U.S. patent application number 10/078677 was filed with the patent office on 2002-07-18 for gene therapy for restenosis using an adenoviral vector.
This patent application is currently assigned to Aventis Pharma S.A.. Invention is credited to Branellec, Didier, Dedieu, Jean-Francois, Denefle, Patrice, Feldman, Laurent, Perricaudet, Michel, Steg, Gabriel.
Application Number | 20020094324 10/078677 |
Document ID | / |
Family ID | 9466347 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094324 |
Kind Code |
A1 |
Branellec, Didier ; et
al. |
July 18, 2002 |
Gene therapy for restenosis using an adenoviral vector
Abstract
A method for treating restenosis by gene therapy is disclosed,
said method comprising delivering a recombinant
suicide-gene-containing adenovirus.
Inventors: |
Branellec, Didier; (La
Varenne Saint-Hilaire, FR) ; Dedieu, Jean-Francois;
(Paris, FR) ; Denefle, Patrice; (Saint Maur,
FR) ; Feldman, Laurent; (Paris, FR) ;
Perricaudet, Michel; (Ecrosnes, FR) ; Steg,
Gabriel; (Paris, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT &
DUNNER LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Aventis Pharma S.A.
|
Family ID: |
9466347 |
Appl. No.: |
10/078677 |
Filed: |
February 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10078677 |
Feb 21, 2002 |
|
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|
08633769 |
Jun 20, 1996 |
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Current U.S.
Class: |
424/93.2 ;
424/93.6 |
Current CPC
Class: |
C12N 2830/15 20130101;
C12Y 305/04001 20130101; A61P 43/00 20180101; C12N 2710/10343
20130101; C12N 15/86 20130101; C12N 2830/60 20130101; A61P 9/10
20180101; A61K 48/00 20130101; A61K 38/50 20130101; C12Y 207/01021
20130101; A61K 38/45 20130101; C12N 2830/00 20130101 |
Class at
Publication: |
424/93.2 ;
424/93.6 |
International
Class: |
A61K 048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 1994 |
FR |
FR94/10083 |
Claims
1. Use of a defective recombinant adenovirus containing a suicide
gene for the preparation of a pharmaceutical composition intended
for the treatment of restenosis.
2. Use of a defective recombinant adenovirus containing a suicide
gene for the preparation of a pharmaceutical composition intended
for the treatment of restenosis by selective transfer of the said
gene into the smooth muscle cells of the atheromatous plaque.
3. Use according to claim 1 or 2, characterized in that the suicide
gene is chosen from the thymidine kinase gene and the cytosine
deaminase gene.
4. Use according to claim 1 or 2, characterized in that the suicide
gene is the human herpesvirus thymidine kinase (HSV-1 TK) gene.
5. Use according to one of the preceding claims, characterized in
that the suicide gene is placed under the control of a promoter
permitting its expression in infected cells.
6. Use according to claim 5, characterized in that the promoter is
chosen from viral promoters, preferably the RSV LTR and CMV
promoter.
7. Use according to one of the preceding claims, characterized in
that the adenovirus comprises the ITRs, a sequence permitting
encapsidation and the suicide gene.
8. Use according to claim 7, characterized in that the adenovirus
comprises the ITRs, a sequence permitting encapsidation and the
suicide gene, and in which the E1 gene and at least one of the
genes E2, E4, L1-L5 is non-functional.
9. Use according to claim 8, characterized in that the adenovirus
comprises the ITRs, a sequence permitting encapsidation and the
suicide gene, and in which the E1 gene and the E4 gene is rendered
nonfunctional.
10. Use according to claim 9, characterized in that the adenovirus
comprises the ITRs, a sequence permitting encapsidation and the
suicide gene, and in which all or part of the E1 and E4 regions are
deleted.
11. Use according to one of the preceding claims, characterized in
that the adenovirus is an adenovirus of human origin, preferably
chosen from the serotypes Ad2 and Ad5.
12. Use according to one of claims 1 to 10, characterized in that
the adenovirus is an adenovirus of animal origin, preferably chosen
from canine adenoviruses.
13. Use according to one of claims 1 to 12, characterized in that
the adenovirus is impregnated in a hydrogel.
14. Use according to claim 13, characterized in that the hydrogel
is deposited on a balloon catheter.
15. Use according to claim 11, characterized in that the adenovirus
is administered via a balloon catheter of the perfusion catheter
type.
16. Use according to claim 15, characterized in that the adenovirus
is administered via a catheter of the channelled balloon catheter
type.
17. Use according to claim 15, characterized in that the adenovirus
perfused via a catheter of the perfusion balloon catheter type is
impregnated in a hydrogel.
18. Use according to one of claims 1 to 12, characterized in that
the adenovirus is impregnated in poloxamer.
19. Use according to claim 15, characterized in that the adenovirus
perfused via a catheter of the perfusion balloon catheter type is
impregnated in poloxamer.
20. Pharmaceutical composition comprising a defective recombinant
adenovirus impregnated in a hydrogel.
21. Pharmaceutical composition according to claim 20, characterized
in that the defective recombinant adenovirus contains a suicide
gene.
22. Device for the percutaneous administration of genes,
characterized in that it comprises a balloon catheter coated with a
hydrogel, the hydrogel being impregnated with a defective
recombinant adenovirus containing the said gene.
23. Device according to claim 22, characterized in that the
administration of genes is carried out selectively at the
atheromatous plaque.
24. Device according to claim 23, characterized in that the
administration of genes is carried out selectively at the smooth
muscle cells.
25. Device according to claim 24, characterized in that, when genes
are administered, this administration takes place with a
selectivity of greater than 95%.
26. Device according to claims 23 to 25, characterized in that the
administration of genes is followed by a treatment with
ganciclovir.
27. Device according to claim 26, characterized in that the
percentage of infected cells is greater than or equal to 0.2%.
28. Method of therapeutic treatment of restenosis, characterized in
that it comprises the percutaneous administration of genes by means
of a balloon catheter coated with a hydrogel, the hydrogel being
impregnated with a defective recombinant adenovirus containing the
said gene.
29. Method of therapeutic treatment of restenosis according to
claim 28, characterized in that the administration of genes takes
place selectively at the atheromatous plaque.
