U.S. patent application number 10/087179 was filed with the patent office on 2002-06-27 for methods, systems, and kits for intravascular nucleic acid delivery.
This patent application is currently assigned to PHARMASONICS, INC.. Invention is credited to Brisken, Axel F., Newman, Christopher M.H..
Application Number | 20020082238 10/087179 |
Document ID | / |
Family ID | 22092957 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082238 |
Kind Code |
A1 |
Newman, Christopher M.H. ;
et al. |
June 27, 2002 |
Methods, systems, and kits for intravascular nucleic acid
delivery
Abstract
Nucleic acid transfection of vascular smooth muscle cells is
enhanced by the application of vibrational energy to the cells. By
applying vibrational energy at frequency in the range from 1 kHz to
10 MHz and at an intensity in the range from 0.01 W/cm.sup.2 to 100
W/cm.sup.2, significant enhancement of the uptake of nucleic acids
into vascular smooth muscle cells can be achieved.
Inventors: |
Newman, Christopher M.H.;
(Dore, GB) ; Brisken, Axel F.; (Fremont,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
PHARMASONICS, INC.
Sunnyvale
CA
|
Family ID: |
22092957 |
Appl. No.: |
10/087179 |
Filed: |
March 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10087179 |
Mar 1, 2002 |
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09223231 |
Dec 30, 1998 |
|
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6372498 |
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60070073 |
Dec 31, 1997 |
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Current U.S.
Class: |
514/44R ;
604/20 |
Current CPC
Class: |
A61M 2210/12 20130101;
A61M 2025/105 20130101; A61B 2017/22082 20130101; A61M 37/0092
20130101; A61B 17/2202 20130101; A61N 1/306 20130101; A61N 1/325
20130101 |
Class at
Publication: |
514/44 ;
604/20 |
International
Class: |
A61K 048/00; A61N
001/30 |
Claims
What is claimed is:
1. A method for intravascular nucleic acid delivery, said method
comprising: providing a flexible catheter having a vibrational
transducer disposed near its distal end; intravascularly
positioning the distal end of the catheter at a target region
within a blood vessel; delivering nucleic acids to vascular smooth
muscle cells which line a wall of the blood vessel; and energizing
the transducer to deliver vibrational energy to the wall at a
frequency and intensity selected to enhance uptake of the nucleic
acids by the smooth muscle cells.
2. A method as in claim 1, wherein the vibratory energy is at a
frequency in the range from 1 kHz to 10 MHz.
3. A method as in claim 2, wherein the vibratory energy has an
intensity in the range from 0.01 W/cm.sup.2 to 100 W/cm.sup.2.
4. A method as in claim 3, wherein the vibratory energy is
delivered with a duty cycle in the range from 1% to 100%.
5. A method as in claim 4, wherein the vibratory energy is applied
for a cumulative treatment time in the range from 10 seconds to 900
seconds.
6. A method as in claim 1, wherein the interface surface directly
contacts the blood vessel wall within the target region.
7. A method as in claim 1, wherein the interface surface is
spaced-apart from the blood vessel wall, wherein ultrasonic energy
is transmitted through a liquid medium containing the nucleic acids
disposed between the interface surface and the wall.
8. A method as in claim 1, wherein the vibrational exciting step
comprises vibrating the surface in a radial direction.
9. A method as in claim 1, wherein the vibrational exciting step
comprises vibrating the surface in an axial direction.
10. A method as in claim 1, further comprising expanding a pair of
axially spaced-apart balloons disposed on either side of the
vibrational surface to localize a solution containing the nucleic
acids as the surface is vibrated.
11. A method as in claim 1, wherein the nucleic acids are delivered
through the flexible catheter.
12. A method as in claim 1, wherein the nucleic acids are selected
from the group consisting of genes, gene fragments, sense
polynucleotides, anti-sense polynucleotides, and
oligonucleotides.
13. A method as in claim 12, wherein the nucleic acids encode an
angiogenic factor eNOS, TIMP, or p21.
14. A method as in claim 1, wherein the nucleic acids are delivered
after the target region has been treated to enlarge or remove an
occlusion.
15. A method as in claim 14, wherein the prior treatment is
selected from the group consisting of angioplasty, atherectomy, and
stenting.
16. A kit comprising: a catheter having a vibratory interface
surface; and instructions for use setting forth a method according
to claim 1.
17. A system comprising: a catheter having a vibratory interface
surface; and a nucleic acid reagent which can be intravascularly
delivered by the catheter.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continutation of serial application
Ser. No. 09/223,231 (Attorney Docket No. 17148-001210) filed Dec.
30, 1998, which was continuation-in-part of provisional application
Ser. No. 60/070,073 (Attorney Docket No. 017148-001200), filed on
Dec. 31, 1997. The full disclosure of each is incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to medical methods
and devices. More particularly, the present invention relates to
methods, systems, and kits for the delivery of nucleic acids to
smooth muscle cells which line the lumen of blood vessels.
