U.S. patent application number 13/887539 was filed with the patent office on 2013-09-19 for controlled laser treatment for non-invasive tissue alteration, treatment and diagnostics with minimal collateral damage.
This patent application is currently assigned to Hadasit Medical Research Services and Development Ltd.. The applicant listed for this patent is HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPM. Invention is credited to Eithan GALUN, Yitzchak Hemo, Artium Khatchatouriants, Aaron Lewis, Alexandra Manevitch, Evelyne Zeira.
Application Number | 20130245618 13/887539 |
Document ID | / |
Family ID | 28454850 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130245618 |
Kind Code |
A1 |
GALUN; Eithan ; et
al. |
September 19, 2013 |
CONTROLLED LASER TREATMENT FOR NON-INVASIVE TISSUE ALTERATION,
TREATMENT AND DIAGNOSTICS WITH MINIMAL COLLATERAL DAMAGE
Abstract
A highly controlled and precise system, device and method for
tissue and cellular alteration and treatment below or at surfaces
with a laser. The present invention is characterized by ultra low
levels of collateral damage as defined by physiologically relevant
tests that measure tissue viability. The operation of the present
invention is based on spectrally confining the interaction between
laser energy and a targeted tissue including an essential element
for physiologically relevant tests for monitoring tissue
viability.
Inventors: |
GALUN; Eithan; (Har Adar,
IL) ; Lewis; Aaron; (Jerusalem, IL) ; Zeira;
Evelyne; (Bet-Shemesh, IL) ; Manevitch;
Alexandra; (Jerusalem, IL) ; Khatchatouriants;
Artium; (Jerusalem, IL) ; Hemo; Yitzchak;
(Jerusalem, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPM |
Jerusalem |
|
IL |
|
|
Assignee: |
Hadasit Medical Research Services
and Development Ltd.
Jerusalem
IL
|
Family ID: |
28454850 |
Appl. No.: |
13/887539 |
Filed: |
May 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10508531 |
Dec 8, 2005 |
8435791 |
|
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PCT/IL03/00260 |
Mar 27, 2003 |
|
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13887539 |
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60367509 |
Mar 27, 2002 |
|
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Current U.S.
Class: |
606/6 ;
607/89 |
Current CPC
Class: |
A61B 2018/00636
20130101; A61N 1/0412 20130101; A61N 5/0613 20130101; A61N 1/327
20130101; A61B 18/20 20130101; A61F 9/00825 20130101 |
Class at
Publication: |
606/6 ;
607/89 |
International
Class: |
A61F 9/008 20060101
A61F009/008; A61N 5/06 20060101 A61N005/06 |
Claims
1. A method for treating an eye, comprising: irradiating the eye
with a laser beam having laser pulses which produce multiphoton
laser tissue effects in the eye; and controlling the laser beam,
such that the eye is selectively affected by said multiphoton laser
tissue effects.
2. A method according to claim 1, wherein said method is for
treating a cataract in an eye; and wherein said controlling
includes controlling the laser beam, such that the cataract is
selectively affected thereby.
3. A method according to claim 2, wherein said laser pulses
produces multiphoton laser tissue effects in the cataract, said
controlling including controlling the laser beam such that the
cataract is selectively affected by said multiphoton laser tissue
effects.
4. A method according to claim 2, wherein said multiphoton laser
tissue effects include mechanical destruction of the cataract.
5. A method according to claim 2, wherein said treating a cataract
includes treating a membrane behind a lens in the eye, said
targeting resulting in selective destruction of the membrane,
wherein collateral damage to tissue surrounding the target area is
below a preselected threshold.
6. A method according to claim 2, whereby at every point other than
at the cataract the laser beam has lower fluence than the fluence
at the cataract.
7. A method according to claim 2, wherein thermal or other
collateral damage to tissue not associated with the cataract is
below a preselected threshold.
8. A method according to claim 1, wherein said method is for
treating age-related macular degeneration (AMD), wherein the
presence of AMD is associated with a pigment that has accumulated
in the eye; and wherein said controlling includes controlling the
laser beam, such that the pigment is selectively affected
thereby.
9. A method according to claim 8, wherein said irradiating includes
irradiating the pigment with a laser beam having laser pulses with
a duration in the range of from femtoseconds to picoseconds.
10. A method according to claim 8, wherein said irradiating
includes irradiating with a laser beam having laser pulses with a
duration of nanoseconds.
11. A method according to claim 8, wherein said irradiating is
performed with a Ti:Sapphire laser.
12. A method according to claim 8, wherein said treating includes
one of bleaching, destroying, and reducing the pigment.
13. A method according to claim 8, wherein said treating includes
bleaching of a certain component of the tissue.
14. A method according to claim 8, wherein said controlling
includes controlling the laser beam such that tissue surrounding
the pigment is not affected thereby.
15. A method according to claim 8, wherein said controlling
includes forming a spot with a resolution of at most microns behind
the retina without touching the overlying retina.
16. A method according to claim 8, wherein said controlling
includes increasing a fluence of the laser beam in a non-linear
fashion to cause multiphoton absorption in a very narrow range
around a focal spot which is under the retina.
17. Apparatus for treating an eye, said apparatus comprising: a
laser configured to produce a laser beam having multiphoton laser
tissue effects; and optics for controlling the laser beam, such
that a targeted portion of the eye is affected by the laser
beam.
18. Apparatus according to claim 17, wherein said laser is a pulsed
laser.
19. Apparatus according to claim 18, wherein said laser is
configured to produce a laser beam having ultrashort pulses.
20. Apparatus according to claim 18, wherein said laser is
configured to produce pulses with a duration in the range of from
femtoseconds to picoseconds.
21. Apparatus according to claim 18, wherein said laser is
configured to produce pulses with a duration of nanoseconds.
22. Apparatus according to claim 17, wherein said laser is
configured to operate at a wavelength selected from 1.5 microns,
800 nm, and 780 nm.
23. Apparatus according to claim 17, further comprising optics
configured to focus the laser beam at the targeted portion of the
eye such that the laser beam produces the multiphoton effects.
24. Apparatus according to claim 23 further comprising of a slit
lamp.
25. Apparatus according to claim 24 wherein said focusing is used
to increase the fluence of said laser to cause multiphoton
absorption in a range around a focal spot in the eye.
26. Apparatus according to claim 17, wherein said laser is
configured to be operated externally to an organ of the body.
27. Apparatus according to claim 17, wherein said laser is
configured to be operated internally in an organ of the body.
28. Apparatus according to claim 17, wherein said multiphoton laser
tissue effects are internal to the eye.
29. Apparatus according to claim 17, wherein said multiphoton laser
tissue effects are external to the eye.
30. Apparatus for treating a body portion other than an eye, said
apparatus comprising: a laser configured to produce a laser beam
having multiphoton laser tissue effects; and optics for controlling
the laser beam, such that a targeted tissue in the body portion is
affected by the laser beam.
31. Apparatus according to claim 30, wherein said multiphoton laser
tissue effects are external to the body portion.
32. Apparatus according to claim 30, wherein said multiphoton laser
tissue effects are internal to the body portion.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/508,531 filed on Dec. 8, 2005, which is a
National Phase of PCT Patent Application No. PCT/IL03/00260 filed
on Mar. 27, 2003, which claims the benefit of priority under 35 USC
.sctn.119(e) of U.S. Provisional Patent Application No. 60/367,509
filed on Mar. 27, 2002. The contents of the above applications are
incorporated herein by reference in their entirety.
FIELD OF INVENTION
[0002] The present invention is of a system, device and method for
tissue and cellular alteration and treatment below or at surfaces
with a laser, and in particular, of such a system, device and
method which at least reduces collateral damage.
BACKGROUND OF THE INVENTION
[0003] All human disease develops as a result from alterations in
genetic or environmental factors or the combination of both.
Cardiovascular diseases, which are the world's leading cause of
death, are the best examples. In this disease genetic and
environmental factors "join" to induce the endothelial pathology
leading to cardiovascular and heart disease. Systemic and local
delivery of genes or gene expression modifying agents could serve
as the future arm of therapy. To achieve this, a temporary
alteration to a particular layer of tissue below the surface of
tissue, which forms pores without perturbing the overlying tissue,
is needed for the facilitated entry of genetic material.
[0004] Some genetically based diseases result in disease states
that can be retarded even without the addition of genetic material.
They require alteration and diagnosis of a specific component below
the surface of the tissue without perturbing the overlying tissue.
The nature of the required alteration can extend from permanent
destruction through a bleaching of a certain component of the
tissue. An example of such a disease is called age related macular
degeneration (AMD) in which a pigment that accumulates behind the
retina has to be bleached under conditions that do not touch the
overlying retinal tissue.
[0005] There are also infections that require a total destruction
of tissue without affecting the overlying and underlying tissue. An
example is a fungal infection in an underlying tissue layer that
must be removed without damaging the overlying tissue. A specific
case is fungal infection under cutaneous tissue like the nail of
the foot that has to be destroyed without damaging the overlying or
underlying tissue.
[0006] All of these problems have been incredibly difficult to
address with existing technology. For example, successful solutions
to these problems require effective calibration with a defined, in
vivo, methodology for depth of penetration and the exact parameters
required for minimal collateral damage. These parameters have to be
checked with a defined diagnostic procedure, other than standard
pathological techniques that are filled with artifacts from the
fixation procedure. Thus, standard pathology is incapable of
defining, with the sensitivity required, the parameters for highly
controlled treatment.
[0007] Laser methods have been applied in the past to each of the
problems previously described, but with limited success. All of
these previous applications have used a linear form of laser tissue
interaction, which cannot highlight a specific tissue layer
selectively without an injection of an external highlighting light
absorbing agent, which is the case for palliative laser attempts
for AMD progress retardation using photodynamic therapy (see H. Sun
and J. Nathans, "The Challenge of Macular Degeneration," Scientific
American, October 2001, p 61). Except for such protocols, in which
injection of a highlighting absorbing substance is required, all
other laser methodologies that have been applied to these problems
have an effect of the laser that is limited to the surface.
Alternatively, these methodologies require transport of the laser
beam to a highly specific area, in a highly limiting fashion, by
way of an invasive intrusion, for example with an optical fiber or
other laser guiding device.
[0008] Thus, for example, no previous solutions of AMD have been
able to bleach the subretinal pigments that are the cause of this
disease without collateral damage to the overlying retina. This is
the case even though higher order laser effects are known in
microscopic analysis and can interrogate specific layers with the
characteristics required (see analysis of T. Wilson and C. J. R.
Sheppard, Theory and Practice of Scanning Optical Microscopy
Academic Press, New York 1984). These effects could not be
effectively be used in therapy without the controlled in vivo
characterization of parameters required for ultralow to zero
collateral damage to the surrounding tissue. Thus, no such
treatments have even been considered because of these problems (see
H. Sun and J. Nathans, "The Challenge of Macular Degeneration,"
Scientific American, October 2001, p 61).
[0009] In addition to AMD, fungal infections have remained
essentially impossible to eradicate in places like the region under
the nails of the feet, because of the lack of accurate
parameterization for the highly specific, highly controlled
treatments that are required.
