U.S. patent application number 16/917809 was filed with the patent office on 2020-10-22 for gene silencing in skin using self-delivery sirna delivered by a meso device.
The applicant listed for this patent is TransDerm, Inc.. Invention is credited to Roger L. Kaspar, Tycho Speaker.
Application Number | 20200330738 16/917809 |
Document ID | / |
Family ID | 1000004931023 |
Filed Date | 2020-10-22 |
United States Patent
Application |
20200330738 |
Kind Code |
A1 |
Kaspar; Roger L. ; et
al. |
October 22, 2020 |
GENE SILENCING IN SKIN USING SELF-DELIVERY siRNA DELIVERED BY A
MESO DEVICE
Abstract
The present invention is drawn to a low-cost "meso" device that
is able to effectively deliver functional self-delivery siRNA to
subject and inhibit expression of a gene in the subject as well as
related methods. In particular, a method of transdermally
delivering nucleic acid material to a subject is provided. The
method includes adapting a motorized meso machine for delivery of
nucleic acid material; introducing nucleic acid material into a
chamber of the motorized meso machine; and contacting the motorized
meso machine to a skin surface of a subject for a period of time
sufficient to deliver the nucleic acid material into the skin
surface of the subject.
Inventors: |
Kaspar; Roger L.; (Santa
Cruz, CA) ; Speaker; Tycho; (Santa Cruz, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TransDerm, Inc. |
Salt Lake City |
UT |
US |
|
|
Family ID: |
1000004931023 |
Appl. No.: |
16/917809 |
Filed: |
June 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15668606 |
Aug 3, 2017 |
10695548 |
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16917809 |
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14181582 |
Feb 14, 2014 |
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15668606 |
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61764928 |
Feb 14, 2013 |
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61887519 |
Oct 7, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61M 37/0092 20130101;
A61M 37/0015 20130101; A61M 2037/0023 20130101; A61M 2037/0061
20130101 |
International
Class: |
A61M 37/00 20060101
A61M037/00 |
Claims
1. A method for delivering nucleic acid material for treatment of a
monogenic skin disorder, the method comprising: introducing a
nucleic acid material into a chamber of a motorized meso machine,
the motorized meso machine comprising microneedles operatively
coupled to the chamber; contacting the microneedles of the
motorized meso machine to a skin surface of a subject in need of
the treatment; and actuating the motorized meso machine such that
the microneedles are inserted into the skin surface of the subject
for a period of time for delivering a therapeutically effective
dose of the nucleic acid material from the chamber into the skin
surface of the subject.
2. The method of claim 1, wherein the nucleic acid material
comprises siRNA or sd-siRNA configured to silence a gene, a
mutation in which results in the monogenic disorder.
3. The method of claim 1, wherein the monogenic disorder is
pachyonychia congenita.
4. The method of claim 5, wherein the nucleic acid material
comprises TD101 siRNA.
5. The method of claim 1, wherein the steps of contacting the
microneedles and actuating the motorized meso machine are repeated
periodically on a same area on the skin surface so as to
periodically deliver the nucleic acid material to the same
area.
6. The method of claim 1, wherein step of contacting the
microneedles and actuating the motorized meso machine are repeated
on an adjacent skin area so as to deliver the nucleic acid material
to the adjacent skin area.
7. The method of claim 1, wherein the microneedles are configured
to deliver the nucleic acid material into the skin surface at a
depth in a range from 25 .mu.m to 3 mm.
8. The method of claim 1, wherein the period of time is at about 5
seconds to about 20 seconds.
9. The method of claim 1, wherein the subject is a human.
10. The method of claim 1, further comprising adjusting, following
an insertion of the microneedles into the skin surface, a rate of
oscillation of the microneedles by the motorized meso machine.
11. A device for treatment of a monogenic skin disorder, the device
comprising: a motorized meso machine having: a chamber configured
to contain a volume of a the nucleic acid material; and
microneedles operatively coupled to the chamber such that upon
contacting the microneedles to a skin surface of a subject and
actuating the motorized meso machine for a period of time, the
nucleic acid material flows from the chamber onto the skin surface
and the microneedles are inserted into the skin surface to deliver
a predetermined volume of the nucleic acid material from the
chamber into the skin surface of the subject at a predetermined
depth.
12. The device of claim 11, wherein the device is configured to
oscillate the microneedles at an adjustable a rate of
oscillation.
13. The device of claim 11, wherein microneedles have a length in a
range from 25 .mu.m to 3 mm.
14. The device of claim 11, wherein the predetermined depth to
which the nucleic acid material is delivered is in a range from 25
.mu.m to 3 mm.
15. The device of claim 11, wherein the predetermined volume is in
a range from 10 .mu.l to 500 .mu.l.
16. The device of claim 11, wherein the period of time is in a
range from 5 seconds to 20 seconds.
