U.S. patent application number 09/760036 was filed with the patent office on 2002-09-19 for transfection system, its preparation and use in somatic gene therapy.
Invention is credited to Schrader, Jurgen.
Application Number | 20020133126 09/760036 |
Document ID | / |
Family ID | 7835409 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020133126 |
Kind Code |
A1 |
Schrader, Jurgen |
September 19, 2002 |
Transfection system, its preparation and use in somatic gene
therapy
Abstract
The present invention relates to a transfection system
comprising one or more Infiltrator catheters, one or more nucleic
acids, and, where appropriate, suitable ancillary substances and/or
additives, and to its preparation and use in somatic gene
therapy.
Inventors: |
Schrader, Jurgen;
(US) |
Correspondence
Address: |
William F. Lawrence
FROMMER LAWRENCE & HAUG LLP
745 Fifth Avenue
New York
NY
10151
US
|
Family ID: |
7835409 |
Appl. No.: |
09/760036 |
Filed: |
January 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09760036 |
Jan 12, 2001 |
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09112519 |
Jul 9, 1998 |
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6200304 |
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Current U.S.
Class: |
604/264 ;
424/529; 604/523 |
Current CPC
Class: |
C12N 2750/14143
20130101; A61K 48/00 20130101; C12N 9/0075 20130101; C12N 15/86
20130101 |
Class at
Publication: |
604/264 ;
604/523; 424/529 |
International
Class: |
A61M 025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 1997 |
DE |
19729769.2 |
Claims
We claim:
1. A transfection system comprising (a) one or more Infiltrator
catheters; (b) one or more nucleic acids in nonviral form; and,
where appropriate, (c) suitable ancillary substances and/or
additives.
2. A transfection system as claimed in claim 1, wherein the
Infiltrator catheter comprises one or more injector ports with a
height of about 100-500 .mu.m.
3. A transfection system as claimed in claim 1, wherein the
Infiltrator catheter comprises about 5.times.7 injector ports.
4. A transfection system as claimed in claim 1, wherein the
Infiltrator catheter is a catheter with more than one lumen.
5. A transfection system as claimed in claim 1, wherein the
Infiltrator catheter comprises a central tubular shaft, an
inflatable balloon which envelops the shaft at a suitable point,
and a tubular sleeve which comprises one or more injector ports at
the point where the inflatable balloon is.
6. A transfection system as claimed in claim 1, wherein the
transfection system additionally comprises a guide wire.
7. A transfection system as claimed in claim 1, wherein the nucleic
acid is selected from the group consisting of a nucleic acid in
naked form and a nucleic acid together with other nonviral
components.
8. A transfection system as claimed in claim 1, wherein the nucleic
acid is selected from the group consisting of a single- or
double-stranded DNA or RNA, a plasmid DNA and an antisense
oligonucleotide.
9. A transfection system as claimed in claim 1, wherein the nucleic
acid is present in the form of a nucleic acid/liposome complex.
10. A transfection system as claimed in claim 9, wherein the
nucleic acid/liposome complex also comprises one or more nucleic
acid binding proteins.
11. A transfection system as claimed in claim 1, wherein the
nucleic acid codes for a therapeutically effective gene
product.
12. A transfection system as claimed in claim 11, wherein the
therapeutically effective gene product is a nitric-oxide
synthase.
13. A transfection system as claimed in claim 11, wherein the
nucleic acid coding for a therapeutically effective gene product
comprises one or more noncoding sequences and/or a polyA
sequence.
14. A process for producing a transfection system comprising (a)
one or more Infiltrator catheters; (b) one or more nucleic acids in
nonviral form; and, where appropriate, (c) suitable ancillary
substances and/or additives, wherein one or more Infiltrator
catheters, one or more nucleic acids in nonviral form and, where
appropriate, suitable ancillary substances and/or additives and,
where appropriate, a guide wire are combined.
15. Method of using of a transfection system comprising (a) one or
more Infiltrator catheters; (b) one or more nucleic acids in
nonviral form; and, where appropriate, (c) suitable ancillary
substances and/or additives, for somatic gene therapy.