30. Method of therapeutic treatment of restenosis according to
claim 29, characterized in that the administration of genes takes
place selectively at the smooth muscle cells.
31. Method of therapeutic treatment of restenosis according to
claim 30, characterized in that the administration of genes takes
place with a selectivity of greater than 95%.
32. Method of therapeutic treatment of restenosis according to
claim 31, characterized in that the administration of TK suicide
genes is followed by a treatment with ganciclovir.
33. Method of therapeutic treatment of restenosis according to
claim 32, characterized in that it induces a "bystander"
effect.
34. Method of therapeutic treatment of restenosis according to
claim 33, characterized in that this induced bystander effect
permits a therapeutic efficacy even with a small percentage of
infected cells.
35. Method of therapeutic treatment of restenosis according to
claim 34, characterized in that the percentage of infected cells is
greater than or equal to 0.02%.
Description
[0001] The present invention relates to a method for treatment of
restenosis by gene therapy, comprising the administration of a
recombinant adenovirus containing a suicide gene. It also relates
to particular pharmaceutical compositions permitting the local and
effective administration of recombinant viruses.
[0002] Atherosclerosis is a complex, polygenic disease which is
defined in histological terms by deposits (lipid or fibrolipid
plaques) of lipids and of other blood derivatives in the wall of
the large arteries (aorta, coronary arteries, carotid). These
plaques, which are more or less calcified according to the degree
of progression of the process, may be coupled with lesions and are
associated with the accumulation in the arteries of fatty deposits
consisting essentially of cholesterol esters. These plaques are
accompanied by a thickening of the arterial wall, with hypertrophy
of the smooth muscle, appearance of foam cells and accumulation of
fibrous tissue. The atheromatous plaque protrudes markedly from the
wall, endowing it with a stenosing character responsible for
vascular occlusions by atheroma, thrombosis or embolism which occur
in those patients who are most affected. These lesions can hence
lead to very serious cardiovascular pathologies such as infarction,
sudden death, cardiac insufficiency, stroke, and the like.
[0003] Since 1977, the technique of angioplasty has been developed
to permit a non-surgical intervention in respect of the
atherosclerosis plaque. However, the treatment of an
atherosclerotic lesion by angioplasty results very frequently (up
to 50% of cases in some studies) in a restenosis following
mechanical injury of the arterial wall. A key event in this
mechanism is the proliferation and migration of vascular smooth
muscle cells (VSMC) from the media to the intima, in particular as
a result of the absence of protection and/or feedback control
exercised by the endothelial cells of the intima.
[0004] The treatment of restenosis by administration of chemical or
proteinaceous substances capable of killing vascular smooth muscle
cells has been proposed in the prior art. Thus, psolaren
derivatives, incorporated by proliferative cells and then
sensitizing these cells to the action of light, have been used
(March et al., 1993, circulation, 87:184191). Similarly, some
cytotoxins consisting of a fusion protein between a plant or
bacterial toxin fragment and a growth factor have also been used
(Pickering et al., J. Clin. Invest., 1993, 91:724-729; Biro et al.,
1992, Circ. Res., 71:640-645; Casscells et al., Proc. Natl. Acad.
Sci. USA, 1992, 89:7159-7163). However, these treatments have many
drawbacks, such as their low specificity, their indifferent
efficacy, a considerable delay in acting and a potential
toxicity.
[0005] The present invention offers an advantageous approach to
this problem. The present invention provides, in effect, an
especially effective and selective method for the treatment of
postangioplasty restenosis by gene therapy. The method of the
present invention consists mainly in administering a recombinant
adenovirus containing a suicide gene, capable of specifically
sensitizing proliferating vascular smooth muscle cells to a
therapeutic agent. Simultaneous or subsequent administration of
this therapeutic agent then brings about the selective death of the
sensitized cells.
[0006] The advantages of the present invention lie, in particular,
in the high capacity of the adenoviruses of the invention to infect
proliferating vascular smooth muscle cells. This enables relatively
small amounts of active principle (recombinant adenovirus) to be
used, and also permits an effective and very rapid action on the
sites to be treated. The adenoviruses of the invention are also
capable of expressing at very high levels the suicide genes
introduced, thereby endowing them with a very effective therapeutic
action. Furthermore, on account of their episomal character, the
adenoviruses of the invention have a limited persistence in
proliferative cells, and hence a transient effect entirely suited
to the desired therapeutic effect. Lastly, the Applicant has also
developed an especially advantageous method of administration,
which enables certain target cells essential to the desired
therapeutic effect to be infected with great efficacy.
[0007] A first subject of the invention hence relates to the use of
a defective recombinant adenovirus containing a suicide gene for
the preparation of a pharmaceutical composition intended for the
treatment of restenosis.
[0008] As mentioned above, for the purposes of the present
invention, suicide gene is understood to mean any gene whose
expression product endows the infected cell with a sensitivity to a
therapeutic agent. As an example, there may be mentioned the
thymidine kinase gene, whose expression product endows mammalian
cells with a sensitivity to certain therapeutic agents such as
ganciclovir or acyclovir, or the cytosine deaminase gene, whose
expression product endows mamalian cells with a sensitivity to
5-fluorocytosine (5-FC).
[0009] Herpes simplex virus thymidine kinase is capable of
phosphorylating nucleoside analogues such as acyclovir and
ganciclovir. These modified molecules may be incorporated in a DNA
chain undergoing elongation, which results in the cessation of DNA
synthesis and brings about the death of the cell (F. L. Moolten,
Cancer Res. 46 (1986) 5276). This strategy thus enables cells
expressing the TK gene to be specifically eliminated. Furthermore,
since the DNA synthesis is the target of the toxicity, only cells
undergoing division are affected.
[0010] More preferably, the human herpesvirus thymidine kinase
(hHSV-1 TK) gene is used in the context of the present invention.
The sequence of this gene has been described in the literature
(see, in particular, McKnight et al., Nucleic Acid. Res. 8 (1980)
5931). It is also possible to use derivatives of this sequence
displaying greater substrate specificity or better kinase activity.
Such derivatives may, in particular, be obtained by mutagenesis at
the binding site, as described previously (Balasubramaniam et al.,
J. Gen. Virol. 71 (1990) 2979; Munir et al., JBC 267 (1992)
6584).