[0004] A number of percutaneous intravascular procedures have been
developed for treating atherosclerotic disease in a patient's
vasculature. The most successful of these treatments is
percutaneous transluminal angioplasty (PTA) which employs a
catheter having an expansible distal end, usually in the form of an
inflatable balloon, to dilate a stenotic region in the vasculature
to restore adequate blood flow beyond the stenosis. Other
procedures for opening stenotic regions include directional
atherectomy, rotational atherectomy, laser angioplasty, stents and
the like. While these procedures, particularly PTA, have gained
wide acceptance, they continue to suffer from the subsequent
occurrence of restenosis.
[0005] Restenosis refers to the re-narrowing of an artery within
weeks or months following an initially successful angioplasty or
other primary treatment. Restenosis afflicts up to 50% of all
angioplasty patients and results at least in part from smooth
muscle cell proliferation in response to the injury caused by the
primary treatment, generally referred to as "hyperplasia." Blood
vessels in which significant restenosis occurs will require further
treatment.
[0006] A number of strategies have been proposed to treat
hyperplasia and reduce restenosis. Such strategies include
prolonged balloon inflation, treatment of the blood vessel with a
heated balloon, treatment of the blood vessel with radiation, the
administration of anti-thrombotic drugs following the primary
treatment, stenting of the region following the primary treatment,
and the like. While enjoying different levels of success, no one of
these procedures has proven to be entirely successful in treating
all occurrences of restenosis and hyperplasia.
[0007] Of particular interest, it has recently been proposed to
deliver nucleic acids to smooth muscle cells within blood vessels
for the treatment of hyperplasia and other disease conditions. See,
e.g. U.S. Pat. No. 5,328,470. Progress in vascular gene therapy,
however, has been hindered by the limited efficiency and/or
toxicity of most currently available transfection materials and
techniques. Current methods used to achieve nucleic acid transfer
into vascular smooth muscle cells comprise the delivery of naked
DNA, cationic liposomes, and specialized adenoviral and retroviral
vectors. Each of these approaches are problematic. While the use of
adenoviral vectors can achieve relatively high transfection
efficiencies, the use of viruses raises concern among many experts
in the field.
[0008] For these reasons, it would be desirable to provide
additional and/or improved methods, systems, kits, and the like for
the delivery of nucleic acids to vascular smooth muscle cells and
other cells which comprise the vascular wall. It would be
particularly desirable if such gene delivery methods were useful
for the treatment of hyperplasia in regions of a blood vessel which
have previously been treated by angioplasty, atherectomy, stenting,
and other primary or secondary treatment modalities for
atherosclerotic disease. Such methods should provide efficient gene
delivery, result in minimum necrosis of the cells lining the
vasculature (particularly smooth muscle cells and endothelial
cells), permit targeting of vascular smooth muscle cells, be
capable of being performed with relatively simple catheters and
other equipment, and suffer from minimum side effects. At least
some of these objectives will be met by the invention described
hereinafter.
[0009] 2. Description of the Background Art
[0010] Catheters and methods for intravascular transfections are
described in U.S. Pat. No. 5,328,470 and published in PCT
applications WO 97/12519; WO 97/11720; WO 95/25807; WO 93/00052;
and WO 90/11734.
[0011] Ultrasound-mediated cellular transfection is described or
suggested in Kim et al. (1996) Hum. Gene Ther. 7:1339-1346; Tata et
al. (1997) Biochem. Biophy. Res. Comm. 234:64-67; and Bao et al.
(1997) Ultrasound in Med. & Biol. 23:953-959. The effects of
ultrasound energy on cell wall permeability and drug delivery are
described in Harrison et al. (1996) Ultrasound Med. Biol.
22:355-362; Gao et al. (1995) Gene Ther. 2:710-722; Pohl et al.
(1993) Biochem. Biophys. Acta. 1145:279-283; Gambihler et al.
(1994) J. Membrane Biol. 141:267-275; Bommannan et al. (1992)
Pharma. Res. 9:559-564; Tata and Dunn (1992) J. Phys. Chem.
96:3548-3555; Levy et al. (1989) J. Clin. Invest. 83:2074-2078;
Feschheimer et al. (1986) Eur. J. Cell Biol. 40:242-247; and
Kaufman et al. (1977) Ultrasound Med. Biol. 3:21-25. A device and
method for transfection, endothelial cells suitable for seeding
vascular prostheses are described in WO 97/13849.
[0012] Local gene delivery for the treatment of restenosis
following intravascular intervention is discussed in Bauters and
Isner (1998) Progr. Cardiovasc. Dis. 40:107-116 and in Baek and
March (1998) Circ. Res. 82:295-305.
[0013] A high frequency ultrasonic catheter employing an air-backed
transducer which may be suitable for performing certain methods
according to the present invention is described in He et al. (1995)
Eur. Heart J. 16:961-966. Other catheters suitable for performing
at least some methods according to the present invention are
described in copending application Ser. Nos. 08/565,575;
08/566,740; 08/566,739; 08/708,589; 08/867,007, and 09/ 09/223,225
(Attorney Docket No. 17148-001400, filed on Dec. 30, 1998),
assigned to the assignee of the present invention, the full
disclosures of which are incorporated herein by reference.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention comprises methods, systems, and kits
for the delivery of nucleic acids to the smooth muscle cells of the
type which line coronary arteries and other blood vessels. The
delivery of nucleic acids to target cells is generally referred to
as "transfection," and the transfection methods of the present
invention are advantageous since they are capable of significantly
increasing transfection efficiency, i.e. the amount of nucleic acid
materials taken up by the smooth muscle cells to which they are
delivered. The methods of the present invention are useful with a
wide variety of nucleic acid types. For example, it has been found
that significant transfection efficiencies can be obtained even
with naked DNA and RNA molecules i.e., nucleic acids which are not
incorporated into liposomes, viral vehicles, plasmids, or other
conventional nucleic acid vehicles. The methods are not limited to
such naked nucleic acids, however, they are also suitable for the
delivery of nucleic acids incorporated into liposomes and other
vesicles; viral vectors, including both adenoviral vectors and
retroviral vectors; plasmids, and the like.
