U.S. patent application number 11/980983 was filed with the patent office on 2008-05-15 for methods for coating stents with dna and expression of recombinant genes from dna coated stents in vivo.
Invention is credited to Elizabeth G. Nabel, Gary J. Natel, Zhi-Yong Yang.
Application Number | 20080112997 11/980983 |
Document ID | / |
Family ID | 25384436 |
Filed Date | 2008-05-15 |
United States Patent
Application |
20080112997 |
Kind Code |
A1 |
Nabel; Elizabeth G. ; et
al. |
May 15, 2008 |
Methods for coating stents with DNA and expression of recombinant
genes from DNA coated stents in vivo
Abstract
The present invention describes DNA coated stents and methods of
using the same to treat or prevent vascular diseases, such as
restenosis.
Inventors: |
Nabel; Elizabeth G.; (Ann
Arbor, MI) ; Natel; Gary J.; (Ann Arbor, MI) ;
Yang; Zhi-Yong; (Ann Arbor, MI) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Family ID: |
25384436 |
Appl. No.: |
11/980983 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10946785 |
Sep 22, 2004 |
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11980983 |
Oct 31, 2007 |
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08884352 |
Jun 27, 1997 |
6818016 |
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10946785 |
Sep 22, 2004 |
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Current U.S.
Class: |
424/423 ;
514/44R |
Current CPC
Class: |
A61K 38/50 20130101;
A61L 31/16 20130101; A61L 2300/606 20130101; A61L 2300/258
20130101; A61K 31/711 20130101; A61K 48/00 20130101; A61K 38/45
20130101; A61L 31/10 20130101; A61L 2300/252 20130101; A61L
2300/416 20130101; A61L 31/10 20130101; A61P 9/00 20180101; A61F
2/82 20130101; C08L 89/00 20130101; A61K 9/0024 20130101 |
Class at
Publication: |
424/423 ;
514/044 |
International
Class: |
A61F 2/82 20060101
A61F002/82; A61K 31/711 20060101 A61K031/711; A61P 9/00 20060101
A61P009/00 |
Claims
1-56. (canceled)
57. A device for delivery of a therapeutic agent, comprising an
implantable device coated with a composition comprising a polymer
and a DNA encoding a therapeutically useful protein.
58. The device of claim 57, wherein the implantable device is a
stent.
59. The device of claim 57, wherein the implantable device is
composed of stainless steel.
60. The device of claim 57, wherein the therapeutically useful
protein is a gene encoding an antiplatelet agent, anticoagulant
agent, antimitotic agent, antioxidant, antimetabolite agent, or
anti-inflammatory agent.
61. The device of claim 60, wherein the therapeutically useful
protein inhibits the proliferation of cells.
62. The device of claim 61, wherein the therapeutically useful
protein is thymidine kinase, p16, p21, p27, p57, retinoblastoma or
cytosine deaminase.
63. The device of claim 62, wherein the therapeutically useful
protein is thymidine kinase or cytosine deaminase.
64. The device of claim 57, wherein the stent is coated with about
50 .mu.g to about 5 mg of DNA.
65. The device of claim 57, wherein the polymer comprises
fibrin.
66. The device of claim 57, wherein the device is coated with a
polymer matrix.
67. The implantable device of claim 57, wherein the device is
coated with a polymer selected from the group consisting of
poly(ethylene terephthalate), polyacetal, and poly(ethylene
oxide)/poly(butylene terephthalate) copolymer.
68. The implantable device of claim 57, wherein the DNA is naked
DNA.
69. The implantable device of claim 57, wherein the DNA is
incorporated into a vector.
70. The implantable device of claim 69, wherein the vector is
selected from the group consisting of shuttle vectors, expression
vectors, retroviral vectors, adenoviral vectors, adeno-associated
vectors and liposomes.
71. The implantable device of claim 66, wherein the polymer matrix
is formed from an aqueous suspension of DNA and liquid monomeric
matrix.
72. The implantable device of claim 57, wherein the DNA comprises
an sm 22.alpha. promoter operatively linked to the DNA encoding the
therapeutically useful protein.
73. The implantable device of claim 57, wherein the therapeutically
useful protein is a fusion protein.
74. The implantable device of claim 58, wherein the stent is an
intravascular stent.
75. The implantable device of claim 57, wherein the device is
coated with a polymer matrix wherein said DNA is uniformly
dispersed within said matrix.