[0010] Furthermore, no previous invention or report had shown site
specific, prolonged expression of genetic material administration
in vivo with any type of laser-related methodology (see for example
Tao et al PNAS (USA) 84:4180-4, 1987; Kurata and Ikawa Cell Struct
Funct 11:205-7, 1986; Paulombo et al J Photochem Photobiol 36:41-6,
1996).
[0011] Laser-related methodologies have been disclosed for example
in U.S. Pat. No. 6,251,099, which teaches the use of pulsed laser
light in order to generate "impulse transients" for delivering
substances through the skin. These impulse transients generate
transient increases in the permeability of epithelial tissue,
thereby enabling the substances to penetrate. However, the laser
light is not described as being useful for administering substances
and/or performing therapeutic treatments within intermediate tissue
layers, as would be required for the treatment of AMD, for
example.
[0012] U.S. Pat. No. 4,775,361 discloses a method for administering
a therapeutic substance through the skin of a patient, by using a
pulsed laser beam of controlled wavelength, pulse length, pulse
energy, pulse number and pulse repetition rate, sufficient to
ablate the stratum corneum (outer layer of the skin) without
damaging the epidermis. The therapeutic substance is then applied
to the area of skin with the ablated stratum corneum. However, the
disclosure still requires destruction of a portion of tissue.
Therefore, the disclosed device of U.S. Pat. No. 4,775,361 could
not be used for treatment of AMD, as it would damage retinal tissue
above the area to be treated.
[0013] U.S. Pat. No. 5,713,845 describes the use of laser to force
drugs into the skin, for example on small graphite particles which
act as an explosive absorber of light energy. The laser beam is
transmitted in very short pulses, which cause small explosions that
force the drug through the skin. Clearly, the disclosed system is
not suitable for applications in which the laser has to penetrate
some distance of tissue before reaching the tissue to be treated.
Thus, the disclosed system could not be used to treat AMD, as it
would also damage retinal tissue above the area to be treated.
[0014] Gene therapy itself faces many obstacles before it will
become a widely available method of treatment. A major obstacle in
applying theoretical and experimental gene therapy methods into
clinical practice is the current complexity of gene delivery
systems. Viral vectors for gene delivery have shown great promise
in relation to their efficiency, longevity and targeting capacities
(1). The use of retroviral vectors for gene delivery in correcting
genetic maladies in children was implemented clinically and
initially, showed promise (2, 3). However, a number of unresolved
issues concerning viral gene delivery remain. These include, among
others: the potential for anti-viral immunological reactions; risk
for development of malignant phenotypes associated with improper
gene integration; size limitations on vector capacity; and
challenges in the production of Good Manufacturing Practice (GMP)
grade genetic material free of replication-competent viruses, that
is suitable for clinical use (4). Hence, attention has focused on
the use of non-viral methods of gene delivery such as cationic
liposomes that transport foreign genes through cell membranes, or
"naked DNA" constructs in which the desired gene is incorporated
into a plasmid that may be injected directly into muscle or other
tissues (5, 6). This latter technique requires physical methods
such as electroporation (EP), that transiently fenestrate the
cellular and nuclear membranes (5, 6). However, the in vivo
efficiency of these methods is often low. Recent modifications such
as the use of ultrasound energy (7) or microfabricated devices (8)
to enhance naked DNA uptake in muscle or the dermis, respectively,
have been successful in specific cases. Other potentially powerful
genetic therapy tools include: anti-sense nucleotides, ribozymes,
intron I and II based nucleic acids, and therapeutic small
interference RNA (RNAi), some of which have been assessed in animal
models and in preliminary clinical trials (9, 10). However, most
methods still face significant obstacles in their specific
applications due to gene delivery problems. One of these is that in
vivo electroporation of naked DNA into large animals, even with
enhancing delivery molecules such as polyethylenimine, will likely
require a high-energy pulse >500 V (11) that while theoretically
efficient for gene transduction, would not be practical as it would
create considerable risk for local tissue injury (burn) or other
deleterious effects (cardiac arrest).
SUMMARY OF THE INVENTION
[0015] The present invention is of a highly controlled and precise
system, device and method for tissue and cellular alteration and
treatment below or at surfaces with a laser. The present invention
is characterized by ultra low levels of collateral damage. The
operation of the present invention is based on spectrally confining
the nature of the interaction that results when a laser spot is
created with a large fluence, or large intensity per square unit of
area, in a short time at a targeted tissue.
[0016] The present invention provides a non-invasive treatment at
any level in the tissue based on the spectral confinement of laser
tissue interaction with critical diagnostic methods for
characterizing the penetration, the level of laser power, the
frequency of the laser light, the focus of the beam and the
determination of the precise three-dimensional area to be treated.
This is a preferred and important component of the invention that
allows for characterizing the in vivo conditions of the laser
tissue alteration and for defining the parameters that are needed
to achieve the desired effects. Preferably, this characterization
is based on the use of higher order laser poration of specific
tissue layers with the site specific administration of nucleic
acids in the form of RNA and DNA, and/or other macromolecules
and/or particles and/or other pharmacological compositions. The
latter compositions can include material that is associated with a
specific disease state and which can be placed at the specific
depth of the tissue being targeted. Since the present invention
also includes an accurate determination of the laser levels that
allow for viability, functional assays of tissue and cellular
systems are also optionally and preferably provided. Although
specific combinations of disease, higher order laser effects and
diagnostic methods for tissue viability are chosen for the target
applications described in this invention, other emulations or other
combinations can be conceived, based on this principle of defined
diagnostics to achieve ultralow to zero collateral damage in
underlying laser tissue interactions, for any type of disease and
not limited only to those diseases and/or pathological conditions
directly addressed herein.
[0017] For example, optionally and preferably, at least one
parameter is monitored in vivo to permit a specific pathological
condition to be treated according to the present invention, in
which the specific pathological condition features a particular
molecular species, according to an effect of the particular
molecular species determined after an injection into a similar live
tissue.
[0018] The combination of defined diagnostics enables higher order
laser effects to be applied to these disease states for the first
time. Thus, the present invention has taken higher order laser
effects that have been previously known (T. Wilson and C. J. R.
Sheppard, Theory and Practice of Scanning Optical Microscopy
(Academic Press, New York 1984)), and have enabled these effects to
be applied to diseases that require ultra low to zero collateral
damage through defined methods of characterization and control
developed in the present invention. The present invention also does
not need either an external highlighting agent or an invasive beam
delivery system for the laser treatment or alteration of tissue at
or below the surface.
[0019] The present invention is applicable to a large number of
therapeutic and other problems, including but not limited to, the
administration of genetic material such as DNA, RNA, or any other
such material, the administration of any biopharmaceutical
composition other than DNA or RNA, treatment of conditions such as
AMD, and other age and non-age related degenerative diseases,
chronic infectious disease, autoimmune diseases, vaccinations and
malignancies, and fungal and other infections.
[0020] The present invention may optionally and preferably be used
to treat infections that require a total destruction of tissue
without affecting the overlying and underlying tissue. An example
is a fungal infection in an underlying tissue layer that is
preferably be removed without damaging the overlying tissue. A
specific case is fungal infection under cutaneous tissue like the
nail of the foot that has to be destroyed without damaging the
overlying or underlying tissue.
[0021] The present invention may also optionally and preferably be
used for separation of cells (with and without genetic markers),
and for diagnostics, with or without dye molecules.
[0022] Hereinafter, the term "ultra-low collateral damage" refers
to collateral damage which still permits surrounding cells to
maintain viability, wherein such viability can be determined
according to appropriate controls for demonstrating cellular
viability.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0024] The foregoing and other objects, aspects and advantages will
be better understood from the following detailed description of a
preferred embodiment of the invention with reference to the
drawings, wherein:
[0025] FIG. 1. The system used in the invention to accomplish the
tissue alteration and gene transduction based characterization is
shown. This instrument is just one example of the geometry of the
interaction with the tissue. Other tissue interaction geometries
could include transmission of the laser for higher order
interactions through the tissue. This is required for such
applications as second or third order effects for functional
diagnosis of tissues and cells in which specific gene expression
occurs as a result of the protocols described herein.
[0026] FIG. 2. Exemplary laser apparatus for gene therapy according
to the present invention. A femtosecond infrared mode-locked
Ti:Sapphire laser (Coherent, Mira 900) was used as an illumination
source. It was pumped by an argon ion laser (Coherent, Innova 200)
that was operated at 12 watt in a multi-line mode. The operating
wavelength was 780 nm, the pulse frequency was 76 MHz, and the
pulse duration was about 200 fsec. The laser beam was transmitted
via an inverted microscope (Zeiss, Axiovert 135) and focused by
.times.50 N.A 0.5 objective (Zeiss) on the sample.
[0027] FIG. 3. Luc expression from the leg muscle of mice following
injection and electroporation of naked pLNC/Luc DNA as control
experiments. 3, 5, 10 and 15 .mu.g of pLNC/Luc were injected
followed by electroporation, to the leg muscle of BALB/c mice
(a-d). Quantification of the light readings is presented in (e).
The contra-lateral leg is always injected with the same dose of DNA
without electroporation.
[0028] FIG. 4. Time kinetics of luc expression following laser beam
application compared to electroporation. BALB/c mice were injected
with 15 .mu.g pLNC/Luc in the muscle of both dorsal legs of the
mice. This was followed by electroporation in the right back leg or
treatment with a laser beam in the left back leg. Luc expression
was followed from day 1 to day 30. Three groups of mice were
assessed based on the length of time that the laser beam was
applied. The laser beam was applied for 1.4, 5 and 10 seconds in
each one of the groups.
[0029] FIGS. 5A-5B. A dose response analysis of laser beam duration
compared to electroporation (A). BALB/c mice were injected with 15
.mu.g pLNC/Luc in the muscle of the dorsal legs of the mice. This
was followed by electroporation or treatment with a laser beam. Luc
expression was assessed on day one. Three groups of mice were
assessed based on the length of time that the laser beam was
applied. The laser beam was applied for 5, 10 or 15 seconds in each
one of the groups. This was compared to the electroporated treated
group over time (B).
[0030] FIGS. 6A-6E. Comparison of Laser (LBGT) to Electroporation
(EP) gene delivery. (A) For a dose response experiment assessing
gene expression on day one, BALB/c mice were injected with 0.5-15
micro-g pLNC/luc DNA. Thirty seconds later the legs were either
electroporated (right leg) or exposed to a laser beam (left leg).
The laser beam was applied at a surface of 95 m.sup.2, at a depth
of 2 mm, for 5, 10 or 15 seconds (in the 15 second laser beam
exposure group no gene expression was seen. One representative
point is depicted). The leg was exposed to the laser beam 10 times
in a rotational manner around the site of injection. Real time in
vivo continuous luc expression was monitored with a
biochemiluminescence CCCD system. The pictures show mice injected
i.m. into the right and left legs with (B) 10 micro-g and (C) 15
micro-g pLNC/luc DNA per leg for 5 seconds exposure for the LBGT
side. As can be seen in parts D and E, despite the significant
advantage to EP (D) on day 23, this advantage disappears over time
and the laser (E) and electroporation methods give the similar
results.