16. Method as claimed in claim 15 for treating vascular disorders,
genetically related disorders and/or disorders which can be treated
by gene transfer, including prevention thereof.
17. Method of using an Infiltrator catheter for producing a
transfection system comprising (a) one or more Infiltrator
catheters; (b) one or more nucleic acids in nonviral form; and,
where appropriate, (c) suitable ancillary substances and/or
additives.
Description
[0001] The present invention relates to a transfection system
comprising one or more Infiltrator catheters, one or more nucleic
acids, and, where appropriate, suitable ancillary substances and/or
additives, and to its preparation and use in somatic gene
therapy.
[0002] Local gene therapeutic treatment of vascular disorders
represents a very promising prospect in interventional cardiology
which might, for example, prevent reocclusion of vessels
(restenosis) after mechanical widening of the blocked vessel with a
balloon catheter (so-called percutaneous transluminal coronary
angioplasty; PTCA), because restenosis still occurs in 30-40% of
all cases after PTCA treatment.
[0003] Systemic administration of drugs has, despite very promising
theoretical ideas, not produced an improvement in the long-term
success of PTCA, presumably because the concentration of the
therapeutic agent in the region of the treated stenosis was
inadequate at the time. These results have led to the development
of various special catheters permitting local treatment of a
damaged section of vessel with specific drugs.
[0004] One disadvantage of the local administration of medicines,
especially of medicines with a low molecular weight, using special
catheters is, however, rapid perfusion out of the treated section
of vessel. The required depot formation persists for only a short
time.
[0005] A prolongation of the therapeutic effect can be achieved,
for example, by therapeutically effective genes being transferred,
by somatic gene transfer, locally into the vessel wall and being
expressed there.
[0006] For gene transfer into the vessel wall, essentially three
components are necessary:
[0007] A therapeutically effective gene which, on local expression,
for example, results in the synthesis of a factor which inhibits
restenosis formation.
[0008] A transfection system which permits
[0009] (a) maximally efficient transfection of the vessel wall with
the therapeutic gene and
[0010] (b) localization of the transfection.
[0011] An efficient catheter technology which allows
[0012] (a) in vivo transfections of specific individual sections of
vessels in a hypo- or atraumatic manner using minimally invasive
techniques,
[0013] (b) in combination with the transfection system, minimal
distribution of the therapeutic gene into the perivascular space or
into the blood circulation to be ensured, and
[0014] (c) a guarantee of short vessel occlusion times.
[0015] Schrader, J. & Godecke, A. (DE 44 11 402) have now found
that transfection with the nitric-oxide synthase gene in the form
of a liposome complex in blood vessels leads to a therapeutically
relevant inhibition of vessel stenosis and restenosis after PTCA.
However, a polyethylene catheter was used for transfection with the
DNA-liposome complexes, which interrupted the blood flow in the
vessel for 15-20 minutes. In addition, transfection led to a
thrombosis after reopening of the vessel in a few cases.
[0016] Some other special catheters have already been described in
the literature with the intention of solving the problem of local
administration of drugs (Wilenksy, R. L. et al. (1993) Trends
Cardiovasc. Med. 3(5), 163-170).
[0017] For example, a double balloon catheter allows a defined
section of vessel to be separated from the circulation by inflating
balloons, one distal and one proximal of the section of vessel to
be transfected (see, for example, Nathan, A. & Edelman E. R.
(1995) in Edelman, E. R. (ed.), "Frontiers in Cardiology; Molecular
Interventions and Local Drug Delivery" Saunders Company Ltd.,
london, GB, 29-52). The lumen isolated in this way is then filled
with the therapeutic agent, which enters the vessel wall by
diffusion. However, the disadvantages of this catheter are the long
vessel occlusion times on use, and essentially only the innermost
vessel cell layers are reached (Flugelman, M. Y. (1995), Thrombosis
and Haemostasis, 74(1), 406-410). This type of catheter is
therefore unsuitable for somatic gene transfer.
