U.S. patent application number 10/813209 was filed with the patent office on 2004-09-30 for devices for needle-free injection and electroporation.
Invention is credited to Hofmann, Gunter A., Rabussay, Dietmar P., Zhang, Lei.
Application Number | 20040193097 10/813209 |
Document ID | / |
Family ID | 32995770 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040193097 |
Kind Code |
A1 |
Hofmann, Gunter A. ; et
al. |
September 30, 2004 |
Devices for needle-free injection and electroporation
Abstract
Methods are proved for introducing a biologically active agent
into cells of a subject by introducing the agent in a form suitable
for electrotransport into a region of tissue of the subject using
one or more needle-free injectors, and applying a pulsed electric
field to the region of tissue, thereby causing electroporation of
the region of tissue. The combination of needle-free injection and
electroporation is sufficient to introduce the agent into cells in
skin, muscle or mucosa. For example, the region of tissue can be
contacted with two oppositely charged injectors, one acting as the
donor electrode and one acting as the counter electrode, or a
single injector and one or more electrodes can be used. In
addition, needle-free injection may be used in combination with
suitable non-invasive electrode configurations. The active agents
delivered into cells using the invention method can be small
molecules, polynucleotides, polypeptides, and the like.
Inventors: |
Hofmann, Gunter A.; (San
Diego, CA) ; Rabussay, Dietmar P.; (Solana Beach,
CA) ; Zhang, Lei; (San Diego, CA) |
Correspondence
Address: |
Daniel M. Chambers
BioTechnology Law Group
658 Marsolan Avenue
Solana Beach
CA
92075-1931
US
|
Family ID: |
32995770 |
Appl. No.: |
10/813209 |
Filed: |
March 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10813209 |
Mar 29, 2004 |
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10339708 |
Jan 8, 2003 |
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10339708 |
Jan 8, 2003 |
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09567404 |
May 8, 2000 |
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6520950 |
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60133265 |
May 10, 1999 |
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Current U.S.
Class: |
604/20 ;
435/173.6; 977/906; 977/920 |
Current CPC
Class: |
A61N 1/0424 20130101;
A61N 1/0428 20130101; A61N 1/325 20130101 |
Class at
Publication: |
604/020 ;
435/173.6 |
International
Class: |
A61N 001/30 |
Claims
1-47. (canceled)
48. An electroporation device, comprising: a. a needle-free
injector configured to serve as a first electroporation electrode
when positioned in contact with a tissue of a patient, wherein the
needle-free injector injects at least one liquid jet to introduce
an agent into or beneath the tissue; b. a second electroporation
electrode disposed in spaced relation to the first electroporation
electrode; and c. electrical connections to electrically connect
the needle-free injector and the second electroporation electrode
with an electrical source for generating electrical current used to
effect electroporation.
49. An electroporation device according to claim 48 wherein the
second electroporation electrode comprises a ring electrode.
50. An electroporation device according to claim 48, wherein the
second electroporation electrode comprises an array of
electrodes.
51. An electroporation device according to claim 50, wherein said
array of electrodes comprises a micropatch electrode.
52. An electroporation device according to claim 51, wherein said
micropatch electrode comprises a meander electrode.
53. An electroporation device according to claim 48, wherein said
electrodes further comprise timing sensors.
54. An electroporation device according to claim 48 wherein the
second electroporation electrode is also a needle-free
injector.
55. An electroporation device according to claim 48 comprising a
plurality of needle-free injectors, each of which is configured to
serve as an electroporation electrode, and wherein the device
comprises electrical connections to electrically connect each
electroporation electrode with the electrical source.
56. An electroporation device according to claim 48 wherein the
needle-free injector serves as the first electroporation electrode
by injecting a conductive fluid comprising the agent and specific
resistivity sufficient to allow application of an electrical field
to effect electroporation of the tissue.
57. An electroporation device according to claim 56, wherein the
liquid jet acts as an electrode.
58. An electroporation device according to claim 56, wherein the
conductive fluid is contained in a partially ionized solvent.
59. An electroporation device according to claim 56, wherein the
application of an electric field takes place without the device
touching the tissue.
60. An electroporation device according to claim 56, wherein the
agent is in a liquid and the injector forces the liquid into the
tissue as a conductive or essentially non-conductive liquid
jet.
61. An electroporation device according to claim 48 wherein the
electrical source is a pulse generator.
62. An electroporation system comprising the electroporation device
of claim 48 in electrical communication with an electrical source
used to effect electroporation.
63. An electroporation system according to claim 62, wherein the
current generated by the electrical source is a wave pulse selected
from the group consisting of a square, rectangular, triangular, and
an exponential decay wave pulse.
64. An electroporation system according to claim 63, wherein the
pulse is monopolar or bipolar.
65. An electroporation system according to claim 63, wherein the
pulse is monopolar or bipolar.
66. An electroporation system according to claim 62, wherein the
electrical source is a pulse generator.
67. An electroporation device, comprising: a. an array electrode
comprising (i) at least one positive electrode and at least one
negative electrode, wherein the electrodes are configured to
generate an electrical field to effect electroporation of a tissue
of a patient when energized, and (ii) an opening through which a
needle-free injector can be inserted, wherein the needle-free
injector injects a liquid jet comprising an agent into or beneath
the tissue; and b. electrical connections to electrically connect
the array electrode with an electrical source for generating
electrical current used to generate the electrical field to effect
electroporation.
Description
RELATED APPLICATION
[0001] This application is a Continuation-In-Part application of
U.S. Ser. No. 09/567,404, filed May 8, 2000, which relies for
priority under 35 U.S.C. .sctn. 119(e), upon U.S. Provisional
Application Serial No. 60/133,265, filed May 10, 1999, each of
which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] This invention generally relates to methods for delivery of
an active agent to a subject and more specifically to the use of
electroporation and needle-free delivery of an active agent to a
subject.
BACKGROUND
[0003] A cell has a natural resistance to the passage of molecules
through its membranes into the cell cytoplasm. Scientists in the
1970's first discovered "electroporation," the use of electrical
fields to create pores in cells without causing permanent damage to
the cells. This discovery made possible the insertion of large
molecules directly into cell cytoplasm. Electroporation was further
developed to aid in the insertion of various molecules into cell
cytoplasm by temporarily creating pores in the cells through which
the molecules pass into the cell.
[0004] Electroporation has been used in both in vitro and in vivo
procedures to introduce foreign material into living cells. With in
vitro applications, a sample of live cells is first mixed with the
agent to be introduced therein and placed between electrodes, such
as parallel plates. Then, the electrodes are used to apply an
electrical field to the mixture containing the cells and the agent
to be introduced therein.
[0005] With in vivo applications of electroporation, electrodes are
provided in various configurations such as, for example, a caliper
that grips the epidermis overlying a region of cells to be treated.
Alternatively, needle-shaped electrodes may be inserted into the
patient, to access more deeply located cells. In either case,
before, simultaneously, or after the agent is injected into the
treatment region, the electrodes are used to apply an electrical
field to the region. See, for example, U.S. Pat. No. 5,019,034,
issued May 28, 1991 and U.S. Pat. No. 5,702,359, issued Dec. 30,
1997.
[0006] Electroporation (both in vitro and in vivo) functions by
causing cell membranes to which a brief high voltage pulse is
administered to temporarily become porous, whereupon molecules can
enter the cells. In some electroporation applications, the electric
field comprises a single square wave pulse on the order of 1000
V/cm, of about 100 microseconds duration. Such a pulse may be
generated, for example, in known applications of the
ElectroSquarePorator T820, made by the BTX Division of Genetronics,
Inc.
[0007] Electroporation has been recently suggested as an alternate
approach to the treatment of certain diseases such as cancer by
introducing a chemotherapeutic drug directly into the cell. For
example, in the treatment of certain types of cancer with
chemotherapy it is necessary to use a high enough dose of a drug to
kill the cancer cells without killing an unacceptably high number
of normal cells. If the chemotherapy drug could be inserted
directly inside the cancer cells, this objective could be achieved.
However, some of the best anti-cancer drugs, for example,
bleomycin, cannot penetrate the membranes of certain cancer cells
effectively under normal circumstances. To overcome this
difficulty, electroporation has been used to cause bleomycin to
penetrate the membranes of cancer cells.
[0008] Electroporation-assisted chemotherapy typically is carried
out by injecting an anticancer drug directly into the tumor and
applying an electric field to the tissue between a pair of
electrodes. The field strength must be adjusted reasonably
accurately so that electroporation of tumor cells occurs without
damage, or at least with minimal damage, to any normal or healthy
cells. Typically, this method is employed with tumors located on
the exterior of the patient's body by applying electrodes to the
body surface on opposite sides of the tumor, thus creating an
electric field between the electrodes. When the field is uniform,
the distance between the electrodes can then be measured and a
suitable voltage, derived according to the formula E=V/d (wherein
E=electric field strength in V/cm; V=voltage in Volts; and
d=distance in cm), can then be applied to the electrodes. However,
when the tumors to be treated are large, irregular in shape, or
located within the body interior, it is more difficult to properly
locate electrodes and measure the distance between them so as to
accurately calculate the voltage that is to be applied. In such
cases, needle array electrodes as, for example, described in U.S.
Pat. No. 5,993,434 (Dev and Hofmann) have proven to be
advantageous.