[0031] FIGS. 7A-7D. Determination of optimal intensity, timing,
focus and surface parameters. Mice were injected with 10 .mu.g
pLNC/luc DNA. Thirty seconds later the left leg was exposed to a
laser beam 10 times in a rotational manner around the site of
injection. The efficiency of each parameter was checked over time.
Intensity (A): Using 10-30 mwatts. Timing (B): The exposure lasted
for 3-10 seconds. Presented are the integrated light units emitted
at the site of injection above background. (C and D). For controls,
10 .mu.g of pLNC/Luc was injected (left leg) followed by
application of laser-beam and pLNC/Luc (right leg) without any
pulses.
[0032] FIGS. 8A-8C. Reporter gene assessment--an
immunohistochemical study: (A) Muscle GFP gene expression: For GFP
gene expression, the muscles were frozen in OCT tissue embedding
medium and sectioned (6 .mu.m) under cryostat. Sections were placed
on polylysine-coated slides, fixed in acetone for 30 sec and GFP
expression was observed directly by fluorescence microscopy. (B)
Muscle Luciferase gene expression: Paraffin-embedded sections were
pretreated by incubation in citrate buffer and heated. After
incubation at room temperature with rabbit polyclonal antibodies
against luciferase, sections were then washed and incubated with
biotin-conjugated goat antibody anti rabbit. After this treatment,
the samples were labeled with peroxidase conjugated streptavidin
and detected using 3-amino-9-ethyl carbazole substrate (see
materials and methods). (C) Muscle gene .beta.-gal expression: The
electroporated, laser-beam-application and
muscle-injected-with-plasmid-only tissues, were excised and
analyzed for the presence of .beta.-gal six days post DNA injection
(see materials and methods). The whole-mount X-gal stained tissues
were then photographed. The tissues were then paraffin embedded and
sections were fixed, washed in PBS containing 2 mM MgCl.sub.2, and
stained. Image analysis was performed using a light microscope
(40.times. magnification). As can be seen with the GFP, .beta.-gal
and luciferase reporter genes, both the EP and LBGT methods
resulted in similar expression. Plasmid-injected-only sections show
extremely weak expression. The arrows indicate single cell plasmid
expression, which can be seen in both luciferase and
.beta.-gal.
[0033] FIGS. 9A-9B. Histology. Extensive and irreversible damage of
skeletal muscle after electrical trauma can be ascribed to
secondary release of myoglobin and CPK because of increased
skeletal muscle cell membrane permeabilization. No tissue
alteration was detected 24 hours after LBGT, and 48 hours post
injection (A), the presence of areas with rare fibers with central
nuclei was the only change in the laser beam stimulated muscles.
These areas are considered as an unspecific sign of disturbance of
muscle function. The damage was transient, and 70 days after
treatment muscles appeared normal (B).
[0034] FIG. 10. To assess the level of muscle injury the CPK serum
enzyme marker for myofiber lysis was determined. CPK in the serum
of the mice collected 2 hours post-injection. Normal mice, mice
injected with the plasmid only and mice injected with the plasmid
were stimulated 30 seconds later with either the laser beam or
electroporation. As seen, the level of CPK activity with the laser
beam method was 6 fold lower compared to electroporation. It is
interesting to note that despite repetition of the experiment, CPK
serum activity levels in the laser beam treated mice were found to
be lower than that of the normal and plasmid-injected-only
mice.
[0035] FIG. 11. To examine the therapeutic potential of the laser
beam method, a plasmid encoding mouse erythropoietin (pcDNA.3mepo)
was used. The pcDNA.3mepo plasmid was injected intramuscularly with
10 .mu.g of either the pcDNA.3mepo plasmid, or the backbone pcDNA.3
(negative control) and followed by laser application. Blood was
collected at various time points, and levels of Hct were
determined.
[0036] FIGS. 12A-12B. Diagrammatic representations of the treatment
of pigments in the in vitro and in vivo preparations and of the
experimental laser system and associated devices used for these
experiments.
[0037] FIG. 13. The bleaching of fluorescein under the retina of
the live eye of a rat. The frames on the left of this figure show a
view of the retina with a slit lamp and its associated
illumination. The arrow indicates a region of multiphoton
fluorescence excited by the ultrashort laser. The frames on the
right are of this spot of fluorescence and its time dependent
photobleaching which is easier to delineate without the presence of
the slit lamp illumination. Nonetheless, even with slit lamp
illumination the photobleaching is also clearly visible. Each frame
corresponds to a point in the graph shown in FIG. 14. and;
[0038] FIG. 14. The relative photobleaching of fluorescein with
time for the experiment shown in FIG. 13. From left to right the
points represent a different frame in FIG. 13 with the point to the
left in this graph corresponding to the top most frame and the
point in the extreme right corresponding to the bottom frame in
FIG. 13.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0039] The present invention is of a highly controlled and precise
system, device and method for tissue and cellular alteration and
treatment below or at surfaces with a laser. The present invention
is characterized by ultra low levels of collateral damage. The
operation of the present invention is based on spectrally confining
the interaction between laser energy and a targeted tissue while
choosing a physiologically relevant parameter to determine the
power levels that can achieve a level of collateral damage that is
consistent with effective function at the cellular level of the
targeted tissue.
[0040] The approach is based on using to advantage the higher order
terms of the polarizability tensor that describes the interaction
of light with matter to limit the laser tissue interaction to a
particular region of tissue. This polarizability tensor, which
defines the interaction between light and matter, consists of a
series of terms to mathematically approximate the polarizability of
the material with the light. Whereas the first term in this series
describes conventional absorption and scattering of laser light,
the higher order terms are preferably used for performing the
present invention.
[0041] For the higher order terms discussed above to have an
appreciable effect, the number of photons per square area per time
has to be very high. This can be arranged using a lens that creates
a beam shape in which there is low fluence at every point other
than at the focus. Non-linear interactions occur when more than one
identical photon occupies the same space at the same time. The
level of fluence at which light has the non-linear interactions
depends on the ultrashort nature of the pulse; the shorter the
pulse, the higher the probability that more than one identical
photon will appear in exactly the same area or location.
[0042] Also, these higher order terms permit the effect of the
geometrical focusing to be increased by the nature of the higher
order phenomena being used to alter the tissue. In addition, the
higher order interaction of the laser with the tissue permits
infrared radiation to be employed and this further reduces the
absorption of tissue in regions that are above and below the
selected region of interest. Finally, if the laser is tunable then
not only is the infrared nature of the radiation used to advantage
but also the wavelength of the infrared radiation can be chosen so
that the higher order interactions can be accomplished with maximal
effect.
[0043] Although specific examples of different types of lasers and
laser systems are described below, the present invention is
generally operable with any type of laser or laser system having
suitable characteristics. These characteristics may also depend
upon the type of treatment, and/or the type of tissue being
treated. For example, the present invention is optionally and
preferably operable with any pulsed laser. The particular pulsed
laser is preferably selected according to particular situations;
for example, the pulse width is optionally and preferably optimized
according to the type of treatment and/or tissue.
[0044] Optionally and preferably, "ultrafast" lasers with
"ultrashort" pulse durations are used for the present invention.
The nature of the ultrashort laser pulses is defined by the
resolution that is required by the depth of penetration; the
present invention is operable with femtosecond lasers as described
below in the seconds, but may also optionally be operable with
picosecond and ultrasecond lasers, for example.
[0045] The range of operating power (in watts) depends on the pulse
width and repetition rate, which is preferably optimized for
specific tissues and/or treatments; the shorter the pulse, the
lower the amount of collateral damage that is caused, and the
higher the resolution in depth of penetration, in terms of
targeting specific tissues.
[0046] The range of suitable operating wavelengths depends upon the
depth of penetration that is required. For example, minimal water
absorption occurs at about 1.5 microns; for highly aqueous tissues,
better penetration is achieved at wavelengths closer to 1.5
microns. Nonetheless, such parameters are preferably adjusted
relative to the non-linear methodology that is chosen for tissue
intervention. For example, even though the depth of penetration is
highest at 1.5 microns in an aqueous medium, the efficacy of this
wavelength for AMD is very low; hence the maximal bleaching of the
drusen droplet can only occur at much shorter wavelengths, such as
800 nm or 0.8 microns. Thus in such a clinical situation, where
depth of penetration is not detrimentally mediated by the tissue in
front of the drusen droplets, a wavelength of 800 nm would be
preferred.
[0047] A suitable beam diameter (or range of beam diameters) is
preferably chosen according to the dimensions of the tissue that is
to be treated. Appropriate lens combinations which allow the light
to be focused are determined according to the numerical aperture,
magnification and working distance of the lens, which are
preferably chosen as appropriate for the size of tissue being
treated, and the depth of that tissue.
[0048] These different parameters are preferably determined in
order to characterize the laser beam according to a plurality of
parameters for permitting the second order term of the higher order
expression of the polarizability tensor for describing the
interaction of light with matter to be used to perform second
harmonic generation substantially without tissue alteration.
Characterization of the Laser Tissue Interaction for Tissue
Alteration for Disease Treatment
[0049] Age related macular degeneration (AMD) currently is not
treatable with regard to the root cause of the disease. Even though
lasers have been applied in ophthalmology for many decades, the
application of the laser to this disease has not been possible,
since the disease originates from the development of deposits of
pigment in retinal pigment epithelium (RPE) cells, slowly killing
them and causing the neighboring light sensitive photoreceptor
cells to die. An important pigment deposit is the pigment known as
A2E ("Isolation and one-step preparation of A2E and iso-A2E,
fluorophores from human retinal pigment epithelium," Proc. Natl.
Acad. Sci. USA 95, 14609 (1998)).
[0050] In fact the total lack of cure or effective retardation of
AMD is evidenced by a review recently published from a center of
AMD research at Johns Hopkins University by one of the leaders in
this field, Jeremy Nathans (see H. Sun and J. Nathans, "The
Challenge of Macular Degeneration," Scientific American, October
2001, p 61). In essence no approach with lasers to cure or to
effectively retard this disease by targeting the pigments that
result in the disease has been successful, and the only approach
that has a palliative effect is photodynamic therapy for the
retardation of blood vessel growth in the Bruch's membrane, which
is a treatment for a symptom or response of the disease rather than
the cause of the disease.
[0051] FIG. 1 shows an exemplary system 10 according to the present
invention for treating AMD. A laser 12 that can be used to cause
higher order laser tissue interactions is shown. Laser 12 is
preferably an ultrafast laser, also termed herein as an ultrashort
laser, which may be for example a near-infrared laser having time
durations of .about.60.times.10.sup.-15 sec, which can be focused
to a point below a surface and can be made to affect only the point
of focus, and not the overlying or underlying material through
which the laser beam is transmitted.
[0052] Optionally and more preferably, laser 12 is a pulsed laser
which has the ultrashort pulse duration of
(.about.60.times.10.sup.-15 secs). Such an ultrashort pulse (for
example, a femtosecond laser) leads to interactions of light with
matter that are highly non-linear. This means that the light that
is focused by a lens 22 above and below the plane of focus does not
have a high enough fluence (intensity per square cm) to result in
these non-linear interactions, i.e. interactions in which the
density of photons is high enough so that more than one photon or
packet is found at the same point in the sample at the same time.