[0011] It is also possible to use the cytosine deaminase gene,
whose expression product endows mammalian cells with a sensitivity
to 5-fluorocytosine (5-FC). Cytosine deaminase is capable of
catalyzing the deamination of cytosine to uracil. Cells which
express this gene are hence capable of converting 5-fluorocytosine
(5-FC) to 5-fluorouracil (5-FU), which is a toxic metabolite. The
sequence of this gene has been described in the literature
(Anderson et al., Arch. Microbiol. 152 (1989) 115).
[0012] More generally, any gene capable of endowing infected cells
with a sensitivity to a therapeutic agent may be used in the
context of the present invention. The thymidine kinase gene
constitutes an especially advantageous embodiment.
[0013] For the construction of the adenoviruses according to the
invention, different serotypes may be used. There are, in effect,
many serotypes of adenovirus, whose structure and properties vary
somewhat. Among these serotypes, it is preferable however to use,
in the context of the present invention, human adenoviruses type 2
or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see
Application FR 93/05954). Among adenoviruses of animal origin which
are usable in the context of the present invention, adenoviruses of
canine, bovine, murine (for example: Mav1, Beard et al., Virology
75 (1990) 81), ovine, porcine, avian or alternatively simian (for
example: SAV) origin may be mentioned. Preferably, the adenovirus
of animal origin is a canine adenovirus, more preferably a CAV2
adenovirus [Manhattan or A26/61 (ATCC VR-800) strain, for example].
It is preferable to use adenoviruses of human or canine or mixed
origin in the context of the invention.
[0014] As stated above, the adenoviruses according to the invention
are defective, that is to say they are incapable of replicating
autonomously in the target cell. Generally, the genome of the
defective adenoviruses used the context of the present invention
hence lacks at least the sequences needed for replication of the
said virus in the infected cell. These regions may be either
removed (in their entirety or partially), or rendered
non-functional, or replaced by other sequences, and in particular
by the suicide gene. Preferably, the defective adenovirus
nevertheless retains the sequences of its genome which are needed
for encapsidation of the viral particles.
[0015] Preferably, the defective adenoviruses of the invention
comprise the ITRs, a sequence permitting encapsidation and the
suicide gene. Still more preferably, in the genome of the
adenoviruses of the invention, the E1 gene and at least one of the
genes E2, E4, L1-L5 are non-functional. The viral gene of interest
may be rendered non-functional by any technique known to a person
skilled in the art, and in particular by total elimination,
substitution, partial deletion or addition of one or more bases in
the gene or genes of interest. Such modifications may be obtained
in vitro (on the isolated DNA) or in situ, for example by means of
genetic engineering techniques, or alternatively by treatment using
mutagenic agents.
[0016] More preferably, a defective adenovirus rendered
non-functional by a total or partial deletion of the E1 region and
a deletion in the E4 region is used. The E4 region comprises 7
reading frames. The deletion in the E4 region may be
transcomplemented by the presence, in the cell line used for
multiplication of the viruses, either simply of the reading frame
ORF6, or of the reading frames ORF6 and ORF6/7.
[0017] Preferred adenoviruses according to the invention are chosen
from the following:
[0018] Defective recombinant adenovirus .DELTA.E1, .DELTA.E4
comprising a deletion of all or part of the E1 region and a
deletion of all or part of the E4 region.
[0019] Defective recombinant adenovirus .DELTA.E1, ORF3.sup.-,
ORF6.sup.- comprising a deletion of all or part of the E1 region
and of nucleotides 34801-34329 and 34115-33126 of the E4
region.
[0020] Defective recombinant adenovirus .DELTA.E1, .DELTA.E4,
ORF1.sup.+ comprising a deletion of all or part of the E1 region
and a deletion of the E4 region except for the reading frame ORF1.
More specifically, the deletion in the E4 region has its 5' end
included in the reading frame ORF7 and its 3' end included in the
reading frame ORF2. For example, in the region covering nucleotides
33093-35053.
[0021] Defective recombinant adenovirus .DELTA.E1, AE4, ORF4.sup.+
comprising a deletion of all or part of the E1 region and a
deletion of the E4 region except for the reading frame ORF4. More
especially, two deletions are carried out, one whose 5' end is
included in the reading frame ORF7 and whose 3' end is located in
the reading frame ORF6, the other whose 5' end is included in the
reading frame ORF3 and whose 3' end is located in the reading frame
ORF1 or in the promoter region of E4. For example, a deletion
covering nucleotides 33093-33695 and a deletion covering
nucleotides 34634-35355.
[0022] Defective recombinant adenovirus .DELTA.E1, .DELTA.E4
comprising a deletion of all or part of the E1 region and a
deletion covering the whole of the E4 region, chosen, for example,
from the following deletions: nucleotides 32720-35835, or
33466-35355, or 33093-35355.
[0023] The construction of these vectors is described in Patents
No. FR 9500749 and No. FR 9506532.
[0024] The defective recombinant adenoviruses according to the
invention may be prepared by any technique known to a person
skilled in the art (Levrero et al., Gene 101 (1991) 195, EP
185,573; Graham, EMBO J. 3 (1984) 2917). In particular, they may be
prepared by homologous recombination between an adenovirus and a
plasmid carrying, inter alia, the suicide gene. Homologous
recombination takes place after cotransfection of the said
adenovirus and said plasmid into a suitable cell line. The cell
line used should preferably (i) be transformable by the said
elements, and (ii) contain the sequences capable of complementing
the portion of the genome of the defective adenovirus, preferably
in integrated form in order to avoid risks of recombination. As an
example of a line, there may be mentioned the human embryonic
kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which
contains, in particular, integrated in its genome, the left-hand
portion of the genome of an Ad5 adenovirus (12%). Strategies of
construction of vectors derived from adenoviruses have also been
described in Applications Nos. FR 93/05954 and FR 93/08596.
[0025] Thereafter, the adenoviruses which have multiplied are
recovered and purified according to standard techniques of
molecular biology, as illustrated in the examples.
