amount of a component, as might be warranted in a
particular
application. For instance, actual dose and schedule can vary depending on
whether the
compositions are administered in combination with other pharmaceutical
compositions, or
depending on interindividual differences in pharmacokinetics, drug
disposition, and metabolism.
Furthermore, the amount of vector to be added per cell will likely vary with
the length and
stability of the gene inserted in the vector, as well as also the nature of
the sequence, and is
particularly a parameter which needs to be determined empirically, and it can
be altered due to
factors not inherent to the methods of the present invention (for instance,
the cost associated
with synthesis). A person skilled in the art can easily make any necessary
adjustments in
accordance with the exigencies of the particular situation. The amount of gene
construct that is
applied to the surrounding tissue or the amount of gene composition that is
applied on the
implant or in the tissue, will be finally determined by the attending
physician or veterinarian
considering various biological and medical factors. For example, one would
wish to consider the
particular EC-SOD and vascular implant material, patient or animal size, age,
sex, diet, time of
administration, as well as any further clinical factors that may affect
inhibition of connective
tissue formation, such as serum levels of different factors and hormones. The
suitable dosage
regimen will therefore be readily determinable by a person skilled in the art
in light of the
coming disclosure, bearing the individual circumstances in mind.
Also, for these embodiments, when one or more different vectors (i.e. each
encoding one or
more different therapeutic genes) are employed in the methods described
herein, the contacting
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36
of cells with various components of the present invention can occur in any
order or can occur
simultaneously. Preferably it occurs simultaneously.
Connective tissue inhibiting tissue
This invention provides advantageous methods for using genes to inhibit
excessive connective
tissue formation and improve endothelialisation. As used here surrounding
tissue refers to any
or all of those cells that have the capacity to ultimately inhibit, or
contribute to the inhibition of,
new connective tissue after tissue trauma or after device implantation. This
includes various
tissues in various forms, such as for example vessel wall, pleura,
pericardium, peritoneum,
omentum, fat and muscle.
The particular type or types of surrounding tissue, which are stimulated with
the methods and
compositions of the invention, are not important, as long as the cells are
stimulated in such a
way that they are activated, and, in the context of in vivo embodiments,
ultimately give rise to
inhibition of unwanted connective tissue growth, neointima formation or
promotion of
endothelialisation and capillarisation of the implant.
The surrounding tissue is also used to particularly refer to those cells that
are located within,
are in contact with, or migrate towards the implant, and which cells directly
or indirectly inhibit
connective tissue formation, neointima formation or stimulate the formation of
endothelium
and/or capillaries. As such, microvascular endothelial cells may be cells that
form capillaries,
that upon stimulation further inhibit connective tissue formation or attract
endothelial cells, are
also considered to be surrounding tissue in the context of this disclosure, as
their stimulation
indirectly leads to inhibition of connective tissue formation or stimulation
of endothelialisation.
Cells affecting connective tissue formation or endothelialisation indirectly
may do so by the
elaboration of various growth factors and cytokines, or by their physical
interaction with other
cell types. Also, cells or tissues that in their natural environment arrive at
an area of active
inhibition of connective tissue formation or stimulation of implant
endothelialisation and
vascularisation may be surrounding tissue. Surrounding tissue cells may also
be cells that are
attracted or recruited to such an area. Although of scientific interest, the
direct or indirect
mechanisms by which surrounding tissue cells inhibit connective tissue
formation or stimulate
endothelialisation is not a consideration in the practising of this invention.
Surrounding tissue cells may be cells or tissues that in their natural
environment arrive at an
area of active connective tissue formation or vascular prosthesis,
endovascular prosthesis
endothelialisation, or tissue implant vascularisation. In terms of surrounding
tissue, these cells
may also be cells that are attracted or recruited to such an area.
According to the invention, the surrounding cells and tissues will be those
cells and tissues that
arrive to the tissues or surfaces of cardiovascular implants where one wishes
to inhibit
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37
connective tissue formation or endothelialisation, or cells or tissues that
arrive to the surface of
tissue implants that one wants to vascularise.
Accordingly, in treatment embodiments there is no difficulty associated with
the identification of
suitable surrounding tissues to which the present therapeutic compositions,
and cardiovascular
and tissue implants or other prosthetic devices should be applied. All that is
required in such
cases is to obtain an appropriate inhibitory and stimulatory composition, as
disclosed herein,
and to contact the cardiovascular or tissue implant or prosthetic device with
the stimulatory
composition and the surrounding tissue. The nature of this biological
environment is such that
the appropriate cells will become activated in the absence of any further
targeting or cellular
identification by the practitioner.
One aspect of the invention involves to generally administer a composition to
contact
surrounding tissues with a composition comprising EC-SOD protein or gene (with
or without
additional genes, proteins, growth factors, drugs or other biomolecules), and
a cardiovascular or
tissue implant or other prosthetic devices to promote expression of said gene
in said cells. As
outlined, cells may be contacted in vivo. This is achieved, in the most direct
manner, by simply
obtaining a functional EC-SOD gene construct, and applying the construct to
the cells.
Contacting the cells with DNA, e.g. a linear DNA molecule, or DNA in the form
of a plasmid, or
some other recombinant vector or artificial chromosome that contains the gene
of interest under
the control of a promoter, along with the appropriate termination signals, is
sufficient to achieve
an uptake and an expression of DNA, with no further steps necessary.
In preferred embodiments, the process of contacting the surrounding tissue
with the EC-SOD
composition is conducted in vivo. Again, a direct consequence of this process
is that the cells
take up and express the gene, and the translational or the transcriptional
product stimulates the
process of decreased connective tissue formation or stimulation of
endothelialisation and/or
capillarisation of the implant without additional steps required by the
practitioner.
Materials used in the devices according to the invention
As used herein, the following terms and words shall have the following
ascribed meanings.
Implantable medical device, which for brevity will be referred to as implant,
device or prosthesis
will refer to an object that is fabricated, at least in part, from a
biomaterial, and is intended for
use in contact with bodily tissues, including bodily fluids. Biomaterial shall
refer to the
composition of the material used to prepare a device, which provides one or
more of its tissue
contacting surfaces. Porosity and inflections thereof (such as pores and
porous), if not specified
otherwise, shall refer to a biomaterial having small channels or passages
which start at an
external (e.g. first major) surface of the biomaterial and extend
substantially through the .
biomaterial to an internal (e.g., second) surface. Rigid and inflections
thereof, will, in case of a
nonabsorbable biomaterial, when fabricated in the form of an implantable
medical device, refer
~,__ _~:~:r.,. a-.. ...i~hetan('~ f~'1P nl'PSCIIYPS PnCOUntered in the course
of its use, e.g. to retain
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38
patency and pore structure in vivo. The surface shall refer to the interface
between the
biomaterial and its environment. The term is intended to include the use of
the word in both its
macroscopic sense (e.g. the two major faces of a sheet of biomaterial), as
well as in its
microscopic sense (e.g. the lining of pores traversing the material). The term
~~attach" and its
derivatives refer to adsorption, such as physisorption, or chemisorption,
ligand/receptor
interaction, covalent bonding, hydrogen bonding, or ionic bonding of a
polymeric substance or
nucleic acids to the implant.