Only in the plane of focus of lens 22 is the density of photons
high enough to allow this to happen. As a result of such a
non-linear event, highly localized absorptions and emissions are
produced.
[0053] Laser 12 is preferably focused by appropriate optics, which
in the case of AMD treatment preferably includes a slit lamp 14 to
microscopically view the organ (shown as an eye 18) and lens 22
that focuses light with the appropriate fluence for the higher
order interactions in the layer of tissue that is to be altered. In
all cases, the organ is located beyond lens 22. Slit lamp 14
preferably also includes a beamsplitter 20.
[0054] Appropriate lens combinations are preferably used to form a
spot with a resolution of several microns behind the retina without
the touching the overlying retina. Next, preferably a filter 24
filters the light received by a CCD camera 16. The information may
optionally be displayed by a television system 26 and/or recorded
by a recorder 28, for the purpose of live, "real time" monitoring
of the process.
[0055] The in vivo diagnostic test is an optional but preferred
part of this invention, in order to verify the power levels that do
not damage the overlying retina. Such in vivo diagnostic tests can
be one of several types, of which two non-limiting examples are
described below. A first example is the online measurement of
electrophysiological parameters in an animal model in which the
newly synthesized pigment A2E has been injected behind the retina.
A second example is the gene expression assay as described
below.
[0056] Without wishing to be limited to a single hypothesis, it is
probable that the lack of the application of this approach was the
lack of understanding of how optics could accomplish this in such a
complicated tissue, even with such a laser, with such a large
distance from the entrance of the eye to the tissue in question. In
addition, a critical aspect has been the inability to define the
associated physiologically relevant measurements developed with
regard to the present invention to determine the power levels that
would permit the essential ultra low collateral damage. This is in
spite of the fact that higher order phenomena have been known for a
long time in microscopy (T. Wilson and C. J. R. Sheppard, Theory
and Practice of Scanning Optical Microscopy (Academic Press, New
York 1984)).
[0057] Thus, the invention includes a concept of treatment which is
based upon the combination of appropriate optical techniques with
lasers that could generate higher order interactions in tissue, and
which have the ability to bleach pigments such as the newly
synthesized A2E without touching the overlying retina as defined by
either physiological or genetic tests.
[0058] In terms of the first example of a physiological test noted
above, optionally and more preferably, the present invention
includes injecting A2E pigment into the tissue behind the retina of
an animal model, in order to set the parameters necessary for the
accurate bleaching of this pigment with higher order effects, while
minimizing damage to the overlying retina as determined by
simultaneous electrophysiological measurements.
[0059] Alternately, genetic expression, as described below, can be
used as a monitor of the surrounding tissues' ability to remain
viable and this can be monitored relative to the power levels that
can bleach the newly synthesized molecule A2E.
[0060] The same or similar arrangement can optionally and more
preferably be used for fungal infections with appropriate
alterations of the arrangement and treatment according to the organ
being modified, more preferably by altering the optical components
described in FIG. 1 as required to cause this modification. The
gene insertion procedure described below can be used to set the
exact thresholds of the higher order laser effects in this geometry
that will not affect the surrounding tissue.
[0061] The same or similar arrangement can also optionally and more
preferably be used for gene insertion, again with the appropriate
alterations, particularly with regard to the energy requirements of
the laser so that the higher order phenomena cause poration of the
cells in the layer of tissue being considered, with minimal or no
collateral damage to surrounding tissue.
[0062] The ability to insert genes that can be expressed in
specific tissue layers is a preferred embodiment of this invention
since the monitoring of gene activity may optionally be used for
characterization of the layer of tissue being altered by the laser,
as well as for determination of the laser intensities required for
keeping the cells in the appropriate layer alive after laser
treatment. Thus, the appropriate energy densities are more
preferably achieved in a more precise and controlled manner after
this critical characterization.
Characterization of the Laser Tissue Interaction for Gene
Transduction
[0063] One important and preferred aspect of the present invention
is the characterization and control of the laser treatment process,
for maintaining viability after the interaction with the laser in a
specific underlying layer of tissue. The process of gene
transduction may optionally be used to monitor and define the
characteristics of this laser tissue interaction for other types of
applications, in addition to gene therapy.
[0064] One of the major limitations in translating the concept of
gene therapy into a routinely used therapeutic approach is the low
efficiency of gene delivery and transduction of target cells. To
overcome these bottlenecks, in recent years a spectrum of new
delivery and gene transduction methods were developed and assessed
in a variety of animal models. In general three approaches were
developed to enable gene transduction into target cells: the use of
viral vectors; cell therapy with or without genetic manipulations;
and non-viral gene therapy.
[0065] The major advantage of using viral vectors is the relatively
efficient transduction efficiency and the possibility for
conducting specific targeting in specific cases. However, viral
vectors harbor major limitations related to the expression of
foreign genes, inducing an immune response or enabling prolonged
expression. To meet these objectives, new modified viral vectors
were developed, one of which is the adenoviral vector. The helper
dependent adenoviral vector is a gutless virus, which infects most
human cells through the CAR receptor. Due to the fact that the
gutless vector doses not express any viral genes, the immune
response against infected cells is very low, enabling prolonged
expression. However, construction of such vectors is very laborious
and needs specific expertise. In addition, the effect of repeated
administration of such vectors for a long period of time was not
assessed until now.
[0066] An alternative viral vector transduction system recently
developed is the HIV based vectors. Here again there are
significant advantages with major limitations. The most significant
advantage of the latest generation of the HIV vectors is their
large genome capacity, as almost all accessory genes have been
deleted. Following transduction, these vectors enable stable
integration of the transgene into the host genome. However, there
are still a number of unresolved limitations. Until now, generating
high titers of the viral vector has proven to be difficult. For
example, there are batch to batch variations due to the fact that
stable cell lines are not yet available, and there are also safety
issues related to this type of vector.
[0067] Other than the adenoviral and the HIV vectors, additional
important advances were reported with the AAV vector as well as
with other viral vectors including the SV40, FIV and other new
approaches. However, for each of these new viral vectors there are
significant limitations hampering their progress application to
enter clinical use.
[0068] The alternative approach to viral vectors in gene
transduction is naked DNA delivery for transgene expression. The
major advantage of naked DNA gene therapy approach is its simple
production process and low or negligible immune response other than
in cases where DNA is injected with specific adjuvant to induce an
immune response in the case naked DNA is used for vaccination. The
use of naked DNA is problematic due to low transfection efficacy of
currently available methods, including liposomes and other
non-immunogenic compounds such as dextran which are mixed with the
DNA. In addition, targeting to specific sites is a barrier.
[0069] To overcome this barrier, DNA can be injected to reachable
organs such as the muscle, which was found to be a sufficient
producer of protein as shown in small animals following muscle
electroporation of DNA. However, naked DNA electroporation,
although an efficient gene transduction approach, is poorly
reproducible in large animals. The efficiency of electroporation in
large animals is very low. In addition, electroporation, which is
in practice an electrical shock, could be unsafe and hazardous for
patients, particularly since much higher voltages (>500 V) are
required in larger animals, and hence, in practice would be
hazardous for human patients, as it could cause serious tissue
damage.
[0070] Previous reports suggested that various methods, which
probably disrupt the cellular membrane transiently, could support
DNA entrance into living cells. Between 1993 and 2001 a single
group had received patents (U.S. Pat. Nos. 5,272,072; 5,330,467;
5,586,982; 6,071,276 and 6,190,380) for the development of a laser
catheter to induce genetic material through introducing a laser
catheter inside blood vessels or by direct application over cells.
Such an approach could be applicable for specific cases but would
need additional manipulations for operation such as an angiographic
monitoring facility and other sophisticated devices.
[0071] The present invention, by contrast, enables the use of a
different laser beam device, which does not require direct contact
with the target tissue. Such an approach enables the laser to be
applied externally, at a location which may be distant from the
target tissue itself. To this end, the present invention preferably
includes a laser beam source which is located outside of the body
to support muscle gene transduction.
[0072] The potential advantage of higher order laser interactions
is that they include energy that is directed and targeted at a
specific layer of tissue, and which can be effectively used for
gene transduction. As described in greater detail below, the laser
treatment of the present invention is suitable for the delivery of
genetic material by using an energy pulse from a laser, which in
the examples below was a femtosecond titanium sapphire
near-infrared laser. This laser system has the capacity to focus
energy at a specific level in the muscle tissue below the surface
of the skin. As these ultrashort laser pulses are in the
near-infrared region, there is less scattering by the tissue and
thus, deeper penetration. Also, by appropriate optical
manipulation, i.e. the use of a long working distance lens, and
also by using a lens having a high numerical aperture, maximal
influence of the laser in the targeted tissue is achievable with
high resolution and minimal collateral tissue damage.
[0073] Without wishing to be limited by a single hypothesis, it
would appear that the cells in the laser-illuminated region undergo
gentle poration, which transiently increases their membrane
permeability without permanent damage. Although the examples below
demonstrate that laser beam gene transduction according to the
present invention may optionally be used to deliver naked DNA
constructs, such as a plasmid expressing the murine erythropoietin
(mEPO) gene, resulting in high and persistent mEPO expression with
negligible tissue damage, in fact the present invention is expected
to be useful for the introduction of many different types of
macromolecules and/or particles into cells. Also, the present
invention is expected to be applicable for gene delivery into a
variety of target tissues. Further, it is anticipated that the
present invention provides a safer and a more effective method than
the "conventional" electroporation techniques for gene delivery in
larger animals and humans due to the larger available muscle tissue
mass over which the laser energy can be applied, and the avoidance
of electrical shocks.
[0074] In terms of suitable parameters for introducing genetic
material, the genetic material itself may optionally be used in any
suitable form for gene therapy. For example, different types of DNA
constructs that could be used for gene therapy may also optionally
be used for gene transduction with the present invention.
Non-limiting illustrative examples of promoters that are suitable
for use with the present invention include E1a, CMV and SV40 for
non-tissue specific expression; Desmin, CK and CAG for specific
muscle expression. The genetic material may also optionally be used
as naked DNA and/or naked viral vectors, and/or with various types
of suitable carriers.
[0075] Furthermore, the present invention is not limited to the
introduction of DNA molecules into cells, as any type of genetic
material, such as RNA for example, could optionally be used. Also,
many different types of macromolecules may also optionally be
introduced into cells with the laser treatment of the present
invention, such as proteins, lipids, and polysaccharides for
example. Also optionally, various suitable carriers may also be
used with these macromolecules. Also, optionally different
combinations of macromolecules could be used, for example to
encapsulate macromolecules such as RNA, DNA and/or proteins for
example, with other macromolecules such as lipids for example, to
provide a carrier. The resultant macromolecular structure may also
optionally be virus-like in terms of the particle structure.
Example 1
Gene Transduction with Higher Order Laser Effects
[0076] The device and system of the present invention was tested
according to the method of the present invention, for determining
the efficacy thereof for gene transduction.
[0077] The objective of these experiments was to assess the
possibility of transducing a DNA expression cassette into muscle
cells by applying an external laser beam source after naked DNA
administration.