17. The device of claim 11, wherein the nucleic acid material
comprises a nucleic acid and an indicator.
18. The device of claim 11, wherein the subject is a mammal.
19. The device of claim 11, wherein the nucleic acid material
comprises siRNA or sd-siRNA.
20. The device of claim 11, wherein the nucleic acid material is
suspended in a solution comprising a phosphate buffer.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/668,606, filed Aug. 3, 2017, which is a
continuation of U.S. patent application Ser. No. 14/181,582, filed
Feb. 14, 2014, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/764,928, filed Feb. 14, 2013, and U.S.
Provisional Patent Application Ser. No. 61/887,519, filed Oct. 7,
2013, each of which are incorporated herein by reference.
BACKGROUND
[0002] Of the 7,000 known monogenic disorders, approximately 2,000
affect the skin. While most of these are individually rare,
together they represent a significant healthcare burden and afflict
up to 1% of the population. For most of these disorders, there are
no effective treatments that target the root cause of the problem.
Nucleic acid therapies, including siRNAs, are a potential way to
modify expression of disease genes in a controlled fashion, and
hold real promise for improving patient lives. While traditional
"small molecule" approaches to drug development have been a
successful model for large pharmaceutical companies, the cost (on
the order of a $1 billion) and the length of development time
(10-12 years) limit their usefulness is rare inherited skin
disorder. Identification of potent and selective siRNAs with
limited off-target effects is now routine in many laboratories and
the cost and time involved is a fraction of what is required for
small molecule drug development. The missing piece in translating
siRNA technology to the clinic is a robust, reproducible,
economical and "patient-friendly" (i.e., little or no pain)
delivery platform. Substantial effort has been invested in a
variety of delivery technologies, with increasing success. However,
the complexity and cost may limit clinical translation and patient
compliance. For example, the first administration of siRNA to skin,
and the first siRNA to target a mutant gene, was for pachyonychia
congenita, a rare genodermatoses caused by mutant keratin alleles.
The intradermal injection of TD101 siRNA (targets a single nt
mutation [N171K] in the keratin 6a gene) resulted in improvement in
the keratoderma and lesion pain, but the painfulness of the
intradermal injection necessitated use of oral pain medication and
a regional nerve block and prevented further enrollment in the
clinical trial.
BRIEF DESCRIPTION OF DRAWINGS
[0003] FIG. 1A shows a handheld motorized meso/microneedles array
device.
[0004] FIG. 1B shows the inner reservoir of the motorized
microneedle array cartridge array contains 300 .mu.L of a red dye
solution for visualization, and shows the needles are set to
protrude 0.1 mm beyond the edge of the chamber.
[0005] FIG. 1C shows channels intermittently located between the
needles allow flow of solution onto the surface of the skin during
treatment.
[0006] FIG. 2A shows Cy3 labeled sd siRNA distribution in mouse
flank skin treated by intradermal injection with a motorized
microneedle array device loaded with 50 .mu.L 0.1 mg/mL Cy3-labeled
sd-siRNA. These sections of hairless mouse flank skin show breaches
in the epidermis center of the injection site. The individual
images were stitched together using ICE software.
[0007] FIG. 2B shows magnification (10.times.) of the meso-treated
and intradermally-injected skin.
[0008] FIG. 2C shows further magnification of images from 2B
showing diffusion of the Cy3-labeled siRNA in the epidermis
originating from the needle penetration site (arrow). Left panels:
4'6-diamidino-2-phenylindole and Cy3 overlay; right panels: Cy3
alone.
[0009] FIG. 2D shows distribution of fluorescently labeled sd-siRNA
in human abdominoplasty skin. Skin breaches due to penetration of
the motorized microneedles are seen in their entirety using a
.times.5 objective. Intradermally injected skin (50 .mu.L of 0.1
mg/ml Cy3-labeled sd-snRNA) was similarly sectioned and imaged.
[0010] FIG. 2E shows magnification of treated skin.
[0011] FIG. 2F shows further magnification of the images from 2E
shows diffusion of the Cy3-labeled sd-siRNA in the epidermis
originating from the needles penetration site (arrow), whereas low
levels of fluorescence are observed in the epidermis following
intradermal injection. Left panels: 4'6-diamidino-2-phenylindole
and Cy3 overlay; right panels: Cy3 alone. Nuclei are visualized by
4'6-diamidino-2-phenylindole stain (blue). Scale bar=200 .mu.m.
[0012] FIG. 3A shows a graphical representation of data showing
that meso-assisted delivery of sd-siRNA inhibits targeted reporter
gene expression from the following procedure. Hairless tg CBL/hMGFP
mouse flank skin was treated daily with the meso device loaded with
50 .mu.L 10 mg/mL CBL3 sd-siRNA or non-specific control sd-siRNA
(sd-TD101) for 10 days. On day 11, the mice were sacrificed and the
treated skin was excised for RTqPCR analysis and fluorescence
imaging. Total RNA was isolated from the epidermis of the excised
skin, reverse transcribed and hMGFP mRNA levels (relative to K14)
were quantified in triplicate by qPCR. Bars indicate standard
error.