The type of cardiovascular, tissue implants and other prosthetic devices that
may be used in the
compositions, devices and methods of the invention is virtually limitless, as
long as they are
tissue compatible. Thus, devices of the present invention include medical
devices intended for
prolonged contact with blood, bodily fluids or tissues, and in particular,
those that can benefit
from inhibition of unwanted or excessive connective tissue growth and fibrosis
or stimulation of
the capillary endothelialisation when used for in vivo applications. Preferred
devices are
implantable in the body, and include cardiovascular implants, tissue implants,
artificial organs,
such as the pancreas, liver, and kidney, and organ implants, such as breast,
penis, skin, nose,
ear and orthopedic implants. The significance of inhibition of connective
tissue formation or
capillary endothelialisation will vary with each particular device, depending
on the type and
purpose of the device. The inhibition of connective tissue formation protects
the device from
excessive scar tissue formation, strictures and fibrosis. Ingrown capillaries
can provide
endothelial cells to line surfaces of vascular implants, protect tissue
implants from infection,
carry nutrients to the cells in the device and make it possible for sensors to
sense substance
levels in circulation. This means that the implant has all the features
commonly associated with
biocompatibility, in that they are in a form that does not produce an adverse,
an allergic, or any
other untoward reaction when administered to a mammal. They are also suitable
for being
placed in contact with the tissue surrounding the implant. The latter
requirement takes factors,
endothelium or to resist unwanted connective tissue formation, into
consideration.
Preferred biomaterials are those that provide sufficient rigidity for their
intended purposes in
vivo. For use in forming a vascular graft and cardiovascular patch, for
instance, the biomaterial
will be of sufficient rigidity to allow the graft to retain graft patency in
the course of its intended
use. The choice of implant material will differ according to the particular
circumstances and the
site where the vascular or tissue implant is implanted. Vascular prosthesises
are made of
biomaterials, selected from the group consisting of e.g. tetrafluoroethylene
polymers,
aromatic/aliphatic polyester resins, polyurethans, and silicone rubbers.
However, any type of
biocompatible microporous mesh may be used. The said biomaterials can be
combined with
each other or other substances, such as polyglycolic acid, polylactic acid,
polydioxone and
polyglyconate. Preferred are expanded polytetrafluorethylene and Dacron.
Dacron may be with
or without velour, or modified in some other way. Dacron is usually woven,
braided or knitted
and suitable yarns are between 10 and 400 deniers. The nodal regions of ePTFE
are composed
of nonporous PTFE that serves to provide tear resistance (e.g. for sutures and
resistance to
.,.,......"....,-,m a;m+.~+~nnl Tha intPrnnrial raninns are composed Of flbreS
Of PTFE, which serve t0
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39
connect the nodes with the spaces between the fibres providing the porosity
referred to herein.
The nodal size can be expressed as the percentage of the tissue-contacting
surface that is
composed of nodal PTFE. The distance between nodes can be expressed as the
average fibril
length. In turn, the porosity is commonly expressed as the internodal distance
(i.e. the average
distance from the middle of one node to the middle of the adjacent node).
Preferred ePTFE
materials have nodes of sufficient size and frequency to provide adequate
strength (e.g., with
respect to aneurysmal dilatation) and internodal regions of sufficient
frequency and fibre length
to provide adequate porosity (to allow for capillary endothelialisation).
Given the present
specification, those skilled in the art will be able to identify and fabricate
devices using
biomaterials having a suitable combination of porosity and rigidity.
Biomaterials are preferably
porous to allow the attachment and migration of cells, which may be followed
by the formation
and growth of capillaries into the surface. Suitable pores can exist in the
form of small channels
or passages, which start at an external surface and extend partially or
completely through the
biomaterial. In such cases, the cross sectional dimensions of the pore
capillary diameter are
greater than 5 microns and typically less than 1 mm. The upper pore size value
is not critical as
long as the biomaterial retains sufficient rigidity, however it is unlikely
that useful devices would
have a pore size greater than about lmm. Such pore dimensions can be
quantified in
microscope. As will be understood by those skilled in the art, several
modifications of the graft
materials and surfaces can be made, such as precoating with, for example,
proteins (see e.g.
5,037,377, 4,319,363), non-heparinised whole blood and platelet rich plasma,
glow-discharge
modifications of surfaces, adding pluronic gel, fibrin glue, fibronectin,
adhesion molecules,
covalent bonding, influencing surface charges, with for example carbon
(5,827,327, 4,164,045),
and treating with a surfactant or cleaning agent, without excluding any other
method. Moreover,
the implant can be constructed as a hybrid of different internodal distances
for the inner and
outer surfaces, such as 60 microns as an outer value and 20 microns as an
inner value, for the
internodal distances (HYBRID PTFE). Also, more layers with different
internodal distances may
be used. They are all intended to fall within the scope of the present
invention when not
inhibiting endothelialisation. Potential biodegradable vascular implants may
be used in
connection with the compositions, devices and methods of this invention. For
example,
biodegradable and chemically defined polylactic acid, polyglycolic acid,
matrices of purified
proteins, semi-purified extracellular matrix compositions and also collagen
can be employed.
Also, naturally occuring autogenic, allogenic and xenogenic material, such as
an umbilical vein,
saphenous vein, native bovine artery or intestinal sub-mucosal tissue may be
used as a vascular
or other implant material. Examples of clinically used grafts are disclosed in
U.S. 4,187,390,
U.S. 5,474,824 and U.S. 5,827,327. Biodegradable or bioabsorbable materials,
such as
homopolymers e.g. poly-paradioxanone, polylysine or polyglycolic acid and
copolymers; e.g.,
polylactic acid and polyglycolic acids or other bio materials, may be used
either alone or in
combination with other materials as the vascular graft or other implant
material, as long as they
provide the required rigidity. Also, other biological materials, such as
intestinal submucosa,
matrices of purified proteins and semi-purified extracellular matrix
compositions may be used.
Appropriate vascular grafts or other prosthetic implants will both deliver the
gene composition
....a .,m" ."-",.~~A a cnrface for new endothelium growth, i.e., will act as
an in situ scaffolding
CA 02483096 2004-10-20
WO 03/092727 PCT/SE03/00713
through which endothelial cells may migrate. It will be understood by a person
skilled in the art
that any material with biocompatibility, rigidity will be acceptable to be
used with the invention.
It will also be understood that inhibition of excessive connective tissue
formation can occur in
connection of an autologous vessel to autologous vessel, allogene vessel to
autologous vessel,
5 autologous vessel to synthetic vessel or any type of vessel to another
vessel either end-to-end,
end-to-side, side-to-side or combination of multiple side-to-side and end-to-
side-anastomosis as
long as there is a connection between the vessels with an anastomotic area.
Also the
hyperplasia can be inhibited in any part of the graft.
10 Background for cardiovascular patches is well described in for example U.S.
5,104,400, U.S.
4,164,045, U.S. 5,037,377. In the case of vascular patches, one side of the
patch engages the
blood while the other side engages other surrounding tissues to promote
transgraft growth of
the endothelial cells. In the case of intracardiac patches, blood engages both
sides of the patch.