76. An implantable device comprising a stent coated with a
composition comprising a polymer and a DNA encoding a
therapeutically useful protein.
77. The implantable device of claim 76, wherein the therapeutically
useful protein is an antiplatelet agent, anticoagulant agent,
antimitotic agent, antioxidant, antimetabolite agent, or
anti-inflammatory agent.
78. The implantable device of claim 76, wherein the therapeutically
useful protein inhibits the proliferation of cells.
79. The implantable device of claim 76, wherein the therapeutically
useful protein is thymidine kinase, p16, p21, p27, p57,
retinoblastoma or cytosine deaminase.
80. The implantable device of claim 76, wherein the stent is coated
with about 50 .mu.g to about 5 mg of DNA.
81. The implantable device of claim 75, wherein the polymer is
selected from the group consisting of poly(ethylene terephthalate),
polyacetal, and poly(ethylene oxide)/poly(butylene terephthalate)
copolymer.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention provides an intravascular DNA coated stent
and methods for expressing recombinant genes in vivo using the DNA
coated stent. DNA coated stents are useful for treating coronary
and peripheral vascular diseases, particularly restenosis.
[0003] 2. Background of the Invention
[0004] Coronary and peripheral angioplasty is routinely performed
to treat obstructive atherosclerotic lesions in the coronary and
peripheral blood vessels. Following balloon dilation of these blood
vessels, 30-40% of patients undergo restenosis.
[0005] Restenosis is the reclosure of a peripheral or coronary
artery following trauma to that artery caused by efforts to open a
stenosed portion of the artery, such as, for example, by balloon
dilation, ablation, atherectomy or laser treatment of the artery.
Restenosis is believed to be a natural healing reaction to the
injury of the arterial wall. The healing reaction begins with the
thrombotic mechanism at the site of the injury. The final result of
the complex steps of the healing process can be intimal
hyperplasia, the uncontrolled migration and proliferation of medial
smooth muscle cells, combined with their extracellular matrix
production, until the artery is again stenosed or occluded. Thus,
restenosis is characterized by both elastic recoil or chronic
constriction of the vessel in addition to abnormal cell
proliferation.
[0006] Currently restenosis must be treated with subsequent
angioplasty procedures. In an attempt to prevent restenosis,
metallic intravascular stents have been permanently implanted in
coronary or peripheral vessels. For example, U.S. Pat. No.
5,304,122 (Schwartz et al.), describe metal stents useful for
treating restenosis after balloon angioplasty or other coronary
interventional procedures. The stent is typically inserted by
catheter into a vascular lumen and expanded into contact with the
diseased portion of the arterial wall, thereby providing mechanical
support for the lumen. However, it has been found that restenosis
can still occur with such stents in place; likely, because although
the stent prevents elastic recoil of the artery, it fails to
prevent the cell proliferation which leads to intimal hyperplasia.
In addition, the stent itself can cause undesirable local
thrombosis. To address the problem of thrombosis, persons receiving
stents also receive extensive systemic treatment with anticoagulant
and antiplatelet drugs.
[0007] Stents coated with various compositions have been proposed.
For example, Dichek et al. (Circulation 1989, 80:1347-1353)
describe coating stainless steel stents with sheep endothelial
cells that had undergone retrovirus-mediated gene transfer for
either bacterial .beta.-galactosidase or human tissue-type
plasminogen activator. The stents were studied ex vivo in tissue
culture dishes only. The feasibility of implanting the stents into
arteries were not explored. This procedure of coating stents with
cells is tedious, cumbersome and costly because cell have to be
derived from a patient.
[0008] Other methods of providing therapeutic substances to the
vascular wall by means of stents have also been proposed. For
example, WO 91/12779, entitled "Intraluminal Drug Eluting
Prosthesis," and WO 90/13332, entitled "Stent With Sustained Drug
Delivery," suggest coating stents with antiplatelet agents,
anticoagulant agents, antimicrobial agents, anti-inflammatory
agents, antimetabolic agents and other drugs to reduce the
incidence of restenosis. Similarly, U.S. Pat. Nos. 5,571,166 and
5,554,182 (both to Dinh et al.) describe intraluminal stents coated
with fibrin and heparin. The stent is used to treat restenosis.
SUMMARY OF THE INVENTION
[0009] Accordingly, one object of this invention is to provide an
intravascular DNA coated stent.