[0026] Advantageously, in the adenoviruses of the invention, the
suicide gene is placed under the control of a promoter permitting
its expression in infected cells. This promoter can be the one
belonging to the suicide gene, a heterologous promoter or a
synthetic promoter. In particular, promoters originating from
eukaryotic or viral genes may be used. For example, it is possible
to use promoter sequences originating from the genome of the cell
which it is desired to infect. Similarly, the promoter sequences
may originate from the genome of a virus, including that of the
virus used. In this connection, the E1A, MLP, CMV, RSV LTR, and the
like, promoters may, for example be mentioned. In addition, these
expression sequences may be modified by adding activation or
regulatory sequences or sequences permitting a tissue-specific
expression. It can, in effect, be especially advantageous to use
expression signals which are active specifically or predominantly
in vascular smooth muscle cells, so that the suicide gene is
expressed and produces its effect only when the virus has actually
infected a vascular smooth muscle cell. Among promoters which are
active specifically or predominantly in vascular smooth muscle
cells, the promoter of .alpha.-actin of smooth muscle may be
mentioned in particular.
[0027] In a particular embodiment of the invention, a defective
recombinant adenovirus is used which comprises a suicide gene under
the control of a viral promoter, preferably chosen from the RSV LTR
and the CMV early promoter.
[0028] According to another advantageous embodiment, the promoter
used is one which is active specifically or predominantly in
vascular smooth muscle cells.
[0029] The present invention thus provides an especially effective
method for the treatment of restenosis. Moreover, to increase
further the efficacy and specificity of the treatment, the
Applicant has developed a method permitting a local administration
of the recombinant adenoviruses at the sites to be treated. More
especially, this method is based on the use of an angioplasty
balloon coated with a hydrophilic film (for example a hydrogel)
impregnated with adenovirus, which may thus be applied precisely to
the site to be treated and permit a local and effective release of
the adenoviruses at the cells to be treated.
[0030] In addition, the Applicant showed that, on healthy arteries,
this method of administration made it possible to infect a high
percentage of cells of the media (up to 9.6%), which are the most
logical targets for the prevention of restenosis.
[0031] In a most particularly advantageous aspect, the Applicant
also showed that the virus and the method of the invention
permitted an effective and selective transfer of genes into an
atheromatous artery. More especially, the Applicant has
demonstrated for the first time the capacity of adenoviruses to
transfer a therapeutically effective gene into an atheromatous
artery. This is absolutely essential, since the therapeutic
efficacy of the treatment of restenosis is conditional on a
demonstration of the capacity to transfer the therapeutic gene,
into the correct cells and with suitable efficacy, under the
physiopathological conditions. Atheromatous arteries are
characterized by the presence in the intima (i) of deposits of
extracellular matrix, (ii) of lipid deposits consisting essentially
of foam cells of the macrophage type and (iii) of proliferating
smooth muscle cells.
[0032] The results presented below show that, in these atheromatous
arteries, the viruses according to the invention permit a lower
percentage of infection but which is of greater specificity (taking
account, in fact, of the presence of macrophage type cells in this
case, macrophage cells not being transduced) and which is
accompanied by a substantial therapeutic efficacy. The results
obtained show, in particular, a very selective transfer of the
adenovirus into the target cells, that is to say the proliferating
smooth muscle cells. Out of the whole cell population present in
the atheromatous zone, more than 95% of the infected cells are
vascular smooth muscle cells. Thus, the macrophage cells present in
the intima are not infected at all (no infected macrophage cell was
detected). As regards the proliferating smooth muscle cells (in the
neointima), the treatment according to the invention enables a
percentage of less than 1% (for example 0.2%) to be infected. This
is much less than the results described previously in healthy
arteries or those possessing lesions of the wall but which do not
represent a physiopatholigical situation of restenosis (endothelial
abrasion of a healthy artery). The Applicant also showed that the
infection of this small percentage of cells nevertheless permitted
a substantial therapeutic effect, demonstrated, in particular, by
measurement of the luminal diameter. This result is especially
surprising and implies the existence of an induced cytotoxic effect
("bystander" effect) in vivo. Hence the invention describes for the
first time a method permitting the selective transfer of genes into
proliferating vascular smooth muscle cells in an atheromatous
artery, comprising the administration into the said artery of a
defective recombinant adenovirus containing the said gene by means
of an angioplasty balloon catheter. The term selective transfer
implies a transfer essentially into the proliferating vascular
smooth muscle cells and no transfer into the surrounding macrophage
cells. This method permits a treatment of restenosis by transfer of
a suicide gene such as the TK gene, followed by treatment with
ganciclovir or acyclovir, for example. This method of treatment is,
in addition, characterized by an effect of toxicity induced in
vivo.
[0033] Another subject of the present invention relates to a
pharmaceutical composition comprising a defective recombinant
adenovirus and a hydrogel. More specifically, the invention relates
to a composition comprising a defective recombinant adenovirus
containing a suicide gene, and a hydrogel. The hydrogel used in the
context of the present invention may be prepared from any
biocompatible and non-cytotoxic (homo- or co-)polymer. Such
polymers have, for example, been described in Application
W093/08845. Some of them, such as, in particular, those obtained
from ethylene oxide and/or propylene oxide, are commercially
available.
[0034] The method of treatment of the invention hence
advantageously consists in introducing, at the site to be treated,
a composition comprising a hydrogel impregnated with recombinant
adenoviruses. The hydrogel may be deposited directly on the surface
of the tissue to be treated, for example during a surgical
intervention. Advantageously, the hydrogel [lacuna] be introduced
into the site to be treated by means of a catheter, for example a
balloon catheter, in particular during angioplasty, thereby
enabling any additional trauma due to a further intervention at the
angioplasty site to be avoided. It is especially advantageous for
the impregnated hydrogel to be introduced into the site to be
treated by means of a balloon catheter protected by a sleeve. As
described in the examples, the hydrogel possesses a large number of
advantages: it enables sliding of the balloon to be improved,
thereby enabling it to pass through heavily stenosed arteries.
Furthermore, the hydrogel can be used with any type of angioplasty
balloon, which enables perfusion balloons to be used in particular.
Thus, according to a particular embodiment, the adenoviruses
according to the invention are administered by means of perfusion
balloons, especially channelled balloon catheters ("channelled
balloon angioplasty catheter", Mansfield Mecical, Boston Scientific
Corp., Watertown, Mass.). The latter consists of a conventional
balloon covered with a layer of 24 perforated channels which are
perfused via an independent lumen through an additional infusion
orifice. These perfusion balloons, which make it possible to
maintain a blood flow and thus to decrease the risks of ischaemia
of the myocardium, on inflation of the balloon, also enable a
medicinal product to be delivered locally at normal pressure for a
relatively long time, more than twenty minutes, which is necessary
for an optimal infection.