[0009] Using these and related techniques (for example, the
molecule can be delivered encapsulated in a liposome),
electroporation has been used to deliver molecules into many
different types of cells. For example, electroporation has been
used to deliver biologically active agents to various human and
mammalian cells, such as egg cells (i.e., oocytes), sperm,
platelets, muscle, liver, skin, and red blood cells. In addition,
electroporation has been used to deliver molecules to plant
protoplasts, plant pollen, bacteria, fungi, and yeast. A variety of
different biologically active molecules and agents have been
delivered to cells using this technique, including DNA, RNA and
various chemical agents.
[0010] Vaccination is the most cost-effective way to prevent
disease. However, there are still many diseases for which no
vaccine exists or for which the currently available vaccines are
inadequate. DNA immunizations, which entail the administration of
DNA encoding an antigen, may offer solutions in at least some of
these cases. Moreover, DNA vaccines offer the use of host cells as
bioreactors for the production of proteins in vivo (Tang, D. C., et
al., (1992) Nature 356:152-4). By doing so, DNA vaccines mimic a
viral infection, improve antigen presentation to the immune system
relative to standard protein vaccines, and work more effectively as
a result (Ulmer, J. B., et al., (1993) Science 259:1745-9).
Moreover, DNA vaccines offer these potential benefits without many
of the safety and stability concerns associated with the
administration of infectious agents.
[0011] DNA immunization has been effective in several small animal
models (Donnelly, J. J., et al., (1997) Annu. Rev. Immunol.
15:617-48). However, demonstrating its effectiveness has been much
more challenging in larger animals and humans. Numerous studies
have shown that the greatest power of DNA vaccines may be their
ability to prime the immune system for responses to other vaccines
(Richmond, J. F., et al., (1998) J. Virol. 72:9092-100; Robinson,
H. L., et al., (1999) Nat. Med. 5:526-34).
[0012] The first hypodermic syringe was developed by a French
surgeon, Charles-Gabriel Pravaz, in 1853 to take advantage of the
highly permeable interstitial tissue below the skin surface to
transport pharmaceuticals to active sites. Although there have been
developments in hypodermic syringes since then, the technology has
remained essentially unchanged for the past 150 years. Needle-free
injection was developed when workers on hydraulic equipment noticed
that high-pressure squirts of hydraulic oil would pierce the skin.
The first description of needle-free injection was in Marshall
Lockhart's 1936 patent for "jet injection." Then, in the early
1940's Higson and others developed high-pressure "guns" using a
very fine jet of liquid medicament to pierce the skin and deposit
it into the tissue underneath. In World War II, needle-free guns
were used extensively to inoculate troops en masse against
infectious disease. Later, needle-free guns were applied more
generally in large-scale vaccination programs.
[0013] However, these early needle-free injectors were used on
multiple patients and fears about the transmission of hepatitis B
and HIV infection by reuse of the injectors led to a sharp decline
in their use. Until recently, the main application of such devices
was veterinary, with a few being used by diabetics for
self-treatment.
[0014] In the past 50 years, over 300 patents have been filed in
the needle-free delivery area. Although various improved products
have come to the market, none has gained wide use and remnants of
the older devices remain to this day. These devices tend to be
expensive to purchase and difficult to use, requiring the user to
perform a series of complicated steps to set up the device for use.
For example, some of these systems require the user to fit a needle
to the delivery device temporarily in order to draw liquid
containing the desired active agent into the device from a vial.
Therefore, even the more modem needle-free delivery systems do not
address the needs of the market for an easy to use, low cost, and
simple system. Consequently, needle-free delivery has not come into
widespread use.
[0015] Despite this apparent failure of needle-free delivery, the
pharmacokinetics and pharmacodynamics of needle-free delivery are
well documented. Accelerating a jet of liquid to high speed
provides power for the liquid to penetrate the stratum corneum as
well as individual cell membranes. Thus, there is a need in the art
for new and better methods for transporting molecules, such as
biologically active agents, across the stratum corneum and/or cell
membranes in treatment of a variety of conditions and diseases.
SUMMARY OF THE INVENTION
[0016] The present invention overcomes such problems in the art by
providing methods for introducing biologically active agents into
cells without use of a hypodermic needle. In one embodiment
according to the present invention, a biologically active agent is
introduced in a form suitable for direct or indirect
electrotransport into a region of tissue of the subject using one
or more needle-free injectors, and an electric field is applied to
the region of tissue, thereby causing electroporation of the region
of tissue prior to, simultaneously with, and/or subsequently to
introducing the agent. Direct electrotransport refers to the
transport of molecules subjected to an electrical or magnetic
force, indirect electrotransport refers to the transport of
molecules facilitated by electric forces which act primarily on
transport barriers, e.g., cell membranes, which become more
permeable as a result of electric forces. The combination of
needle-free injection and electroporation is sufficient to
introduce the active agent into the cell and allows for delivery of
pharmaceutical compounds, nucleic acid constructs, or other agents
into cells contained within the tissue region so treated.
[0017] In another embodiment according to the present invention, a
biologically active agent is introduced into cells in a region of
tissue of a subject by contacting the region of tissue or adjacent
tissue with two or more spaced apart needle-free injectors while
injecting a biologically active agent into the tissue, and applying
an electrical field to the tissue via the two or more injectors
prior to, simultaneously with, and/or subsequently to injection of
the agent so as to electroporate the region of tissue, whereby the
combination of needle-free injection and electroporation is
sufficient to introduce the agent into the cell.
[0018] In yet another embodiment according to the present
invention, a biologically active agent is introduced into cells in
a region of tissue of a subject by contacting the region of tissue
with at least one needle-free injector while injecting an agent
suitable for direct or indirect electrotransport into the region of
tissue, and applying an electrical field across the region of
tissue using the at least one injector prior to, simultaneously
with, and/or subsequently to injection of the agent, whereby the
combination of needle-free injection and electroporation is
sufficient to introduce the agent into the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A and B show diagrams illustrating the invention
method wherein electrically conducting needle-free injectors are
used as the electrodes for delivering an electrical impulse to a
region of tissue. In FIG. 1A, two needle-free injectors are
disposed in spaced apart relation to one another and in contact
with the surface of a region of tissue of the subject. The
oppositely charged injectors act as electrodes for conducting
electroporation, being connected with an electrical source, such as
a pulse generator, such that an electrical current is delivered
through the region of tissue by completing the circuit between the
two electrically conducting injector tips. One injector is the
active or donor electrode and the second, oppositely charged,
injector is the counter or return electrode. In FIG. 1B, one
needle-free injector contacts the surface of a region of tissue
while providing an electrical current in conjunction with two
oppositely charged electrodes. The injector acts as the active or
donor electrode and the two ring electrodes act as counter or
return electrodes.
[0020] FIG. 2A is a schematic drawing showing a needle-free
injector that is not in contact with the skin injecting a liquid
into tissue through an opening in an array electrode containing
multiple positive and negative electrodes.
[0021] FIG. 2B is a schematic drawing showing a needle-free
injector with array electrode attached to the nozzle area and an
opening in the array electrode allowing the liquid jet to go
through the electrode into the skin.
[0022] FIG. 2C is a schematic drawing showing a needle-free
injector with an array electrode attached to the nozzle area. The
array electrode has multiple openings to allow multiple liquid jets
to pass through the array electrode into the skin.
[0023] FIG. 3 is a graph showing electroporation used in
conjunction with intradermal injection of DNA vaccine improves gene
expression levels. Intradermal expression levels are shown for DNA
vaccine injected using needle-free BioJect (b.j.) and needle
(i.d.n.) injections, without or with electroporation, at the
voltages indicated. Error bars represent standard error of the mean
(SEM).
[0024] FIG. 4 is a graph showing immune responses to Hepatitis B
after various combinations of DNA vaccine followed by protein
booster immunizations. HBsAg antibody titers at 10 weeks were
determined using the AUSAB EIA and titers shown are the geometric
mean of 5 animals for all the groups except for the i.m. subunit
group, which had 4 animals. Error bars are SEM.
[0025] FIGS. 5A-5D are graphs showing antibody isotype responses
elicited by Hepatitis B immunizations according to the invention
methods. Data represent Hepatitis B specific IgG1 and IgG2 titers
at 8 and 10 weeks for individual animals. The bar represents the
geometric mean. Group 1=needle-free injection of DNA, Group
2=needle-free injection of DNA+electroporation (EP), Group
3=intradermal injection of DNA, Group 4=intradermal injection of
DNA+EP, Group 5=needle-free administration of the subunit vaccine
control, and Group 6=intramuscular injection of the subunit vaccine
control.
DETAILED DESCRIPTION OF THE INVENTION
[0026] According to the present invention, there are provided
methods for introducing a biologically active agent into cells in a
region of tissue of a subject by injecting the agent in a form
suitable for direct or indirect electrotransport into a region of
tissue of the subject using one or more needle-free injectors, and
applying an electric field to the region of tissue, thereby causing
electroporation of the region of tissue prior to, simultaneously
with, and/or subsequently to injection of the agent. The
combination of needle-free injection and electroporation is
sufficient to introduce the agent into the cell.
[0027] A "needle-free injector," as the term is used herein, refers
to a device that injects an agent into tissue without the use of a
needle, for example as a small stream or jet, with such force
(usually provided by expansion of a compressed gas, such as carbon
dioxide through a micro-orifice within a fraction of a second) that
the agent pierces the surface of the tissue and enters underlying
tissue and/or muscle. In one embodiment, the injector creates a
very high-speed jet of liquid that painlessly pierces the tissue.