Preferred biomaterials are those that provide sufficient rigidity in vivo. A
vascular patch
15 biomaterial will be of sufficient rigidity to allow the patch to retain its
form and pore-structure in
the course of its intended use. The choice of patch material will differ
according to the particular
circumstances and site where the vascular patch is implanted. Vascular patch
is made of
synthetic biomaterial, such materials include, but are not limited to,
tetrafluoroethylene
polymers, aromatic/aliphatic polyester resins, polyurethans, and silicone
rubbers, however any
20 type of biocompatible microporous mesh may be used. The said biomaterials
can be combined
with each other or other substances such as polyglycolic acid. Preferred are
expanded
polytetrafluorethylene and Dacron. Dacron is usually woven, braided or
knitted, and with or
without velour, and suitable yarns are between 10 and 400 deniers. The nodal
regions of ePTFE
are composed of nonporous PTFE that serves to provide tear resistance (e.g.
for sutures and
25 resistance to aneurysmal dilatation). The internodal regions are composed
of fibres of PTFE,
which serve to connect the nodes, with the spaces between the fibres providing
the porosity
referred to herein. The nodal size can be expressed as the percentage of the
tissue-contacting
surface that is composed of nodal PTFE. The distance between nodes can be
expressed as the
average fibril length. In turn the porosity is commonly expressed as the
internodal distance (i.e.
30 the average distance from the middle of one node to the middle of adjacent
node). Preferred
ePTFE materials have nodes of sufficient size and frequency to provide
adequate strength (e.g.,
with respect to aneurysmal dilatation) and internodal regions of sufficient
frequency and fibre
length to provide adequate porosity (to allow for capillary
endothelialisation). Such materials
will provide fewer though thicker nodes, which will in turn confer
significantly greater strength in
35 vivo. Given the present specification, those skilled in the art will be
able to identify and fabricate
devices using biomaterials having a suitable combination of porosity and
rigidity. Biomaterials
are preferably porous to allow the attachment and migration of cells, which
may be followed by
the formation and growth of capillaries into the luminal surface. Suitable
pores can exist in the
form of small channels or passages, which start at an external surface and
extend through the
40 biomaterial. In such cases, the cross sectional dimensions of the pores are
larger than the
diameter of a capillary 5 microns and are typically less than 1 mm. Upper pore
size value is not
_..:~.:....~ .". ~,..", a~ the hi~matPrial retains sufficient rigidity.
However, it is unlikely that useful
CA 02483096 2004-10-20
WO 03/092727 PCT/SE03/00713
41
devices would have pore size greater than about 1 mm. Such pore dimensions can
be quantified
in microscope. As will be understood by a person skilled in the art, several
modifications of graft
materials and surfaces can be made, such as precoating with for example
proteins (for example,
U.S. 5,037,377, U.S. 4,319,363), non-heparinised whole blood and platelet rich
plasma, glow-
s discharge modifications of surfaces, adding poloxemers, fibrin glue,
adhesion molecules,
covalent bonding, influencing surface charges with for example carbon (U.S.
5,827,327, U.S.
4,164,045), treating with a surfactant or cleaning agent, without excluding
any other method.
Also the implant can be constructed as a hybrid of different internodal
distances in inner and
outer surface, such as outer 60 microns and inner 20 microns in internodal
distance (HYBRID
PTFE). Even more layers with different internodal distances may be used. They
all are intended
to fall in the scope of present invention when not inhibiting
endothelialisation. Potential
biodegradable materials may be used in connection with the compositions,
devices and methods
of this invention, for example homopolymers e.g. poly-paradioxanone,
polylysine or polyglycolic
acid and copolymers e.g., polylactic acid and polyglycolic acids or other bio
materials, such as
matrices of purified proteins and semi-purified extracellular matrix
compositions may be used
either alone or in combination with other materials as cardiovascular patch
material, as long as
they provide the required rigidity. Naturally occurring autogenic, allogenic
and xenogenic
material such as an umbilical vein, saphenous vein, native bovine artery,
pericardium or
intestinal submucosal tissue may also be used as cardiovascular patch
material. Examples of
clinically used vascular patches are disclosed in U.S. 5,037,377, U.S.
5,456,711, U.S.
5,104,400, U.S. 4,164,045. Appropriate vascular patches will both deliver the
gene composition
and also provide a surface for new endothelium growth, i.e., will act as an in
situ scaffolding on
which and through which endothelial cells may migrate. Preferably, nucleic
acids are attached to
the side engaging the tissues surrounding the vessel. Appropriate intracardiac
patches will both
deliver the gene composition to the surrounding tissues and provide a surface
for new
endothelium growth, i.e., will act as an in situ scaffolding on which and
through which
endothelial cells may migrate. Preferably, nucleic acids are attached to both
intracardiac patch
surfaces. Alternatively, nucleic acids may be attached to one of the
intracardiac patch surfaces.
It will be understood by a person skilled in the art, that any material with
biocompatibility and
rigidity will be acceptable to be used with the invention.
Stent herein means a medical implant in the form of a hollow cylinder, which
will provide
support for the body lumen when it is implanted in contact with a site in the
wall of a lumen to
be treated. They can be of several different designs such as tubular, conical
or bifurcated. The
configuration can be such as a coiled spring, braided filament, perforated
tube, slit tube, and
zigzag, or any other variant. Preferably, in the case of vascular stents it is
adapted for use in
blood vessels in a way that the stent has an outer, lumen-contacting surface,
and an inner,
blood-contacting surface. Many stents of the art are formed of individual
member(s), such as
wire, plastic, metal strips, or mesh, which are bent, woven, interlaced or
otherwise fabricated
into a generally cylindrical configuration. The stent can also have underlying
polymeric or
metallic structural elements, onto which elements, a film is applied (U.S.
5,951.586). Stents
. _ ._ __.. ..m~~~f~o,~ ~n~" A~+hAr ~Pif-Pxnandina or pressure expandable. The
terms expand,
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42
expanding, and expandable are used herein to refer to diametrically adjustable
intraluminal
stents. When the self expanding stents are positioned at the treatment site
with a delivery
catheter, they are supposed to radially expand to a larger diameter after
being released from a
constraining force, which force restricts them to a smaller diameter and
conform a surface
contact with a blood vessel wall or other tissue without exertion of outwardly
directed radial
force upon stent. Stents of this type include stents of braided or formed
wire. Self-expanding
stents may also expand to a size as defined by thermal memory. The pressure-
expandable
stents are fabricated of malleable or plastically deformable material,
typically formed of metal
wire or metal strips. The collapsed stent is taken to the treatment site with
a delivery catheter,
and is then radially expanded with a balloon or other stent-expansion
apparatus to its intended
operative diameter. Thread elements or strands formed of metal are generally
favoured, for
applications requiring flexibility and effective resistance to radial
compression after implantation.
The favourable combination of strength and flexibility is largely due to the
properties of the
strands after they have been age hardened, or otherwise thermally treated in
the case of
polymeric strands. The braiding angle of the helical strands and the axial
spacing between
adjacent strands also contribute to strength and flexibility.