[0018] The porous balloon was developed in order to force a
therapeutic agent under pressure through pores in an inflated
balloon into the vessel wall and thus to achieve transmural
distribution of the therapeutic agent. The high pressures required
for the injection (2-5 atm.) frequently resulted, however, in
serious mechanical damage to the vessel wall, which was manifested
either by dissection or the development of necrotic zones in the
media. In addition, the transfection efficiency is extremely low
and not localized (Flugelman, M. Y. (1995), supra; Flugelman, M. Y.
et al. (1992) Circulation, 85(3), 1110-1117). This type of catheter
was therefore also found to be unsuitable for somatic gene
transfer.
[0019] The principle of the functioning of the Dispatch catheter
resembles in principle the double balloon catheter described above
(see, for example, McKay, R. G. et al. (1994), Catheterization and
Cardiovascular Diagnosis, 33, 181-188). The section of vessel to be
treated is, however, in this case not separated from the remainder
of the circulation by two peripheral balloons but is separated by a
coil which makes contact with the vessel wall after inflation. The
vectors are injected into the closed chamber through orifices in
the catheter shaft between the helical elements. Transfection takes
place by diffusion. In order to make the required long transfection
time of about 30 minutes possible without cutting off the distal
regions from the blood flow, a conduit passing through the shaft of
the inflated catheter was fitted to make blood flow possible.
[0020] The needle injection catheter (NIC) is characterized by
three injection needles which can be advanced out of the rounded
tip of a catheter and then penetrate into the vessel wall (see, for
example, Gonschior, P. et al. (1995) Coronary Artery Disease, 6,
329-344). It has already been possible to inject drugs through
these needles into the vessel wall.
[0021] However, manipulation of the NIC is difficult because it is
not easily possible to check how far the needles have emerged from
the head of the catheter, although this defines the depth of
injection. This entails the risk of perforation of the vessel wall
and thus transfection of perivascular tissue, but also of bleeding
from the vessel. This danger applies particularly on transfection
of eccentric plaques.
[0022] Janssens, S. et al., The Second Annual International
Symposium, Oct. 13-15, 1996, Cambridge, Mass., describe gene
transfer with the aid of an adenoviral vector and of an Infiltrator
catheter which is not described in detail. However, the
disadvantage of this transfection system is that cytotoxic effects
have been observed with adenoviral vectors (Flugelman, M. Y.
(1995), supra).
[0023] It was therefore an object of the present invention to find
a transfection system which makes it possible to perform somatic
gene transfer into vessels efficiently and with minimum damage.
[0024] The present invention therefore relates firstly to a
transfection system comprising one or more Infiltrator catheters,
one or more nucleic acids in nonviral form and, where appropriate,
suitable ancillary substances and/or additives.
[0025] The Infiltrator catheter is a special catheter developed for
intravascular injection of drugs into the vessel wall. It takes the
form of a balloon catheter from whose surface injector ports
(tubular, stud-like extensions for administering one or more active
substances) project. The height of the injector ports is normally
about 100-500 .mu.m, preferably about 100-250 .mu.m, in particular
about 100 .mu.m, and the number of injector ports per balloon is
normally about 5.times.7, preferably about 3.times.7. Inflation of
the balloon to, normally, about 2 atm forces these injector ports
into the vessel wall. It is then possible to inject through the
injector ports in general up to about 500 .mu.l, preferably about
250-300 .mu.l, of active substance or active substances under low
pressure, preferably about 100-200 mm Hg, in particular about 150
mm Hg, into the vessel wall.
[0026] The catheter normally has more than one lumen and is, in
particular, a double lumen, particularly preferably a triple lumen,
catheter.
[0027] The double lumen catheter generally consists of a tubular
shaft and the inflatable balloon which has been mentioned and which
envelops a suitable point on the shaft. The shaft comprises at the
point where the balloon is one or more orifices to which the
balloon can be inflated and the active substance can enter the
balloon. The active substance passes from there through the
injector port into the vessel wall. A double lumen Infiltrator
catheter as described in U.S. Pat. No. 5,112,305 or U.S. Pat. No.
5,242,397 is particularly preferred.