Such needle-free injectors are commercially available and can be
used by those having ordinary skill in the art to introduce agents
(i.e. by injection) into tissues of a subject. Examples of
needle-free injectors that can be utilized in practice of the
invention methods include those described in U.S. Pat. Nos.
3,805,783; 4,447,223; 5,505,697; and 4,342,310.
[0028] As used herein, the term "introduce," "inject" or
"injecting," and grammatical equivalents thereof, as applied to the
action of a needle-free injector means that the agent is forced
through at least the surface of the tissue (e.g., the mucosa or
epidermis, stratum corneum, or dermis of skin) and, preferably,
delivered into underlying tissue and/or musculature using a
needle-free injector as described herein.
[0029] A desired agent in a form suitable for direct or indirect
electrotransport is introduced (e.g., injected) using a needle-free
injector into the tissue to be treated, usually by contacting the
tissue surface with the injector so as to actuate delivery of a jet
of the agent, with sufficient force to cause penetration of the
agent into the tissue. For example, if the tissue to be treated is
mucosa, skin or muscle, the agent is projected towards the mucosal
or skin surface with sufficient force to cause the agent to
penetrate through the stratum corneum and into dermal layers, or
into underlying tissue and muscle, respectively.
[0030] Needle-free injectors are well suited to deliver active
agents to all types of tissues, particularly to skin and mucosa. In
some embodiments, a needle-free injector may be used to propel a
liquid that contains DNA molecules or a drug toward the surface and
into the subject's skin or mucosa. Representative examples of the
various types of tissues that can be treated using the invention
methods include pancreas, larynx, nasopharynx, hypopharynx,
oropharynx, lip, throat, lung, heart, kidney, muscle, breast,
colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood
vessels, or any combination thereof.
[0031] In addition to their function in introducing the active
agent, two or more needle-free injectors can also be used to apply
an electric field to the tissue for electroporation of cell
membranes therein. As shown in FIG. 1A, two needle-free injectors 2
and 4, each project a jet of liquid 6 and 8 containing the
biologically active agent. The injectors are disposed in spaced
relation to one another and in close contact with the surface 10 of
a region of tissue 12 of the subject. The portion of the injectors
in contact with the tissue surface are electrically conductive and
are in electrical connection with an electrical source (not shown),
such as a pulse generator, such that electroporation is
accomplished by delivering an electrical current through the region
of tissue by completing the circuit between the two electrically
conducting injector tips. As shown in FIG. 1A, injector 2 is the
active or donor electrode and injector 4 is the counter or return
electrode. In other embodiments, both injectors can act as donor
electrodes. Usually, although not always, the injectors are also in
contact with the tissue surface while the active agent is
introduced.
[0032] Another embodiment of the invention method wherein the
injector is utilized to apply an electrical field to the surface of
a subject is shown in FIG. 1B. In this embodiment of the invention
method, at least one injector contacts the surface of the tissue
and provides an electrical current in conjunction with one or more
electrodes, such as, for example, a ring electrode(s). As shown in
FIG. 1B, injector 2 contacts surface 10 of a region of tissue 12 so
as to act as the active or donor electrode while charged ring
electrode 14 acts as the counter or return electrode.
[0033] In yet another embodiment shown in FIG. 2, the needle-free
injector introduces a conductive fluid as a jet through an opening
16 in an array electrode 18, which contains multiple positive and
negative electrodes. e.g., a micropatch electrode as described in
U.S. patent application Ser. No. 09/134,245, filed on Aug. 14,
1998, which is hereby incorporated herein in its entirety by
reference). By example, the electrode can be a meander electrode
that consists of an array of interweaving electrode fingers with
alternating polarity. The width of individual electrodes about 2 mm
and the gap between electrodes is about 0.2 mm. Alternatively, the
electrode can be made of a porous material such that e.g.,
polyacrylamide hydrogels the liquid jet from the injector passes
through the pores of the electrode to the target layers of the
tissue.
[0034] Various shapes and compositions of the needle-free injector
tip that delivers the electric pulse (or electrode, if used) can be
used so long as it is capable of delivering a sufficient electric
pulse as set forth herein. Optionally, at least the portion of the
needle-free injector that is pressed against the tissue in practice
of the invention methods is insulated to protect against excess
heat or burning, current leakage, shock, etc. Appropriate electric
pulsing parameters are set forth herein or can be determined using
the teachings herein and, in view of these parameters, the skilled
artisan can select among various suitable materials (e.g., ceramic,
metal, etc.) and configurations (ring, solid disc, etc.) for
manufacture of the portion of the needle-free injector (and
electrodes, if used) that contact the tissue to be
electroporated.
[0035] In addition, to the injectors, optional electrodes, and
electrical source, the apparatus used in practice of the invention
method typically further includes a means for controlling the
amount of current passing from the device and through the contacted
surface, as well as additional control elements typical of
electroporation systems as are known in the art.
[0036] The liquid jets themselves from the injector(s) can be made
highly electrically conductive by using a conductive suspension or
solution of the agent, e.g., in an ionic solution, such as a saline
solution. As used herein, the term "conductive" means that the
fluid has a specific resistivity sufficient to allow the
application of an effective electrical field without unacceptable
heating of the liquid jet occurring during that electrical pulse.
The jets of conductive fluid then can act, not only as liquid
needles, but also as electrodes. Thus, when conductive jets of
liquid are introduced into tissue, the injector device does not
need to touch the tissue into which the active agent is introduced.
Rather, the injector can be placed in proximity to the surface of
the tissue and the conductive jet from a needle-free injector
device in combination with such another jet, or in combination with
one or more surface electrodes is sufficient to complete the
electrical circuit through the tissue. As one of skill in the art
will appreciate, therefore, in the invention methods the active
agent can be introduced either as a jet of conductive fluid from a
needle-free injector (without touching the surface of the tissue
with an electrically conductive injector device), or a low
conductivity jet of fluid can be introduced while contacting the
tissue surface with an electrically conductive injector device in
any of the combinations of injector(s) and electrode(s) described
herein.
[0037] Typically in this situation, the electroporation pulse would
be administered "substantially contemporaneously" with the
injection of the agent into the tissue. As used herein, the term
"substantially contemporaneously" can mean that the electroporation
pulse is delivered during the time that the jet remains intact
(i.e., has not broken up). For example, the electroporation pulse
can be timed, (e.g., mechanically or electronically) to coincide
with the jet driving mechanism in the injector. If electrodes are
placed on the surface of the tissue for the purpose of promoting
current flow (see FIG. 1B for example), timing sensors can be
incorporated into the electrodes to coordinate the electroporation
pulse and the jet driving mechanism. Alternatively, the term
"substantially contemporaneously" can mean that the needle-free
injector is activated to inject the agent and the electric pulse is
applied to the region of skin or mucosa to be treated reasonably
close together in time. Alternatively, an electrical current can be
provided before or following introduction of a therapeutic agent to
the tissue of the subject. When multiple electrical impulses are
applied, the agent can be administered in a form suitable for
direct or indirect electrotransport before or after each of the
pulses, or at any time between the electrical pulses.
[0038] The active agent can include ionic species, molecules having
charged functionalities, or molecules of neutral charge. The agent
may be completely charged (i.e., 100% ionized), completely
uncharged, or partly charged and partly uncharged. Alternatively,
two or more agents of differing charge (or % ionization) can be
combined to arrive at a desired level of charge for the
combination, or an uncharged active agent can be contained in a
medium suitable for direct or indirect electrotransport, such as a
charged liquid (e.g., a solvent). Various degrees of ionization of
the medium containing the active agent can be employed to produce
the agent in a form suitable for electrotransport. For example, the
liquid medium containing the active agent can be ionized from about
5% to about 95% by volume, or the liquid medium can be ionized from
about 10% to about 75%, or from about 30% to about 50% by
volume.
[0039] Electroporation as utilized in the invention method is a
method of increasing the permeability of tissue and cell membranes
which allows transport, or migration, of an agent through tissue or
across cell membranes into cells. For example, electroporation can
include applying a voltage across tissue to increase the
permeability of the tissue and at least a portion of the cell
membranes of cells in the tissue. If the tissue is in the presence
of an agent in a form suitable for electrotransport, as described
herein, the agent migrates across the tissue and into cells of the
tissue.
[0040] The electric field applied in practice of the invention
method is determined by the nature of the tissue, the size of the
selected tissue, and its location. It is desirable that the field
be as homogeneous as possible and of the correct amplitude.
Excessive field strength results in lysing of cells, whereas a low
field strength results in reduced efficacy. When the region of
tissue being treated is skin or mucosa, during electroporation a
voltage sufficient to cause that region of the epidermis to become
electroporated is applied to the portion of the epidermis or tissue
into which the active agent is introduced.