[0015] The methods of the present invention are particularly
suitable for delivering nucleic acids incorporated into liposomes
often referred to as "lipofection," to the vascular smooth muscle
cells. As is demonstrated in the Experimental section hereinafter,
transfection of vascular smooth muscle cells with naked DNA is
enhanced significantly by vibratory energy (by a factor of 7.5 in
the particular data shown), but overall transfection efficiency
still remains at a relatively low level. In contrast, lipofection
enhanced with vibratory energy according to the present invention
shows a lesser enhancement over lipofection without vibrational
energy (by a factor of three in the particular data which are
shown), but the overall transfection efficiency, is substantially
greater than that which can be achieved with naked nucleic acids,
even with vibrational energy enhancement. Thus, the combination of
lipofection with vibrational energy enhancement will frequently be
preferred. While similar overall transfection efficiencies may be
achieved with vibrational enhancement of viral vectors, the use of
viral vectors will often not be preferred because of the safety
concerns which have been raised with respect to such delivery
vehicles. Additionally, as other delivery vehicles are developed as
alternatives for variations of the liposome and viral vehicles
which presently find use, it will be expected that the vibratory
enhancements of the present invention will find use with such
methods. A significant advantage of the present invention, however,
is that such delivery vehicles are not essential for efficient
uptake.
[0016] While the methods, systems, and kits of the present
invention will preferably be used with in vivo transfection
techniques described above, they will also find use with in vitro
techniques for transfecting vascular smooth muscle cells in
culture. Such in vitro methods will find use in many contexts, such
as in the testing of different structural and regulatory genes to
determine their effect on vascular smooth muscle cells, the
transformation of vascular smooth muscle cells to other predictable
phenotypes research purposes, and the like. In other instances, it
may be desirable to transfect autologous or heterologous vascular
smooth muscle cells in vitro so that the cells can later be
"seeded" back into a patient for a particular therapeutic purpose.
For example, vascular smooth muscle cells can be transfected to
produce therapeutic proteins which can be released by the
transfected cells after they are implanted or otherwise introduced
to a patient.
[0017] The nucleic acids may be in the form of genes, gene
fragments, sense oligonucleotides and polynucleotides, anti-sense
oligonucleotides and polynucleotides, and any other type of nucleic
acid having biological activity or benefit. Exemplary genes that
may be delivered for treating cardiovascular disease and
hyperplasia include angiogenic factors, such as vascular
endothelial growth factor (VEGF), endothelial nitric oxide synthase
(eNOS), tissue inhibitor matrix matallio-proteinase (TIMP), p21,
and the like.
[0018] Smooth muscle and other vascular cells are transfected
according to the present invention by delivering nucleic acids to
the cells located, for example, in a target region within a blood
vessel or in cell culture. The cells are exposed to vibratory
energy at a frequency and intensity selected to enhance the uptake
of the nucleic acids by the smooth muscle cells, which line the
blood vessel wall. The exposure of the cells to the vibratory
energy can occur before exposure or introduction of the nucleic
acids, after exposure or introduction of the nucleic acids, or
simultaneously with such exposure or introduction. Preferably,
exposure of the cells to the vibratory energy will continue for at
least a time (total elapsed time) following the introduction or
exposure of the cells to the nucleic acids, typically for at least
10 seconds, preferably for at least 60 seconds, more preferably for
at least 300 seconds, and still more preferably for at least 900
seconds, usually being in the range from 10 seconds to 900
seconds.
[0019] Preferably, the vibratory energy is delivered at a frequency
in the range from 1 kHz to 10 MHz, preferably in the range from 20
kHz to 3 MHz, usually from 100 kHz to 2 MHz. The intensity of the
vibrational energy will usually be in the range from 20 W/cm.sup.2
to 100 W/cm.sup.2, preferably in the range from 0.1 W/cm.sup.2 to
10 W/cm.sup.2, usually from 0.5 W/cm.sup.2 to 5 W/cm.sup.2. The
vibratory energy may be delivered continuously during the
transfection event, or alternatively may be delivered
intermittently, e.g. with a duty cycle within the range from 1% to
100%, usually from 5% to 95%, preferably from 10% to 50%.
[0020] The duration of exposure of the cells to the vibration
energy will be a function of total elapsed time (usually within the
range and limits set forth above), the duty cycle (percentage of
the total elapsed time in which the vibrational energy is turned
on), and pulse repetition frequency (PRF; the frequency at which
the vibrational energy is turned off and on, typically in the range
from 1 Hz to 1000 Hz). Generally, the duty cycle and/or PRF can be
controlled to permit heat dissipation to maintain a temperature in
the treated artery or cell culture below 45.degree. C., preferably
below 42.degree. C., and more preferably below 40.degree. C. Higher
temperatures can be deleterious to the viability of the vascular
smooth muscle cells.