[0028] A triple lumen Infiltrator catheter which comprises a
central tubular shaft, an inflatable balloon which envelops the
shaft at a suitable point, and a tubular sleeve which comprises the
said injector ports at the point where the inflatable balloon is,
is particularly preferred. After positioning of the catheter, the
balloon is inflated, whereupon the said sleeve with the injector
ports is pressed against the vessel wall. The active substance is
then introduced into the tubular sleeve in order to pass from there
through the injector ports into the vessel wall. A particularly
preferred triple lumen Infiltrator catheter is a catheter as
described in EP 0 753 322 A1 or EP 0 768 098 A2.
[0029] The transfection system according to the invention generally
additionally comprises a guide wire which makes correct positioning
of the catheter possible.
[0030] The nucleic acid of the transfection system according to the
invention is present in nonviral form, preferably as single- or
double-stranded DNA or as RNA, for example as naked nucleic acid or
together with other nonviral components.
[0031] The term "nonviral" means according to the present invention
that the nucleic acid is not transfected with the aid of
genetically manipulated viral vectors such as, for example,
retroviral, adenoviral or adeno-associated viral vectors. The use
of, for example, Sendai viruses in the form of virosomes (see
below) is not precluded by the term "nonviral".
[0032] A suitable naked nucleic acid is, for example, a nucleic
acid in the form of a plasmid DNA or of a so-called antisense
oligonucleotide (see, for example, Uhlmann, E. & Peyman, A.
(1990) Chemical Reviews, 90, 543-584, No. 4).
[0033] Nucleic acids effective for gene therapy can also be
obtained by complexation of the required nucleic acid with other
nonviral components, preferably liposomes, since this makes it
possible to achieve a very high transfection efficiency, in
particular of the vessel wall (see, for example, DE 44 11 402 A1).
Transfection with nucleic acid/liposome complexes using Sendai
viruses in the form of so-called HVJ liposomes (virosomes) is
particularly advantageous because this makes it possible to
increase the transfection rate still further.
[0034] In lipofection, small unilamellar vesicles of, for example,
cationic lipids are prepared by ultrasound treatment of the
liposome suspension. The nucleic acid is ionically bound to the
surface of the liposomes, in particular in a ratio such that a net
positive charge remains and the nucleic acid is 100% complexed by
the liposomes. Besides the lipid mixtures employed by Felgner et
al. (Feigner, P. L. et al. (1987), Proc. Natl. Acad. Sci. USA, 84,
7413-7414), DOTMA (1,2-dioleyloxy-3-propyltrimethylammonium
bromide) and DOPE (dioleylphosphatidylethanolamine), numerous new
lipid formulations have now been synthesized and tested for their
efficiency in transfecting various cell lines (Behr, J. P. et al.
(1989), Proc. Natl. Acad. Sci. USA, 86, 6982-6986; Felgner, J. H.
et al. (1994) J. Biol. Chem., 269, 2550-2561; Gao, X. & Huang,
L. (1991), Biochim. Biophys. Acta, 1189, 195-203). Examples of the
new lipid formulations are DOTAP
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl
sulphate or DOGS (TRANSFECTAM; dioctadecylamidoglycylspermine). One
example of the preparation of DNA/liposome complexes from
phosphatidylcholine, phosphatidylserine and cholesterol and
successful use thereof in the transfection of vessel walls using
Sendai viruses is described in DE 44 11 402.
[0035] It is particularly advantageous for the nucleic
acid/liposome complex to comprise nucleic acid binding proteins,
for example chromosomal proteins, preferably HMG proteins (high
mobility group proteins), in particular HMG-1 or HMG-2, or
nucleosomal histones such as H2A, H2B, H3 or H4, because this makes
it possible to increase the expression of the required nucleic acid
by at least 3-10-fold. The chromosomal proteins can, for example,
be isolated from calf thymus or rat liver by generally known
methods or be prepared by genetic manipulation. Human HMG-1 can,
for example, be prepared particularly straightforwardly by genetic
manipulation by methods known to the skilled person using the human
cDNA sequence from Wen, L. et al. (1989) Nucleic Acids Res., 17(3),
1197-1214.
[0036] The required nucleic acid is generally a nucleic acid which
codes for a therapeutically effective gene product.