Methods
Ultrashort (Femtosecond) Laser Apparatus
[0078] The interaction of the laser beam with the tissue depends on
laser power, pulse duration, surface area illuminated, and the
depth and the nature of focal parameters of the laser beam at the
layer of tissue being targeted. For these experiments, a Coherent
Radiation Mira Titanium Saphire mode-locked laser emitting 200 fsec
pulses with a 76 MHz repetition rate, was pumped by an argon ion
laser (Coherent, Innova 200) that was operated at 12 watts in a
multi-line mode. This particular laser is an example of a
femtosecond laser, but may be more generally considered as a
preferred but non-limiting example of an ultrashort laser for use
with the present invention. It should also be noted that
optionally, the method of pumping a pulsed laser could be performed
according to any of the generally accepted methodologies, including
but not limited to, single or multi-line optical pumping,
electrical pumping or chemical pumping.
[0079] For this example, the operating wavelength of the laser was
780 nm. The laser beam was transmitted via an inverted microscope
(Zeiss, Axiovert 135) and focused by .times.50 N.A 0.5 objective
(Zeiss) onto an anaesthetized animal that was placed securely on
the microscope stage. The tissue was irradiated at depths 1, 2 or 3
mm under the skin using a beam scanning system used in confocal
microscopy over a tissue region (95 micro-m.sup.2) which had been
injected 30 seconds before the irradiation (see FIG. 2). The dwell
time at each of approximately 250,000 pixels in the scan was tens
of microseconds with the entire scan time being 5-20 seconds. The
laser powers that were used varied between 10 to 30 mwatt with an
optimum (for these experimental conditions) being about 20 mwatts
(FIG. 2). It should be noted that these parameters are illustrative
only and are not intending to be limited in any way.
[0080] As shown in more detail with regard to FIG. 2, a system 30
was used for the experiments. It should be noted that system 30
shows an exemplary, non-limiting configuration of the present
invention. System 30 features a laser 32, which is preferably an
ultrashort laser, such as a femtosecond laser for example. For the
example below but without any intention of being limiting, laser 32
was a femtosecond Ti:Sapphire laser. Laser 32 may be the type of
laser which is activated or "pumped" by another laser, shown as a
pump laser 34. For the example below but without any intention of
being limiting, pump laser 34 was an argon ion pump laser. The beam
from pump laser 34 was bent with a mirror 36 as shown, as is well
known in the art.
[0081] The beam from laser 32 was then transmitted to a slit lamp
38, featuring a beamsplitter 40. The beam was focused by the optics
of slit lamp 38 and also preferably by a lens 42, onto a sample,
shown herein as a mouse 44 for the purposes of illustration only
and without any intention of being limiting.
[0082] A filter 46, for this example a green filter, filtered light
being transmitted to a CCD camera 48 and hence to a monitor 50
and/or a recorder 52, for the purpose of live "real time"
monitoring of the process.
Electroporation
[0083] Caliper electrodes were used for EP (BTX Caliper electrode
Model 384 (1 cm.sup.2). Before EP, a conductive gel was applied to
the shaved skin on either side of the marked injection point, and
the calipers were closed to a gap of about 4 mm, so that the muscle
was between the electrode plate extensions and electrical contact
with the skin was maximized. Consecutive square-wave electrical
pulses were administered using a BTX eCM 2001 pulse generator (BTX,
San Diego, Calif.) at an interval of one second between pulses. The
EP settings were at a chosen mode of LV (500 v/99 msec). The
voltage was set at 100 V. The desired field strength was 200 v/cm.
The setting for the pulse length was 20 msec, and 4 pulses were
given, after which the polarity was reversed. A total of 16 pulses
were given.
Intramuscular Injections and Animals
[0084] Female BALB/c mice (obtained from Harlan Laboratories,
Jerusalem, Israel), aged 4-6 weeks, were anesthetized by
intraperitoneal injection (IP) of 0.2 ml of 4% chloral
hydrate/saline solution (Fluka-Sigma, Israel cat no 23100). The
point of injection was marked and 10 or 15 micro-g of plasmids
pLNC/Luc, or pcDNA.3/Luc or pcDNA.3mepo in a volume of 30 micro-1
of 0.9% NaCl, were administered into the tibia cranial muscle using
a 271/2-gauge needle and a 0.5 cc insulin syringe. The
Institutional Animal Welfare Committee approved all animal
experiments. All animals were given humane care in compliance with
institutional guidelines. All animals drank tap water and were fed
rat chow ad libitum. Animals were kept with a 12 h light-dark cycle
at constant temperature and humidity.
Plasmid Vectors
[0085] The pLNC/Luc was constructed by introducing the firefly
luciferase gene (luc) downstream to the CMV promoter in the plasmid
vector pLNC. The plasmid pcDNA.3mepo containing the mouse
erythropoietin (Epo) ORF was provided. It was constructed by
inserting the mouse Epo cDNA into a unique BamH1 site between the
human CMV immediate early promoter/enhancer and a 3'-flanking
sequence of the bovine growth hormone gene polyadenylation signal
from the pcDNA.3 expression vector. This mouse Epo cDNA contained
the entire 630-base pair (630-bp) Epo-coding sequence. The empty
pcDNA.3 backbone plasmid was used as a control. Plasmids were
amplified in Escherichia coli JM109, and prepared with a Qiagen
Endo-Free plasmid Giga kit (Qiagen GmbH, Germany).
Blood Sampling and Serum Biochemical Analysis
[0086] Blood samples were collected from the retro-orbital plexus
from anesthetized animals. Blood samples were obtained on day -2
(preinjection time point) and on the indicated times following in
vivo electroporation (EP) or laser beam gene therapy (LBGT). For
the measurement of muscle damage following laser beam gene therapy
application, or electroporation (EP), creatine phosphokinase (CPK)
levels were measured in the serum of mice. For the assessment of
the effect of transduced mEPO genes, blood hematocrit (Hct) was
measured by using a Coulter STKS electronic counter for standard
analysis.
In Vivo Imaging and Quantification of Gene Expression.
[0087] For the detection and quantification of gene expression
continuously in the live animals the CCCD imaging system as
described in Honigman, A., Zeira, E., Ohana, P., Abramovitz, R.,
Tavor, E., Bar, I., Zilberman, Y., Rabinovsky, R., Gazit, D.,
Joseph, A., Panet, A., Shai, E., Palmon, A., Laster, M., and Galun,
E. (2001). Imaging transgene expression in live animals. Mol Ther
4: 239-249, hereby incorporated by reference as if fully set forth
herein. In brief, the Roper Chemiluminescence Imaging System was
used. This system contains the cooled CCCD model LN/CCD-1300EB
equipped with ST-133 controller and a 50 mm Nikon lens (Roper
scientific, Princeton instrument, Trenton, N.J.). In all
experiments mice were anesthetized before light detection, and 5
minutes before monitoring light emission, the animals were injected
i.p. with Beetle luciferin (Promega Corp., Madison, Wis.) in PBS at
126 mg/kg body weight. The animals were placed in a dark box,
supplemented with a controlled light in order to take pictures of
the background image. The light measurements were taken at the same
conditions, including time (2 min) and distance of lenses from the
mice.
Histology and Immunohistochemistry
[0088] For routine histological analysis formalin fixed paraffin
embedded muscle samples were cut into sections 4 micro-m in
thickness, deparaffinized in xylene and rehydrated through a series
of decreasing concentrations of ethanol. Sections were stained with
hematoxylin and eosin.
[0089] For immunochemical detection of luciferase (performed as
described in Lavon, I., Goldberg, I., Amit, S., Landsman, L., Jung,
S., Tsuberi, B. Z., Barshack, I., Kopolovic, J., Galun, E., Bujard,
H., and Ben-Neriah, Y. (2000). High susceptibility to bacterial
infection, but no liver dysfunction, in mice compromised for
hepatocyte NF-kappaB activation. Nat Med 6: 573-577, hereby
incorporated by reference as if fully set forth herein),
paraffin-embedded sections were pretreated by incubation in 0.01 M
citrate buffer and heated in a microwave twice for 5 min. Samples
were then incubated for 60 min at room temperature with rabbit
polyclonal antibodies against luciferase (1:100 dilution; Cortex
Biochem, San Leandro, Calif.), washed and incubated with
biotin-conjugated goat antibody anti-rabbit (1:100 dilution;
Jackson Immunoresearch, West Grove, Pa.). Afterward, the samples
were labeled with peroxidase-conjugated streptavidin and detected
using 3-amino-9-ethyl carbazole substrate.
[0090] For the detection of .beta.-galactosidase gene expression, 6
days after DNA injection of DNA, tibial muscles were excised from
all treatment groups and .beta.-galactosidase activity was measured
using the whole-mount method as previously described (as described
in Duguez, S., Feasson, L., Denis, C., and Freyssenet, D. (2002).
Mitochondrial biogenesis during skeletal muscle regeneration. Am J
Physiol Endocrinol Metab 282: E802-809, hereby incorporated by
reference as if fully set forth herein). The
5-bromo-4-chloro-3-indolyl .beta.-D-galactoside (x-gal) stained
tissues were digitally photographed. Subsequently, the tissues were
paraffin embedded and 5 micro-m sections were postfixed by
incubating the sections at 4.degree. C. for 1 h in PBS/1%
gluteraldehyde. The sections were then washed 3 times in cold PBS
containing 2 mM MgCl.sub.2, stained with a solution consisting of 1
mg/mL of x-gal solution in PBS, 5 mM K.sub.3Fe(CN).sub.6, 5 mM
K.sub.4Fe(CN).sub.6, and incubated at 37.degree. C. overnight.
[0091] Image analysis was performed using a light microscope at
40.times. magnification. To detect green fluorescence protein (GFP)
gene expression, the muscles were frozen in OCT tissue embedding
medium and sectioned (6 micro-m) with a cryostat. Sections were
placed on polylysine-coated slides, fixed in acetone for 30 seconds
and GFP expression was observed by direct fluorescence microscopy
using a Nikon eclipse E600 microscope and photographed with a
digital camera (as described in Blair-Parks, K., Weston, B. C., and
Dean, D. A. (2002). High-level gene transfer to the cornea using
electroporation. J Gene Med 4: 92-100, hereby incorporated by
reference as if fully set forth herein).
Results
[0092] Part 1--Control Study
[0093] First the effect of naked DNA injection (negative control)
was assessed in the experimental model system, in comparison with
naked DNA injected followed by electroporation (positive
control).
[0094] The animal model featured BALB/c mice (female, 4-7 week old)
which received a pLNC/luc construct injected into the muscle. These
experiments were conducted with mice in groups of three mice, and
were performed between two to three times repeatedly. The pLNC/luc
DNA vector has a backbone of a retroviral vector. The luc gene was
derived from the CMV promoter and the construct also included the
Neo resistance gene. Fifteen micrograms of plasmid pLNC/Luc, in a
volume of 30 microliters of 0.9% NaCl was injected into the tibia
cranial muscle of anesthetized BALB/c mice. The leg skin was shaved
and a conductive gel was applied in order to ensure the electrical
contact. Thirty seconds after the DNA injection, electric pulses
were applied using plate electrodes at each side of the leg. The
leg skin was shaved and a conductive gel was applied in order to
ensure the electrical contact. A BTX electroporator (ECM 2001, BMX,
San Diego, Calif.) was used to deliver pulses for 1-99 ms. The
electroporation settings were: 100V for 20 ms for a total of 16
pulses. The results of this experiment are shown in FIG. 3.