[0021] The vibrational energy will usually be ultrasonic energy and
may be delivered in a variety of ways. For example, the vibrational
energy may be delivered from an external source, e.g. by focused
ultrasonic systems, such as high intensity focused ultrasound
(HIFU) systems which are commercially available. Usually, however,
the ultrasonic energy will be delivered intravascularly using an
interface surface which is disposed within the region within the
blood vessel. The interface surface is vibrationally excited to
radiate ultrasonic energy directly or indirectly (as defined below)
into the blood vessel wall. Typically, the ultrasonic surface is
carried on a flexible catheter having a vibrational transducer or
other oscillator disposed on the catheter near the surface. The
transducer is then energized to vibrate the surface within the
desired frequency range and at the desired intensity.
Alternatively, ultrasonic or other vibrational energy can be
delivered from an external source down a transmission member
through the catheter to the interface surface. For in vitro
methods, a variety of hand-held probes and transducers could be
employed. A particular transducer useful for imparting vibratory
energy to cultures of vascular smooth muscle cells is described in
the Experimental section hereinafter.
[0022] The vibrational energy may be delivered directly into the
blood vessel wall, e.g. by contacting the interface surface
directly against a portion of the wall within the target region.
Alternatively, the vibrational energy can be delivered indirectly
by vibrating the surface within blood or other liquid medium within
the blood vessel. Usually, the nucleic acids will be released or
disposed in the liquid medium. In an exemplary embodiment, the
nucleic acids are contained within a suitable transfection medium
which is localized within the target region by a pair of axially
spaced-apart balloons. The interface surface is also disposed
between the balloons, and energy is applied to the entrapped medium
via the interface surface. Alternatively, the nucleic acid medium
may be delivered to the interior of a porous balloon and/or to
fluid delivery conduits secured to the outside of a balloon, where
in both cases the vibrational transducer can be mounted on the
catheter body within the balloon. Conveniently, the medium
containing the nucleic acids can be delivered to the region via the
same catheter, optionally being recirculated or replenished via the
catheter during the treatment.
[0023] Alternatively, the nucleic acids can be delivered to the
patient systemically while the vibrational energy is applied
locally and/or from an external source as described above.
Optionally, the nucleic acids may be delivered to a vascular target
site in the presence of microbubbles of gas or other cavitation
nucleation components. It is believed that low intensity vibration
of the type preferably employed in the methods of the present
invention will generally not induce cavitation in a vascular
environment devoid of cavitation nucleii. As cavitation is
presently believed to contribute to the formation of pores in the
walls of the smooth muscle cells (and thus enhance nucleic acid
uptake), the introduction of microbubbles or other cavitation
nucleii together with the nucleic acids, e.g. from the same
delivery catheter, may significantly enhance the nucleic acid
uptake. For example, the nucleic acids may be delivered in a liquid
medium to which dissolved gases have been added as cavitation
nucleii.
[0024] The nucleic acids can be delivered to the smooth muscle or
other vascular cells for a variety of purposes. In a preferred
example, the nucleic acids are delivered to a region of the blood
vessel which has previously been treated by a primary intravascular
technique for treating cardiovascular disease, such as balloon
angioplasty, directional atherectomy, rotational atherectomy,
stenting, or the like. The methods of the present invention for
inhibiting intimal hyperplasia in vascular smooth muscle cells will
find particular use following stenting procedures in order to
prevent or inhibit hyperplasia which can occur following stenting.
The nucleic acids delivered will be intended to inhibit hyperplasia
and/or promote angiogenesis following such primary treatment.
Methods for promoting angiogenesis, of course, need not be
performed in conjunction with a primary treatment. Suitable genes
for such treatments have been described above.
[0025] Kits according to the present invention will comprise a
catheter having an interface surface which may be vibrated. The
kits will further include instructions for use setting forth a
method as described above. Optionally, the kits will further
include packaging suitable for containing the catheter and the
instructions for use. Exemplary containers include pouches, trays,
boxes, tubes, and the like. The instructions for use may be
provided on a separate sheet of paper or other medium. Optionally,
the instructions may be printed in whole or in part on the
packaging. Usually, at least the catheter will be provided in a
sterilized condition. Other kit components, such as the nucleic
acids to be delivered, may also be included.
[0026] Systems according the present invention will comprise a
catheter having an interface surface which may be vibrated at the
frequencies and power levels described above. Such systems may
further include nucleic acids in a form suitable for the
transfection or lipofection of vascular smooth muscle cells lining
an artery or other blood vessel. The nucleic acids may be naked,
viral-associated, but will preferably be incorporated within
liposome vesicles in order to enhance transfection efficiency when
delivered in the presence of vibratory energy according to the
methods of the present invention. The catheter will usually be
packaged in a sterile tray, pouch, or other conventional container,
while the nucleic acid reagent will be incorporated in an ampoule,
bottle, or other conventional liquid pharmaceutical container.
