U.S. patent application number 15/596820 was filed with the patent office on 2017-08-31 for nanoparticles for use in medical and research applications.
The applicant listed for this patent is Genesegues, Inc.. Invention is credited to Gretchen Unger.
Application Number | 20170247253 15/596820 |
Document ID | / |
Family ID | 46753458 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170247253 |
Kind Code |
A1 |
Unger; Gretchen |
August 31, 2017 |
Nanoparticles for Use in Medical and Research Applications
Abstract
Disclosed are drug delivery systems and methods for
extravascular administration of drug, vaccine, and/or diagnostic
agents, for use in research and medical applications.
Inventors: |
Unger; Gretchen; (Chaska,
MN) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Genesegues, Inc. |
Chaska |
MN |
US |
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Family ID: |
46753458 |
Appl. No.: |
15/596820 |
Filed: |
May 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13218165 |
Aug 25, 2011 |
9650244 |
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15596820 |
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13071067 |
Mar 24, 2011 |
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13218165 |
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61403491 |
Sep 15, 2010 |
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61402202 |
Aug 25, 2010 |
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61340902 |
Mar 24, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55555
20130101; A61K 31/713 20130101; A61K 9/5115 20130101; A61K
2039/55505 20130101; B82Y 5/00 20130101; A61K 9/5161 20130101; A61K
9/5123 20130101 |
International
Class: |
B82Y 5/00 20060101
B82Y005/00; A61K 31/713 20060101 A61K031/713; A61K 9/51 20060101
A61K009/51 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT RIGHTS
[0003] The U.S. Government may have certain rights in this
application pursuant to Grant Nos. 1R43DK084644-01 and
1R43AI06268-01 and Contract No. HHSN261200800027C awarded by the
National Institutes of Health.
Claims
1. A method of treating a disease or disorder, comprising
administering to a subject in need of such treatment, a
therapeutically effective amount of non-viral nanoparticles,
wherein said nanoparticles comprise a micelle core comprising a
bioactive macromolecule and a surfactant with an HLB value of less
than or equal to about 6.0, a shell adsorbed to the micelle core
and comprising a ligand and lithium, and having a mean diameter of
less than about 50 nanometers, wherein; (a) the bioactive
macromolecule comprises an oligonucleotide, a protein, a peptide, a
carbohydrate, or an antibody; (b) the ligand comprises a protein, a
peptide, a carbohydrate, an antibody, or a small molecule; and (c)
the nanoparticles are administered subcutaneously.
2. A pharmaceutical composition comprising non-viral nanoparticles
comprising a micelle core comprising an antigen and a surfactant
with an HLB value of less than or equal to about 6.0, a shell
adsorbed to the micelle core and comprising a ligand, lithium, and
an adjuvant, and having a mean diameter of less than about 50
nanometers, wherein; (a) the antigen is hydrophilic and comprises a
DNA plasmid, a protein, or a peptide; (b) the adjuvant comprises
nickel; and (c) the ligand comprises hyaluronan, wherein said
hyaluronan ligand has an average molecular weight of less than
about 50,000 Daltons and is non-covalently attached to the
nanoparticles.
3. A method of inducing an immune response in a subject, comprising
administering to the subject a therapeutically effective amount of
the composition of claim 2, wherein the composition is topically
administered.
4. A composition for imaging a subject, said composition comprising
non-viral nanoparticles comprising a micelle core comprising an
imaging agent, wherein the imaging agent is dysprosium, and a
surfactant with an HLB value of less than or equal to about 6.0, a
shell adsorbed to the micelle core and comprising a ligand and
lithium, and having a mean diameter of less than about 50
nanometers.
Description
RELATED APPLICATIONS AND INCORPORATIONS BY REFERENCE
[0001] This application is a Continuation of U.S. patent
application Ser. No. 13/218,165, filed Aug. 25, 2011, which is a
Continuation-in-Part of U.S. patent application Ser. No.
13/071,067, filed Mar. 24, 2011, now abandoned, and claims priority
to U.S. Provisional Patent Application Nos. 61/402,202, filed Aug.
25, 2010; 61/403,491, filed Sep. 15, 2010; and 61/340,902, filed
Mar. 24, 2010, each of which is incorporated herein by reference in
its entirety.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference and may be employed in the practice of the
invention. More generally, documents or references are cited in
this text, either in a Reference List before the claims, or in the
text itself; and, each of these documents or references ("herein
cited references"), as well as each document or reference cited in
each of the herein cited references (including any manufacturer's
specifications, instructions, etc.), is hereby expressly
incorporated herein by reference.
TECHNICAL FIELD
[0004] The invention relates to compositions for and methods of
treating, diagnosing, and/or vaccinating a subject comprising
extravascularly administering nanoparticles comprising a drug,
vaccine, and/or diagnostic agent.
BACKGROUND
[0005] Many drugs and vaccines are administered intravenously, so
that the agent is directly available in the bloodstream and reaches
its intended site at a significant concentration. The chief
obstacle to treatment strategies involving macromolecules is their
generally poor bioavailability, resulting in either ineffective
treatment or in high dosages that significantly limit their
therapeutic applications due to cost.
SUMMARY OF THE INVENTION
[0006] It would be advantageous if therapeutic agents, including
vaccines, and diagnostic agents could be efficaciously administered
using methods that are easier to handle for the subject and/or are
amenable to repeat dosing. For example, subcutaneous administration
does not require venous access, can often be self-administered, and
typically requires a smaller needle size. Topical administration is
minimally invasive or non-invasive, can be self-administered, and,
with regard to vaccine applications, provides more direct access to
the highly specialized immune system located in the skin.