[0041] The electric pulse can be provided by any electronic device
or electric pulse generator that provides an appropriate electric
pulse sufficient for introducing an active agent (e.g., a
therapeutic agent) in a form suitable for direct or indirect
electrotransport into target cells. The waveform of the electrical
signal provided by the pulse generator during electroporation can
be an exponentially decaying pulse, a square pulse, a unipolar
oscillating pulse train, or a bipolar oscillating pulse train, or
any combination of these forms. The nominal electric field strength
can be from about 10 V/cm to about 20 kV/cm. The nominal electric
field strength is determined by computing the voltage between any
two injectors (injector and one or more electrodes) divided by the
distance between the injectors (or injector and one or more
electrodes). The pulse length is generally in the range from about
ten .mu.s to 100 ms. There can be any desired number of pulses,
typically one to about 100 pulses per second. The interval between
pulse sets can be any suitable time, such as one second. The
waveform, electric field strength and pulse duration may also
depend upon the type of cells or tissue and the type of agents that
are to enter the cells during electroporation.
[0042] Each pulse wave form has particular advantages; square wave
form pulses provide increased efficiencies in transporting
compounds into the cells in comparison to exponential decay wave
form pulses, and the ease of optimization over a broad range of
voltages, as described, for example, in Saunders, Guide to
Electroporation and Electrofusion, 1991, pp 227-47. Preferably the
waveform used is an exponential or a square wave pulse. Other wave
forms such as rectangular or triangular will be known in the art
and are included herein.
[0043] The electric fields needed for in vivo cell electroporation
of various cell types are generally similar in magnitude to the
fields required for cells in vitro and are well known in the art.
Presently preferred magnitudes are in the range of from 10 V/cm to
about 1300 V/cm. The higher end of this range, over about 600 V/cm,
has been verified by in vivo experiments of others reported in
scientific publications.
[0044] The nominal electric fields can be designated either "high"
or "low." It is presently preferred that, when high voltage fields
are used, the nominal electric field is from about 700 V/cm to 1300
V/cm and more preferably from about 1000 V/cm to 1300 V/cm. It is
presently preferred that, when low fields are used, the nominal
electric field is from about 10 V/cm to 200 V/cm, and more
preferably from about 25 V/cm to 75 V/cm.
[0045] In a particular embodiment of the present invention, it is
presently preferred that when the electric field is low, the pulse
length is long, i.e., the "low voltage long pulse" mode of
electroporation. For example, when the nominal electric field is
about 25 V/cm to 75 V/cm, it is preferred that the pulse length is
about 1 to 80 msec. For this type of low voltage long pulse
electroporation, a square wave pulse is preferably used. Square
wave electroporation systems deliver controlled electric pulses
that rise quickly to a set voltage, stay at that level for a set
length of time (pulse length), and then quickly drop to zero.
Square wave electroporation pulses have a gentler effect on the
cells than an exponential decay pulse, and therefore, yield higher
cell viability and better transformation efficiency for the
electroporation of plant and mammalian tissues. Exemplary pulse
generators capable of generating a square pulsed electric field
include, for example, the ElectroSquarePorator (T820) pulse
generator (BTX division of Genetronics, Inc., San Diego, Calif.),
which can generate a square wave form of up to 3000 volts and a
pulse length from about 5 .mu.sec to about 99 msec. The T820
ElectroSquarePorator is active in both the High Voltage Mode (HVM)
(100-3000 Volts) and the Low Voltage Mode (LVM) (10-500 Volts). The
pulse length for LVM is about 0.3 msec to 99 msec and for HVM,
about 5 .mu.sec to 99 .mu.sec, with multiple pulsing capability
from about 1 pulse to 99 pulses. Additional electroporation
apparatus are commercially available and can be used in practice of
the invention methods, for example, the ECM600 (BTX division,
Genetronics, Inc.), which can generate an exponential wave
form.
[0046] Although electroporation of the region of tissue treated can
be prior to, simultaneously with, and/or subsequently to injection
of the agent, the chemical composition of the agent will dictate
the most appropriate time to administer the agent in relation to
the administration of the electric pulse for electroporation. For
example, while not wanting to be bound by a particular theory, it
is believed that a drug having a low isoelectric point (e.g.,
neocarcinostatin, IEP=3.78), would likely be more effective if
administered post-electroporation in order to avoid electrostatic
interaction of the highly charged drug within the field. Another
group of drugs (such as bleomycin) has a very negative log P, (P
being the partition coefficient between octanol and water), are
very large in size (MW about 1400), and/or are hydrophilic, thereby
associating closely with the lipid membrane. Such drugs diffuse
very slowly into a tumor cell. Therefore, in practice of the
invention method, drugs having such characteristics are typically
administered prior to or substantially simultaneously with the
electric pulse.
[0047] In addition, certain biologically active agents may require
chemical modification in order to facilitate more efficient entry
into the cells and/or electrotransport. For example, an agent with
poor water solubility, such as taxol, can be chemically modified
using methods known in the art, to increase solubility in
water.
[0048] The agent (and medium) may undergo electrotransport through
pores created in cell membranes (e.g., during electroporation) by
electromigration, electroosmosis, or a combination of the two.
(Electroosmosis has also been referred to as electrohydrokinesis,
electro-convection, and electrically-induced osmosis.) In general,
electroosmosis of a therapeutic species into a tissue results from
the migration of a liquid in a non-conducting capillary system in
which the species is contained, as a result of the application of
electromotive force to the therapeutic species reservoir., i.e.,
solvent flow induced by electromigration of other ionic species (C.
Morris and P. Morris, Separation Methods in Biochemistry, New York
Interscience Publishers, Great Britain, 1964, pp 632, 639).
[0049] In conjunction with any of the above-described procedures, a
brief period of iontophoresis may optionally be applied to
distribute the agent between the electrodes (e.g., the injectors)
before, during, or after pulsing for electroporation. Iontophoresis
is a process that can be used to transport molecules across tissue
without necessarily causing electroporation, especially once
enhanced electroporation has occurred. For iontophoresis, an
electrical potential of much lower voltage and greater duration
than is used for electroporation is applied to the region of tissue
treated. For example, electroporation of the stratum corneum is
caused by large pulses (between about 50 volts and about 500 volts
at the electrodes), while iontophoresis is often caused by
application of essentially steady (direct current), relatively
small voltages (between about 0.1 Volt and about 5 Volt) or
currents, which transport molecules through pre-existing pathways
(see, for example, B. H. Sage, "Iontophoresis" in Percutaneous
Penetration Enhancers E. W. Smith and H. I. Maibach, Eds., CRC
Press, pp. 351-368, 1995). Therefore, in one embodiment,
iontophoresis through skin tissue is practiced in conjunction with
the invention methods by maintaining a constant current of about 1
mA for 30 seconds. Those of skill in the art will know how to
select appropriate parameters to be used for iontophoresis of other
types of tissues.
[0050] During iontophoresis, ions present in a sustained low
voltage field will migrate toward sources of opposite charge. Thus,
an active agent having at least some percent ionization will
migrate towards an oppositely charged electrode through an
electroporated membrane into subcutaneous, interstitial fluids.
Neutral molecules can also be moved via iontophoresis by repeated
contact of charged particles moving in one direction, such that net
transport of the neutral molecular species occurs because of the
transport of the electrically charged species. Iontophoresis is
most efficient when the low voltage field for the iontophoresis is
temporarily interrupted when the pores have retracted to a size at
which the transport rate drops below a selected level (or is
maintained) while a new electrical pulse having the characteristics
to induce electroporation is applied.
[0051] During iontophoresis, the skin resistance changes much more
slowly, and in lesser magnitude than during electroporation, and
this skin resistance behavior is believed to be due to changes of
ionic composition of solutions within pre-existing aqueous pathways
(see, for example, S. M. Dinh, C-W. Luo and B. Berner "Upper and
Lower Limits of Human Skin Electrical Resistance in Iontophoresis"
AIChe J. 39:2011-2018, 1993). Thus, the larger skin resistance
during iontophoresis means that the electric field is more confined
to the surface of the tissue than during electroporation.
[0052] The term "iontophoresis" as used herein refers to (1) the
delivery or transport of charged drugs or agents by
electromigration, (2) the transport and/or delivery of uncharged
drugs or agents by the process of electroosmosis, (3) the transport
and/or delivery of charged drugs or agents by the combined
processes of electromigration and electroosmosis, and/or (4) the
transport and/or delivery of a mixture of charged and uncharged
drugs or agents by the combined processes of electromigration and
electroosmosis.
[0053] During the electrotransport process certain modifications or
alterations of the skin or mucosal tissue may occur, such as
increased ionic content, hydration, dielectric breakdown,
extraction of endogenous substances, and electroporation. Any
electrically assisted transport of species enhanced by
modifications or alterations to a body surface (e.g., formation of
pores in the skin) are also included in the term electrotransport
as used herein.
[0054] The biologically active agents and active agents introduced
according to the invention methods include drugs (e.g.,
chemotherapeutic agents), nucleic acids (e.g., polynucleotides),
peptides and polypeptides, including antibodies and other molecules
for delivery to a subject. For example, the polypeptide can be an
antigen introduced for the purpose of raising an immune response in
the subject into whose cells it is introduced. Alternatively, the
polypeptide can be a hormone, such as calcitonin, parathyroid
hormone, erythropoietin, insulin, a cytokine, a lymphokine, a
growth hormone, a growth factor, and the like, or a combination of
any two or more thereof. Additional illustrative polypeptides that
can be introduced into cells using the invention method include
blood coagulation factors and lymphokines, such as tumor necrosis
factor, interleukins 1, 2 and 3, lymphotoxin, macrophage activating
factor, migration inhibition factor, colony stimulating factor,
.chi.-interferon, .beta.-interferon, .chi.-interferon (and subtypes
thereof), and the like.