Stent wires may be of metal, inorganic fibres, ceramic or organic polymers.
They should be
elastic, strong, biocompatible, and fatigue and corrosion resistant. For
example, core wires
made of metals, such as stainless steel or gold or other relatively pliable
non-toxic metals and
alloys that do not degrade during the time of implantation or are not subject
to severe
degradation (corrosion) under the influence of an electric current, are
usually chosen. Such
metals include, but are not limited to, platinum, platinum-iridium alloys,
copper alloys, with tin
or titanium, nickel-chrome-cobalt alloys, cobalt based alloys, molybdenium
alloys, nickel-
titanium alloys. The strands need not be of metal and may for example be of a
polymeric
material such as PET, polypropylene, PEEK, HDPE, polysulfone, acetyl, PTFE,
FEP, and
polyurethane without excluding any other substance (other variants:
polytetrafluorethylene,
fluorinated ethylene propylene, polytetrafluorethylene-perfluoroalkyl vinyl
ether copolymer,
polyvinyl chloride, polypropylene, polyethylene terephthalate, broad fluoride
and other
biocompatible plastics). Also, a biodegradable or bioabsorbable material, such
as homopolymers
e.g. poly-paradioxanone, polylysine or polyglycolic acid and copolymers, e.g.
polylactic acid and
polyglycolic acids, polyurethane, or other biomaterials, may be used either
alone or in
combination with other materials as the stent material. Such monofilament
strands range from
0,002 to 0,015 inches in diameter but of course the diameter could vary
depending on the
lumen size and the degree of support needed. Also antithrombotic, anti-
platelet, vasodilative,
antiproliferative, antimigratory, antifibrotic, anti-inflammatory agents and
more specifically,
heparin, hirudin, hirulog, etritinate, freskolin, rapamycin, sirolimus,
paclitaxel, tacrolimus,
dexamethasone, cytochalasine D and Actinomycin C and the like, may be attached
the stent.
Examples of clinically used stents are disclosed in U.S. 4,733,665, U.S.
4,800,882, U.S.
4,886,062 incorporated here by reference.
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43
Stent grafts, also called covered stents, for transluminal implantations
include a resilient tubular
interbraided latticework of metal or polymeric monofilaments, a tubular
interbraided sleeve
formed of a plurality of interwoven textile strands, and an attachment
component that fixes the
latticework and the sleeve together, in a selected axial alignment with one
another, engaged
with one another and with a selected one of the latticework and the sleeve
surrounding the
other, whereby the latticework structurally supports the sleeve. It is ensured
that the
latticework and the sleeve behave according to substantially the same
relationship governing
the amount of radial reduction that accompanies a given axial elongation. The
sleeve may be
exterior or interior to the latticework, or the latticework may be integrated
in the sleeve, and it
can be continuous or discontinuous. Several prosthesis constructions have been
suggested for
composite braided structures that combine different types of strands, e.g.
multifilament yarns,
monofilaments, fusible, materials and collagens. Examples are found in
W091/10766. Textile
strands are preferably multifilament yarns, even though they can be
monofilaments. In either
case the textile strands are much finer than the structural strands, ranging
from about 10
denier to 400 denier. Individual filaments of the multifilament yarns can
range from about 0.25
to about 10 denier. Multifilament yarns can be composed of various materials,
such as PET,
polypropylen, polyethylen, polyurethane, HDPE, silicone, PTFE, polyolefins and
ePTFE. By
modifying the yarns it is possible to modify sleeve qualities, for example
untwisted flat filaments
provide thinner walls, smaller intersticies between yarns so achieving lower
permeability, and
higher yarn cross-section porosity for capillary transgraft growth. Porous
expanded PTFE film
has a microstructure of nodes interconnected by fibrils and may be made as
taught by for
example U.S. Pat. Nos. 3,953,566, 4,187,390 and 4,482,516. Suitable pores can
exist in the
form of small channels or passages starting at an external surface and
extending through the
biomaterial. In such cases the cross-sectional dimensions of the pores are
larger than the
diameter of a capillary 5 microns, and are typically less than 1 mm. Upper
pore size value is not
critical so long as the biomaterial retains sufficient rigidity, however it is
unlikely that useful
devices would have pore size greater than about lmm. Such pore dimensions can
be quantified
in microscope. As will be understood by those in the art several modifications
of stent graft
materials and surfaces can be made such as precoating with proteins, non-
heparinised whole
blood and platelet rich plasma, glow-discharge modifications of surfaces,
adding pluronic gel,
fibronectin, fibrin glue, adhesion molecules, covalent bonding, influencing
surface charges with
for example carbon (U.S. 5,827,327, U.S. 4,164,045), treating with a
surfactant or cleaning
agent, mechanically changing the characteristics, such as drilling holes,
adding grooves and
changing the end angles without excluding any other method. Also the implant
can be
constructed as a hybrid of different internodal distances in inner and outer
surface such as outer
60 microns and inner 20 microns in internodal distance (HYBRID PTFE). Even
more layers with
different internodal distances can be used. They all are intended to fall in
the scope of present
invention when not promoting unwanted connective tissue growth or inhibiting
endothelialisation. The fibrils can be uni-axially oriented, that is oriented
in primarily one
direction, or multiaxially oriented, that is oriented in more than one
direction. The term
expanded is used herein to refer to porous expanded PTFE. It will be
understood by a person
W.incrl in tho ari- that anv matPrl81 with biocompatibility will be acceptable
to be used with the
CA 02483096 2004-10-20
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44
invention. Examples of clinically used stent grafts are disclosed in U.S.
5,957,974, U.S.
5,928,279, U.S. 5,925,075, and U.S. 5,916,264.
Also, naturally occuring autologous, allogenic or xenogenic materials, such as
arteries, veins
and intestinal submucosal can be used in stent grafts, such as an umbilical
vein, saphenous
vein, or native bovine artery. Potential biodegradable vascular implants may
be used as stent
grafts in connection with the compositions, devices and methods of this
invention, for example
biodegradable and chemically defined polylactic acid, polyglycolic acid,
matrices of purified
proteins, semi-purified extracellular matrix compositions. Appropriate
vascular grafts and stent
grafts will both deliver the gene composition and also provide a surface for
new endothelium
growth, i.e., will act as an in situ scaffolding through which endothelial
cells may migrate and
preferably inhibit unwanted tissue growth or restenosis. The particular design
of the implants
that are implanted using the methods and compositions of the invention are not
important, as
long as connective tissue formation can be inhibited and they act as scaffolds
through which
endothelium can migrate, in the context of in vivo embodiments, and ultimately
give rise to
endothelialisation of the implant.
A variety of catheter systems are useful for delivering the interventional
stents and stent grafts
into the desired site. The chosen type is not important as long as the methods
of present
invention are used.