[0037] Examples of nucleic acids which code for a therapeutically
effective gene product are the nitric-oxide synthase gene,
especially a gene which codes for inducible nitric-oxide synthase
(see, for example, DE 44 11 402 A1), the erythropoietin gene (see,
for example, EP 0 148 605 B1), the insulin gene (see, for example,
EP 0 001 929 B1) or the genes coding for blood coagulation factors,
interferons, cytokines, hormones, growth factors etc. Particularly
preferred genes are those coding for proteins which occur in blood.
The somatic gene therapy according to the invention of the vessel
wall can eliminate or alleviate in a particularly simple and
lasting manner for example a pathological deficiency phenomenon
such as, for example, a deficiency of insulin in diabetics, a
deficiency of factor VIII in haemophiliacs, a deficiency of
erythropoietin in kidney patients, a deficiency of thrombopoietin
or a deficiency of somatostatin associated with stunted growth, by
increasing the plasma concentrations of the particular active
substance. The present invention is therefore not limited just to
the therapy of purely vascular disorders such as, for example,
arteriosclerosis, stenosis or restenosis, but is generally
applicable.
[0038] It is also advantageous for the described use for gene
therapy if the part of the nucleic acid which codes for the
polypeptide comprises one or more non-coding sequences including
intron sequences, preferably between promoter and the polypeptide
start codon, and/or a polyA sequence, in particular the naturally
occurring polyA sequence or an SV40 virus polyA sequence,
especially at the 3' end of the gene, because this can achieve
stabilization of the mRNA in the cell (Jackson, R. J. (1993) Cell,
74, 9-14 and Palmiter, R. D. et al. (1991) Proc. Natl. Acad. Sci.
USA, 88, 478-482).
[0039] The transfection system according to the invention can be
produced in a simple manner by combining one or more of the
Infiltrator catheters which are described above or can be
purchased, one or more of the nucleic acids described above, where
appropriate suitable ancillary substances and/or additives and,
where appropriate, a guide wire.
[0040] Examples of suitable ancillary substances and/or additives
are those known to a skilled person in the form of stabilizers,
such as nuclease inhibitors, preferably complexing agents such as
EDTA, protease inhibitors or alkaline earth metal solutions,
especially on use of Sendai viruses in the transfection system
according to the invention, such as, for example, a CaCl.sub.2
solution, which is preferably added before transfection with
virosomes.
[0041] The described Infiltrator catheter is suitable and
advantageous in the form of the transfection system according to
the invention for somatic gene therapy, especially for treating
vascular disorders, genetic disorders and/or disorders which can be
treated by gene transfer, including prevention thereof, as already
described in detail above by way of example.
[0042] The present invention therefore also extends to the use of
an Infiltrator catheter for producing a transfection system
according to the invention, preferably for somatic gene therapy.
The present invention further extends to the use of the
transfection system according to the invention for somatic gene
transfer, in particular for the treatment or prevention of the
disorders described.
[0043] The unexpected advantage of the present invention derives
from the fact that, in contrast to other transfection systems
described in the literature, which have been developed for local
administration of pharmaceuticals, an Infiltrator catheter is
particularly suitable according to the present invention for the
transfer of one or more nucleic acids in somatic gene therapy. It
was possible to position the Infiltrator catheter accurately in the
vessel and, on inflation of the balloon, it led to leak-free
occlusion of the vessel so that the injector ports were pressed by
the balloon into the vessel wall, and the injection of the nucleic
acid(s) could take place effectively under low pressure, which
resulted in only minimal changes in the vessel wall. This made it
possible to achieve, with short vessel occlusion times (maximally
about 30 seconds), efficient transfection of the vessel wall and
high and permanent expression of the required nucleic acid.
[0044] The following figures and examples are intended to
illustrate the invention in detail without restricting it
thereto.
DESCRIPTION OF THE FIGURES
[0045] FIG. 1 shows a radiograph of the iliac vascular system of a
pig during transfection of the left iliac artery. The film shows an
inflated Infiltrator catheter (B) in the iliac artery (A) of the
pig. The guide wire used to introduce the catheter (C) is evident
distal of the catheter. The leak-proof occlusion of the vessel by
the inflated balloon is evident from the lack of contrast in the
distal femoral arteries (compare right-hand side).