[0035] It is especially advantageous to use a perfusion balloon
catheter coated with hydrogel. In this case, the advantages of
both, that is to say the possibility of keeping the balloon
inflated for a longer period of time by retaining the properties of
facilitated sliding and of site-specificity of the hydrogel, are
gained simultaneously. In this case, an optimal efficacy of
infection is obtained.
[0036] The results presented in the examples demonstrate, in fact,
the efficacy of this system for the percutaneous transfer of genes
into the arterial walls.
[0037] Another subject of the present invention relates to a
pharmaceutical composition comprising a defective recombinant
adenovirus and poloxamer. More specifically, the invention relates
to a composition comprising a defective recombinant adenovirus
containing a suicide gene, and poloxamer. Poloxamer 407 is a
non-toxic, biocompatible polyol; it is commercially available
(BASF, Parsippany, N.J.).
[0038] Hence a method of treatment of the invention advantageously
consists in introducing, into the site to be treated, a composition
comprising poloxamer impregnated with recombinant adenoviruses. The
poloxamer may be deposited directly on the surface of the tissue to
be treated, for example during a surgical intervention.
Advantageously, the poloxamer may be introduced into the site to be
treated by means of a catheter, for example a balloon catheter, in
particular during angioplasty, thereby enabling any additional
trauma due to a further intervention at the angioplasty site to be
avoided. It is especially advantageous for the impregnated
poloxamer to be introduced into the site to be treated by means of
a balloon catheter protected by a sleeve. Poloxamer possesses
essentially the same advantages as hydrogel while having a lower
viscosity.
[0039] It is especially advantageous to use a perfusion balloon
catheter coated with poloxamer, especially channelled balloon
catheters. In this case, the advantages of both, that is to say the
possibility of keeping the balloon inflated for a longer period of
time while retaining the properties of facilitated sliding and of
site-specificity of the poloxamer, are gained simultaneously. In
this case also, an optimal efficacy of infection is obtained.
[0040] The present invention will be described more completely by
means of the examples which follow, which are to be considered to
be illustrative and non-limiting.
LEGEND TO THE FIGURES
[0041] FIG. 1: Diagram of the vector pONT-tk
[0042] FIG. 2: Diagram of the vector pRSV-tk
[0043] FIG. 3: Cytotoxic effect of the ganciclovir/Ad-LTR-tk
combination on smooth muscle cells in culture.
[0044] FIG. 4: Reduction of restenosis by adenoviral transfer of
the tk gene and administration of ganciclovir.
GENERAL TECHNIQUES OF MOLECULAR BIOLOGY
[0045] The methods traditionally used in molecular biology, such as
preparative extractions of plasmid DNA, centrifugation of plasmid
DNA in a caesium chloride gradient, agarose or acrylamide gel
electrophoresis, purification of DNA fragments by electroelution,
phenol or phenol/chloroform extraction of proteins, ethanol or
isopropanol precipitation of DNA in a saline medium, transformation
in Escherichia coli, and the like, are well known to a person
skilled in the art and are amply described in the literature
[Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1982); Ausubel
F.M. et al. (eds), "Current Protocols in Molecular Biology", John
Wiley & Sons, New York, (1987)].
[0046] Plasmids of the pBR322 and pUC type and phages of the M13
series are of commercial origin (Bethesda Research
Laboratories).
[0047] To carry out ligation, the DNA fragments may be separated
according to their size by agarose or acrylamide gel
electrophoresis, extracted with phenol or with a phenol/chloroform
mixture, precipitated with ethanol and then incubated in the
presence of phage T4 DNA ligase (Biolabs) according to the
supplier's recommendations.
[0048] The filling in of 5' protruding ends may be performed with
the Klenow fragment of E.coli DNA polymerase I (Biolabs) according
to the supplier's specifications. The destruction of 3' protruding
ends is performed in the presence of phage T4 DNA polymerase
(Biolabs) used according to the manufacturer's recommendations. The
destruction of 5' protruding ends is performed by a controlled
treatment with S1 nuclease.
[0049] In vitro site-directed mutagenesis using synthetic
oligodeoxynucleotides may be performed according to the method
developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764]
using the kit distributed by Amersham.
[0050] The enzymatic amplification of DNA fragments by the
so-called PCR [Polymerase-catalyzed Chain Reaction, Saiki R. K. et
al., Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A.,
Meth. Enzym. 155 (1987) 335-350] technique may be performed using a
"DNA thermal cycler" (Perkin Elmer Cetus) according to the
manufacturer's specifications.
[0051] The verification of the nucleotide sequences may be
performed by the method developed by Sanger et al. [Proc. Natl.
Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by
Amersham.
EXAMPLES
Example 1
Construction of the Vector Ad-LTR-TK Carrying the TK Gene Under the
Control of the Rous Sarcoma Virus LTR (RSV LTR) Promoter (FIG.
1)
[0052] This example describes the construction of a recombinant
adenovirus comprising the herpes simplex virus thymidine kinase
(tk) gene under the control of a viral promoter (RSV LTR promoter).
This adenovirus was constructed by homologous recombination between
the defective adenovirus Ad-dl1324 and plasmid pRSVtk carrying the
tk gene under the control of the RSV promoter (Example 1.3.). This
plasmid was constructed from plasmid pONTtk (Example 1.1.) by
substituting the RSV promoter for the promoter which can be
transactivated by EBNA1 (Example 1.2.).
[0053] 1.1. Construction of plasmid pONTtk
[0054] a) Construction of plasmid p7tk1
[0055] This example describes the construction of plasmid p7tk1
containing the 1131-base pair open reading frame of the tk gene
(ATG 114-116 and stop codon TGA 1242-1244), inserted into a
multicloning site.
[0056] The BglII-NcoI fragment containing the herpes simplex type 1
virus thymidine kinase (tk) gene was isolated from plasmid pHSV-106
(marketed by Gibco BRL), repaired by the action of the Klenow
fragment and then inserted at the SmaI site of plasmid pGEM7zf(+)
(marketed by Promega). The SmaI and BgIII sites are destroyed
during this step, the NcoI site is preserved.