Heart valves are well known in the art and operate hemodynamically as a result
of the pumping
action of the heart. Generally, there is an annular body having an interior
surface, which defines
a blood flow passageway, and which has one or multiple occluders supported
thereon, for
alternately blocking, and then allowing the blood flow in a predetermined
direction. Heart valve
prostheses are of various different designs, and of autologous, allogenic,
xenogenic or synthetic
material. The mechanical valve annular housing, also called annular body, and
the valuing
members, can be made of any biocompatible and nonthrombogenic material, that
also will take
the wear they will be subjected to. There are various different designs, such
as a circular valve
housing and a valuing member, such as a spherical member or ball, pivoting
disc, poppet disc,
and leaflet members, such as single or multiple leaflet constructs, for
example two flat leaflets,
leaflets with conical, semiconical and cylindrical surfaces. The orifice ring
can be made of
various materials, such as a pyrocarbon coated surface, a silver coated
surface or from solid
pyrolytic carbon (described in U.S. 4,443,894), and leaflets may be made of
one substrate, such
as polycristalline graphite, plastic, metal or any other rigid material, and
then coated with
another, such as pyrolytic carbon (e.g. in U.S. 3,546,711, and U.S.
3,579,645). Circular valve
housing can be porous, (here referred as having a porous surface and a network
of
interconnected interstitial pores below the surface in fluid flow
communication with the pores,
see U.S. 4,936,317), or nonporous, and suitable means, such as peripheral
groove or a pair of
4p flats can be provided for attaching a suturing ring to the annular body to
facilitate sewing or
suturing of the heart valve to the heart tissue. The suturing member may have
a rigid annular
..,.,o~.",,p,. ~,. ~iPPwP cnrroundina the base. The sleeve may be of a rigid
material, such as metal,
CA 02483096 2004-10-20
WO 03/092727 PCT/SE03/00713
plastic or alike. The sleeve may have collars of fabric, such as Teflon or
Dacron (RE31,400). The
valve may have further members, such as a cushioning member and a shock-
absorbing
member. Examples of mechanical heart valves are described in U.S. 3,546,711,
U.S. 4,011,601,
U.S. 4,425,670, U.S. 3,824,629, U.S. 4,725,275, U.S. 4,078,268, U.S.
4,159,543, U.S.
5 4,535,484, U.S. 4,692,165, U.S. 5,035,709, and U.S. 5,037,434.
Xenografts, allografts or autografts can be used as tissue valves. When an
autologous graft is
used, usually the pulmonary valve is operated to the aortic position - a Ross
operation.
Allografts, also called homografts, are of cadaveric origin. Xenograft
bioprosthetic heart valves
10 are usually of porcine origin. They can be stented or stentless. The
traditional stented valves
may be designed to have a valuing element, stent assembly and a suture ring.
The stent may
be cloth covered. All the known stent materials can be used in the stent,
including but not
limited to titanium, Delrin, polyacetal, polypropylene, and Elgiloy. As is
known by a person
skilled in the art, there are several ways to manipulate tissue valves. For
example a
15 bioprosthesis may be made acellular (Wilson, Ann. Thorac Surg., 1995;60 (2
supply: S353-8) or
preserved in various ways, such as with glutaraldehyde, glycerol (Hoffman),
dye-mediated
photooxidation (Schoen, J. Heart Valve Dis., 1998; 7(2):174-9), and if
preserved with
glutaraldehyde, glutaraldehyde can be neutralised by aminoreagents (e.g. U.S.
4,405,327).
Homografts can be deendothelialised. Examples of tissue heart valves are
described in U.S.
20 3,755,823, U.S. 4,441,216, U.S. 4,172,295, U.S. 4,192,020, U.S. 4,106,129,
U.S. 4,501,030,
and U.S. 4,648,881. Also, there exists an extensive scientific literature in
the subject. It will be
understood by a person skilled in the art, that any material or tissue with
biocompatibility to
inhibit unwanted connective tissue growth or allow endothelial growth will be
acceptable. Genes
can be attached to the heart valve prostheses by various methods but the
method is not
25 important as long as gene is taken up by the surrounding tissue and EC-SOD
is produced and
excessive connective tissue formation and fibrosis inhibited or angiogenesis
is stimulated, which
results in endothelialisation of the orifice ring and/or the valuing member
surface. Nucleic acids
or a composition comprising nucleic acids may be attached to whole or parts of
the heart valves.
Preferably, in tissue valves nucleic acids will be attached to the whole
surface and stent
30 assembly, and in mechanical valves to annular body and sewing ring.
Tissue implants can be made of various materials, such as polyethylene,
polypropylene,
polytetrafluorethylene (PTFE), cellulose acetate, cellulose nitrate,
polycarbonate, polyester,
nylon, polysulfone, mixed esters of cellulose, polyvinylidene difluoride,
silicone, collagen and
35 polyacrylonitrile. Preferred support materials for tissue engineering are
synthetic polymers,
including oligomers, homopolymers, and copolymers resulting from either
addition or
condensation polymerisations. Examples of tissue implants are described in
e.g. U.S. 5,314,471,
U.S. 5,882,354, U.S. 5,874,099, U.S. 5,776,747, and U.S. 5,855,613. It will be
understood by
the person skilled in the art that any material with biocompatibility to allow
endothelial growth
40 and/or capillarisation will be acceptable. EC-SOD genes can be attached to
the implant by
various methods, but the method is not important as long as gene is taken up
by the
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46
surrounding tissue and EC-SOD is produced and fibrosis is inhibited or
angiogenesis is
stimulated resulting in endothelialisation and/or capillarisation of the
implant.
Background for anastomotic devices also called graft connectors is well
described in U.S.
5,904,697 and U.S. 5,868,763. Generally, anastomotic devices are employed
either in end-to-
end anastomosis or end-to-side anastomosis. This invention comprises end-to-
side anastomotic
devices, preferably those anastomotic devices that have an anchoring member
being implanted
intraluminally to the target vessel and exposed to blood, such as SOLEM
GraftConnectorTM. The
term "anchoring member°' is here referred to the member forming the
attachment with the
target vessel. The term "coupling member" or "connecting member" here refers
to the member
that forms attachment with the bypass graft vessel. Anchoring member and
coupling member
may form one single unit or be separated being connected during the procedure.
Additional
members such as a handle and pins may be comprised. The intraluminal anchoring
member
may be of various design, preferably it is a tubular structure. The
intraluminal anchoring
member may be made of any biocompatible material such as metal, ceramic,
plastic, polymer,
PTFE, DACRON, PET, polypropylen, polyethylen, polyurethane, HDPE, silicone,
polyolefins and
ePTFE or combination of several structures. Also a biodegradable or
bioabsorbable material such
as homopolymers e.g. poly-paradioxanone, polylysine or polyglycolic acid and
copolymers e.g.