[0013] FIG. 3B shows representative fluorescence images (bottom)
with bright field overlay (top) of frozen skin sections (10 .mu.m)
prepared from treated mice showed knockdown of hMGFP signal
fluorescence signal in the skin treated with specific sd-siRNA over
control sd-siRNA. Nuclei are visualized by
4',6-diamidino-2-phenylindole stain (blue). Scale bar is 100
.mu.m.
[0014] FIG. 4A-4C show confocal fluorescence imaging of Cy3-labeled
sd-siRNA in human skin xenografts. A,B. Following meso assisted
delivery (30-60 min) of 100 .mu.L 0.5 mg/mL Cy3-labeled sd-siRNA
(in Phosphate Buffered Saline, hereinafter "PBS") into human skin,
reflectance (panel A) and red fluorescence (panel B, C) were imaged
with the Lucid Vivascope system)
[0015] FIG. 5A shows a graphical representation of a procedure
where the meso chamber was loaded with the indicated volume of PBS
solution and applied to mouse flank skin at the 0.1 mm depth
setting for 10 s. The solution remaining in the chamber and on the
surface of the skin was collected and measured (red bars).
[0016] FIG. 5B shows images from the following procedure. To
confirm the volume delivered as described in A, 50 .mu.L of labeled
siRNA was loaded in the chamber and an equivalent volume of the
predicted delivery volume using the meso device (16 .mu.L) was
intradermally injected adjacent to the meso treatment site and
imaged with the IVIS Lumina II.
[0017] FIG. 6A shows distribution of fluorescently-labeled siRNA in
frozen sections prepared from meso-treated skin, specifically,
Cy3-labeled siRNA distribution in human skin. Human abdominoplasty
skin was treated with the meso device loaded with 50 .mu.L 0.1
mg/mL Cy3-labeled sd-siRNA (Accell) or injected intradermally with
50 .mu.L of the same solution. Skin breaches due to meso needle
penetration are seen in their entirety using a 5.times. objective
as well as a distribution gradient in signal from the delivery
site. Intradermally-injected skin was sectioned to the center of
the injection and similarly imaged. The individual images (stitched
together using ICE software, see Materials and Methods) show
similar levels of fluorescence when comparing the meso-treated and
ID-injected skin sites.
[0018] FIG. 6B shows magnification (10.times. objective) of the
meso-treated skin showed diffusion of the Cy3-labeled siRNA in the
epidermis originating from the needle penetration site, whereas low
levels of fluorescence was observed in the epidermis following
intradermal injection. Left panels: DAPI and Cy3 overlay; right
panels: Cy3 alone. Scale bar=400 .mu.m.
SUMMARY
[0019] As set forth herein, the present invention is drawn to a
low-cost "meso" device that is able to effectively deliver
functional self-delivery siRNA to a subject and inhibit expression
of a gene in the subject as well as related methods. Accordingly, a
method of transdermally delivering nucleic acid material to a
subject is provided. The method includes adapting a motorized meso
machine for delivery of nucleic acid material; introducing nucleic
acid material into a chamber of the motorized meso machine; and
contacting the motorized meso machine to a skin surface of a
subject for a period of time sufficient to deliver the nucleic acid
material into the skin surface of the subject.
DETAILED DESCRIPTION OF EXAMPLE EMBODIMENT(S)
[0020] Before the present devices, formulations, systems and
methods for the delivery and use of nucleic acid materials are
disclosed and described, it is to be understood that this invention
is not limited to the particular process steps and materials
disclosed herein, but is extended to equivalents thereof, as would
be recognized by those ordinarily skilled in the relevant arts. It
should also be understood that terminology employed herein is used
for the purpose of describing particular embodiments only and is
not intended to be limiting.
[0021] It should be noted that, the singular forms "a," "an," and,
"the" include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a nucleic acid
material" includes reference to one or more of such nucleic acids
materials, and reference to "the motorized meso machine" includes
reference to one or more of such motorized meso machines.
[0022] As used herein, "subject" refers to a mammal in having a
condition for which rapamycin is a therapeutically effective
treatment. In some aspects, such subject may be a human.
[0023] As used herein, the term "motorized meso machine" or
"motorized meso device" are used interchangeably and refers to a
motorized microneedle device that when placed on a skin surface can
cause the microneedles to penetrate a skin surface due to vibration
caused by the device. Such devices are well known in the cosmetic
and dermal arts. A commercially available example of such a device
is the Tiple-M or TriM by Bomtech Electronic Co, Seoul, South
Korea).