Optionally, the catheter and reagent will be packaged together in a
box, bag, or other suitable package. Further optionally, the
systems may include instructions for use as described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a perspective view of a catheter suitable for use
in the methods of the present invention.
[0028] FIG. 2 is a cross-sectional view taken along line 2-2 of
FIG. 1.
[0029] FIGS. 3 and 4 are alternative cross-sectional views for the
catheter of FIG. 1.
[0030] FIGS. 5A-5C illustrate use of the catheter of FIG. 1 in
performing nucleic acid transfection within a blood vessel.
[0031] FIG. 6 illustrates a kit constructed in accordance with the
principles of the present invention.
[0032] FIGS. 7-10 are charts and graphs comparing transfection
according to the present invention with controls. Porcine VSMCs
(FIGS. 7 and 9) and ECs (FIGS. 8 and 10) were transfected for 3 h
with naked or liposome (Promega Tfx-50) complexed luciferase DNA
(n=12) and luciferase activity in cell lysates was determined after
48 h at 37.degree. C. (FIGS. 7 and 8). Parallel adherent cell
counts were performed at baseline (time 0) and at 3, 18 and 48 h
after transfection (FIGS. 9 and 10). Where applicable ultrasonic
energy (1 MHz, CW, 0.4 W/cm.sup.2, 60 s) was applied for 30 minutes
into the 3 h transfection period. Asterisks indicate significant
differences between control and ultrasound-exposed cells
(p<0.05).
[0033] FIG. 11 compares cumulative mitosis in two wells of
subconfluent porcine VSMCs which were observed concurrently by TLVM
for 48 h and the cumulative rate of mitosis was analyzed (n=3). One
well was exposed to ultrasound (1 MHz, CW, 0.4 W/cm.sup.2, 60 s)
prior to filming. Asterisks indicate significant differences
between control and ultrasound-exposed cells (p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The nucleic acids delivered by the methods and devices of
the present invention will comprise nucleic acid molecules in a
form suitable for uptake into target cells within a host tissue,
usually smooth muscle cells lining the blood vessels. The nucleic
acids will usually be in the form of bare DNA or RNA molecules,
where the molecules may comprise one or more structural genes, one
or more regulatory genes, antisense strands, strands capable of
triplex formation, or the like. Commonly, such nucleic acid
constructs will include at least one structural gene under the
transcriptional and translational control of a suitable regulatory
region. Optionally, but not necessarily, the nucleic acids may be
incorporated in a viral, plasmid, or liposome vesicle delivery
vehicle to improve transfection efficiency.
[0035] If viral delivery vehicles are employed, they may comprise
viral vectors, such as retroviruses, adenoviruses, and
adeno-associated viruses, which have been inactivated to prevent
self-replication but which maintain the native viral ability to
bind a target host cell, deliver genetic material into the
cytoplasm of the target host cell, and promote expression of
structural or other genes which have been incorporated in the
particle. Suitable retrovirus vectors for mediated gene transfer
are described in Kahn et al. (1992) Circ. Res. 71:1508-1517, the
disclosure of which is incorporated herein by reference. A suitable
adenovirus gene delivery is described in Rosenfeld et al. (1991)
Science 252:431-434, the disclosure of which is incorporated herein
by reference. Both retroviral and adenovirus delivery systems are
described in Friedman (1989) Science 244:1275-1281, the disclosure
of which is also incorporated herein by reference. The nucleic
acids may preferably be present in a lipid delivery vehicle which
enhances delivery of the genes to target smooth muscle cells within
the vascular epithelia or elsewhere. Transfection in a lipid
delivery vehicle is often referred to as "lipofection." Such
delivery vesicles may be in the form of a liposome where an outer
lipid bilayer surrounds and encapsulates the nucleic acid
materials. Alternatively, the nucleic complexes may be in the form
of a nucleic acid-lipid dispersion, nucleic acid-lipid emulsion, or
other combination. In particular, the complexes may comprise
liposomal transfection vesicles, including both anionic and
cationic liposomal constructs. The use of anionic liposomes
requires that the nucleic acids be entrapped within the liposome.
Cationic liposomes do not require nucleic acid entrapment and
instead may be formed by simple mixing of the nucleic acids and
liposomes. The cationic liposomes avidly bind to the negatively
charged nucleic acid molecules, including both DNA and RNA, to
yield complexes which give reasonable transfection efficiency in
many cell types. See, Farhood et al. (1992) Biochem. Biophys. Acta.
1111:239-246, the disclosure of which is incorporated herein by
reference. A particularly preferred material for forming liposomal
vesicles is lipofection which is composed of an equimolar mixture
of dioleylphosphatidyl ethanolamine (DOPE) and
dioleyloxypropyl-triethylammo- nium (DOTMA), as described in
Felgner and Ringold (1989) Nature 337:387-388, the disclosure of
which is incorporated herein by reference.
[0036] It is also possible to combine these two types of delivery
systems. For example, Kahn et al. (1992), supra., teaches that a
retrovirus vector may be combined in a cationic DEAE-dextran
vesicle to further enhance transformation efficiency. It is also
possible to incorporate nuclear proteins into viral and/or
liposomal delivery vesicles to even further improve transfection
efficiencies. See, Kaneda et al. (1989) Science 243:375-378, the
disclosure of which is incorporated herein by reference.