[0057] The plasmid obtained was designated p7tkl.
[0058] b) Construction of plasmid pONT1.
[0059] This example describes the construction of a plasmid
containing a chimeric promoter consisting of a sequence needed for
transactivation by the antigen EBNA1 and of the EBV virus TP1
promoter.
[0060] The EcoRI(7315)-SmaI(8191) fragment of the EBV virus was
isolated from the strain B95-8. The complete sequence of the EBV
virus has been described by Baer et al. (Nature 310 (1984) 207).
This fragment contains the sequences needed for transactivation by
the nuclear antigen 1 (EBNA1) (D. Reisman & B. Sugden, 1986,
Molecular and Cellular Biology, vol. 6 pp. 3838-3846). This
fragment was then fused to the NruI(166 241)-PstI(166 559) fragment
of EBV B95-8 (the PstI site was digested with T4 polymerase),
containing the TP1 promoter. The chimeric promoter thereby obtained
was then inserted into the multicloning site of the plasmid
pBluescript II SK to generate plasmid pONT1.
[0061] c) Construction of plasmid pONTtk
[0062] Plasmid pONTtk contains the herpes simplex virus thymidine
kinase (tk) gene cloned into plasmid p7tk1, under the control of
the chimeric promoter EBNA1-RE/TP1 cloned into plasmid pONT1.
[0063] To construct this plasmid, the BamHI-XhoI fragment of pONT1,
which contains the chimeric promoter transactivated by EBNA-1 and
EBNA-2, and the XhoI-ClaI fragment of p7tkl, which contains the
open reading frame of tk, were cloned at the BamHI (478) and ClaI
(4550) sites of plasmid pAd.RSVbgal. Plasmid pAd.RSVbGal contains,
in the 5'.fwdarw.3' orientation,
[0064] the PvuII fragment corresponding to the left-hand end of the
Ad5 adenovirus comprising: the ITR sequence, the origin of
replication, the encapsidation signals and the E1A enhancer;
[0065] the gene coding for b-galactosidase under the control of the
RSV (Rous sarcoma virus) promoter,
[0066] a second fragment of the Ad5 adenovirus genome, which
permits homologous recombination between plasmid pAd.RSVbGal and
the adenovirus d1324. Plasmid pAd.RSVbGal has been described by
Stratford-Perricaudet et al., (J. Clin. Invest. 90 (1992) 626).
[0067] All the cloning sites are preserved. The plasmid obtained
was designated.pONTtk.
[0068] 1.2. Construction of plasmid pRSVtk
[0069] This plasmid was constructed from plasmid poNTtk (Example
1.1.) by substituting the RSV promoter for the promoter which can
be transactivated by EBNA1. To this end, the RSV promoter was
isolated in the form of a BamHI-SalI fragment from plasmid
pAd.RSV..beta.gal (Stratford-Perricaudet et al., J. Clin. Invest.
90 (1992) 626), and then cloned at the BamHI(478) and SalI(1700)
sites of plasmid pONTtk. The resulting plasmid was designated
pRSVtk (FIG. 1).
[0070] 1.3. Construction of the recombinant adenovirus
Ad-RSV-tk
[0071] The vector pRSVtk was linearized and cotransfected with a
defective adenoviral vector into helper cells (line 293), providing
in trans the functions encoded by the adenovirus E1 (E1A and E11B)
regions.
[0072] More precisely, the adenovirus Ad-RSV-tk was obtained by in
vivo homologous recombination between the mutant adenovirus
Ad-dl1324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector
pRSVtk, according to the following protocol: plasmid pRSVtk,
linearized with XmnI, and the adenovirus Ad-dl1324, linearized with
the enzyme ClaI, were cotransfected into line 293 in the presence
of calcium phosphate, to permit homologous recombination. The
recombinant adenoviruses thus generated were selected by plaque
purification. After isolation, the recombinant adenovirus DNA was
amplified in cell line 293, leading to a culture supernatant
containing the unpurified defective recombinant adenovirus having a
titre of approximately 10.sup.10 pfu/ml.
[0073] The viral particles were then purified by centrifugation on
a caesium chloride gradient according to known techniques (see, in
particular, Graham et al., Virology 52 (1973) 456). The adenovirus
Ad-RSV-tk may be stored at -80.degree. C. in 20% glycerol.
Example 2
Activity of an Adenovirus According to the Invention in the
Presence of Ganciclovir on Smooth Muscle Cells in Culture
[0074] The activity of the adenovirus containing the TK gene,
prepared in Example 1, was checked on in vitro models of smooth
muscle cells. To this end, smooth muscle cells isolated from rat
and rabbit aorta were infected with the recombinant adenovirus
Ad-RSV-tk and incubated in the presence of ganciclovir. The effect
of the Ad-RSV-tk/ganciclovir combination on cell viability is then
confirmed by the calorimetric test employing MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
according to the technique described by Mosman (J. Immunol. Meth.
65 (1983) 55), or more accurately by cell counting. Briefly,
vascular smooth muscle cells (VSMC) are set up in culture by
enzymatic digestion of NZW rabbit aorta according to a method
adapted from Chamley et al. (Cell Tissue Res. 177: 503-522 1977).
The cells are maintained in the presence of 20% of foetal calf
serum and used for all the tests (see below) before the tenth
passage. In all our experiments, smooth muscle cells are
characterized by immunolabelling using anti-.alpha.SM-actin
antibody (Sigma).
[0075] In order to measure the cytotoxic activity of the
Ad-RSV-TK/ganciclovir combination, rabbit aorta VSMC are incubated
in the presence of the adenovirus diluted in culture medium (DMEM,
0.5% FCS). After approximately one hour at 37.degree. C. in a humid
atmosphere, the medium containing the adenoviral solution is
aspirated off and replaced by culture medium (DMEM, 0.5% FCS) for a
period of 18 to 24 hours. Different concentrations of ganciclovir
are then added in an FCS-rich (10%) medium. Four days after the
addition of ganciclovir, the cells are counted (100% cell viability
corresponding to cells not transduced with Ad-RSV-TK and not
treated with ganciclovir).