[0095] Briefly, FIG. 3 shows Luc expression from the leg muscle of
mice following injection and electroporation of naked pLNC/Luc DNA
as control experiments. As shown in FIG. 3, 3, 5, 10 and 15 g of
pLNC/Luc were injected followed by electroporation, to the leg
muscle of BALB/c mice FIG. 3a-d). Quantification of the light
readings is presented in FIG. 3e). The contra-lateral leg was
always injected with the same dose of DNA without
electroporation.
[0096] The conclusion from this experiment, which is in the same
line as reported by other groups in recent publications, is that
naked DNA alone is not sufficient to induce gene expression and an
additional DNA cellular entrance facilitator is needed. Due to the
fact that electroporation might not be applicable to large animals
and humans, and also given the lack of a complete effect, the
suitability of the present invention for enabling DNA cellular
entrance and expression was examined.
[0097] Part 2--Laser Interaction Study
[0098] The effect of an external laser beam was assessed for its
potential to support DNA transduction in muscle cells after naked
DNA injection. Side by side in the same animal, laser beam
transduction was compared to DNA electroporation into muscle cells.
BALB/c animals were used in this experiment; as for Part 1 above,
these experiments were conducted with mice in groups of three mice,
and were performed between two to three times repeatedly. The luc
expression cassette and detection system was also applied as
described in Part 1 above; the results are shown in FIG. 4. FIG. 4
shows time kinetics of luc expression following laser beam
application compared to electroporation. Luc expression was
followed from day 1 to day 30, although FIG. 4 only shows results
at days 1, 11 and 17. The laser beam was applied for 1.4, 5 and 10
seconds to mice in each one of three groups of mice. Comparison of
the electroporation method to the laser beam application after one
day revealed that the level of expression following the 10 second
laser beam application is in the same range of the electroporation
transduction. Laser beam application of 15 seconds did not induce
sufficient luc expression.
[0099] For the assessment of laser beam gene transduction, luc
expression was determined by using the same animal model, comparing
again electroporation to the laser beam application. FIG. 5 shows a
dose response analysis of laser beam duration compared to
electroporation. Luc expression was assessed on day one (FIG. 5A),
or from 1-18 days (FIG. 5B). The laser beam was again applied for
5, 10 or 15 seconds in each one of the groups. This was compared to
the electroporated treated group. As shown in FIG. 5, laser beam
application of 5 seconds had a similar and even a significant
higher transduction effect than the electroporation approach, both
on day one but even more strongly on the results obtained 17 days
after administration.
[0100] Part 3--Additional Studies of Laser Treatment for Gene
Therapy
[0101] As described in greater detail below, the potential clinical
application of the laser treatment of the present invention was
assessed, by using it to transfer the murine erythropoietin (mEPO)
gene into mice. Laser treatment-mediated mEPO gene delivery
resulted in elevated (>22%) hematocrit levels that were
sustained for 8 weeks. Gene expression following laser treatment
was detected for >100 days. Hence, the laser treatment of the
present invention is a simple, safe, effective and reproducible
method for therapeutic gene delivery with significant clinical
potential.
[0102] These studies also enable system testing to be performed,
which revealed that injection of 10 .mu.g naked DNA to the tibial
muscle of mice followed by application of the laser beam for 5 sec,
focused to 2 mm depth upon an area of 95 micro-m.sup.2, resulted in
the highest intensity and duration of gene expression with no
histological or biochemical evidence of muscle damage, for this
particular example.
[0103] The results of these additional studies are described in
greater detail in the sections below.
Gene Expression and Dose Response Experiments Following Femtosecond
Laser Beam Application
[0104] As described above, the model system used a femtosecond
infrared mode-locked Ti:Sapphire laser as an illumination source
(see Materials and Methods). The energy source was an argon ion
laser and the beam was focused onto the tissue by an inverted
microscope. Laser beam interaction with the tissue depends on
current intensity, pulse number, pulse duration, surface laser beam
area, and the depth of focus. For this example, the duration of the
laser beam pulse was from about 1 to about 25 sec, the focus was
for depths of from about 0.1 to about 15 mm, and the laser beam
area was about 95 mm.sup.2. It should be noted that these are
intended as illustrative examples only.
[0105] To optimize these parameters for these conditions, the
tibial muscles of mice were injected with a DNA plasmid (pLNC/Luc)
encoding the firefly luciferase gene. The plasmid DNA (1 micro-g to
15 micro-g dissolved in 0.9% saline) was administered by injection
in a volume of 30 micro-1 followed 30 seconds later with either
electroporation (EP) on the right leg (16 consecutive 20
millisecond square-wave pulses at a field strength of 200
volts/centimeter), or by application of an ultrashort pulsed laser
beam (LBGT) on the left leg.
[0106] Pulse timing was also optimized for these conditions, by
comparing durations of 5 to 15 seconds. Luciferase gene expression
was followed by monitoring light emission using a bioluminescence
cooled charged-coupled device (CCCD) detection system (see
Honigman, A., Zeira, E., Ohana, P., Abramovitz, R., Tavor, E., Bar,
I., Zilberman, Y., Rabinovsky, R., Gazit, D., Joseph, A., Panet,
A., Shai, E., Palmon, A., Laster, M., and Galun, E. (2001). Imaging
transgene expression in live animals. Mol Ther 4: 239-249,
previously incorporated by reference). It was observed that
injection of 10 micro-g DNA was optimal and resulted in similar
levels of luciferase expression with both the EP and the LBGT
methods (FIGS. 6a-6c). FIG. 6A shows that for a dose response
experiment assessing gene expression on day one, BALB/c mice were
injected with 0.5-15 micro-g pLNC/luc DNA. Thirty seconds later the
legs were either electroporated (right leg) or exposed to a laser
beam (left leg). The laser beam was applied at a surface of 95
m.sup.2, at a depth of 2 mm, for 5, 10 or 15 seconds (in the 15
second laser beam exposure group no gene expression was seen. One
representative point is depicted). The leg was exposed to the laser
beam 10 times in a rotational manner around the site of injection.
Real time in vivo continuous luc expression was monitored with a
biochemiluminescence CCCD system. FIGS. 6B and 6C show mice
injected i.m. into the right and left legs with (B) 10 micro-g and
(C) 15 micro-g pLNC/luc DNA per leg for 5 seconds exposure for the
LBGT side.
[0107] The optimal pulse duration was 10 seconds for gene
expression after one day, however, subsequent experiments revealed
a significant advantage of using a 5 second pulse for inducing
longer expression (see following results).
Intramuscular Gene Delivery Efficiency of the Femtosecond Laser Vs.
Electroporation Compared Over Time
[0108] To comparatively assess the duration of the plasmid
expression with LBGT and EP, the same naked DNA construct was
injected into the tibial muscles of BALB/c mice followed 30 seconds
later by electroporation, or by application of laser beam pulses
(10 pulses rotationally, surface of 95 micro-m.sup.2, focus of 2
mm, pulses duration of 5 seconds, current intensity of 30 mwatt).
Luciferase expression was monitored for over 60 days (FIGS. 6d and
e). While gene expression mediated by EP was higher on day 23
(shown in FIG. 6d), the observed differences between EP and LBGT
disappeared over time and on day 52, expression levels were similar
for both treatment methods (see FIG. 6e for the results with laser
treatment).
Testing of Laser Beam Parameters
[0109] The laser treatment according to the present invention was
further examined with regard to laser current strength (10 mwatt-30
mwatt) to determine its effect on gene expression. Luciferase
expression increased by up to 3-fold at an intensity of 20 mwatt,
but declined thereafter (FIG. 7a). Using the optimized conditions
for these experimental conditions (20 mwatt current strength,
surface area of 95 micro-m.sup.2 and depth of 2 mm), pulse duration
was varied between 3-10 seconds, to examine the relationship
between these parameters. The highest luciferase expression levels
(integrated light units) were achieved with pulses of 5 seconds as
shown in FIG. 7b. Expression efficiency was examined as an outcome
of varying the muscle surface area exposed to the laser beam.
Surface areas ranging from 40 micro-m.sup.2 to 120 micro-m.sup.2
were evaluated, and the optimal surface for these experimental
conditions was determined to be 95 micro-m.sup.2 at a current
intensity of 20 mwatt with a 5-second pulse (data not shown). In
the same experiment laser beam depth of focus into the muscle was
also optimized for these experimental conditions, varying it from 1
to 3 mm, adjusted to the depth of the muscle tissue, and observed
that 2 mm was optimal for these conditions (data not shown).
[0110] It is likely that the application of LBGT to other animal
species will require similar optimization steps, such that
preferably the conditions for laser treatment according to the
present invention are optimized according to the method described
herein for human treatment or for treatment of lower mammals.
Similar processes as described above could optionally and
preferably be performed for different species, and/or treatments
and/or tissues being treated.
[0111] In all subsequent experiments in mice LBGT the optimized
conditions used were: laser beam intensity of 20 mwatt; surface
area of 95 micro-m.sup.2; pulse duration of 5 seconds; and focus of
2 mm. LBGT-assisted gene delivery was compared to naked DNA
administration without assistance, in mice. Both left and right
tibial muscles were injected with the luciferase pLNC/Luc vector,
but only the left leg underwent LBGT (using the conditions
described above). FIGS. 7c and 7d show two controls, for which 10
.mu.g of pLNC/Luc was injected (left leg) followed by application
of laser-beam and pLNC/Luc (right leg) without any pulses. FIGS. 7c
and 7d demonstrate that on both day 1 and day 48, light was emitted
only from the laser treated left leg. These results were
reproducible and consistent in replicate experiments (data not
shown).
Localization and Comparative Evaluation of Gene Expression after EP
and LBGT
[0112] The tissue distribution and localization of reporter genes
was assessed following injection with naked DNA alone, or with EP,
or with LBGT (laser). the reporter genes used were one of green
fluorescence protein (GFP, FIG. 8a), luciferase (FIG. 8b) or
.beta.-galactosidase (FIG. 8c). Plasmids were injected into the
muscle without any pulsed assistance, or with EP or LBGT. Six days
later mice were sacrificed, the injected muscles were excised, and
reporter gene expression levels were determined semi-quantitatively
by direct fluorescence or immunostaining.
[0113] FIG. 8A shows two different electroporation (EP) examples
for muscle injected with GFP as the reporter gene, one plasmid only
(injection of naked DNA only without further treatment) example and
three different laser examples. FIG. 8B shows one negative control
(e.g. no action taken), two positive controls (injection of naked
DNA only without further treatment), two examples of treatment with
EP and injection of naked DNA, and two examples of treatment with
laser and injection of naked DNA, all with luciferase as the
reporter gene, again using muscle samples. FIG. 8C shows three
examples of treatment with EP, two positive controls (injection of
naked DNA only without further treatment), and three examples of
treatment with laser and injection of naked DNA, all with
.beta.-galactosidase as the reporter gene, again using muscle
samples. It should be noted that the above description is provided
according to the order in which the photographs appear within each
figure.