[0037] The nucleic acids will usually be incorporated into a
suitable carrier to facilitate delivery and release, into the blood
vessels according to the present invention. The carriers will
usually be liquids or low viscosity gels, where the nucleic acids
will be dissolved, suspended, or otherwise combined in the carrier
so that the combination may be delivered through the catheter
and/or carried by the catheter and released intravascularly at the
treatment site. Alternatively, the nucleic acids may be provided in
a dry or solid form and coated onto or otherwise carried by the
catheter or the vibrational surface. An exemplary catheter 10
suitable for use in the methods of the present invention is
illustrated in FIGS. 1 and 2. The catheter 10 comprises a catheter
body 12 having a proximal end 14, a distal end 16, and a
vibrational interface surface 18 near the distal end. The
vibrational interface surface 18 comprises a piezoelectric ceramic
20 disposed between an insulating layer 22 and an aluminum shim 24.
An air gap 26 is behind the shim, and the transducer assembly is
suitable for a high frequency oscillation. The catheter further
includes a central lumen 28 to enable the catheter to be delivered
over a guidewire in a conventional manner. The catheter further
includes a pair of axially spaced-apart balloons 30 and 32 on
either side of the vibrational interface surface 18. The balloons
30 and 32 may be inflated via an inflation port 33 on a proximal
hub 23 secured to the proximal end 14 of the catheter body 12. The
proximal hub also includes an infusion port 34 which can deliver an
infusate, usually comprising the nucleic acids to be delivered,
through a port 35 between balloons 30 and 32 and proximate the
vibrational interface surface 18. The hub further includes wires 36
which permit the transducer 24 to be connected to a suitable
driver, e.g. a commercially available signal generator and power
amplifier capable of exciting the transducer within the target
frequency ranges and intensities.
[0038] While a single transducer 24 is illustrated in FIGS. 1 and
2, it will frequently be desirable to provide multiple transducers
20, as illustrated in FIG. 3, or a circularly symmetric transducer
40, as illustrated in FIG. 4. As illustrated in FIG. 3, a plurality
of transducers 20 could be circumferentially spaced-apart about the
exterior of the catheter body 12. In this way, energy can be
transmitted radially outwardly in multiple directions at once. In
order to enhance the uniformity of the treatment, the catheter
could optionally be rotated while the energy is being delivered. In
order to further enhance the uniformity of ultrasonic energy being
radiated outwardly, the multiple transducer embodiments can be
driven by a multiplexd power source. To still further enhance the
uniformity of ultrasonic energy being delivered, a piezoelectric
transducer 40 can be formed in a cylindrical geometry, as
illustrated in FIG. 4. The transducer ceramic 40 can be driven by
inner and outer electrodes 42 and 44, and the outer electrodes
coated by a thin insulating layer 46. A transducer ceramic can be
supported on a suitable cylinder, such as an aluminum cylinder 48,
and for high frequency operation an air gap 50 may be provided. The
transducer can be mounted symmetrically about catheter body 52
having a conventional guidewire lumen 54. In all of the above
cases, the dimensions will depend in large part on the frequency of
operation as well as the catheter size. The width of the
transducers will typically be in the range from 0.1 mm to 6 mm,
usually from 0.5 mm to 3 mm. The length of the transducer may vary
from 1 mm to 2 or more cm, with the length being primarily limited
by loss of flexibility of the distal end of the catheter. Multiple
transducer elements could also be provided along the length of the
catheter, i.e. being axially spaced-apart. Other transducer designs
may be employed, such as those disclosed in the copending
applications cited above.
[0039] Catheter 10 may be used to deliver nucleic acids to a target
region within a blood vessel BV, as illustrated in FIGS. 5A-5C. The
catheter 10 is intravascularly introduced to the target region in a
conventional manner. Once at the desired target region, as shown in
FIG. 5A, the balloons 30 and 32 will be inflated, as illustrated in
FIG. 5B, to define an isolated region R within the blood vessel
lumen. A suitable liquid medium containing the nucleic acids to be
delivered can then be introduced via port 34 and orifice 35 into
the isolated region R until a desired concentration of the nucleic
acid is achieved. Optionally, the nucleic acid may be replenished
and/or recirculated within the region if desired. After sufficient
nucleic acid has been introduced, the vibrating interface surface
18 is actuated in order to transmit ultrasonic energy through the
medium into the blood vessel wall to enhance uptake of the nucleic
acids, as illustrated in FIG. 5C. The vibrational energy is
delivered within the frequency ranges and at the intensities
described above, typically for periods of time from 10 sec. to 10
min., usually from 20 sec. to 3 min.
[0040] Referring now to FIG. 6, the catheters 10 of the present
invention will usually be packaged in kits. In addition to the
catheter 10, such kits will include at least instructions for use
50 (IFU). The catheter and instructions for use will usually be
packaged together within a single enclosure, such as a pouch, tray,
box, tube, or the like 52. At least some of the components may be
sterilized within the container. Instructions for use 50 will set
forth any of the methods described above.
[0041] The following examples are offered by way of illustration,
not by way of limitation.