[0010] A second object of this invention is to provide methods for
expressing recombinant genes in vivo using the DNA coated
stents.
[0011] A third object of this invention is to provide methods for
treating coronary and peripheral vascular diseases, particularly
restenosis and vein by-pass grafts, using the DNA coated
stents.
[0012] The present inventors have now realized these and other
objects through their discovery of methods for coating DNA on the
outside surface of a stent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 are restriction maps of plasmid pCMV-CAT
(VR1332).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
DNA Coated Stents
[0014] Stents are devices which can be delivered percutaneously to
treat coronary artery occlusions and to seal dissections or
aneurysms of splenic, carotid, iliac and popliteal vessels.
Suitable stents useful in the invention are polymeric or metallic.
Examples of polymeric stents include stents made with biostable or
bioabsorbable polymers such as poly(ethylene terephthalate),
polyacetal, poly(lactic acid), and poly(ethylene
oxide)/poly(butylene terephthalate) copolymer. Examples of metallic
stents include stents made from tantalum or stainless steel. Stents
are available in myriad designs; all of which can be used in the
present invention and are either commercially available or
described in the literature. For example, a self-expanding stent of
resilient polymeric material is described in WO 91/12779, entitled
"Intraluminal Drug Eluting Prosthesis." Alternatively, U.S. Pat.
No. 4,886,062 describes a deformable metal wire stent. Commercial
sources of stents include Johnson & Johnson, Boston Scientific,
Cordis, Advanced Catheter Systems, and U.S. Catheter, Inc.
[0015] Suitable genes which encode for therapeutic proteins useful
in the invention include genes which encode antiplatelet agents,
anticoagulant agents, antimitotic agents, antioxidants,
antimetabolite agents, and anti-inflammatory agents. Preferred
genes which encode therapeutic proteins include proteins which can
inhibit proliferation of cells (particular of vascular smooth
muscle cells (vsmc), including:
[0016] HSV thymidine kinase (McKnight, 1980, Nucleic Acids Res.
8:5949; Mansour et al., 1988, Nature 336:348-352),
[0017] .beta.-galactosidase,
[0018] p16 (Chan et al., 1995, Mol. Cell. Biol. 15:2682-2688; Guan
et al., Genes & Dev. 8:2939-2952),
[0019] p21 (Harper et al., 1993, Cell 75:805; Xiong et al., 1993,
Nature 366:701),
[0020] p27 (Toyoshima et al., 1994, Cell 78:67-74; Polyak et al.,
1994, Cell 78:59-66),
[0021] p57 (Lee et al., 1995, Genes & Dev. 9:639-649; Matsuoka
et al., 1995, Genes & Dev. 9:650-662),
[0022] retinoblastoma (Rb) (see Chang et al., 1995, Science,
267:518) or its mutants (see for example, Hamel et al., 1992, Mol.
Cell. Biol. 12:3431), and
[0023] cytosine deaminase (WO 9428143; Wang et al., 1988, Can. Soc.
Petrol. Geol. Mem., 14:71).
[0024] The sequences of these gene products are known in the
literature. Any DNA encoding these gene products can be used,
including the cDNA sequences that are described in the literature.
Alternatively, fusion proteins of the above can be used. The
preferred genes encode thymidine kinase (HSV-tk) or cytosine
deaminase gene.
[0025] Any DNA encoding the above therapeutic proteins can be used.
Preferably, the DNA sequence of the human cDNA encoding those
proteins are used. The DNA can be naked or can be incorporated into
a vector. Suitable vectors include shuttle vectors, expression
vectors, retroviral vectors, adenoviral vectors, adeno-associated
vectors and liposomes. Preferably a replication-defective
adenovirus vector is used, such as pAd-BglII as described by
Davidson et al. (1993, Nature Genet. 3:219-223). These vectors have
been demonstrated to program high levels of expression of genes in
balloon-injured rat carotid, rabbit coronary and porcine femoral
arteries (Ohno et al., Science 255:781 (1994); Guzman et al.,
Circulation 88:2838 (1993) and Barr et al., Gene Ther. 1:51
(1994)).
[0026] Various DNA constructs encoding HSV tk genes are available
from American Type Culture Collection, Rockville, Md., including
ATCC 39371, ATCC 39369 and VR-2036. Construction of adenoviral
constructs containing HSV-tk is described in co-pending application
Ser. No. 08/210,902, Example 1.