[0114] In the group receiving the naked DNA plasmid expression
cassettes without EP or LBGT, very little gene expression was
detected (FIG. 8a, b, and c). In contrast, when the plasmids were
injected into the muscle, and followed with either EP or LBGT,
groups of muscle cells expressed the reporter genes (FIGS. 8a, b
and c).
Muscle Histological and Biochemical Changes Related to pLNC/Luc
Gene Delivery
[0115] EP is known to induce muscle tissue injury (6, 13) evidenced
histologically by necrosis with intense interstitial edema,
inflammatory cell infiltration, myophagy, in addition to other
signs of muscle degeneration and regeneration (13, 14). To evaluate
the impact of LBGT on muscle tissue of mice, 10 micro-g of plasmid
pLNC/Luc was injected into the tibia muscle followed 30 seconds
later with a pulse from the femtosecond laser using the optimal
conditions described above. Twenty-four, 48 hours and 70 days
later, the mice were sacrificed and the muscle tissues were
examined histologically. No tissue alterations were seen 24 hours
after LBGT. At 48 hours, rare fibers with central nuclei was the
only change observed in the LBGT group, suggesting mild tissue
disturbance. This appeared to be transient and was not observed at
70 days after LBGT when the muscle tissue appeared normal. FIG. 9a
shows muscle tissue after 48 hours; FIG. 9b shows muscle tissue
after 70 days.
[0116] Naked DNA administration to muscle may, in itself, cause
direct muscle injury. To further assess the level of muscle damage
following naked DNA administration, EP or LBGT (laser), creatine
phosphokinase (CPK) levels were determined in the serum of mice two
hours after DNA administration. FIG. 10 shows that the level of CPK
activity levels in mice treated with LBGT was six fold lower
compared to those treated with EP. Surprisingly, CPK levels
observed in the LBGT group were lower than in mice receiving only
naked DNA. This observation was reproducible in a subsequent
experiment (data not shown).
Erythropoietin Expression after LBGT-Assisted Delivery of Naked DNA
Carrying the mEPO Gene.
[0117] To test the therapeutic potential of LBGT method of gene
delivery, a plasmid (pcDNA.3mepo) encoding the mouse erythropoietin
(mEPO) gene was used. In a dose response experiment, the plasmid
was injected into the tibial muscle in a volume of 30 micro-1. The
most effective dose was found to be 10 micro-g (data not shown).
Mice were injected intramuscularly with 10 micro-g of either the
pcDNA.3mepo plasmid, or the backbone plasmid, pcDNA.3 (negative
control) with or without LBGT. Blood was collected from the orbital
plexus at various intervals after treatment, and hematocrit (Hct)
levels were determined. In mice that received pcDNA.3mepo without
LBGT, Hct levels were not different from those of negative control
mice (data not shown). In the group who received pcDNA.3mepo with
LBGT, Hct levels were higher than the levels of those receiving the
negative control plasmid with LBGT, throughout the period of
evaluation, and were significantly (p=0.05) higher at 22% at 8
weeks (FIG. 11).
[0118] These experiments indicate that the laser beam gene
transduction method could be use for inducing gene expression after
naked DNA administration into the muscle and probably to other
organs.
Discussion
[0119] Gene therapy is currently at a critical turning point (15)
from the "proof of principle era," in which some viral based
protocols and therapeutic platforms had shown some promise (16),
into a period where safety issues of the delivery have emerged as
prominent factors in the choice of therapeutic modalities. A recent
report (http://www.esgt.org/; and http://www.asgt.org/) concerning
the development of a lymphoproliferative disorder in a child
treated for X-SCID with an integrating retroviral vector
spotlighted vector safety issues. These risks occur when the viral
and non-viral-integrating (such as the use of transposable
elements) gene delivery methods are utilized (17). While various
novel tools have recently been developed for site-specific
correction of point mutations (18) no level of risk is considered
clinically acceptable. Non-viral gene delivery approaches while
encouraging in some experimental situations, have generally been
relatively unsatisfactory for clinical applications. Various ways
of overcoming the problems associated with non-viral gene delivery
have been explored including: linking viral proteins to DNA to
enhance penetration of cell membranes (19); and novel techniques
which enhance the transport of DNA from the cytosol to the nucleus
such as the use of nuclear targeted receptors like the,
glucocorticoid receptor (20). Another approach that has met with
some success experimentally in small animal models, is
intra-arterial injection of naked DNA (plasmids expressing the gene
of interest) into limb muscle (21). Intrauterine injection of DNA
with microbubble-enhanced ultrasound (22) has also been used
experimentally in fetal gene therapy. However, all of these systems
fall short of their targets, as they are too complicated for
clinical use.
[0120] Currently, the most promising non-viral gene delivery
approach for high protein expression is electroporation (EP) of
muscle into which naked DNA has been injected. In general, two
types of approaches have been tried: high voltage/short pulse; or
low voltage/long pulse. So far, the highest plasma levels of an
encoded protein after muscle EP have been obtained with repeated
low voltage (.about.200 V/cm) at long pulses (20 ms) (23). A recent
report (24), compared adverse outcomes and tissue toxicity of the
EP and adenoviral gene transfer methods. Following muscle EP,
histological examination revealed muscle necrosis with severe
polynuclear and mast cell infiltration, which was maximal at day 7
after treatment. Histological changes were accompanied by elevation
in CPK between day 7 and 14, suggesting the possible development
rhabdomyolysis, which in humans could lead to kidney failure. The
degree of muscle damage and necrosis was correlated with the
voltage intensity. Unfortunately, at lower transducing voltages of
50-100 V/cm gene expression was found to be very low. Furthermore,
the degree of muscle injury was comparable to that observed after
adenovirus-mediated gene administration to the muscle. Hence,
better gene delivery methods are needed to enhance DNA delivery
into muscle tissue as well to other organs. The present invention
provides a simple, more effective and safe gene delivery method
which uses laser pulsed gene transduction.
[0121] In this study, successful expression of a foreign gene in
the tibial muscle of mice was demonstrated, following a femtosecond
laser beam application after injection of naked DNA. This
expression is higher than that observed with naked DNA
administration per se and comparable to that observed when naked
DNA administration combined with EP. The application of the laser
beam to the muscle tissue of the mice proved to be gentle and could
be specifically focused to a small area of a muscle injected with a
low amount of DNA and in a small volume. Moreover, it should be
possible to apply this technique of gene transduction to larger
surface areas of muscle tissue with larger volumes and higher
amounts of DNA. This would simplify targeting of the laser beam to
the desired area, allowing this method to be used for gene delivery
in larger animals or humans. As the laser was not found to be
damaging to muscle tissue, this technique should allow scanning of
a larger skin area of the skin to maximize the number of muscle
fibers that are transfected. Additional technologies could also be
combined with laser beam gene transfer to enhance DNA accessibility
to the muscle such as the use of hyaluronidase and other DNA
uptake-enhancing components that could be mixed with the DNA
solution.
[0122] An alternative and easier target for this laser beam
technology is the skin, itself. Recent reports have suggested
various methods to enhance gene expression in the skin. Some use
retroviral vectors (25) or other integrating technologies (14), and
hence, impose safety considerations. EP protocols were also used
for skin gene transfer for vaccination. Some tissue damage was
apparent with even relatively low energy pulses (26). Hence, naked
DNA injection into the cutaneous tissue followed by the application
of the femtosecond laser beam could also be useful for
vaccination.
[0123] The novel method described in this report is based on the
potential of the femtosecond beam to enhance the transfer of
plasmid DNA across mammalian cell membrane as recently reported
(27). The mechanism by which electric pulses or infrared laser beam
application (femtosecond) induces DNA movement across cell
membranes is not understood at this time. Electrophoretic forces or
laser beam minimal heating effects caused by multiphoton disruption
may cause DNA accumulation at the cell interface, facilitating DNA
insertion though the membrane. The laser beam may also "porate" the
cell membranes, enabling a transient transfer of DNA into the
cellular cytosol and later, into the nuclear compartment. As shown
herein, this method is effective, simple, and safe for use in
muscle tissues of mice. The prolonged expression reporter genes in
mice observed in this study is expected to be reproducible in
studies with larger animals. The focus of the laser beam developed
could be adjusted to between 0 to 10 mm below the skin level. This
could enable expression of the transduced gene in either one of two
tissues, skin or muscle, in humans. Future developments would
probably also open the way for the application of this method to
other organs and indications.
[0124] In conclusion, femtosecond lasers have great potential as a
therapeutic delivery tool in medicine. Their application allows for
tissue effects with minimal collateral damage where the effects of
the light can be confined to a specific layer of tissue under the
skin or for that matter on the skin surface. This can be
accomplished at depths of several millimeters below the surface
with indications in the experiments described herein that such
effects of the fundamental beam can be as deep as a centimeter.
These lasers also provide a good connection to diagnostic
techniques such as optical coherence tomography, which allows for
in situ cytological analysis of the effects of the laser. Thus,
where cold laser approaches are called for, these results indicate
that the femtosecond laser has premier capabilities and will have
great potential in therapeutic protocols. The results of this study
have shown that femtosecond laser based gene transduction may,
indeed, be a breakthrough in the current hiatus of
non-viral-mediated gene therapy.
[0125] The present invention has a number of clear technical
advantages. For example, the present invention includes the
characterization of the optics that is required to accomplish the
specific tissue alterations previously described, while also
allowing for a new modality in gene therapy. The ability to
characterize the higher order laser tissue interaction also permits
the definition of the critical parameters needed for using other
terms in the higher order expression discussed above to allow for
functional imaging without destruction.
[0126] Specifically, the second order term of the higher order
expression can now be used to perform second harmonic generation
without tissue alteration, based on the in vivo parameters that
have been defined by the operation of the present invention with
gene transfection. Second harmonic generation enables membrane
potential to be determined. The in vivo application in cellular
systems or in tissue systems has been difficult without an in vivo
assay for accurately determining the conditions of laser
irradiation for keeping the tissue or cellular system functionally
viable. As a result of this invention, this technique can now be
used for effective diagnosis of membrane potential changes as a
diagnostic of a variety of cellular phenomena, and for defining the
basis of specific cell separation protocols using membrane
potential as the basis of the separation.
[0127] The background art has not taught the user of higher order
laser tissue interaction and/or has not applied this technology
with the characterization methodology that is a preferred
embodiment of the present invention. Such a characterization
methodology is critical to the application of such technologies;
the lack of such characterization is the reason that no therapeutic
application of these technologies is currently available.
Example 2
Characterization of the Laser Tissue Interaction for AMD
Treatment
[0128] The present invention was also tested for the treatment of
AMD. The present invention uses a ultrashort laser in which
focusing can be used to increase the fluence of the laser in a
non-linear fashion to cause multiphoton absorption in a very narrow
range around the focal spot which is under the retina and in the
RPE where the pigment containing droplets form. The optional but
preferred example of such a laser is a femtosecond (for example, 10
-15 sec) laser, as described herein.
[0129] The objective is to cause such multiphoton absorption only
in the tissue containing the fluorescent droplets while leaving the
surrounding tissue untouched by the effects of the laser beam.