[0024] As used herein, the term "about" is used to provide
flexibility to a numerical range endpoint by providing that a given
value may be "a little above" or "a little below" the endpoint.
[0025] The terms, "comprises," "comprising," "containing" and
"having" and the like can have the meaning ascribed to them in U.S.
Patent law and can mean "includes," "including," and the like, and
are generally interpreted to be open ended terms. The terms
"consisting of" or "consists of" are closed terms, and include only
the components, structures, steps, or the like specifically listed
in conjunction with such terms, as well as that which is in
accordance with U.S. Patent law. "Consisting essentially of" or
"consists essentially of" have the meaning generally ascribed to
them by U.S. Patent law. In particular, such terms are generally
closed terms, with the exception of allowing inclusion of
additional items, materials, components, steps, or elements, that
do not materially affect the basic and novel characteristics or
function of the item(s) used in connection therewith. For example,
trace elements present in a composition, but not affecting the
compositions nature or characteristics would be permissible if
present under the "consisting essentially of" language, even though
not expressly recited in a list of items following such
terminology. When using an open ended term, like "comprising" or
"including," it is understood that direct support should be
afforded also to "consisting essentially of" language as well as
"consisting of" language as if stated explicitly.
[0026] As used herein, compounds, formulations, or other items may
be presented in a common list for convenience. However, these lists
should be construed as though each member of the list is
individually identified as a separate and unique member. Thus, no
individual member of such list should be construed as a de facto
equivalent of any other member of the same list solely based on
their presentation in a common group without indications to the
contrary.
[0027] Concentrations, amounts, and other numerical data may be
expressed or presented herein in a range format. It is to be
understood that such a range format is used merely for convenience
and brevity and thus should be interpreted flexibly to include not
only the numerical values explicitly recited as the limits of the
range, but also to include all the individual numerical values or
sub-ranges encompassed within that range as if each numerical value
and sub-range is explicitly recited. As an illustration, a
numerical range of "about 0.5 to 10 g" should be interpreted to
include not only the explicitly recited values of about 0.5 g to
about 10.0 g, but also include individual values and sub-ranges
within the indicated range. Thus, included in this numerical range
are individual values such as 2, 5, and 7, and sub-ranges such as
from 2 to 8, 4 to 6, etc. This same principle applies to ranges
reciting only one numerical value. Furthermore, such an
interpretation should apply regardless of the breadth of the range
or the characteristics being described.
[0028] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, representative methods, devices, and materials are
described below.
[0029] Reference will now be made in detail to preferred
embodiments of the invention. While the invention will be described
in conjunction with the preferred embodiments, it will be
understood that it is not intended to limit the invention to those
preferred embodiments. To the contrary, it is intended to cover
alternatives, variants, modifications, and equivalents as may be
included within the spirit and scope of the invention as defined by
the appended claims.
[0030] Despite the development of potent siRNA molecules that
effectively target genes responsible for skin disorders,
translation to the clinic has been hampered by the difficulty of
efficient delivery through the stratum corneum barrier into the
live skin cells. Although intradermal injection of siRNA using
hypodermic needles results in reproducible gene silencing, this
approach is limited by the size of a single injection and is
painful. The use of microneedle arrays is a less painful method for
siRNA delivery, but limited payload capacity limits this approach
to highly potent molecules. In the present case, a device that
combines useful elements of both hypodermic needles and array
technologies was used to effectively deliver functional siRNA to
mouse and human skin. This commercially-available device utilizes
an array of vibrating, adjustable-height needles, facilitating
delivery of cargo solution through the stratum corneum with little
to no pain. Treatment of both human and murine skin resulted in
distribution throughout the treated skin (including the epidermis).
Efficient silencing (58% reduction) of reporter gene expression was
achieved in a transgenic reporter mouse skin model.
[0031] siRNAs are promising agents for treating monogenic skin
disorders particularly those caused by dominant mutations, if
delivery concerns can be overcome. The use of siRNAs as
therapeutics has made substantial progress in recent years and
clinical trials are underway for treatment of a variety of
disorders in eye, liver, kidney and skin. Due to the accessibility
of skin, direct injection of "naked" nucleic acids has been
suggested as the most simple, safe and efficient delivery method.
However, direct injections are limited to a highly localized region
of the epidermis coincident to the injection site, and large number
of injections may be needed to achieve uniform delivery and a
therapeutic outcome.
[0032] The first siRNA used in skin, TD101, targeted a mutant
version (N171K) of keratin 6a, which is one of the mutations
responsible for the dominant negative monogenic skin disorder
pachyonychia congenita (PC). TD101 was also the first siRNA used in
the clinic to target a mutant gene. Improvements in PC symptoms
were observed at the plantar site of siRNA intradermal injection in
the single patient initially enrolled in the study, but not the
paired injection site on the opposite foot that received vehicle
alone. Intradermal injections of either siRNA or vehicle alone were
accompanied by severe pain, necessitating nerve blocks as well as
oral pain medication on the treatment days. Additional patients
were not enrolled due to the intense pain associated with these
injections. The intense pain associated with intradermal injection
leads to the need to explore alternative "patient-friendly"
delivery technologies (i.e. effective delivery of functional siRNA
with little or no pain).