[0027] A list of preferred vectors is shown below in Table I.
TABLE-US-00001 TABLE 1 Plasmid Description CMVtkcitep27 CMVDSacIItk
with cite-p27 (EcoRI-Xbal fragment from pcitep27) inserted at the
BglII site CMVtkcitep27rev CMVDSacIItk with CITE-p27rev (EcoRI-Xbal
fragment from CITE p27rev) inserted into the BglII site CMVp27tk
pCMVp27citetk with the AatII-Ncol fragment (containing cite)
deleted. Tk and p27 are still active CMVp27citetk plasmid resulting
from the ligation of 3 fragments: (1) HindIII- EcoRI from
1332DSacII (=CMVtk DSacII) + (2) SaII-Ncol from p27 cite + (3)
Ncol-HindIII from 1012-tk CMVp27revcitetk results from the ligation
of 3 fragments: (1) HindIII-EcoRI from 1332 DSacII + (2) SaII-Ncol
from p27revcite + (3) Ncol-HindIII from 1012-tk CMVp27Sfcitetk
CMVp27citetk with the fragment SacII-Fspl deleted. (region of p27
between the cdk2 binding site and the putative NLS) CMVp27Nfcitetk
CMVp27 citetk with the fragment Narl-Fspl deleted. (region of p27
between the cdk2 binding site and the putative NLS) CMVp27Afcitetk
CMVp27citetk with the fragment AvaII-Fspl deleted. (region of p27
between the cdk2 binding site and the putative NLS) CMVp27cdccitetk
CMVp27citetk with the cdc2 kinase consensus site mutated (TPKK to
AAGG) CMVp27SFtk CMVp27tk with the SacII-Fspl fragment deleted
(that contains the region of p27 between the cdk2 binding site and
the putative NLS) CMVp27NFtk CMVp27tk with the Narl-Fspl fragment
deleted (that contains the region of p27 between the cdk2 binding
site and the putative NLS) CMVp27Aftk CMVp27tk with the AvaII-Fspl
fragment deleted (that contains the region of p27 between the cdk2
binding site and the putative NLS) CMVp27SNtk CMVp27citetk with the
SacII-Ncol fragment deleted (containing the C-terminus of p27)
CMVp27Sp21Ftk CMVp27tk with the HindIII-Ncol fragment from
1012-p21N inserted between the SacII and Fspl sites CMVp27Np21Ftk
CMVp27tk with the HindIII-Ncol fragment from 1012-p21N inserted
between the Narl and Fspl sites CMVp27Sp21Fcitetk CMVp27citetk with
the HindIII-Ncol fragment from 1012-p21N (containing the N-terminal
part of p21 coding sequence) inserted between the SacII and Fspl
sites in the p27 coding region CMVp27Np21Fcitetk CMVp27citetk with
the HindIII-Ncol fragment from 1012-p21N inserted between the Narl
and Fspl sites CMVp27Sp21 Clal-SacII fragment from CMVp27citetk
fused to the Ncol-Clal fragment of VR 1012-p21N (giving a fusion
between p27N and p21N) CMVp27Np21 Clal-Narl fragment from CMVp27
citetk fused to the Ncol-Clal fragment of VR 1012-p21N (giving a
fusion between p27N and p21N) CMVp27Dkcitetk CMVp27citetk with all
K mutated to R between ATG and SacII of p27. There is an additional
`c` before the SacII site CMVp27Ncitetk CMVp27citetk with a stop
codon between SacII and Xbal in p27 (only the N-terminus of p27
remains) CMVp27NLScitetk CMVp27citetk with a NLS (GRRRRA = ATF2
NLS) and a stop codon between SacII and Xbal in p27 (only the
N-terminus of p27 remains) CMVp27DKNcitetk CMVp27Dkcitetk with a
stop codon between SacII and Xbal in p27 (only the N-terminus of
p27 remains) CMVp27DKNLScitetk CMVp27Dkcitetk with a NLS (GRRRRA =
ATF2 NLS) and a stop codon between SacII and Xbal in p27 (only the
N-terminus of p27 remains)
[0028] The stent can optionally be coated with other therapeutic
proteins such as heparin, hirudin, angiopeptin, ACE inhibitors,
growth factors (such as IL.sub.2-10), nitric oxide or with DNA
encoding the same.