polylactic acid and polyglycolic acids or other bio materials may be used
either alone or in
combination with other materials. Anastomotic device may be porous, partly
porous, or
nonporous. Preferably the connecting member is nonporous and the anchoring
member is
porous. Alternatively both the connecting member and anchoring member are
porous. If porous,
the cross sectional dimensions of the pore capillary diameter are greater than
5 microns and
typically less than 1 mm. Upper pore size value is not critical so long as the
biomaterial retains
sufficient rigidity, however it is unlikely that useful devices would have
pore size greater than
about imm. Such pore dimensions can be quantified in microscope. Suitable
pores can exist in
the form of channels or passages starting at the external surface and extend
through the
biomaterial. As will be understood by those in the art several modifications
of graft connector
design materials and surfaces can be made such as precoating with proteins,
non-heparinised
whole blood and platelet rich plasma, glow-discharge modifications of
surfaces, adding pluronic
gel, fibrin glue, adhesion molecules, covalent bonding, influencing surface
charges with for
example carbon (U.S. 5,827,327, and U.S. 4,164,045), treating with a
surfactant or cleaning
agent, mechanically changing the surface characteristics, such as adding
grooves and changing
the end angles without excluding any other method. Also the implant can be
constructed as a
hybrid of different internodal distances in inner and outer surface, such as
outer 60 microns and
inner 20 microns in internodal distance (such as HYBRID PTFE). Even more
layers with different
internodal distances may be used. They all are intended to fall in the scope
of present invention
when not inhibiting endothelialisation. Genes can be attached to the implant
by various
methods, but the method is not important as long as the gene is taken up by
the surrounding
tissue and EC-SOD produced, connective tissue formation inhibited and
angiogenesis is
stimulated resulting in endothelialisation and/or capillarisation of the
implant. Appropriate graft
connectors will both deliver the gene composition and also provide a surface
for new
CA 02483096 2004-10-20
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47
endothelium growth, i.e., will act as an in situ scaffolding through which
endothelial cells may
migrate. It will be understood by a person skilled in the art that any
material with
biocompatibility, rigidity and porosity to allow transgraft growth will be
acceptable.
Suture materials are well known in the art. "Filament" is here referred a
single, long, thin
flexible structure of a non-absorbable or absorbable material. It may be
continuos or staple.
"Absorbable" filament is here referred one, which is absorbed, that is
digested or dissolved, in
mammalian tissue. Sutures may be monofilament i.e. single filament strands or
multifilament
i.e. several strands in a braided, twisted or other multifilament construction
and are made of
wide variety of materials both natural, such as metal, silk, linen, cotton and
catgut, and
synthetic, such as nylon, polypropylene, polyester, polyethylene,
polyurethane, polylactide,
polyglycolide, copolymers of lactide and glycolide. Sutures may be porous
(U.S. 4,905,367, U.S.
4,281,669) or nonporous and they can be coated with various materials
described in for
example in U.S. 4,185,637, U.S. 4,649,920, U.S. 4,201,216, U.S. 4,983,180, and
U.S.
4,711,241 or uncoated. Nucleic acids may be attached to any suture material to
inhibit
unwanted connective tissue growth or promote endothelialisation of sutures.
Attachment of the
nucleic acids is particularly useful with synthetic non-absorbable vascular
sutures. If
multifilament suture is to be coated, it is not necessary that every filament
within the suture be
individually or completely coated. Sizes of suture materials usually range
between 12-0 U.S.P.
size 0,001 mm to size 2 U.S.P. with outer diameter 0,599 mm. Suture materials
may be with or
without needle in one or both ends and needle may be attached to the suture
material by any of
the methods known in the art, such as by defining a blind hole, i.e, a
cylindrical recess,
extending from a proximal end face of the suture needle along the axis
thereof. The length of
the suture-mounting portion is generally equal to or slightly greater than the
length of the hole.
A suture is inserted into the hole and then the suture-mounting portion is
crimped, i.e.
deformed or compressed, to hold the suture. Alternatively, the suture may be
secured by
addition of cement material to such blind hole (for example in U.S.
1,558,037). Also adhesive
and bonding agents may be used, such as in U.S. 2,928,395, U.S. 3,394,704.
Also other
modifications may be employed such as in U.S. 4,910,377, U.S. 4,901,722, U.S.
4,890,614,
U.S. 4,805,292, and U.S. 5,102,418. The surgical needle itself may be made of
various
materials, such as medically acceptable stainless steel of required diameter.
The suture
attachment to the needle may be standard i.e. the suture is securely attached
and is not
intended to be separable therefrom, except by cutting or severing the suture,
or detachable or
removable i.e. be separable in response to a force exerted by the surgeon
(U.S. 3,890,975, U.S.
3,980,177, and U.S. 5,102,418). Surgical needles may be of various form such
as'/a circle, 3/8
circle,'/a curve,'/z circle, 5/8 circle, or straight and the needle distal
point may be taper point,
taper cut, reverse cutting, precision point, spatula-type, and the like. The
amount of nucleic acid
attached to the suture material or to the composition coating the suture will
vary depending
upon the construction of the fibre, e.g. the number of filaments and tightness
of braid or twist
and the composition, solid or solution applied. It will be understood by the
skilled person that
any material with biocompatibility to allow inhibition of connective tissue
hyperplasia will be
arrantahlc~_ c;enes can be attached to the sutures by any of the methods
described in this
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48
disclosure or any other method if so preferred. After gene has been taken up
by the surrounding
tissue, EC-SOD is produced and excessive connective tissue growth is inhibited
and
endothelialisation stimulated resulting in endothelialisation of the suture
material surface.
Surgical pledgets are well known in the art. It will be understood by a person
skilled in the art,
that any material with biocompatibility to allow endothelial growth will be
acceptable. Genes can
be attached to the surgical pledgets by any method included in this
disclosure, or any other
method. After gene is taken up by the surrounding tissue, EC-SOD is produced
and excessive
connective tissue growth inhibited and angiogenesis is stimulated resulting in
endothelialisation
of the implant.
Physical and chemical characteristics, such as e.g. biocompatibility,
biodegradability, strength,
rigidity, porosity, interface properties, durability and even cosmetic
appearance may be
considered in choosing the said vascular or tissue implant, as is well known
for those skilled in
the art. Also, an important aspect of the present invention is its use in
connection with other
implants having the advantage of avoidance of excessive connective or
fibromuscular tissue
growth or vascularisation of the interface with the tissues, including
implants themselves and
functional parts of the implant, such as tissue chambers, pacemaker wires,
indwelling vascular
catheters for long time use and the like. The surface may be coated or pores
filled with nucleic
acids or with a material having an affinity for nucleic acids, and then the
coated-surface may be
further coated with the gene or nucleic acid that one wishes to transfer. The
available chemical
groups of the adsorptive, may be readily manipulated to control its affinity
for nucleic acids, as
is known to those skilled in the art.