[0055] Polynucleotides or oligonucleotides that can be introduced
according to the invention methods include DNA, cDNA, and RNA
sequences of all types. For example, the DNA can be double stranded
DNA, single-stranded DNA, complexed DNA, encapsulated DNA, naked
RNA, encapsulated RNA, and combinations thereof. Such agents are
introduced by needle-free injection and electroporation as
described herein in an amount to modulate cell proliferation or to
elicit an immune response, either against the nucleic acid or a
protein product encoded by the nucleic acid.
[0056] The polynucleotides can also be DNA constructs, such as
expression vectors, expression vectors encoding a desired gene
product (e.g., a gene product homologous or heterologous to the
subject into which it is to be introduced), and the like. A
therapeutic polypeptide (one encoding a therapeutic gene product)
may be operably linked with a regulatory sequence such that the
cells of the subject are transfected with the therapeutic
polypeptide, which is expressed in cells into which it is
introduced according to the invention methods. The polynucleotide
may further encode a selectable marker polypeptide, such as is
known in the art, useful in detecting transformation of cells with
active agents according to the invention method.
[0057] In various embodiments of the invention method, the active
agent can be a "proliferation-modulating agent," which alters the
proliferative abilities of cells. Proliferation modulating agents
include, but are not limited to, cytotoxic agents, agents toxic or
becoming toxic in the presence of a protein, and chemotherapeutic
agents. The term "cytotoxic agent" refers to a protein or other
molecule having the ability to inhibit, kill, or lyse a particular
cell. Cytotoxic agents include proteins such as ricin, abrin,
diphtheria toxin, saporin, or the like. In one embodiment, the
cytotoxic agent is only effective when it can gain access to the
cell, such as by the introduction of the agent into the cell by
needle-free injection in combination with electroporation. The
introduction of such agents intracellularly, or the expression of
nucleic acids encoding polypeptides intracellularly, results in
inhibition of protein synthesis or death of the cell. Illustrative
toxic subunits include the A chains of diphtheria toxin,
enzymatically active proteolytic fragments from Pseudomonas
aeruginosa exotoxin-A, ricin A-chain, abrin A-chain, modeccin
A-chain, and proteins having similar activity found in various
plants, such as the plants Gelonium multiflorum, Phytolacca
Americana, Croton, Tiglium, Jatropha, Curcas, Momordic, Charantia,
Reachan, the toxin saporin from Saponaria officinalis (Thorpe et
al. J. National Cancer Institute (1985) 75:151), the Chinese
cucumber toxin, trichosanthin (Yeung et al. Intl. J. of Peptide
Protein Res. (1985) 27:325-333), and the like. Mutant species of
the toxins also may be used, for example, CRM 45 (Boquet et al.
Proc. Natl. Acad. Sci. USA (1976) 73:4449-4453).
[0058] In other embodiments, the active agent can be a
"chemotherapeutic agent," having an antitumor or cytotoxic effect.
Such agents can be "exogenous" agents, which are not normally found
in the subject (e.g., chemical compounds and drugs).
Chemotherapeutic agents can also be "endogenous" agents, which are
native to the subject, including suitable naturally occurring
agents, such as biological response modifiers (i.e., cytokines,
hormones, and the like). Specific chemotherapeutic
proliferation-modulating agents include, but are not limited to
daunomycin, mitomycin C, daunorubicin, doxorubicin, 5-FU, cytosine
arabinoside, colchicine, cytochalasin B, bleomycin, vincristine,
vinblastine, methotrexate, and the like. Additional active agents
that act as chemotherapeutic agents are cytotoxic agents, such as
those derived from microorganism or plant sources.
[0059] Drugs contemplated for use in the invention method as the
active agent include antibiotics such as are known in the art and
chemotherapeutic agents having an antitumor or cytotoxic effect.
Such drugs or agents include bleomycin, neocarcinostatin, suramin,
doxorubicin, carboplatin, taxol, mitomycin C, cisplatin, and the
like. Other chemotherapeutic agents will be known to those of skill
in the art (see for example The Merck Index). In addition, agents
that are "membrane-acting" agents can also be introduced into cells
according to the invention method. Membrane acting agents are a
subset of chemotherapeutic agents that act primarily by damaging
the cell membrane, such as N-alkylmelamide, para-chloro mercury
benzoate, and the like. Alternatively, the composition can include
a deoxyribonucleotide analog, such as azidodeoxythymidine,
dideoxyinosine, dideoxycytosine, gancyclovir, acyclovir,
vidarabine, ribavirin, or any chemotherapeutic known to those of
average skill in the art.
[0060] Vaccination is an effective form of preventative care
against infectious diseases. Safe and effective vaccines are
available to protect against a variety of bacterial and viral
diseases. These vaccines may consist of inactivated pathogens,
recombinant or natural subunits, and live attenuated or live
recombinant microorganisms. Accordingly, in another aspect, an
agent or composition introduced to the epidermis of a subject can
be a vaccine, such as a vaccine that includes a polynucleotide or a
protein component.
[0061] DNA immunization, a method to induce protective immune
responses using "naked" DNA, complexed DNA or encapsulated DNA, is
effective as shown in U.S. Pat. No. 5,589,466. DNA immunization
entails the direct, in vivo administration of vector-based DNA or
non-vector DNA that encodes the production of defined microbial or
cellular antigens, for example, and cytokines (e.g., IL and IFN),
for example. The de novo production of these antigens in the host's
own cells results in the elicitation of antibody and cellular
immune responses that provide protection against challenge and
persist for extended periods in the absence of further
immunizations. The unique advantage of this technology is its
ability to mimic the effects of live attenuated vaccines without
the safety and stability concerns associated with the parenteral
administration of live infectious agents. Because of these
advantages, considerable research efforts have focused on refining
in vivo delivery systems for naked DNA that result in, for example,
maximal antigen production and resultant immune responses. Such
systems also include liposomes and other encapsulated means for
delivery of DNA.
[0062] Accordingly, it is presently preferred that the DNA or RNA
molecule introduced as a vaccine to induce a protective immune
response encodes not only the gene product (i.e., active agent) to
be expressed, but also initiation and termination signals operably
linked to regulatory elements including a promoter and
polyadenylation signal capable of directing expression in the cells
of the vaccinated subject. The vaccine polynucleotide can
optionally be included in a pharmaceutically acceptable carrier as
described herein.
[0063] As used herein, the term "gene product" refers to a protein
resulting from expression of a polynucleotide within the treated
cell. The gene product can be, for example, an immunogenic protein
that shares at least an epitope with a protein from the pathogen or
undesirable cell-type, such as a cancer cell or cells involved in
autoimmune disease against which immunization is required. Such
proteins are antigens and share epitopes with either
pathogen-associated proteins, proteins associated with
hyperproliferating cells, or proteins associated with autoimmune
disorders, depending upon the type of genetic vaccine employed. The
immune response directed against the antigenic epitope will protect
the subject against the specific infection or disease with which
the antigenic epitope is associated. For example, a polynucleotide
that encodes a pathogen-associated gene product can be used to
elicit an immune response that will protect the subject from
infection by the pathogen.
[0064] Likewise, a polynucleotide that encodes a gene product
containing an antigenic epitope associated with a
hyperproliferative disease such as, for example, a tumor-associated
protein, can be used to elicit an immune response directed at
hyperproliferating cells. A polynucleotide that encodes a gene
product that is associated with T cell receptors or antibodies
involved in autoimmune diseases can be used to elicit an immune
response that will combat the autoimmune disease by eliminating
cells in which the natural form of target protein is being
produced. Antigenic gene products introduced into cells as active
agents according to the present invention may be either
pathogen-associated proteins, proteins associated with
hyperproliferating cells, proteins associated with auto-immune
disorders or any other protein known to those of average skill in
the art.
[0065] In addition, it may be desirable to introduce into cells of
a subject a polynucleotide that modulates the expression of a gene,
such as an endogenous gene, in cells. The term "modulate" envisions
the suppression of expression of a gene when it is over-expressed,
as well as augmentation of expression when it is under-expressed.
Where a cell proliferative disorder is associated with the
expression of a gene, nucleic acid sequences that interfere with
the gene's expression at the translational level can be used to
modulate gene expression. This approach introduces into the cells
of a subject such active agents as antisense nucleic acid
sequences, ribozymes, or triplex agents to block transcription or
translation of a specific mRNA, either by masking that mRNA with an
antisense nucleic acid or triplex agent, or by cleaving it with a
ribozyme.
[0066] Antisense nucleic acid sequences are DNA or RNA molecules
that are complementary to at least a portion of a specific mRNA
molecule (Weintraub, Scientific American, 262:40, 1990). In the
cell, the antisense nucleic acid hybridizes to the corresponding
mRNA, forming a double-stranded molecule. The antisense nucleic
acid interferes with the translation of the mRNA, since the cell
will not translate a mRNA that is double-stranded. Antisense
oligomers of about 15 nucleotides are preferred, since they are
easily synthesized and are less likely than larger molecules to
cause problems when introduced into the target cell. The use of
antisense methods to inhibit the in vitro translation of genes is
well known in the art (Marcus-Sakura, Anal. Biochem., 172:289,
1988).