[0029] Suitable polymerizable matrix useful for binding the DNA to
the stent include any monomeric biocompatible material which can be
suspended in water, mixed with DNA and subsequently polymerized to
form a biocompatible solid coating. Thrombin polymerized fibrinogen
(fibrin) is preferred.
[0030] The stent is preferably coated with about 50 .mu.g to about
5 mg of DNA. The thickness of the polymerizable matrix containing
the DNA is typically about 5-500 .mu.m. The matrix preferably
covers the entire surface of the stent.
Methods for Coating a Stent with DNA
[0031] Methods for coating surfaces are well known in the art and
include, for example, spray coating, immersion coating, etc. Any of
these methods can be used in the invention. For example, a liquid
monomeric matrix can be mixed with the DNA and polymerization
initiated. The stent can then be added to the polymerizing
solution, such that polymer forms over its entire surface. The
coated stent is then removed and dried. Multiple application steps
can be used to provide improved coating uniformity and improved
control over the amount of DNA applied to the stent.
[0032] In a preferred embodiment, an aqueous mixture of DNA and
human thrombin is added to an aqueous suspension of fibrinogen. The
fibrinogen concentration of the suspension is typically between
about 10-50, preferably about 20-40, more preferably about 30
mg/ml. The concentration of the DNA in the aqueous mixture is
typically about 1-20, preferably about 5-15, more preferably about
10 .mu.g/ml. The amount of human thrombin in the aqueous mixture
about 0.5 to 5, preferably about 1 U. The DNA and human thrombin
are first added together to form a mixture and that mixture is then
added to the fibrinogen suspension. Thereafter, a stent is dipped
into the polymerizing solution. After the mixture solidifies, the
stent is removed.
Methods for Placing the DNA Coated Stent Within the Vasculature
[0033] The stent can be placed onto the balloon at a distal end of
a balloon catheter and delivered by conventional percutaneous means
(e.g. as in an angioplasty procedure) to the site of the
restriction or closure to be treated where it can then be expanded
into contact with the body lumen by inflating the balloon. The
catheter can then be withdrawn, leaving the stent of the present
invention in place at the treatment site. The stent may therefore
provide both a supporting structure for the lumen at the site of
treatment and also a site for instillation of DNA at the lumen
wall. The site of instillation can be either an arterial or venous
wall.
[0034] Site specific instillation of a solution of DNA at an
arterial wall using a balloon catheter has previously been
described by the present inventors in U.S. Ser. No. 08/376,522, now
allowed. Thus, the viability of incorporation of "naked DNA" into
arterial cells and subsequent expression of that DNA has previously
been demonstrated.
[0035] The stent can be placed in any peripheral or coronary artery
or vein. The stent is preferably placed at the site of injury
either immediately or soon after mechanical vessel injury.
Methods for Expressing Recombinant Genes In Vivo Using the DNA
Coated Stents
[0036] Recombinant genes can be expressed in vivo by implanting the
DNA coated stents of the present invention in an artery or vein of
a patient. Gene expression is continuous and can optionally be
controlled with viral promoters or cell specific promoters such as
smc, in particular sm 22.varies..
[0037] SM 22.varies. is a putative calcium-binding protein that is
expressed in cardiac, smooth and skeletal muscle lineages during
mouse embryogenesis and in adult smcs (Lees-Miller et al., 1987, J.
Biol. Chem. 262:2988; Duband et al., 1993, Differentiation, 55:1;
Shanahan et al., 1993, Circ. Res. 73:193). Promoters of smcs are of
particular interest because they direct transgene expression
specifically in vascular and not visceral smooth muscle cells.
Method of Treating Coronary and Peripheral Vascular Diseases with
the DNA Coated Stents
[0038] Coronary and peripheral diseases, including restenosis,
atherosclerosis, coronary artery bypass graft stenosis, vein bypass
graft stenosis or restenosis, arterio-venous fistula stenosis or
restenosis, peripheral artery stenosis or restenosis, can be
treated by implanting the DNA coated stent of the present
invention, into a coronary or peripheral artery or vein of a
patient. Suitable patients include mammals such as dogs, horses,
cattle, humans, etc. Humans are preferred patients.