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Experimental section
Example 1
Rabbits and stent
Methods
Example
EC-SOD expression plasmid
A rabbit lung cDNA library (Clontech # TL1010a) was screened by plaque
hybridization using a
partial rabbit EC-SOD cDNA (genbank X78139; EC-SOD encoding bases 126-465) as
a probe
(Hiltunen et al.., 1995, Hiltunen, T., Luoma, J., Nikkari, T. and Yla-
Herttuala, S.: Induction of
15-lipoxygenase mRNA and protein in early atherosclerotic lesions. Circulation
92 (1995) 3297-
3303). Positive clones were purified by a standard method (Ausubel, F.M.,
Brent, R., Kingston,
R.E., Moore, D.D., Seidman, J.G., Smith, A.J. and Struhl, K. (eds.): Current
protocols in
molecular biology. John Wiley & Sons, Inc., USA, 1995) and were found to
contain the 3'
regions of the EC-SOD cDNA. The 5' end of the coding sequence was amplified
from rabbit
genomic DNA by PCR using primers specific to EC-SOD gene (genbank AJ007044);
5'-GAT GCT
GGC GTT GGT GTG CTC-3' / 5'-GCA CGG CCA GCG GGT TGT AGT-3'. The 5' and 3'
fragments of
the cDNA were subsequently ligated to produce the entire open reading frame of
EC-SOD gene
which was further subcloned into pHHT631 expression vector (Mizushima, S, and
Nagata, S.:
pEF-BOS, a powerful mammalian expression vector. Nucleic. Acids. Res. 18
(1990) 5322) under
elongation factor 1a promoter (pEC-SODIa). DNA sequencing was done using ALF
automated
DNA sequencer (Pharmacia), and the sequence analyses were performed with the
GCG program
package (Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of
sequence analysis
programs for the VAX. Nucleic. Acids. Res. 12 (1984) 387-395). The expression
cassette of
pEC-SOD 1a was further cloned into an adenovirus vector (AdBgIII) for
adenovirus construction
as described previously (Kozarsky, K.F. and Wilson, J.M.: Gene therapy:
adenovirus vectors.
Curr. Opin. Genet. Dev. 3 (1993) 499-503).
Adenovirus Production
The EC-SOD expression cassette containing elongation factor 1-a (Laukanen et
al., Gene 2000,
Vol 254, pag 173-179) promoter was cloned into an adenovirus vector (AdBgIII)
see Fig.2.
Nuclear-targeted LacZ adenovirus was used as a control (Laitinen et al., Hum
Gene Ther. 1998,
Vol 9, pg 1481-1486). Clinical-grade adenoviruses were produced in 293 cells
and analyzed to
be free of microbiological contaminants, mycoplasma, endotoxin, and
replication-competent
viruses.
Animal Experiments
Adult homozygous Watanabe Heritable Hyperlipidemic (WHHL) rabbits (Finnish
National
Experimental Animal Center, Kuopio, Finland) were kept on a standard diet (K2
special,
I actamin AB, Sweden). Acetylsalicylic acid was added to drinking water to
give an estimated
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daily ASA dose of 10 mg/day. The animals were followed for 4 weeks after gene
transfer with
either EC-SOD (n=8) or LacZ (n=8). The animals were anesthetized with a
combination of 0.25
ml/kg (fentanyl 0,315 mg/mL fluanosine 10 mg/mL mixture, Hypnorm~, Janssen)
s.c. and 0.25
ml/kg i.m. (midazolam 5 mg/ml, Dormicum~, Roche). The entire aorta was denuded
from
5 endothelium by repeated passage of an endarterectomy catheter (3F Sorin
Medical) from a
femoral artery cutdown. Three days later the animals were anesthetized again.
After iv.
heparinization a 2 cm infra-renal aortic segment was transfected (3x109
pfu/kg) with
InfiltratorO catheter (Boston Scientific) and an intravascular stent applied
(1.1 stent to artery
ratio, Guidant Corporation). After the follow-up the animals were sacrificed
with an overdose of
10 the anesthetic agents and the stented and a proximal unstented vessel
section was collected
and processed for further analysis.
Histological Analysis
Intact vessel segments were removed, flushed gently with saline, and immersion-
fixed en bloc
15 in 4% neutral buffered formalin for 24 hours. Fixed samples were dehydrated
in a graded series
of ethanol and infiltrated with a 1:1 solution of methyl methacrylate (MMA)
and xylene and,
finally, with MMA (4°C, 12 hours each). Afterwards, the samples were
polymerized in plastic
tubes at -4°C. These primary methacrylate blocks were sawed into 4 mm
thick segments
(resulting in 4 to 6 segments per animal). Segments were placed into embedding
molds, and
20 polymerization of blocks was again performed at -4°C. Polymerized
blocks were initially ground
to bring the tissue components closer to the cutting surface. Serial sections
(2-5 Nm) of the
MMA blocks were cut on a Microm rotating microtome with hard tissue blades.
After immersion
in a drop of 80% ethanol, sections were stretched to a fold-free state on
Superfrost glass slides
(Menzel-Glaser), covered with a polyethylene sheet and several layers of
filter paper, and tightly
25 pressed on the glass slides, followed by overnight drying at 42°C
under pressure. Deplastination
was carried out in 2-methoxyethyl acetate 5 times for 15 minutes. Rehydration
of the sections
was performed in graded ethanol solutions and 1 mmol/L PBS. Toluidine blue,
hematoxylin and
eosin, Masson's trichrome and Elastics van Gieson stainings were performed
according to
standard histopathologic methods. ( Cotran RS, Kumar V, Robbins SL. Robbins
Pathologic Basis
30 of Disease. Philadelphia, Pa: WB Saunders Co; 1994.)
Thickness of neointima, pre-existing atherosclerotic lesions and media was
measured on
digitalized pictures using a Zeiss microscope (Axioplan), MRC digital camera
and KS software
(Rel. 4.0). The ratio of neointima to total intima was calculated. Endothelial
cell (PECAM)
35 coverage and inflammatory cell (macrophage) infiltration was quantified as
percent of luminal
cross sectional surface and percent of intimal cross sectional area,
respectively. Mean values of
treated and control group was calculated from median values of each animal.
The following
antibodies were used: PECAM (endothelium, dilution 1:50, Santa Cruz), RAM 11
(macrophages,
dilution 1:200, DAKO), HHF35 (smooth muscle cells [SMCs], dilution 1:50,
DAKO). An avidin-
40 biotin-horseradish peroxidase system was used with DAB for signal detection
(Vector Elite Kit).
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Autopsy Analysis
Autopsy analysis was done at the National Veterinary and Food Research
Institute, Kuopio
Department, Finland, by P. Syrjala, DVM.
Statistical analyses
Differences between the groups were analyzed by nonparametric Mann-Whitney U-
test or
Students T test, when appropriate. Spearman (or Pearson, when appropriate)
correlation
coefficients were calculated to evaluate relationships between variables. All
data represent
means ~ standard deviation (SD) and/or standard error (SE). Differences were
considered
significant if the p-value was < 0.05. Analysis of distribution fitting was
performed by
Kolmogorov-Smirnov and the Chi-Square test.
Results
Histological Analysis
The gene transfer site was analyzed histologically to determine the effect of
local EC-SOD gene
transfer on neointima formation, re-endothelialization and inflammatory cell
infiltration at a 4-
week time point. There was no between group difference in extent of pre-
existing
atherosclerotic disease and pre-existing macrophage content therein. The
neointima formation
in EC-SOD animals was significantly (P<0.05) reduced compared with control
animals; both in
vessel segments that were relatively unaffected by the atherosclerotic lesion
formation before
stenting as well as segments which had a pre-existing atherosclerotic lesion
(Table 1, Figure 1).
ecSOD
restenosis (pm, struts on media) 115.8 t 34.4 ~ 8.9 t 20.5
(Nm, struts on lesion) 228.9 t 92.5 ~ 61.6 t 10
restenosis/lesion ratio 5.4 ~ 4.6 ~ 1.4 t 0.6
Table 1: Results of restenosis formation
Example 2
pigs and plasmids
Methods
Plasmid Production
The sequence for human EC-SOD was cloned into a pNGVL3 plasmid vector
(National Gene
Vectror Laboratories, USA). Plasmid Production
The coding sequence for human EC-SOD cDNA was obtained from a pOTB7 plasmid
vector
_ . _ _ . . _ . ..~,._."",~~ T~~~r~.n~n~~~a~ nsina EcoRI and PstI restriction
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enzymes and further cloned into a pNGVL3 plasmid vector (National Gene Vectror
Laboratories,
USA).