[0067] Use of a short oligonucleotide sequence (i.e., "triplex
agent") to stall transcription is known as the triplex strategy,
since the oligomer winds around double-helical DNA, forming a
three-strand helix. Therefore, such triplex agents can be designed
to recognize a unique site on a chosen gene (Maher, et al.,
Antisense Res. and Dev., 1(3):227, 1991; Helene, C., Anticancer
Drug Design, 6(6):569, 1991).
[0068] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer.Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0069] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
that are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences that are 11-18 bases in length. The longer
the recognition sequence, the greater the likelihood that the
sequence will occur exclusively in the target mRNA species.
Consequently, it is preferred to employ hammerhead-type ribozymes
over tetrahymena-type ribozymes for inactivating a specific mRNA
species, and 18-based recognition sequences are preferable to
shorter recognition sequences as active agents in practice of the
invention methods.
[0070] The active agent introduced according to the invention
methods can also be a therapeutic peptide or polypeptide. For
example, immunomodulatory agents and other biological response
modifiers can be administered for incorporation by cells. The term
"biological response modifiers" is meant to encompass substances
which are involved in modifying the immune response. Examples of
immune response modifiers include such compounds as lymphokines.
Lymphokines include tumor necrosis factor, interleukins 1, 2, and
3, lymphotoxin, macrophage activating factor, migration inhibition
factor, colony stimulating factor, and alpha-interferon,
beta-interferon, and gamma-interferon, their subtypes and the
like.
[0071] Also included are polynucleotides which encode metabolic
enzymes and proteins, including anti-angiogenesis compounds, e.g.,
Factor VIII or Factor IX. The active agent introduced according to
the invention methods can also be an antibody. The term "antibody"
as used herein is meant to include intact molecules as well as
fragments thereof, such as Fab and F(ab').sub.2, and the like, as
are known in the art.
[0072] In addition, the composition can include a detectable
marker, such as a radioactive label. Alternatively, the composition
can include a photoactive modification, such as Psoralin C2.
Further, the composition can include a phosphoramidate linkage,
such as butylamidate, piperazidate, and morpholidate.
Alternatively, the composition can include a phosphothiolate
linkage or ribonucleic acid. These linkages decrease the
susceptibility of oligonucleotides and polynucleotides to
degradation in vivo.
[0073] The term "pharmaceutical agent" or "pharmaceutically active
agent" as used herein encompasses any substance that will produce a
therapeutically beneficial pharmacological response when
administered to a subject, including both humans and animals. More
than one pharmaceutically active substance may be included, if
desired, in a pharmaceutical composition used in the method of the
present invention.
[0074] The pharmaceutically active agent can be employed in the
present invention in various forms, such as molecular complexes or
pharmaceutically acceptable salts. Representative examples of such
salts are succinate, hydrochloride, hydrobromide, sulfate,
phosphate, nitrate, borate, acetate, maleate, tartrate, salicylate,
metal salts (e.g., alkali or alkaline earth), ammonium or amine
salts (e.g., quaternary ammonium) and the like. Furthermore,
derivatives of the active substances such as esters, amides, and
ethers which have desirable retention and release characteristics
but which are readily hydrolyzed in vivo by physiological pH or
enzymes can also be employed.
[0075] As used herein, the term "therapeutically effective amount"
or "effective amount" means that the amount of the biologically
active or pharmaceutically active substance is of sufficient
quantity and activity to induce a desired pharmacological effect.
The amount of substance can vary greatly according to the
effectiveness of a particular active substance, the age, weight,
and response of the individual subject as well as the nature and
severity of the subject's condition or symptoms. Accordingly, there
is no upper or lower critical limitation upon the amount of the
active agent introduced into the cells of the subject although it
is generally a greater amount than would be delivered by passive
absorption or diffusion, but should not be so large as to cause
excessive adverse side effects to the cell or tissue containing
such cell, such as cytotoxicity, or tissue damage. The amount
required for transformation of cells will vary from cell type to
cell type and from tissue to tissue and can readily be determined
by those of ordinary skill in the art using the teachings herein.
The required quantity to be employed in practice of invention
methods can readily be determined by those skilled in the art.
[0076] In one embodiment of the invention method, the amount of
active agent such as a nucleic acid sequence encoding a gene
product introduced into the cells is a "transforming amount." A
transforming amount is an amount of the active agent effective to
modify a cell function, such as mitosis or gene expression, or to
cause at least some expression of a gene product encoded by the
nucleic acid sequence.
[0077] Introduction of active agents across the natural barrier
layer of skin or mucous membrane can be enhanced by encapsulating
the active agent in a controlled release vehicle or mixed with a
lipid. As used herein with respect to preparations or formulations
of active agents, the term "controlled release" means that the
preparation or formulation requires at least an hour to release a
major portion of the active substance into the surrounding medium,
for example, about 1-24 hours, or even longer.
[0078] Preferred controlled release vehicles that are suitable for
electrotransport are colloidal dispersion systems, which include
macromolecular complexes, nanocapsules, microcapsules,
microspheres, beads, and lipid-based systems, including
oil-in-water emulsions, micelles, mixed micelles, liposomes, and
the like. For example, in one embodiment, the controlled release
vehicle used to contain the active agent for injection is a
biodegradable microsphere. Microspheres wherein a pharmaceutically
active agent is encapsulated by a coating of coacervates is called
a "microcapsule."
[0079] Liposomes, which may typically bear a cationic charge, are
artificial membrane vesicles useful as delivery vehicles in vitro
and in vivo. It has been shown that large unilamellar vesicles
(LUV), which range in size from about 0.2 to 4.0 .mu.m, can
encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules, such as DNA.
[0080] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations, making them
suitable vehicles for encapsulating an active agent intended to
undergo electrotransport according to the invention methods.
[0081] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, gangliosides, and the like.
Particularly useful are diacylphosphatidylglycerols, where the
lipid moiety contains from 14-18 carbon atoms, particularly from
16-18 carbon atoms, and is saturated. Illustrative phospholipids
include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoyl-phos-phatidylcholine.
[0082] Preparations suitable for electrotransport may also include
a "pharmaceutically acceptable carrier." Such carriers include
sterile aqueous or non-aqueous solutions, suspensions and
emulsions. Examples of non-aqueous solvents include propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, fixed oils, and
the like. Vehicles suitable for intercellular or intracellular
injection may also include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose,
and the like. Preservatives and other additives may also be present
such as, for example, antimicrobials, anti-oxidants, chelating
agents, and inert gases, and the like.
[0083] The invention method may optionally further comprise
pretreatment of the tissue surface with compounds or compositions
that facilitate injection of the active agent into cells underlying
the tissue surface. Examples of components of a composition
suitable for pretreatment of the epidermis of the subject include,
for example, a reducing agent, such as a charged reducing agent
(e.g., DMSO) that disrupts cross linked keratin within
keratinocytes of the epidermis. Alternatively, the epidermis can be
pretreated by application of a proteinase, such as keratinase,
papain, or reducing agents or compounds, to overcome possible
hindrance of DNA transport during injection and electroporation
that might be caused by the dense keratin matrix of the
epidermis.
[0084] As used herein, the term "subject" refers to any animal. It
is envisioned that the methods for delivering an agent into cells
of a subject can be performed on any animal, including domesticated
animals kept as pets, as well as animals raised as workers or as a
providers or sources of food. Preferably, the subject is a
human.
[0085] As used herein, the term "local," when used in reference to
an active agent introduced by a needle-free injector according to
the invention method, refers to activity within the region of
tissue treated (e.g., the region electroporated). Thus, an agent
injected into skin or mucosal tissue is believed to be taken up by
cells underlying or contiguous with the skin or mucosal tissue and
to exert its biological or pharmaceutical activity within the cells
of the tissue or muscle directly underlying the skin. Nevertheless,
the skilled artisan will recognize that some biologically active
agents introduced according to the invention method may have a
systemic effect or activity, such that, after being injected into a
particular region of tissue according to the invention method, the
agent may be distributed at least in part to other areas of the
subject, thereby producing or contributing to a systemic
effect.
[0086] The invention methods for introducing an agent into cells
are useful in treatment of a variety of conditions and diseases
ranging from diabetes to psoriasis and baldness. Like other types
of transdermal drug delivery, the invention methods have
application in treatment of conditions that have a large potential
market, such as pain management (acute and chronic), treatment of
erectile dysfunction, skin aging, and the like. For example, in one
aspect, the invention method is useful in treating undesired cells.
An "undesired cell" is any cell targeted for removal due to its
location, genotypic and/or phenotypic properties, and the like.
Examples of conditions exhibiting undesired cells that can be
treated using the invention methods include, but are not limited
to, the presence of excess fat cells, endometrial tissue in
endometriosis, excess tissue caused by psoriasis, birth marks such
as port wine stains, adhesions or scar tissue from injury or
surgery, moles, and the like.
[0087] The methods of the invention are useful in treating cell
proliferative disorders or other disorders of the various organ
systems, particularly, for example, cells in the skin, mucosal
tissue uterus, prostate and lung, and also including cells of
heart, kidney, muscle, breast, colon, prostate, thymus, testis,
ovary, blood vessel and the like. The term "cell proliferative
disorder" refers to a disease or condition characterized by
inappropriate cell proliferation, and includes neoplasia. Concepts
describing normal tissue growth are applicable to malignant tissue
since normal and malignant tissues can share similar growth
characteristics, both at the level of the single cell and at the
level of the tissue. In tumors, production of new cells exceeds
cell death. For instance, a neoplastic event tends to produce an
increase in the proportion of stem cells undergoing self-renewal
and a corresponding decrease in the proportion progressing to
maturation (McCulloch, E. A., et al., Blood 59:601-608, 1982).