Lasers can provide extreme control of such non-linear optical,
multiphoton processes for microscopic illumination [T. Wilson and
C. J. R. Sheppard, Theory and Practice of Scanning Optical
Microscopy (Academic Press, New York 1984]. With such multiphoton
absorption the excited state of the molecule in created and various
studies have shown that a rule of thumb for the life of molecule
before photochemical destruction or photobleaching is approximately
10 5 excursions through a molecular singlet state which is most
probably the excited of the autofluorescent pigments excited in the
RPE. Such singlet states last for approximately 10 -9 secs and
depending on the fluence the time for photochemical destruction of
the molecule is effectively controlled.
[0130] In terms of ophthalmology this is similar to destroying the
membrane that forms behind the lens in approximately 30% of the
cataract removal procedures that are performed. In the case of
cataract, the laser that is used to destroy this membrane is a
nanosecond (10 -9 sec) neodymium yittrium aluminum garnet laser and
the region in the focus of the beam where the fluence is sufficient
to destroy the membrane is much larger than what can be achieved
with present state of the art ultrafast lasers. In addition, in the
case of nanosecond lasers the multiphoton effect that is employed
is dielectric breakdown and associated mechanical destruction of
the membrane and not multiphoton excited state absorption, which is
a much gentler, photochemical, process.
[0131] Photochemical bleaching of the pigments related to AMD could
delay the destructive effects of AMD. Even an alteration of a few
years in the progress of the disease could have a significant
effect on the onset of blindness in these patients who are
significantly older than the average population.
[0132] Materials and Methods
[0133] Biological Preparations
[0134] In-Vitro Investigation
[0135] A series of in-vitro investigations were performed with post
mortem fresh bovine and sheep eyes, which were obtained from a
local slaughterhouse. The anterior segments of the eyes were
removed. The eyes were prepared as an eyecup preparation: the
vitreous was removed and the eyecup was filled with physiological
media. A standard eye irrigation solution was used as the
physiological medium. Fluorescein solution (30-50 .mu.l, 10 mg/ml)
and the synthetic AMD pigment, A2-E, solution, prepared by reported
procedures [R. X.-F. Ren, N. Sakai and K. Nakanishi, J. Am. Chem.
Soc. 119, 3619 (1997)], (30 .mu.l, 0.16 mg/ml) was injected behind
the pigment epithelium and the Bruks membrane of the eyes.
[0136] In-Vivo Investigation
[0137] All investigations involving animals were conformed to the
ARVO resolutions on the Use of Animals in Research. White rats were
used in these experiments. During the experiments, the animals were
anaesthetized by injecting imalgene 1000 (ketamine) (Rhone Merieux,
0.1 ml per 100 g of animal's weight, 100 mg/ml). For opening the
diaphragm of the animal's eye during the procedure, one drop of
mydramide (Fisher, sterile eye drops) and one drop of Efrin-10
(Fisher, sterile ophthalmic solution) was added to the animal's
eye. Also, a contact lens was placed on an animal's eye in order to
allow for viewing the retina. A drop of methylcellulose 2% (oily
eye drops) was put between the animal's cornea and the contact lens
in order to improve contact between the lens and the cornea and to
prevent drying and possible damage to the anterior tissues of an
eye.
[0138] Fluorescein solution (10 .mu.l, 10 mg/ml) was injected
behind the pigment epithelium and the Bruks membrane of the eyes.
After the treatment the animals were sacrificed and the eyes were
taken for pathological examination in the Pathology Division of the
Ophthalmology Department in the Hadassah Hospital. The eyes were
fixed in 4% buffered formaldehyde for at least 48 hours. The tissue
was embedded in paraffin and processed. Sections of 5-6 micron
thickness were produced and were stained with
hematoxylin-eosin.
[0139] Ultra Fast Laser System
[0140] As shown in FIG. 12A, another embodiment of a system 60
features both an in vitro 64 and an in vivo component 66. An
ultrashort laser beam 62, or beam from an ultrashort laser, was
produced by an ultrafast infrared mode-locked Ti:Sapphire laser
(Coherent, Mira 900), as a non-limiting example of an ultrashort
laser. It was pumped by an argon ion laser (Coherent, Innova 200)
that was operated at 12 W in a multi-line mode (not shown). The
operating wavelength was 800 nm, the pulse frequency was 76 MHz,
and the pulse duration was about 0.200 psec. Beam diameter was 0.7
mm.
[0141] For the in vitro research (shown as component 64), laser
beam 62 was transmitted via an upright microscope (Zeiss, Axioskop)
68 and was focused by .times.40 N.A. 0.75 objective (Zeiss) on a
sample 70 as described above. A CCD camera 74 (in this example a
Sony camera, model SSC-C374) was attached to microscope 68 in order
to allow for monitoring the procedure on-line. A filter 72, in this
example a green filter, was placed in front of CCD camera 74 in
order to cut off the infrared illumination of beam 62 (from the
Ti:Sapphire laser). CCD camera 74 was connected to a TV
(television) system 76 with a video recorder 78 where the relevant
eye tissues and the dynamics of the green fluorescent excitation of
the fluorescein or synthetic drusen were observed and recorded.
[0142] For the in vivo research (shown as component 66), laser beam
62 was transmitted via a slit-lamp 80, in this example a standard
slit-lamp widely used in ophthalmological diagnosis. The slit-lamp
magnification used was 16, 25 and 40. Slit-lamp also featured a
beamsplitter 82. The infrared beam was focused under the retina of
a rat's eye 86 using the slit-lamp lens (not shown) and a contact
lens 84. The process was monitored and recorded as mentioned above,
using (for the purpose of this example) the same CCD camera with
the green filter attached to the slit-lamp and connected to the
recording system, shown herein as a filter 88, a CCD camera 90, a
TV system 92 and a recorder 94. Electrophysiological measurements
can optionally be performed at the same time in this
arrangement.
[0143] FIG. 12 B shows a diagrammatic representation of a few
components of the system of FIG. 12A, with more details of the eye
in the sample. As shown, a laser 96, optionally and preferably the
ultrashort laser of FIG. 12A, such as a femtosecond laser for
example, produces a beam that is focused by a lens 98 onto a retina
102 of an eye 100 in the sample. The beam then destroys or at least
reduces AMD related pigments (shown as reference number 104) which
interfere with the vision of the subject.
[0144] Results and Discussion
[0145] In-Vitro Investigation
[0146] The experiments were performed on six bovine and six sheep
eyes using fluorescin as a fluorescent material injected under the
retina. The Ti:Sapphire laser beam intensity was 25-30 mW in these
initial experiments where the objective was simply to address the
geometric issues and the general feasibility of the experiment.
With A2-E two bovine and three sheep eyes were investigated. The
results of photobleaching are shown in Table 1. The duration of the
photobleaching varied from case to case with the minimum being a
few seconds, and the maximum being close to 3 minutes. This
variation may probably be caused by parameters such as local
concentration of the fluorescent material, exact location of the
illuminated point behind the retina, etc.
TABLE-US-00001 TABLE 1 Number Type Duration Type of the of case of
the eye of bleaching fluorescent material 1 cow 0 min 6 sec
fluorescein 2 cow 0 min 10 sec fluorescein 3 cow 2 min 50 sec
fluorescein 4 cow 2 min 56 sec fluorescein 5 cow 0 min 27 sec
fluorescein 6 cow 0 min 36 sec fluorescein 7 sheep 2 min 47 sec
fluorescein 8 sheep 2 min 12 sec fluorescein 9 sheep 0 min 22 sec
fluorescein 10 sheep 0 min 23 sec fluorescein 11 sheep 0 min 49 sec
fluorescein 12 sheep 0 min 28 sec fluorescein 13 cow 1 min 05 sec
A2E 14 cow 0 min 33 sec A2E 15 sheep 0 min 07 sec A2E 16 sheep 0
min 04 sec A2E 17 sheep 0 min 04 sec A2E
[0147] In-Vivo Investigation
[0148] Further experiments were performed with live animals (rats).
These experiments were performed in a manner that would simulate as
close as possible the real situation that is associated with human
eyes. In order to do this, fluorescein was finely injected through
the back tissues of the rat's eye under the Bruks membrane. The
objective was to cause the absorbing material to tightly associate
with the relevant tissues under the animal's retina, where the
droplets of the AMD pigments would be formed in the case of a human
eye. The beam of Ti: Sapphire Ultrafast laser, with an intensity of
40-65 mW, that exited from the slit lamp was then focused under the
animal's retina in order to see the bright green spot of
fluorescein or A2-E fluorescence.
[0149] The goal was, firstly, to see the fluorescent spot from the
region under the retina, which is a result of multiphoton excited
fluorescence, and secondly, to follow the dynamics of the bleaching
of this spot and to see whether it decreases in its intensity as a
result of photobleaching. Indeed, in experiments described below,
the bright fluorescence spot from the relevant region was seen,
with a gradual decrease of the spot intensity.
[0150] In order to check for permanent bleaching, control
experiments were performed, where the laser beam was allowed to
strike the stained fluorescein tissue for a few seconds, and was
allowed to illuminate the spot again after it was blocked for as
much as 15-20 min in order to view the effect of dye diffusion in
the stained tissue. The fluorescent spot brightness did not change,
indicating that there was no fluorescein diffusion to or from the
adjacent tissues, and that the only reason that could be
responsible for the intensity decrease was photobleaching. The time
required for nearly complete photobleaching was on the order of
5-10 min but in seconds the intensity of the fluorescence began to
drop as a result of the action of the laser. In the following
frames from the simultaneously recorded video, shown in FIG. 13,
the typical dynamics of the decrease in the fluorescence intensity
is seen (the frames proceed sequentially in time from top left to
bottom right), as photobleaching occurs. The graph shown in FIG. 14
is representative of the typical rate of bleaching for this case.
Total intensity is shown at the y-axis, while time is shown in
seconds at the x-axis; clearly the intensity of the fluorescence
decreased over time due to bleaching. Table 1 shows that the
photobleaching rate of A2-E was 100 times faster than that of
fluorescein.
[0151] Each different type of eye can optionally be monitored with
the on-line measurements of physiological viability mentioned
above, although it is important to note that these are not the only
possible tests for viability. These measurements readily show that
there is little or no damage caused to the retina and the adjacent
tissues.
[0152] A control experiment was also performed, in which the
intensity of the Ti:Sapphire laser beam was only increased up to
400 mW, and then focused under the retina as for the experiments
described above. Four points under the retina around the central
optical nerve were illuminated by the laser beam. The exposure time
for each point was 10 min. Also, a larger magnification factor of
the slit lamp was used (.times.25 instead of .times.16 used
previously) in order to decrease the beam spot size at the retina
and to further increase the energy density at these points. Under
these conditions, various physiologically relevant tests may
optionally be performed for determining the ultra low collateral
damage levels that are relevant for disease perturbation (the above
exemplary test was performed on these samples; data not shown). The
results clearly demonstrated that even for much stronger
intensities than in the previous series of experiments, the laser
beam intensity caused little or no damage.
[0153] In summary, it has been shown that effective photobleaching
of dyes including A2-E beneath the retina can be induced, while
causing little or no change and/or damage in the retina itself and
the surrounding tissue.
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