Nuclear-targeted LacZ plasmid (pNGVL3) was used as a controll.
Animal Experiments
The animal work was approved by the ethics committee of Gothenburg University,
Sweden. 8
stents were implanted in the right and left external iliac arteries of 4
domestic swine (weight, 20
to 30 kg) fed a normal chow diet. The animals were premeditated with a loading
dose ASA and
clopidrogel by mouth the day before stent placement and the daily drug therapy
was continued
throughout the 14 day follow-up period. Ketamine (20 mg/kg IM) and xylazine (4
mg/kg IM)
were used for induction of anesthesia. General anesthesia was maintained by
use of inhalational
anesthetics. A 9F sheath was placed retrograde in the right carotid artery. A
bolus of heparin
(150 U/kg) was administered intraarterially. Distal aortic and iliac
angiography was performed
with and the proximal iliac artery was engaged with the guiding catheter. A
0.014-in high-
torque floppy guide wire (Advanced Cardiovascular Systems) was advanced to the
femoral
artery under fluoroscopy. A 5-cm artery segment was transfected (2000 Ng
plasmid in PBS
divided in 3 injections) with Infiltrator~ catheter (Boston Scientific). The
animals were
subjected to stenting with either EC-SOD (n=2) or control treatment (n=2). To
achieve a brisk
neointima formation at the stented vessel segment a long stent was selected
and The guiding
catheter was used as a reference for stent sizing in an attempt to
overdilatate the vessel
segment and achieve a balloon (stent) to artery ratio of 1.5. Placement of an
intravascular stent
(50 mm length) was completed. The procedure was then repeated in the opposite
iliac artery.
Angiography was completed after placement to confirm patency. The animals were
allowed to
recover and were returned to care facilities. All animals remained on a normal
laboratory diet.
The animals were returned to the research catheterization laboratory for
angiography 14 days
after implantation. After completion of follow-up angiography, the animals
were euthanatized
with an overdose of the anesthetic agents. The angiographic findings were
confirmed by
macroscopic pathological examination of the vessel segments.
Results
In the EC-SOD treated animals 4 of 4 stented vessels remained open after the 2-
week follow-up
period. In contrast, 2 of 4 stents (one in each control treated animal), were
occluded in control
animals.
Example 3
Vein graft experiments
Animals
New Zealand White rabbits are maintained on a normal or atherogenic diet with
water ad
libitum. Anesthesia for surgery is induced with a combination of 0.25 ml/kg
(fentanyl 0,315
,.r~ ci.,~".,~ir,o in mn/ml mixture. Hvonorm~, Janssen) s.c. and 0.25 ml/kg
i.m.
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(midazolam 5 mg/ml, Dormicump, Roche).. All procedures are approved by
institutional ethics
committee. The jugular vein segment is harvested and the vessel segment is
transfected ex vivo
with EC-SOD or control vector and anastomosed into the ipsilateral carotid
artery in a reverse
end-to-end fashion. At various time points after the procedure, the animals
are killed by
overdose of the anesthetic agens.
Vessel Harvesting and Analysis
The vein grafts are harvested at the predetermined time points after surgery.
Animals are
anesthetized, and vessels excised. Fresh vessel segments are carefully rinsed
withouth
removing any present thrombi and processed further for superoxide, transgene
expression and
histological analysis. Vein-graft thickening, endothelial cell integrity and
inflammatory infiltration
are quantified by computerized morphometry on stained and immunostained vessel
sections
performed according to standard histopathologic methods.( Cotran RS, Kumar V,
Robbins SL.
Bobbins Pathologic Basis of Disease. Philadelphia, Pa: WB Saunders Co; 1994.).
Example 4
Synthetic graft experiments
Animals
New Zealand White rabbits are maintained on a normal or atherogenic diet with
water ad
libitum. Anesthesia for surgery is induced with a combination of 0.25 ml/kg
(fentanyl 0,315
mg/mL fluanosine 10 mg/mL mixture, Hypnorm~, Janssen) s.c. and 0.25 ml/kg i.m.
(midazolam 5 mg/ml, DormicumO, Roche).All procedures are approved by
institutional ethics
committee. Infra-renal aortic segment is exposed and a synthetic vascular
graft is implanted in
a end-to-end fashion. The surrounding tissues are transfected with EC-SOD or
control vector. At
various time points after the procedure, the animals are killed by overdose of
the anesthetic
agens.
Harvesting and Analysis
The grafted vessel segments are harvested at the predetermined time points
after surgery.
Animals are anesthetized, and vessels excised. Fresh vessel segments are
carefully rinsed
withouth removing any present thrombi and processed further for superoxide,
transgene
expression and histological analysis. Neointima formation, endothelial cell
integrity and
inflammatory infiltration are quantified by computerized morphometry on
stained and
immunostained vessel sections performed according to standard histopathologic
methods.(
Cotran RS, Kumar V, Bobbins SL. Bobbins Pathologic Basis of Disease.
Philadelphia, Pa: WB
Saunders Co; 1994.).
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Example 5
Prosthetic implant experiment
This example showes faster endothelialisation of heart valve surfaces with or
without fibronectin
precoating when VEGF plasmid was administered. Also increased capillarisation
of the implant
was noticed.
Methods
Prosthetic material, used or intended for use in medical implants is tinted in
sterile saline
solution (0,9 %). Thereafter the material is divided in two approximately 1
cmz pieces under
sterile conditions.
Surgical procedure and sacrifice:
Animals are anesthesized with mixture of Hypnorm~ (1 part), Dormicum~ (1 part)
and 2 parts
of sterile water (0.33m1 / 1008 weight rat) administered intraperitoneally.
Abdomen is shaved
and opened. 2 mL of bupivacain is administered in the wound area. The
materials is sewn to the
peritoneum on both sides of midline with continuous 5-0 nylon. Each piece is
attached
separately on the wall with running 5-0 monofilament suture. The tissues
surrounding the first
half is transfected with EC-SOD, and the tissues surrounding the second half
are transfected
with control (LacZ) vector. Abdomen is closed in layers with 3-0. Animals are
sacrificed in
anesthesia after explanting abdominal wall with implant material pieces.
Analysis
Every piece are divided in the middle. Half of the material is sent to
electron microscopy after
preservation in 2% paraformaldehyde/2% glutaraldehyde. Second half is examined
in light
microscopy after preservation in 4% formalin and immunostained with
endothelial, smooth
muscle cell, connective tissue specific and inflammatory cell markers. The
tissue response is
quantified by computer assisted morphometry for capillary endothelialization
and connective
tissue formation.