[0039] In an alternate embodiment, the DNA coated stent is
implanted into the patient and an antiplatelet agent, anticoagulant
agent, antimicrobial agent, anti-inflammatory agent, antimetabolic
agent, antimitotic agent or other drug is administered to reduce
the incidence of restenosis. Suitable anticoagulant agents can
include drugs such as heparin, coumadin, protamine, hirudin and
tick anticoagulant protein. Suitable antimitotic agents and
antimetabolite agents can include drugs such as colchicine,
methotrexate, azathioprine, vincristine, vinblastine, fluorouracil,
adriamycin and mutamycin. Ganciclovir or acyclovir is preferably
administered.
[0040] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
Examples
Procedure for Coating the Stents Using Thrombin Polymerized
Fibrinogen (Fibrin)
[0041] Human fibrinogen was dissolved in water at concentrations of
30 mg/ml. 100 .mu.l of different concentrations of fibrinogen were
used in the preparation. Fibrinogen was diluted in water when
necessary and transferred to an Eppendorf tube.
[0042] Plasmid CAT (pCMV-CAT) was dissolved in water at
concentrations of 10 mg/ml. The DNA was diluted in water in an
Eppendorf tube to a final volume of 100 .mu.g/ml. 1 U of human
thrombin was added in the DNA solution and mixed gently.
[0043] The mixture of DNA and thrombin was added to the fibrinogen
solution. After brief mixing, the mixture was loaded into Tygon
tubing (1/8'' ID; 1'' to 11/4'' long, Formulation S-50-HL) which
was sealed at one end. A Johnson & Johnson metallic stent, 5.0
mm, was immediately inserted into the DNA/fibrinogen/thrombin
mixture in the tubing, and incubated until the mixture solidified.
The fibrin-coated stent was removed and air dried.
[0044] The coated stent was installed into the left and right pig
iliac femoral arteries using routine surgical procedures.
[0045] Three days after installment of the stents, the arteries
were excised, and homogenized using glass dowels. The protein
extract was freeze-thawed 3.times., heat-inactivated for 15 minutes
at 65.degree. C. and the supernatant was collected. 300 .mu.g of
the soluble protein was used for CAT assays. The results were read
using a Betagen machine which measures the acetylation of CAT.
Implantation of the DNA Coated Stents in the Vasculature
[0046] Juvenile domestic pigs (3 months, 15-20 kg) of either sex
are given aspirin (10 mg/kg) orally two days prior to surgery and
three times weekly for the duration of the study.
[0047] Pigs were anesthetized using Telazol (6.0 mg/kg IM) and
xylazine (2.2 mg/kg IM) and intubated with an endotracheal tube. 1%
isofluane is administered throughout the surgical procedure. 150
units/kg of heparin were administered via IV prior to surgery.
[0048] Following prepping and draping, a midline abdominal incision
was made, extending caudally to the pubis through the skin and
fascia, and the abdominal musculature was divided in the midline.
The peritoneal cavity was opened and the intestines retracted
cranially using a Balfour retractor. Using a combination of blunt
and sharp dissection, each iliac and femoral artery was isolated
from their cranial extent, caudally to beyond the bifurcation of
the femoral artery.
[0049] The internal iliac artery was ligated at its most caudal
point with 2-0 silk. Ties were looped around the proximal iliac and
femoral arteries, then temporarily secured. An arteriotomy of the
internal iliac artery was made just proximal to the ligature. The
balloon-expandable stent was advanced to the iliac artery and the
balloon inflated using an inflation device at pressure of 6
atmospheres. The balloon was deflated and the balloon catheter
removed, then the internal iliac artery was ligated followed by
release of the loops. Restoration of arterial blood flow was
confirmed. The peritoneum and the muscle were closed with 1-0
vicryl continuous sutures, and the fascial layer closed with 1-0
vicryl interrupted sutures. The skin was closed with staples.
Results
[0050] The following data demonstrate the expression of the
reporter gene, CA T in porcine arteries in vivo following
implantation of the DNA coated stent. TABLE-US-00002 Reporter DNA
days after stent Fibrinogen (mg) (.mu.g) % CAT activity placement 1
15 100 8.4, 23.1, 6.2 3 15 500 7.5, 3.9 3 15 1000 2.0 3 2 15 100
3.4 7 3 15 100 2.54 10 4 10 100 2.8 3 5 10 100 0.9 10
The above data was used to determine the optimal dose of DNA and
fibrinogen. This data supports the principle that DNA coated stents
can be implanted in a patient, the gene is expressed as a protein,
and sufficient quantities of protein are produced to allow
measurement thereof.
[0051] Having now fully described the invention, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention as set forth herein.
* * * * *