Thus, the term "cell proliferative disorder" denotes malignant as
well as non-malignant cell populations, which often appear to
differ from the surrounding tissue both morphologically and
genotypically. Specific non-limiting examples of non-malignant cell
proliferative disorders include warts, benign prostatic
hyperplasia, skin tags, and non-malignant tumors. For example, the
invention can be used to treat such cell proliferative disorders as
benign prostatic hyperplasia or unwanted genital warts by targeting
the undesirable cells that characterize such conditions for
removal.
[0088] The methods of the invention are advantageous in several
respects. First, the invention methods allow, for example, topical
treatment of skin or mucosal lesions, such as melanoma. Such
treatment is not invasive and delivery of pharmaceutical compounds,
polynucleotides or other agents can be localized to the site of the
lesion. Further, the amount of agent necessary to treat a
particular lesion is significantly reduced by localized application
of the agent, thereby substantially diminishing the cost of
treatment and side effects. In addition, risk of infection and
mechanical trauma, such as that caused by subcutaneous injections,
is avoided by using electroporation in combination with needle-free
injection. Further, risk associated with disrupting cancer cells,
such that they are dislodged from a primary location, thereby
spreading the cancer, is lessened. In addition, systemic illnesses
can be treated by delivery of pharmaceuticals, polynucleotides,
such as antisense oligonucleotides, or other agents, to control
expression of a targeted gene associated with the illness over an
extended period of time.
[0089] One therapeutic application of electroporation includes
needle-free introduction of a cytotoxic agent into tissue and
electroporation of the agent into cells by applying voltage pulses
between electrodes or electrically conductive needle-free injectors
disposed on opposite sides of or within the tissue. Another
therapeutic application of the invention methods includes
needle-free injection of a nucleic acid encoding a cytotoxic agent
into tissue having undesirable cell types (i.e. cells proliferating
in an unnatural manner) and electroporation of the nucleic acid
into the cells of the tissue by applying voltage pulses between
electrodes strategically located on opposite sides or within the
tissue containing undesirable cells. As disclosed herein, it is
preferred that the needle-free jet injection device itself serves
as an electrode. (See FIGS. 1A and B). However, when the injector
is not used as an electrode, caliper or surface electrodes are
utilized.
[0090] The invention methods can also be used in practice of gene
therapy for the treatment of cell proliferative or immunologic
disorders mediated by a particular gene or absence thereof. Such
therapy would achieve its therapeutic effect by introduction of a
specific sense or antisense polynucleotide into cells having the
disorder. Polynucleotides intended for introduction into cells of a
subject for the purpose of gene therapy can be contained in a
recombinant expression vector such as a chimeric virus, or the
polynucleotide can be delivered as "naked" DNA as described
herein.
[0091] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (-MoMuLV), Harvey murine sarcoma virus (HaMuS-V),
murine mammary tumor virus (-MuMTV), and Rous Sarcoma Virus (RSV).
When the subject is a human, a vector such as the gibbon ape
leukemia virus (GaLV) can be utilized. A number of additional
retroviral vectors can incorporate multiple genes. All of these
vectors can transfer or incorporate a gene for a selectable marker
so that transduced cells can be identified and generated.
[0092] The invention will now be described in greater detail by
reference to the following, non-limiting examples.
EXAMPLE 1
[0093] The following study was conducted to compare quantatively
the level of gene expression when a DNA-containing plasmid is
administered by intradermal injection and by needle-free injector.
Electroporation of the injection site at various voltage levels was
used to determine and compare electroporation enhancement of gene
expression when DNA is injected by the two methods tested.
[0094] I. Luciferase reporter gene. To quantify the level of gene
expression, the luciferase reporter gene was used in the first
study.
[0095] Animals. Four to six week old male and female outbred pigs
weighing 20 to 40 pounds were purchased from the Prairie Swine
Center (University of Saskatchewan, Saskatoon, Saskatchewan). The
animals were housed and treated in compliance with the Canadian
Council for Animal Care. Animals were randomly assigned to six
groups of five animals each.
[0096] Electroporation. To determine the effect of electroporation
on expression of plasmid DNA, electroporation was performed using
the BTX ECM 830 Pulse Generator with the needle-free micropatch
round electrode mounted on a handle (model MP 35) (Genetronics, San
Diego, Calif.). Six square-wave pulses were applied at 60, 70, or
80 V, with pulse duration of 60 msec, pulse interval of 200 msec,
and reversal of polarity after three pulses.
[0097] Luciferase expression and assay. A luciferase encoding
plasmid (Pmas-luc) under the control of the CMV promoter in the
pMAS backbone (Krieg, A. M., et al., Proc. Natl. Acad. Sci. USA.
95:12631-6.) was a gift from Dr. Heather Davis (University of
Ottawa, Ontario, Canada) (Weeratna, R., et al., Antisense Nucleic
Acid Drug Dev. 8:351-356). Plasmid DNA encoding luciferase was
injected intradermally using a 26-gauge needle or a DERMAL
BIOJECT.TM. needle-free injection system (BioJect, Inc., Portland,
Oreg.), followed by electroporation with various voltages.
Intradermal needle injection was tested with no electroporation and
with electroporation at voltages of 60V and 80V. Needle-free using
the Bioject device was tested with no electroporation and with
electroporation at voltages of 60 V, 70 V, and 80 V.
[0098] For each injection, a dose of 100 .mu.g of pMAS-luc
(Weeratna et al., supra) in 100 .mu.l phosphate buffered saline
(PBS) was administered into the skin of the shaved abdomens (eight
sites per pig), with each treatment group having four sites from
the same pig. The luciferase-encoding plasmid was injected at eight
distinct sites, and electroporation was applied to four of those
sites. Forty-eight hours after the administration of the plasmid,
the area surrounding each injection site was biopsied with an 8-mm
diameter puncher at a depth of approximately 8-mm. Skin was
homogenized in 500 .mu.l lysis buffer (Promega, Madison, Wis.) with
a POLYTRON.RTM. homogenizer (Brinkmann Instruments, Rexdale,
Ontario) to produce protein extracts. Luciferase activity in the
protein extracts was determined using Promega's luciferase assay
system. On a Packard PICOLITE.RTM. luminometer (Packard Instruments
Canada LTD, Mississauga, Ontario), the bioluminescence of each
500-.mu.l sample was counted for 30 seconds and recorded as
relative light units (RLUs). Untreated or PBS-injected tissues were
used to determine the background luminescence levels.
[0099] Histological examination of skin. Forty-eight hours after
intradermal injection of 100 .mu.l PBS alone or PBS containing 100
.mu.g of DNA using the needle-free injection system and
electroporation with 60 V, 70 V, or 80 V, skin biopsies Were fixed
in 10% formalin. Tissues were embedded in paraffin and 2-.mu.m
sections were stained with hematoxylin/eosin (H&E).
[0100] Histological examination of tissue sections was used to
determine how voltage could influence gene expression. The results
of histological examination showed that increasing the voltage
increases the amount of tissue damage and cellular infiltration of
the skin. The optimal voltages determined were 60V for intradermal
needle injection and 70V for the needle-free deliveries. Thus,
proper voltage selection for the DNA immunizations enhanced
cellular uptake of plasmid and minimized tissue damage.
[0101] The luciferase assay results indicated that delivery by
needle-free injector consistently outperformed needle injections,
with or without electroporation. However, electroporation
significantly enhanced the level of expression with both types of
injection (FIG. 3). From the results of these studies, it can be
concluded that the optimal voltage to accompany needle injection is
lower than the optimal voltage to accompany injection with a
needle-free injector.
[0102] II. GFP gene expression. To analyze localization of gene
expression a plasmid encoding green fluorescent protein (GFP) was
used. A plasmid encoding GFP under the control of the CMV promoter
was obtained through Quantum Biotechnologies, (Montreal, Que.). 100
.mu.g of the plasmid in 100 .mu.l PBS was administered
intradermally at the sites on the shaved abdomens of the pigs using
a syringe or needle-free injector in combination with
electroporation (60 V for syringe injection and 70 V for
needle-free administration). Twenty-four hours after administration
the injection site was biopsied using an 8-mm punch. Skin samples
were frozen in liquid nitrogen and stored at -70.degree. C. until
they were sectioned. Skin samples were cut transversally with an
IEC MINITOME.RTM. microtome cryostat (Damon, Needham, Miss.) into
7-.mu.m sections. Sections containing GFP expressing cells were
photographed with an Olympus AH2-RFL microscope using standard
light together with blue fluorescent light. The results were based
on four skin punch biopsies per treatment.
[0103] The results of histological examination of tissue samples
showed that delivery of plasmid by either intradermal injection or
needle-free injector resulted in GFP expression surrounding the
injection site in the epidermis. However, delivery with the
needle-free injector resulted in consistent GFP expression in skin
biopsies with GFP expression in all four biopsies; whereas the
results of intradermal needle injection was not consistent, since
GFP was only detected in one out of four biopsies. In addition,
administration by needle-free injector resulted in GFP expression
in cells surrounding the hair follicles, where dendritic cells are
known to be numerous. Since dendritic cells are active sites of
immunological activity, it can be expected that needle free
injection will be a good route of administration DNA vaccines.