[0046] FIGS. 2a and b show the immunohistochemical detection of
iNOS expression in iliac arteries of the pig after transfection
with the control vector (FIG. 2a) and with the pSCMV-iNOS vector
(FIG. 2b). The dark colour in FIG. 2b (arrow) indicates strong iNOS
expression.
[0047] FIGS. 3 A and B show detection of NOx accumulation in
culture supernatant from vessels transfected either with the
pSCMV-iNOS vector (A) or with the control vector (B). Whereas no
NOx signal is detectable in the control segment (FIG. 3 B), there
is clear NOx release in the transfected vessel (FIG. 3 A).
(ppB=parts per billion)
EXAMPLE
Expression of the inducible nitric-oxide synthase gene in the pig
femoral artery
[0048] 1. Transfection protocol
[0049] 1.1 Preparation of the DNA
[0050] 200 .mu.g of the gene transfer vector pSCMV-iNOS which codes
for the inducible mouse nitric-oxide synthase (DE 44 11 402 A1), in
a concentration of 2 .mu.g/.mu.l dissolved in TE buffer (10 mM
Tris.multidot.HCl, pH 7.4), were mixed with 64 pg of HMG-1 protein
from calf thymus, dissolved in BSS buffer (140 mM NaCl, 5.4 mM KCl,
10 mM Tris.multidot.C1, pH 7.6), and the solution was adjusted to a
final volume of 200 .mu.l with BSS buffer. The resulting solution
was incubated at 37.degree. C. for 30 min.
[0051] 1.2 Liposome preparation
[0052] 5 mg portions of a lipid mixture consisting of
phosphatidylcholine (PC), cholesterol (C) and phosphatidylserine
(PS) with a ratio by weight in a mixture of 4.8:2:1 were dissolved
in 2 ml of diethyl ether. 200 .mu.l of the DNA solution prepared in
Example 1.1 were added to the dissolved lipids. The mixture was
then homogenized by vortexing for 2 min and subsequently sonicated
in an ultrasonic bath for 10 sec. The ether was then evaporated in
a rotary evaporator at 37.degree. C. The remaining emulsion was
subsequently vortexed for about 2 min until an opalescence
appeared.
[0053] 1.3 Preparation of the Sendai viruses
[0054] Sendai viruses were grown by standard methods in
chorioallantoic fluid from fertilized chicken eggs (Nakanishi, M.
et al. (1985) Exp. Cell. Res., 159(2), 399-409). The viruses were
subsequently purified by the following centrifugation method in a
Sorvall GSA rotor at 4.degree. C.:
1 1. Chorioallantoic fluid: 10 min at 3000 rpm 2. Supernatant: 30
min at 13,000 rpm 3. Pellet: suspended in BSS buffer 4. Suspension:
10 min at 3000 rpm 5. Supernatant: 30 min at 13,000 rpm 6. Pellet:
resuspended in BSS buffer.
[0055] The titre was then adjusted to 16,000 haem-agglutination
units (HAU)/ml. Shortly before use, the viruses were inactivated by
UV irradiation with 11 J/m.sup.2 .multidot.s.
[0056] 1.4 Preparation and purification of the virosomes (HVJ
liposomes)
[0057] 1 ml of the UV-inactivated viruses prepared as in Example
1.3 (16,000 HAU) were mixed with the liposomes prepared as in
Example 1.2 and incubated on ice for 10 min. The suspension was
then shaken for fusion of the viruses with the liposomes on an
orbital shaker (120 rpm, 37.degree. C.) for 60 min. The virosomes
which were formed were then removed from the unincorporated viruses
by ultracentrifugation through a sucrose cushion (30% sucrose) in a
TH641 rotor, Sorvall Instruments, at 28,000 rpm and at 4.degree. C.
The virosomes in this case banded on the sucrose cushion, and the
unincorporated viruses sedimented to the bottom of the tube. The
virosomes were then removed and stored on ice until transfected.
Shortly before carrying out the transfection, CaCl.sub.2 was added
to a final concentration of 2 mM.