[0076] The Ad-RSV-TK/ganciclovir combination induces a cytotoxic
effect with respect to rabbit VSMC (see FIG. 3). This cytotoxicity
varies in accordance with the ganciclovir concentration and the
multiplicity of Ad-RSV-TK infection. Under our experimental
conditions, i.e. four days of incubation in the presence of 10%
FCS, the Ad-RSV-TK (M.O.I. 1000)/ganciclovir (25 .mu.M) combination
brings about complete cytolysis. Under these experimental
conditions where a high multiplicity of infection enables the
majority of the cells to be transduced, the IC.sub.50 is 0.3 .mu.M.
At a low multiplicity of infection (M.O.I. 10), the IC.sub.50 is
less than 5 .mu.M. Thus, the concentrations of ganciclovir which
are active in vitro on SMC are compatible with a therapeutic use.
In effect, in patients treated for viral infection with a non-toxic
dose of ganciclovir, it is possible to reach plasma concentrations
above 15 .mu.M (Paul and Dummer, Am. J. Med. Sci. 4: 272-277,
1992).
[0077] In addition, this in vitro study demonstrates that it is
possible to induce a major cytotoxic effect in spite of a small
percentage of transduction by the Ad-RSV-TK adenovirus. The
presence of the HSV-TK protein was demonstrated by indirect
immunofluorescence using monoclonal antibodies specific for HSV-TK
(monoclonal antibody 4C8, Yale University). In rabbit (and human)
VSMC treated with Ad-RSV-TK, the localization of the TK protein is
cytoplasmic but also nuclear. We have shown in this way that the
use of a multiplicity of infection of 10 is associated with a
transduction of fewer than 5% of the VSMC. Generally speaking, at
an equivalent multiplicity of infection, the percentage of cells
transduced by Ad-RSV-TK is similar to that obtained using a control
adenovirus coding for .beta.-galactosidase (e.g. more than 90% of
cells transduced at a multiplicity of infection of 1000). These
data hence demonstrate that the transduction of fewer than 5% of
VSMC brings about a substantial cytotoxicity in the presence of an
optimal concentration of ganciclovir (see FIG. 3: 80% drop in cell
viability at 25 .mu.M). Immunodetection of the HSV-TK protein hence
illustrates the magnitude of the "bystander" effect observed on
VSMC treated with the Ad-RSV-TK/ganciclovir combination. This
"bystander" effect may have its counterpart in vivo. In particular,
these data strongly suggest that a limited transfer of Ad-RSV-TK
adenovirus, in particular into a pathological artery, may lead to a
significant reduction of the neointimal mass, which is rich in SMC
and responsible for restenosis in the patient.
[0078] Moreover, the cytolytic effect is selective, since neither
simple treatment with ganciclovir nor transduction with Ad-RSV-TK
per se is associated with cell death. The cytotoxicity of the
Ad-RSV-TK/ganciclovir combination has been confirmed by the
calorimetric test employing MTT. Lastly, similar results, namely a
selective toxicity in the presence of ganciclovir and adenovirus,
have been observed on a primary culture of human smooth muscle
cells.
[0079] Hence these data demonstrate the effective blocking of the
proliferation of VSMC in vitro by Ad-RSV-TK.
Example 3
Arterial Transfer of a Recombinant Adenovirus via the Percutaneous
Route
[0080] This example describes the development of an especially
effective technique for gene transfer via the percutaneous route.
This technique is based on the use of a balloon catheter with
hydrogel. The results presented show that, most advantageously,
this technique enables certain favoured cell populations to be
infected effectively, in particular for the treatment of
restenosis.
[0081] This example was carried out using a defective recombinant
adenovirus comprising the E. coli .mu.-galactosidase gene under the
control of the RSV RSV promoter and of a nuclear localization
signal. The construction of this adenovirus has been described, in
particular, in Stratford-Perricaudet et al., (J. Clin. Invest. 90
(1992) 626).
[0082] The experiments were carried out on New Zealand white
rabbits anaesthetized with acepromazine and maintained under
pentobarbital. Gene transfer was performed at the external iliac
artery.
[0083] The adenovirus Ad-RSV..beta.-Gal (1-2.times.10.sup.10 pfu in
100 .mu.l of phosphate buffer) was deposited on a balloon catheter
previously coated with hydrogel (Hydroplus, Mansfield Medical,
Boston Scientific Corp., Watertown, Mass.) (Riessen et al., Hum.
Gene Ther. 4 (1993) 749). The catheter used is a balloon catheter 2
cm in length and between 2.5 and 3 mm in diameter. The catheter was
then introduced, using a protective sleeve, into the right femoral
artery. A pressure of one atmosphere was then applied, and the
catheter was thereafter directed into the external iliac artery
where a pressure of 6 atmospheres was then applied to the balloon
for 30 minutes. This experiment was carried out on 27 rabbits. 3 to
28 days after administration, the animals were sacrificed by
overdosing with pentobarbital.
[0084] Transfer of the Gene into the Arterial Wall and its
Expression Therein
[0085] The arteries of the sacrificed animals were isolated, and
the expression of .beta.-galactosidase was detected by staining in
the presence of X-gal according to the technique of Sanes et al.,
(EMBO J. 5 (1986) 3133). For each animal, at least two arterial
segments were either mounted on OCT (Miles Laboratories Inc.; IL)
for frozen-section experiments, or coated with paraffin, cut into 6
.mu.m sections and counterstained with haematoxylin and eosin.
Expression was considered positive only when a deep blue
colouration was observed in the nucleus. The results obtained show
clearly that the arteries of the infected animals display a blue
colouration characteristic of .mu.-gal. A microscopic analysis
discloses that there is no residual intact endothelium, but that
the continuity of the internal elastic lamina is preserved.
Microscopic analysis also shows that the cells of the media have
been infected with the adenoviruses and express the transferred
gene. More precisely, whereas, in the case of an administration by
double-balloon catheter, only 0.4% of the cells of the media have
been infected, 9.6% are infected using a balloon catheter coated
with hydrogel (see morphometric analysis below). Furthermore, the
9.6% are calculated relative to the total thickness of the media
but, in the superficial layers of the media, 100% of the cells are
infected. These results are far better than those obtained with
double-balloon catheters, or by transfer of naked genes or by means
of liposomes. These experiments demonstrate the extent to which
adenoviruses can constitute an especially advantageous vector for
the administration of suicide genes for the purpose of treatment of
restenosis.