[0033] Multiple physical approaches have been reported in the
literature to facilitate delivery across the stratum corneum
barrier including ultrasound, erbium:YAG laser, gene gun (ref),
iontophores is, electroporation and microneedles. Once the siRNA
passes the stratum corneum barrier, however, the affected cells
must still internalize the siRNA in a manner that allows for
incorporation into the RNA-induced silencing complex (RISC). Naked
siRNA is not normally taken up by keratinocytes in the absence of
transfection unless the siRNA administration is accompanied with
pressure ("pressure-fection"). It has been shown that covalent
"self-delivery" modifications (including Dharmacon's Accell
modifications), facilitate cellular uptake in vitro and in vivo
without the need for tranfection reagents. Additionally, it has
been previously reported that administration of these self-delivery
siRNA by dissolvable microneedle arrays could reduce target gene
expression up to 50% in both mouse and human skin models. The
nearly 60% reduction in target gene expression reported herein
delivering sd-siRNAs with the meso device warrants additional study
for use in patients and represents an alternative path to using
microneedle arrays for delivery across the stratum corneum
barrier.
[0034] With the above in mind, the present invention is drawn to a
low-cost "meso" device that is able to effectively deliver
functional self-delivery siRNA to subject and inhibit expression of
a gene in the subject as well as related methods. Accordingly, a
method of transdermally delivering nucleic acid material to a
subject is provided. The method includes adapting a motorized meso
machine for delivery of nucleic acid material; introducing nucleic
acid material into a chamber of the motorized meso machine; and
contacting the motorized meso machine to a skin surface of a
subject for a period of time sufficient to deliver the nucleic acid
material into the skin surface of the subject. The adapting of the
motorized meso device can include adjusting the depth of needle
penetration for the device and/or adjusting the oscillation rate of
the needles. It is noteworthy that it is theoretically possible
that the "adapting" step of the claimed invention may not require
any affirmative adjustment of the device, rather a mere checking of
the settings of the device to assure that they are at the desired
setting for a given application. In one aspect, the needles of the
motorized meso machine includes deliver the nucleic acid material
into the skin surface at a depth of about 25 microns to about 3
mm.
[0035] In one embodiment, the nucleic acid material delivered by
the motorized meso machine can be siRNA. In another embodiment, the
nucleic acid material can be sd-siRNA. In some aspects, it can be
useful to utilize nucleic acid material that is suspended in a
solution. In one embodiment, the nucleic acid material can be
suspected in a PBS solution. Other solvents or liquid carriers
known in the art to be compatible with nucleic acid material can
also be used.
[0036] Once the motorized meso machine is applied loaded with the
nucleic acid material, the machine can contact the skin of a
subject being treated for a period of time sufficient to allow for
delivery of the nucleic acid. In some embodiments, the period of
time of contacting can be for a period of about 5 seconds to about
20 seconds. In another embodiment, the period of time can be about
7 seconds to about 15 seconds. The contacting can be repeated in
the same skin area or can be repeated on other skin areas, e.g.
adjacent skin areas in order to provide the desired delivery.
[0037] The disclosed invention provides a strong alternative to
traditional microneedles and hypodermic needle injections.
EXAMPLES
[0038] The following examples are provided to promote a more clear
understanding of certain embodiments of the present invention, and
are in no way meant as a limitation thereon. The compositions may
be suitably modified by a person skilled in the art. The following
materials and methods were utilized in the Examples described
herein.
[0039] Animals
[0040] Hairless mice (6-8 weeks old) were purchased from Charles
River Laboratories (Wilmington, Mass.) and housed at TransDerm.
Hairless Tg CBL/hMGFP mice were generated by breeding Tg CBL/hMGFP
mice on a Hairless background and maintained at Stanford
University. Animals were treated according to the guidelines of the
National Institutes of Health (NIH), TransDerm and Stanford
University.
[0041] siRNA
[0042] Unmodifed specific (CBL3 and non-specific siRNAs (TD101
K6a3'UTR.1 NSC4 and "self-delivery" Accell.RTM. specific (sd-CBL3
and non-specific (sd-TD101 sd-CD44 and Cy3-labeled (ref)) siRNAs
containing Dharmacon-proprietary modifications allowing for
cellular uptake without traditional transfection reagent) were
synthesized by Thermo Fisher Scientific, Dharmacon Products
(Lafayette, Colo.); Accell siRNAs are available commercially from
this source.