[0058] 1.5 Transfection
[0059] Transfection took place by injecting 300 .mu.l of the
virosomes prepared as in Example 1.4 (8-10 .mu.g of pSCMV-iNoS
entrapped in HVJ liposomes, about 1000 HAU) through the described
catheter into the iliac artery of Munich minipigs. The injection
took about 10 sec. Virosomes which contained the vector pSCMV2 (DE
44 11 402 A1) were employed as control.
[0060] 2. Surgical procedures
[0061] The left carotid artery was exposed by a surgical procedure
and a 7-F or 9-F support was inserted by a modified Seldinger
technique. All further angiographic or procedural steps took place
via this support. Firstly, a survey angiography of the distal aorta
and of the iliac vessels was performed via a calibration pigtail
catheter. The catheter for local transfection was then advanced
over a guide wire as far as the femoral arteries. The transfection
solution was then introduced through a lateral injector port in
accordance with the manufacturer's instructions for the local
administration of drugs into the vessel. In this case, expression
vector and control vector were respectively injected into the right
and left femoral arteries of the same animal. After removal of the
catheter, the carotid artery was closed by suturing the vessel. The
wound was then closed.
[0062] 3. Immunohistochemical detection
[0063] Frozen sections (8 .mu.m) of the transfected vessel wall
sections were reacted with monoclonal anti-iNOS antibodies
(Transduction Laboratories, #N32020). Antibody binding was detected
via a streptavidin/ peroxidase complex using biotinylated
anti-mouse anti-bodies (Vector Laboratories, #BA-2000) with the
Vecta Stain Elite kit (Vector Laboratories, #PK-6100;).
[0064] 4. Biochemical detection
[0065] In addition to the immunohistochemistry, increased NO
formation by the transfected vessels was directly detected on the
basis of the accumulation of nitrate/nitrite (NOx) in the culture
supernatant from explanted transfected vessel sections. Transfected
vessel sections were incubated in Krebs-Hensel light buffer (in
mmol/L: 116 NaCl; 4.6 KCl; 1.1 MgSO.sub.4; 24.9 NaHCO.sub.3; 2.5
CaCl.sub.2; 1.2 KH.sub.2PO.sub.4; 10 glucose and 0.5 EDTA
equilibrated with 95% O.sub.2 and 5% CO.sub.2 (pH 7.4; 37.degree.
C.)). Incubation took place at 37.degree. C. for 24 h. The NOx
accumulation was determined after reducing in nitrate +nitrite to
NO by a chemiluminescence method (nitric oxide analyzer NOA 280;
Sievers Inc., USA).
[0066] 5. Results
[0067] 5.1 The Dispatch catheter
[0068] Use of a Dispatch catheter did not result in efficient
transfection because it was not possible to produce a tightly
closed isolated chamber by inflation of the helical elements.
Injection of the vectors through the catheter shaft does not lead
to a rise in pressure, which would be expected with a closed
chamber. The leakiness of the chamber was demonstrated by the rapid
flowing out of X-ray contrast medium from the chamber. The Dispatch
catheter is therefore unsuitable for local gene transfer.
[0069] 5.2 The Infiltrator catheter
[0070] In contrast to the Dispatch catheter or needle injection
catheter, accurate positioning (see FIG. 1) was possible with the
Infiltrator catheter (Barath drug delivery catheter with 3.times.7
injector ports in the longitudinal axis with the cylindrical shape
of the ports, which have a slight upward conical taper, model No.
DD140015, Interventional Technologies Europe Ltd., Ireland), and
inflation of the balloon resulted in tight closure of the
vessel.
[0071] As shown in FIG. 2, a pronounced immune reactivity was
detectable in the transfected vessel sections and extended up to
50% of the vessel wall diameter. The control-transfected vessels
lacked the corresponding stain, so that this detection specifically
showed expression of iNOS.
[0072] FIG. 3 shows that a distinctly increased NOx release was
obtained after transfection by comparison with sections transfected
with the control vector.
[0073] It was thus possible with the Infiltrator catheter to show
efficient in vivo transfection of the vessel wall, which extended
up to 50% of the vessel wall diameter, with short vessel occlusion
times (max. 30 s).
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