[0086] MorPhometric Analysis
[0087] The efficacy of transfer was determined on 7 treated
rabbits. All these animals received 5.times.10.sup.9 pfu of
adenovirus to infect an arterial segment 2 cm in length, so that
the multiplicity of infection is similar for each animal. For each
iliac artery transfected, two serial segments 5 mm in length were
taken from the target zone and, for each segment, at least three
sections at random were examined under the light microscope after
staining with X-gal. On each section, the efficacy of transfer was
determined from the ratio of stained media cells to the total
number of media cells. In all, more than 30.times.10.sup.3 cells
originating from arteries infected with the adenoviruses (48
sections) were counted. The mean percentage of infected media cells
is 4.02%, with values which can reach 9.6%. In the case of a
transfer with a double-balloon cathether, the mean percentage is
only 0.18%.
[0088] Kinetics of Expression
[0089] To determine the duration of expression of the gene
transferred by the adenoviruses according to the invention, a study
of the expression of .beta.-gal was carried out over time on 20
rabbits treated either using a double-balloon catheter (10 animals)
or using a balloon catheter impregnated with hydrogel (10 animals).
For each group, 2 animals were sacrificed on days 3, 7, 14, 21 and
28. Expression was detected by macroscopic and microscopic
examination of arteries stained with X-gal as described above. The
results obtained show, for each group, a stable expression for 14
days, followed by a drop at 21 days. No expression is detected at
28 days. The same kinetics could be demonstrated in a pathological
artery in the atheromatous rabbit model. These results confirm the
transient effect of the vectors of the invention, which is
especially advantageous and suited to the treatment of restenosis,
in particular on the atheromatous walls.
[0090] Selectivity of the Transfer and Expression with Respect to
Arterial Walls
[0091] In order to check the possible dissemination to other
tissues of the adenoviruses injected, in all the animals sacrificed
3 days after injection, tissue samples originating from the liver,
brain, testicles, heart, lung, kidney and skeletal muscle, as well
as an arterial segment adjacent to the site treated, were removed
immediately after sacrifice. On each sample, gene transfer and
expression were demonstrated by PCR (by means of probes directed
against the gene coding for adenovirus protein IX and against the
lacZ gene) and histochemistry. The results obtained show that none
of the samples removed from the animals treated with a balloon
catheter coated with hydrogel displays staining in the tissues
tested. Similarly, no presence of virus could be detected by PCR in
the samples tested, even using an optimized and very sensitive
protocol of 45 amplification cycles.
[0092] These results demonstrate the efficacy of the mode of
administration according to the invention for delivering
therapeutic genes very locally.
Example 4
Arterial Transfer of the Adenovirus Ad-RSV-TK
[0093] This example demonstrates the properties of the TK
adenovirus of the invention for the treatment of restenosis by
selective transfer into an atheromatous artery.
[0094] Animal Model
[0095] The efficacy of the arterial transfer was evaluated in a
model of restenosis in New Zealand white rabbits. The rabbits were
subjected beforehand to a cholesterol-rich (1%) diet for two weeks.
The iliac artery was abraded by five successive inflations using a
latex balloon (4F). The animals were subjected again to a
hypercholesterolaemia-inducin- g diet for six weeks. Arterial
transfer was performed percutaneously according to the procedure
described above, at the damaged artery. The adenovirus Ad-RSV-TK
(4.times.10.sup.9 pfu in 40 .mu.l of phosphate buffer) was
accordingly deposited on a balloon catheter previously coated with
hydrogel (Hydroplus, Mansfield Medical, Boston Scientific Corp.,
Watertown, Mass.) (Riessen et al., Hum. Gene Ther. 4 (1993) 749).
The catheter used is a balloon catheter 2 cm in length and 2.5 mm
in diameter.
[0096] Based on a double lesion following abrasion and angioplasty,
this model of restenosis, and not only of stenosis, enables the
efficacy of adenoviral transfer of a suicide gene into an
atheromatous artery to be evaluated.
[0097] In the light of the in vitro experimental data, and in order
to verify the selectivity of the treatment with Ad-RSV-TK, the
animals were divided into two groups, treated or otherwise with
ganciclovir. The treatment with ganc-clovir was prolonged for five
days (from the second to the seventh day following angioplasty) at
the rate of 2.times.25 mg/kg/day.
[0098] Morphometric Analysis
[0099] Six weeks after angioplasty, the animals were sacrificed
with pentabarbital, and the iliac arteries were fixed and removed
for the purpose of morphometric analysis. The contours of the lumen
as well as those of the internal/external elastic limiting
membranes were evaluated after staining with orcein/haematoxylin.
In all, six blocks were analysed per artery, including 4 lying
within the angioplasty zone and two immediately upstream and
downstream of this zone. For each block, three adjacent sections
were analysed. Briefly, different parameters such as luminal
diameter and intima/media ratio were calculated. Samples for which
these contours could not be revealed as a result of a rupture of
the internal elastic limiting membrane or of a thrombotic occlusion
were excluded.
[0100] Morphometric analysis reveals a high intima/media ratio in
the control group which has been subjected to adenoviral transfer
by angioplasty but not treated with ganciclovir (5.73+/-0.81, n=3).
The severity of the lesion hence enables the efficacy of a gene
transfer treatment to be evaluated on a pathological artery. In
addition, as shown by the immunohistochemical study, the lesions
induced in this animal model are rich in macrophages, but also rich
in smooth muscle cells which constitute the therapeutic target of
the gene transfer. These data collectively bring out the value of
the model used, which is not based on a simple endothelial abrasion
in a healthy artery, and consequently mimics at least partially the
pathology of postangioplasty restenosis in man.
[0101] Morphometric analysis shows that the intima/media ratio is
reduced by 42% (p<0.05) in the group of animals subjected to the
Ad-RSV-TK/ganciclovir combination (3.30 +/-1.26, n=6). The
significant reduction in this parameter demonstrates that local
transfer of the TK gene by a recombinant adenovirus, in combination
with the administration of ganciclovir, constitutes a promising
therapeutic approach as a preventive treatment for postangioplasty
restenosis.
* * * * *