Example 1--Delivery of siRNA Cargo into the Epidermis by the Meso
Device
[0043] Despite the promise of siRNA for use as skin therapeutics, a
major obstacle is delivery of these molecules through the stratum
corneum barrier due to their size (.about.13,000 MW) and
polyanionic nature. Meso devices can be used to deliver a variety
of molecules across this barrier through direct penetration using
vibrating needles that are provided as a single use sterile
disposable cartridge (FIG. 1). As the depth of needle penetration
can be adjusted, this device has the potential to deliver cargo to
different skin types ranging from thin mouse skin (<50 um) to
thick human plantar skin (>1 mm). Furthermore, the ability to
adjust depth allows for deposition in the epidermis, avoiding the
pain nerve fibers that are prevalent in the dermis.
[0044] To determine the amount of potential cargo that can be
delivered to skin, the meso device was loaded with 50, 100, 200 or
300 .mu.L of PBS solution. After application to murine flank skin
for 10 s (set to deliver at a depth of 0.1 mm), the solution
remaining in the meso device and on the surface of the skin was
collected and measured and used to calculate the total volume
delivered. Once a threshold volume of 100 .mu.L was loaded into the
device, adding additional solution did not result in increased
cargo delivery (FIG. 5A). Thus, in mouse flank skin, a maximal
delivered volume of 40 .mu.L occurred when 100 .mu.l of solution
was loaded into the cartridge. In order to confirm that delivery
was occurring, murine flank skin was treated with the meso device
loaded with 50 .mu.L of 0.1 mg/mL fluorescently-tagged sd-siRNA
(Cy3-Accell siRNA, see Materials and Methods). The calculated
volume delivered by meso (16 .mu.L) of the same solution was then
intradermally injected adjacent to the meso-treated area.
Fluorescence was measured by in vivo imaging and resulted in
similar intensities (FIG. 5B).
[0045] In order to visualize siRNA distribution in the skin, mice
were sacrificed 1 h following treatment with the
fluorescently-tagged siRNA, and the treated skin was embedded in
OCT, sectioned (10 .mu.m) and analyzed by fluorescence microscopy.
Meso-assisted delivery resulted in a gradient of
fluorescently-tagged siRNA distribution throughout treated area of
the skin with peak intensity observed at the site of needle
injection (FIG. 2A). Importantly, the bulk of fluorescent signal
was observed migrating laterally through the epidermis from the
needle penetration site (FIG. 2B). Intradermal injection of the
fluorescently-tagged siRNA also resulted in distribution of signal
throughout the dermis and epidermis (FIG. 2A and data not shown).
The distribution of labeled sd-si-RNA in human skin was similarly
analyzed (FIG. 2D). As in mouse skin, the fluorescent signal was
observed in a gradient pattern from the site of needle penetration
including lateral distribution through the epidermis (FIG. 2E, F).
In contrast to the distribution pattern observed upon intradermal
injection in mouse flank (FIG. 2C), less labeled siRNA was detected
in the epidermis of the human skin following intradermal injection
with a hypodermic needles (FIG. 2E, F), consistent with previous
experiments in both human abdominal explant skin and mouse footpad
skin.
Example 2--Silencing of CBL/hMGFP Reporter Gene in Transgenic Mouse
Epidermis
[0046] It has been previously reported silencing of a fluorescent
reporter gene in a tg mouse skin model (Tg CBL/hMGFP) after
administration of unmodified and self-delivery CBL3 siRNA by
intradermal injection and microneedle application, respectively. In
order to evaluate the ability of the meso device to deliver
functional sd-CBL3 siRNA in this model, Tg CBL/hMGFP mouse flank
skin was treated and reporter gene expression analyzed. Flank skin
was treated every day for 10 days. On day 11, the mice were
sacrificed and flank skin was excised for RNA isolation and
histology. Reporter mRNA (CBL/hMGFP) levels were measured by
RT-qPCR (FIG. 3A). A significant reduction (averaging 58.+-.5%) of
reporter expression was detected in skin treated with the specific
CBL3 sd-siRNA compared to the contralateral flank skin treated with
non-specific control sd-siRNA (sd-CD44 siRNA). This experiment was
repeated comparing CBL3 sd-siRNA to non-specific sd-TD101 siRNA
with similar results (data not shown). The decreased hMGFP levels
were corraborated by fluorescence microscopy of CBL3
sd-siRNA-treated skin compared to control sd-siRNA treatment (FIG.
3B). Fluorescence images of hMGFP were overlaid with
4',6-diamidino-2-phenylindole (DAPI) and bright field images to
locate the basal layer and stratum corneum.
Example 3--Distribution of Fluorescently-Tagged siRNA Following
Meso-Assisted Delivery in Human Skin
[0047] In order to visualize delivery of sd-siRNA in a human model,
freshly-obtained explant skin (from plastic surgery procedures) was
treated for 10 s with the meso device set at a depth of 100 .mu.m.