[0042] Experimental
[0043] 1. Methods
[0044] A. Cell Culture
[0045] Explant-derived porcine medial vascular smooth muscle cells
(VSMCs) and enzyme-dispersed luminal endothelial cells (ECs) were
obtained from the thoracic aorta of Yorkshire White cross pigs aged
under 6 months and cultured on gelatin-coated tissue culture flasks
(Costar) in Dulbecco's Modified Eagle Medium (DMEM) containing 10%
porcine serum; EC cultures were supplemented with EC growth factor
(20 .mu.g/ml; Sigma) and heparin (90 .mu.g/ml; Sigma).
[0046] B. Transfection Conditions
[0047] All transfections were for 3 h at 37.degree. C. in 24-well
plates with cells at 60-70% confluence, and were stopped by
dilution with 1 ml of fresh culture medium. Naked DNA transfections
were carried out in 200 .mu.l DMEM containing 10% porcine serum and
7.5 .mu.g/ml luciferase plasmid DNA (pGL3, Promega) per well.
Lipofections were carried out using Promega Tfx-50 (which contains
DOPE), according to conditions optimized for VSMCs (200 .mu.l DMEM
containing 10% porcine serum; DNA:lipid charge ratio of 4:1; 7.5
.mu.g/ml final DNA concentration) and ECs (200 .mu.l serum-free
DMEM; DNA:lipid charge ration 3:1; 5 .mu.g/ml final DNA
concentration).
[0048] Where applicable, ultrasound exposure (USE) was performed 30
min. into the transfection period using a 10 mm diameter air-backed
piezoelectric flat plate ceramic transducer activated to produce
continuous wave (CW) 1 MHz ultrasound at 0.4 W/cm.sup.2 using a
multifunctional signal generator (DS 345, Stanford Research
Systems) working through a Krohn-Hite 7500 power amplifier,
monitored continuously using an oscilloscope (TDS 220, Tektronix).
The 24-well plates were suspended in a 2 cm-deep polystyrene water
bath at 37.degree. C. during USE, which was performed for 60 s with
the transducer within the transfection medium 2 mm above the cell
monolayer. This level of USE caused only minor acute damage to the
cell monolayer and had no effect on naked or liposome-complexed
plasmid DNA integrity as accessed by agarose gel electrophoresis
(data not shown). Temperature was recorded continuously using a
custom-built computerized probe placed adjacent to the ultrasound
transducer. A 10 mm diameter heating probe was constructed in-house
and used to mimic the rate of rise and final temperature achieved
during USE.
[0049] C. Assays for Luciferase Activity, Adherent Cell Number and
Validity
[0050] Luciferase activity in VSMC and EC lysates 48 h after
transfection was measured using the GenGlow kit and 1253
Luminometer (BioOrbit) and expressed as light units per microgram
total cell protein (assayed in parallel using the Bradford method
(BioRad)). Parallel wells were trypsinised at 0, 3, 18 and 48 h
after treatment. Cell counts and viability were assayed by
Coulter.TM. counter and FACS analysis of propidium-iodide and
fluorescein diacetate exclusion.
[0051] D. Time Lapse Video Microscopy (TLVM)
[0052] Identically seeded subconfluent VSMCs in 24-well plates were
observed by TLVM using a Leitz DM 1RB inverted microscope (Leica UK
Ltd) within a 37.degree. C. environment chamber. One frame of a
high-power field was recorded every 2.4 min. at for 48 h beginning
3 h after USE (where applicable) using a monochrome video camera
(Sony), Super-VHS video recorder (Panasonic) and a BAC900 animation
controller (EOS electronics AV Ltd, Barry, UK). A mitotic event was
recorded when 2 daughter cells appeared from a single dividing
cell. An apoptotic event was recorded when an individual cell
underwent the typical morphological changes of membrane blebbing,
cytoplasmic shrinkage, nuclear condensation and dislodgement.
[0053] E. Statistical Analysis
[0054] All data are presented as mean .+-.SEM. Treatments were
compared using the Friedman ANOVA test, and the Wilcoxon signed
rank test for post hoc comparisons. Values were considered to be
significantly different if p<0.05, applying the Bonferroni
correction for multiple comparisons were appropriate. The n numbers
quoted refer to the number of separate experiments; on each
occasion each treatment was performed in triplicate wells.
[0055] F. Results
[0056] Luciferase activity was barely detectable in VSMC lysates 48
h after transfection with naked plasmid alone (0.4.+-.0.2
LU/.mu.g), but was 7.5-fold higher in parallel wells exposed to 1
MHz ultrasound (3.0.+-.2.0 LU/.mu.g; n=12; p<0.02 cf naked DNA
alone), equivalent to 11% of that achieved following optimal
lipofection alone (27.6.+-.6.9 LU/.mu.g) (FIG. 7). USE during
lipofection further enhanced reporter gene expression, by almost
3-fold (72.8.+-.17 LU/.mu.g; n=12; p<0.002 cf lipofection alone)
(FIG. 7). The temperature of the culture medium increased
progressively during USE, reaching 14.+-.1.degree. C. above
baseline after 60 s. To exclude the possibility that
ultrasound-induced heating may be responsible for the observed
effects on reporter gene expression, VSMCs were exposed to an
identical rate and final temperature rise over 60 s in the absence
of 1 MHz ultrasound. No effect on luciferase activity in VSMC
lysates after 48 h was observed (FIG. 7).