[0104] Electroporation influenced the localization of GFP with a
much greater dispersion of GFP in the dermis away from the
injection site following both intradermal needle administration and
needle-free injector administration. Delivery by needle-free
injector in combination with electroporation (70 volts) resulted in
GFP expression only in the deeper layers of the skin and was not
detected under the damaged stratum corneum. In a single isolated
biopsy, GFP expression following intradermal needle injection
looked more robust and wide spread compared to needle-free
administration. All other biopsies showed that needle-free
injection administration resulted in gene expression and, and more
importantly, the gene expression was distributed in a much wider
area than resulted from intradermal needle injection.
EXAMPLE 2
[0105] To study the effects of electroporation in the context of a
DNA vaccine, a DNA-prime/DNA-boost/protein-boost strategy was
evaluated for enhancement of immune responses in large animals, and
compared the responses achieved following standard protein
vaccination. The strategy used resembled DNA-prime/protein-boost
strategies previously used in non-human primates, which yielded
outstanding results for both malaria (12) and HIV vaccinations
(1).
[0106] Immunization protocols. For immunization studies, animals
were selected and care for as above. Pigs were randomly assigned to
six groups of five animals each. The pigs were anesthetized with
halothane prior to DNA injection and electroporation and treated as
follows: The animals in Group 1 each received 250 .mu.g pHBsAg in
100 .mu.l 0.1 M phosphate buffered saline (PBS) by a dermal BIOJECT
B 2000.RTM. needle-free injection device (Bioject, Inc. Portland,
Oreg.) at each of two abdominal sites for a total of 500 .mu.g
pHBsAg. The animals in Group 2 were treated identically to those in
Group 1, except that the injection sites were also treated with 70
V electroporation, immediately following plasmid injection. The
animals in Group 3 each received 250 .mu.g pHBsAg in 100 .mu.l PBS
by an intradermal injection at each of two abdominal sites for a
total of 500 .mu.g pHBsAg. The animals in Group 4 were treated
identically to those in Group 3 except that injection sites were
treated with a 60 V electroporation, immediately following plasmid
administration. Animals in Group 5 received 500 .mu.l of the
commercial hepatitis B vaccine (Engerix-B, SmithKline Beecham
Pharma, Oakville, Ont.) injected intradermally with the Bioject
device in two 250 .mu.l doses on the abdomen and Group 6 was
immunized with Engerix-B by an intramucular injection. All animals
were boosted with the same injection conditions after four weeks.
All treatment groups were boosted at week eight with Engerix B
vaccine by intramuscular injection for all groups except group 5,
which was injected with Engerix using the Bioject device as
previously described (Table 1).
1TABLE 1 EXPERIMENTAL DESIGN 3.sup.rd immunization.sup.3 1.sup.st
and 2.sup.nd immunizations.sup.2 (Engerix-B) Group.sup.1 Vaccine
Route/Method Voltage (V) Route/Method 1 pHBsAg i.d./BioJect --
i.m./needle 2 pHBsAg i.d./BioJect 70 V i.m./needle 3 pHBsAg
i.d./needle -- i.m./needle 4 pHBsAg i.d./needle 60 V i.m./needle 5
Engerix-B i.d./BioJect -- i.d./BioJect 6 Engerix-B i.m./needle --
i.m./needle .sup.1Each group consisted of five animals. .sup.2First
and second immunizations were administered on day zero and four
weeks later. .sup.3The third immunization was given eight weeks
after the first immunization i.d., intradermal; i.m.,
intramuscular.
[0107] To assess efficacy of DNA vaccination in priming the immune
system, animals in all experimental groups were boosted with a
protein vaccine at week 8 post initial injection.
[0108] Measurement of humoral responses. At four, eight, and ten
weeks after the first immunization serum was collected from
anesthetized animals and centrifuged for analysis. Anti-HBsAg
antibodies were quantitatively measured using the AUSAB EIA
Diagnostic Kit, and quantification in milli-International Units/ml
was performed in parallel with the AUSAB Quantification Panel,
according to the manufacturer's instructions (Abbott Laboratories,
North Chicago, Ill.).
[0109] Anti-hepatitis B IgG.sub.1 and IgG.sub.2 isotypes were
identified by ELISA as follows. IMMUNLON 2.RTM. ELISA plates
(Dynex, Chantilly, Va.) were coated with HBsAg (BioDesign
International, Saco, Me.) (1 .mu.g/ml in 20 mM Na.sub.2CO.sub.3)
and stored overnight at 4.degree. C. Then, the plates were washed
with phosphate-buffered saline-Tween (PBST) (PBS, 0.05% TWEEN
20.RTM.; Sigma Chemical Co., St. Louis, Mo.). Serum was diluted in
diluent (PBST, 0.5% gelatin) (Sigma) 20-fold, followed by serial
4-fold dilutions, and incubated overnight at 4.degree. C. Plates
were washed six times in PBST. Porcine IgG.sub.1 and IgG.sub.2
isotypes were detected using mouse anti-porcine antibodies specific
against IgG.sub.1 and IgG.sub.2 isotypes (Serotec, Hornby,
Ontario). Following incubating at room temperature for one hour,
plates were washed six times in PBST. Anti-mouse IgG.sub.1
biotinylated antibodies (Caltag, Toronto, Ontario), diluted in
diluent, were added and incubated for one hour. Plates were washed
six times in PBST, and streptavidin-alkaline phosphatase (Jackson
Immuno-Research Labs, West Grove, Pa.) was added to the plates and
incubated for one hour. The alkaline phosphatase activity was
measured by the conversion of p-nitrophenol phosphate (PNPP)
(Sigma). The absorbance was read after 15 to 20 minutes at 405-nm
wavelength (Bio-Rad, Hercules, Calif.).
[0110] Immune Responses in Immunized Pigs. Although there was
variability in the immune response among the animals in each group
described above, the animals treated with electroporation showed
better immune responses than the non-electroporated animals, based
on both the number of animals responding and the level of response
recorded by AUSAB (Table 2 below and FIG. 4).
2TABLE 2 Responses in Pigs to Various Hepatitis B
Vaccinations.sup.1 Number of Animals Responding Week 4 Week 8 Week
10 Vaccine Group AUSAB ELISA AUSAB ELISA AUSAB ELISA (1) DNA, b.j.
0/5 0/5 1/5 1/5 4/5 3/5 (2) DNA, 0/5 0/5 2/5 5/5 5/5 5/5 b.j. + EP
(3) DNA, i.d.n. 0/5 0/5 0/5 0/5 5/5 3/5 (4) DNA, 0/5 0/5 0/5 1/5
5/5 5/5 i.d.n. + EP (5) Engerix B, 0/5 0/5 4/5 4/5 5/5 5/5 b.j. (6)
Engerix B, 0/5 0/5 5/5 5/5 4/4 4/4 i.m.n. .sup.1The number of
animals that showed an immune response out of the total number of
animals in a group is indicated. b.j. = Bioject needle-free
injection; EP = electroporation; i.d.n. = intradermal needle;
i.m.n. = intramuscular needle.
[0111] The results summarized in Table 2 are consistent with the
results of the luciferase experiments of Example 1 herein, which
indicated that electroporation enhances gene expression, and that
needle-free delivery is more effective than needle delivery. These
results also demonstrate the superiority of immune responses
elicited with the combination of needle-free delivery and
electroporation over responses achieved by intradermal needle
injections, both with respect to the number of animals responding
and the magnitude of the response.
[0112] As a positive control for these studies, two groups of
animals were immunized with a subunit vaccine injected either
intramuscularly with needle and syringe (the prior art route) or
intradermally with the needle-free injector. Results of the control
studies indicate that the intramuscular needle injection resulted
in more robust immune responses than injection with the needle-free
injector.
[0113] Results in Table 2 also demonstrate that even though immune
responses were undetectable in most of the animals after two rounds
of DNA immunization, the majority responded rapidly to a protein
boost. The fact that nearly all DNA immunized animals responded
within two weeks of the protein boost demonstrates an anamnestic
response, since the protein vaccine alone did not induce immune
responses 4 weeks post immunization (Table 2).
[0114] To determine whether the experimental manipulations had an
impact on the antibody isotypes generated, the sera were analyzed
for the presence of anti-HBsAg IgG.sub.1 and IgG.sub.2 using an
ELISA. As shown in FIGS. 5A-5D, in those animals mounting an early
response, most produced primarily IgG.sub.1 at 8 weeks. However,
after boosting with protein, a much more balanced response with
approximately equivalent levels of IgG.sub.1 and IgG.sub.2 were
evident. Additionally, the groups injected with DNA and treated
with electroporation (Groups 2 and 4) produced antibodies similar
in titer to those in the cohorts receiving multiple protein
injections (Groups 5 and 6). In contrast, those groups that
received DNA without electroporation (Groups 1 and 3) produced
antibodies of lower titer, even after the protein boost.
[0115] It will be apparent to those skilled in the art that various
modifications and variations can be made to the compounds and
processes of this invention. Thus, it is intended that the present
invention cover such modifications and variations, provided they
come within the scope of the appended claims. Accordingly, the
invention is limited only by the following claims.
* * * * *