50 .mu.L of 0.1 mg/mL Cy3-labeled sd-siRNA was delivered to skin
for cryosectioning while 100 .mu.L of 0.5 mg/mL Cy3-labeled
sd-siRNA was delivered to skin for in vivo confocal imaging. For
cryosectioning, the skin was embedded in OCT 1 h post-treatment,
sectioned and imaged by fluorescent microscopy (FIG. 6A). Similar
to delivery of labeled sd-siRNA to mouse skin, the fluorescent
signal was observed in a gradient pattern from the site of needle
penetration including diffusion laterally through the epidermis
(FIG. 6B). Interestingly, lower signal was detected in the
epidermis of intradermally-injected skin (FIG. 6B and data not
shown).
[0048] siRNA distribution was also visualized in human skin using
confocal imaging. Skin removed during a rhytidectomy procedure was
treated with 100 .mu.L 0.5 mg/mL Cy3-labeled sd-siRNA and imaged
using the Lucid Vivascope 30-60 min post-treatment. Reflectance
images collected at 658 nm show the needle penetration site (FIG.
4A) at 26 .mu.m depth. Red fluorescence (ex. 532 nm; em. 607 nm) at
59 .mu.m depth shows radial distrubution from the needle
penetration side (FIG. 4B) and interaction with individual cells
(FIG. 4C).
Example 3--Meso-Assisted Delivery of sd-siRNA
[0049] A Motorized Meso Machine (Triple M) was adapted for delivery
of siRNA to mouse and human skin. sd-siRNA solution (up to 300
.mu.L) was introduced into the chamber of the disposable meso
needle cartridge using a standard P-200 pipet tip. For treating
mice, a fold of skin was laid flat on a plastic surface and held in
place with the tip of the meso device. With the meso device
oriented vertically (perpendicular to the fold of skin) the device
was turned on and held in place for 10 seconds. For treating human
skin, fresh abdominal skin (obtained from abdominoplasty procedure)
was manually stretched and pinned to a cork platform prior to
treatment as described above.
Example 4--Histological Analysis of Fluorescently-Labeled sd-siRNA
Distribution in Murine and Human Skin
[0050] Cy3-Accell Non-Targeting siRNA (Dharmacon Products, Thermo
Fisher Scientific, Lafayette, Colo.) was loaded into the chamber of
the meso device (50 .mu.L 0.1 mg/mL). Mouse flank skin or
de-identified human abdominal skin from an abdominoplasty procedure
was treated as described above and imaged in an IVIS Lumina imaging
system (Xenogen product from Caliper LifeSciences, Alameda, Calif.)
using the 535 nm excitation and DsRed emissions settings (1-10 s
acquisition time). The data were quantified using Livinglmage
software (Caliper LifeSciences) and presented as an overlay with
the brightfield data. Fluorescent background from an untreated area
of the same animal or tissue sample was subtracted and values were
reported as radiant efficiency. Skin was then embedded in OCT and
sectioned for analysis by fluorescence microscopy using a Zeiss
Axio Observer Inverted Fluorescence Microscope equipped with Cy3
and DAPI filter sets as previously described.
Example 5--Confocal Microscopy of Fluorescently-Labeled sd-siRNA
Distribution in Murine and Human Skin
[0051] Cy3-Accell Non-Targeting siRNA was loaded into the chamber
of the meso device (100 .mu.L 0.5 mg/mL). De-identified human
facial skin from a face lift procedure was treated as described
above and imaged using a modified Lucid VivaScope 2500 System
(Lucid Inc., Rochester, N.Y.) 30-60 min following treatment as
previously described. Briefly, image z-stacks were generated by
image acquisition at successive z-depths using native VivaScan
software (v. VS008.01.09), and then post-processed using public
domain Fiji java-based image processing software, Images were
acquired in reflectance mode using a 658 nm laser source, and
duplicate stacks were acquired using a 532 nm excitation laser and
with a long pass filter to collect 607 nm emission.
[0052] To increase the effective resolution of the VivaScope
images, 10 nominal duplicate images were taken at each z-step, and
these 10 image sets were averaged to produce z-step-averaged
images, resulting in the final image stack. Because in vivo imaging
is influenced by respiration and other minor subject motion,
successive frames were co-registered using an affine transform;
distributed with Fiji software as the StackReg plugin) prior to any
frame-averaging. Images were further intensity-scaled to maximize
contrast with the Fiji software using a global constraint such that
the highest intensity 0.1% of image pixels in each frame were
scaled to a pixel intensity value of 256.