[0057] Luciferase activity was almost undetectable in EC lysates 48
h after transfection with naked DNA (0.7.+-.0.1 LU/.mu.g) but, in
contrast to the results with VSMCs, was not enhanced by adjunctive
USE; (1.2.+-.0.2 LU/.mu.g; n=4; p=NS of naked DNA alone) (FIG. 8).
USE during lipofection, however, enhanced reporter gene expression
in ECs by more than 3-fold, from 17.7.+-.1.1 to 57.8.+-.20.2
LU/.mu.g (n=4; p<0.04).
[0058] USE had no significant effect on adherent cell number at 3 h
but was associated with much smaller subsequent increases compared
with either untreated control wells or those exposed to a
temperature rise alone (FIG. 9). This effect was not observed in
ultrasound-exposed EC, which increased in number in identical
fashion to control cells (FIG. 10). Adherent VSMC and EC viability
was identical in control, heat-exposed and 1 MHz-treated wells and
remained unchanged throughout (p=NS for each treatment and at all
timepoints, data not shown). We did not observe an excess of
detached VSMCs in the culture medium following USE, either by eye
or by performing cell counts on the culture medium itself (data not
shown). TVLM analysis of identically prepared, randomly cycling,
subconfluent VSMCs showed that ultrasound exposure significantly
reduced the rate of mitosis (FIG. 11). In contrast, ultrasound had
no effect on the rate of apoptosis in the same cultures (cumulative
percent apoptosis in control wells; 9.9.+-.4.2% at 24 h,
11.6.+-.5.0% at 48 h. In ultrasound-exposed wells; 4.8.+-.4.3% a 24
h, 7.6.+-.5.2% at 48 h; n=3; p=NS for all comparisons).
[0059] 3. Discussion
[0060] In the present study we demonstrate that adjunctive USE
enhances reporter gene expression following optimal naked DNA
and/or lipofection of primary vascular smooth muscle cells. A
number of recent reports have shown that ultrasound also enhances
reporter gene expression following transfection of non-vascular,
mainly immortalized, cells in vitro, including human prostate
cancer (Tata et al. (1997) Biochem. Biophys. Res. Comm. 234:64-67),
chondrocyte (Greenleaf et al. (1998) Ultrasound Med. Biol.
24:587-595), Chinese Hamster Ovary (Bao et al. (1997) Ultrasound
Med. Biol. 23:953-959), and HeLa cell lines (Unger et al. (1997)
Invest. Radiol. 32:723-727), primary rat fibroblasts and
chondrocytes (Kim et al. (1996) Hum. Gene. Ther. 7:1339-1346), and
mouse NIH/3T3 and mammary tumor cell lines (Unger et al. (1997)
Invest. Radiol. 32:723-727). Transfection rates of up to 15% of
surviving immortalized human chondrocytes using naked DNA have been
reported following exposure to CW 1 MHz ultrasound, and two to
1000-fold enhancements in lipofection efficiency have been reported
in a number of immortalized cell lines, in each case independent of
heat. The three to 7.5-fold enhancements recorded herein probably
underestimate the effects of ultrasound for several reasons. First,
USE was performed from above to minimize standing wave formation
resulting from reflection at fluid/air and plastic/air interfaces.
This constrained transducer design such that only one-third of each
cell monolayer was covered by the transducer. Secondly, the choice
of ultrasound parameters may not have been optimal. We used 1 MHz
ultrasound at less than 1 W/cm.sup.2 as this corresponds to the
mean output of diagnostic transducers and is clinically safe
(Henderson et al. (1995) Ultrasound Med. Biol. 21:699-705; Barnett
et al. (1996) Ultrasound Med. Biol. 24:i-xv, S1-58). Furthermore,
USE at this level had no effect on DNA integrity or vascular cell
viability in vitro. Additionally, the small number of cells
physically dislodged acutely during USE may have been those most
likely to have been transfected. These cells were lost to analysis
under these tissue culture conditions, although this situation may
not pertain to VSMCs and ECs within the intact vessel wall in
vivo.
[0061] Low intensity ultrasound requires pre-formed microbubbles or
nucleation sites to generate cavitation, and these conditions
certainly exist in the non-degassed culture media used in our
experiments. The effects of ultrasound in vitro may be further
enhanced in the presence of additional microbubbles in the form of
the echocontrast agent AlbunexTM, and naked DNA transfection rates
approaching those achieved with lipofection have been reported
(Tata et al. (1997) Biochem. Biophys. Res. Comm. 234:64-67). There
is relatively little evidence for the existence of microbubbles or
cavitation nuclei, however, in blood. Thus, methods according to
the present invention which combine low intensity USE and local
delivery of DNA mixed with (or even within) microbubbles would be
useful not only to focus but also restrict gene delivery to a
desired target site in a blood vessel.
[0062] While the above is a complete description of the preferred
embodiments of the invention, various alternatives, modifications,
and equivalents may be used. Therefore, the above description
should not be taken as limiting the scope of the invention which is
defined by the appended claims.
* * * * *