Example 6--Meso-Assisted Delivery of sd-siRNAs and Analysis of Gene
Silencing
[0053] Two cohorts of anesthetized tg-CBL/hMGFP mice were treated
with 50 .mu.L of 10 mg/mL solution in PBS of either sd-CBL3 siRNA
or a non-specific control sd-siRNA (CD44 or TD101 sd-siRNA) as
described above every day for 10 days. The day following the last
treatment, mice were euthanized and the treated area was excised
for analysis by both fluorescence microscopy and RTqPCR as
described in with the following modifications. The epidermis was
separated from the dermis by incubation in dispase II (10 mg/mL in
PBS, Roche, Indianapolis, Ind.) for 2-4 hours at 21.degree. C.
prior to RNA isolation.
Example 7--Histological Analysis of Treated Skin
[0054] To assess potential tissue damage due to penetration of the
array microneedles alone versus inflammation caused by deposition
of sd-siRNA, PBS, or sd-siRNA (in PBS) was administered to hairless
tg-CBL/hMGFP mice with MMNA device. The skin was harvested 24 hours
after treatment and immediately fixed in formalin and embedded in
paraffin. Histology of the treated skin revealed areas consistent
with needle penetration and associated skin damage. Acute
inflammation was observed with prominent polymorphonuclear
infiltrate in the papillary dermis extending down into, but not
through the reticular dermal layer, primarily at eh site of needle
penetration through the epidermis but also throughout the dermis.
Scattered macrophages and chronic inflammatory cells were also
present, consistent with classic wound healing, as would be
expended from a standard hypodermic needle injection. Inflammation
was generally localized around wounded regions. There were no
visual differences in wound response in skin treated with vehicle
alone as compared with skin treated with TD101 or CBL3 sd-siRNA
(data not shown), suggesting that observed acute inflammation is
not due to the presence of sd-siRNA.
Example 8--Analysis and Discussion of the Use of Meso-Devices to
Deliver sd-siRNA's
[0055] Due to its accessibility, skin is an attractive target for
siRNA therapeutics, and direct injection of "naked" nucleic acids
are thought as simple, safe, and efficient delivery method.
However, direct injections are limited to a highly localized region
of the epidermis coincident with the injection site, and large
number of injections may be needed to achieve the uniform delivery
required for a favorable therapeutic outcome. Indeed, although some
efficacy may result from intradermal injection of siRNA, generally
the efficacy can be limited to the area immediately surrounding the
plantar injection site. Further intradermal injections of either
siRNA or vehicle alone are accompanied by severe pain,
necessitating nerve blocks as well as oral pain medication before
treatment. This pain is likely due, at least in part, to the large
volume (up to 2 ml) of drug injected into the lesion. The high
pressure required for siRNA delivery is also likely at least
partially responsible for the intense pain experienced with these
injections. Thus, the disclosed invention is an alternative
"patient-friendly" (i.e. little or no pain) delivery
technology.
[0056] For functional delivery, siRNA must not only transit the
stratum corneum barrier, but also be internalized into cells in a
manner that allows for incorporation into the RNA-induced silencing
complex (RISC). In addition to direct injection with hypodermic
needle, multiple physical approaches have been evaluated the
reportedly facilitate delivery of nucleic acids across the stratum
corneum barrier including ultrasound, erbium:YAG laser, gene gun,
iontophoresis, electroporation, microneedles, and now motorized
microneedles. However, unmodified nucleic acids are not normally
taken up by keratinocytes in the absence of transfection agents
unless the administration is accompanied with pressure
("pressure-fection"). Covalent "sd" siRNA modifications (e.g.,
Dharmacon's Accell modifications) facilitate a cellular uptake in
vitro and in vivo without the need for transfection reagents.
Administration of sd-siRNA by dissolvable microneedle arrays can
reduce target gene expression up to 50% in both mouse and human
skin models. The nearly 90% average reduction in target gene
expression provided by the devices and techniques disclosed herein
exceeds with the threshold of 50% target gene expression reported
via the use of dissolvable microneedles.
[0057] The results set forth in the above examples indicate that
disclosed meso devices effectively deliver siRNA to relevant
regions of the skin with an efficiency (up to 80% inhibition) that,
if translatable to human subjects, may offer relief to patients
suffering from debilitating monogenic skin disorders. In contrast
to this, direct injection of unmodified siRNA with a hypodermic
needle results in 33% decrease in reporter gene expression.
Generally it is known that the use of microneedles significantly
decreases pain associated as compared with intradermal injections
inhuman studies.
[0058] It has to be understood that the above-described various
types of compositions, are only illustrative of preferred
embodiments of the present invention. Numerous modifications and
alternative arrangements may be devised by those skilled in the art
without departing from the spirit and scope of the present
invention and the appended claims are intended to cover such
modifications and arrangements. Thus, while the present invention
has been described above with particularity and detail in
connection with what is presently deemed to be the most practical
and preferred embodiments of the invention, it will be apparent to
those of ordinary skill in the art that variations including, may
be made without departing from the principles and concepts set
forth herein.
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