[0007] In one embodiment, the invention provides a non-viral drug
delivery system and in another, a method for improved delivery of
cargo, including therapeutics, vaccines, and imaging agents, by
non-intravenous administration. It has been found that
non-intravenous administration of low dosages of targeted
nanoparticles results in target modulation equivalent or superior
to that resulting upon intravenous administration. These unexpected
results allow for advantages, compared to intravenous
administration, which include greater convenience (patients can be
treated at home or in a physician's office, rather than at
hospitals or infusion centers), increased compliance, reduced
treatment cost, and more efficient use of healthcare resources.
[0008] Disclosed herein are compositions and methods that
demonstrate the flexibility of nanoparticles to successfully
accommodate ligands and cargoes of choice, for extravascular,
including subcutaneous and topical, delivery. There are several
diseases and classes of drugs for which the availability of
targeted nanoparticles for extravascular delivery would enhance
treatment and diagnostic options. For example, the class of drugs
known as RNA interference (RNAi) agents holds the potential to make
any gene into a drug target, a significant advance over
conventional therapies that can target only a relatively small
portion of the genome. However, despite tremendously broad interest
and investment in RNAi, there are currently no subcutaneous
applications of the same in the clinic.
[0009] Delivery modes for RNAi-based therapies in the clinic can be
characterized as either intravenous (e.g., Alnylam's ALN-VSP for
treatment of liver cancers and solid tumors; Calando's CALAA-01 for
solid tumors; Quark's I5NP for acute kidney injuries; Silence
Therapeutics' Atu027/Atu093 for lung cancers; and Tekmira's
liposomal treatment for hypercholesterolemia) or local (e.g.,
Alnylam's intranasal RSV-P for respiratory syncytial virus; Duke
University's intradermal treatment using siRNA-transfected
dendritic cells to treat melanoma; and Sylentis' intraocular
treatment for glaucoma).
[0010] Disclosed herein are nanoparticle compositions bearing drug,
vaccine, and/or diagnostic agents, methods for synthesis, and
methods for extravascular administration and use in research and
medical applications. The disclosed compositions include a
nanoparticle comprising a core comprised of drug, vaccine, and/or
diagnostic agent; a surfactant substantially surrounding the core
to form a surfactant-coated complex, wherein the surfactant has an
HLB value of less than about 6.0 units; and a shell that
non-covalently adheres to and substantially surrounds the
surfactant-coated complex, wherein the shell comprises a targeting
moiety and a cationic precipitating agent comprising lithium. The
mean diameter of the resulting nanoparticle is less than about 50
nanometers.
[0011] Additionally disclosed are methods for synthesizing the
nanoparticles described herein, including introducing into an
aqueous composition a plurality of drug, vaccine, and/or diagnostic
agents to create a core mixture, disposing into the core mixture a
hydrophobic surfactant to create a surfactant complex mixture,
wherein the hydrophobic surfactant has an HLB value of less than
about 6.0 units, and mixing the surfactant complex mixture with a
precipitating solution comprising a targeting moiety and a cationic
precipitating agent comprised of lithium to create a precipitated
nanoparticle mixture, resulting in nanoparticles comprising the
therapeutic or diagnostic core substantially surrounded by the
hydrophobic surfactant, which is substantially surrounded by the
targeting moiety and cationic precipitating agent.
[0012] The disclosed nanoparticles provide specific targeting and
intact delivery of cargo in an efficient composition. In one
embodiment, the therapeutic cargo comprises more than about
two-thirds of the total weight of the nanoparticle. In addition,
the disclosed nanoparticles are compatible with a range of drug,
vaccine, and diagnostic cargoes. Furthermore, the disclosed
nanoparticles provide a modular targeting component that can be
readily synthesized for a given cellular target, without the steps
of chelating, conjugating, or covalently attaching the targeting
moiety to the nanoparticle. With judicious selection of targeting
moiety and/or particle size, the nanoparticles are capable of
delivering cargo to targeted tissue and cells of the epidermis,
dermis, or subcutaneous tissues, or muscle, or to other targeted
tissue and cells reached via the lymph nodes or the systemic
circulation.
[0013] In another embodiment, the invention provides methods of
treatment comprising administering to a subject in need thereof a
therapeutically effective amount of a formulated composition
according to the invention, including via extravascular, for
example, subcutaneous and/or topical, administration.
[0014] In one embodiment, the invention provides a method of
treating a disease or disorder, comprising administering to a
subject in need of such treatment, a therapeutically effective
amount of non-viral nanoparticles, wherein said nanoparticles
comprise a micelle core comprising a bioactive macromolecule and a
surfactant with an HLB value of less than or equal to about 6.0, a
shell adsorbed to the micelle core and comprising a ligand and
lithium, and having a mean diameter of less than about 50
nanometers, wherein; (a) the bioactive macromolecule comprises an
oligonucleotide, a protein, a peptide, a carbohydrate, or an
antibody; (b) the ligand comprises a protein, a peptide, a
carbohydrate, an antibody, or a small molecule; and (c) the
nanoparticles are administered subcutaneously.
[0015] In another embodiment, the bioactive macromolecule comprises
an oligonucleotide or a therapeutic plasmid. In still another
embodiment, the bioactive macromolecule comprises an
oligonucleotide, and the dose administered of the oligonucleotide
is less than or equal to about 5 mg/kg. In another embodiment, the
ligand is Asialoorosomucoid (ASOR).
[0016] In another embodiment, the bioactive macromolecule comprises
a therapeutic plasmid, and the administering comprises repeat
dosing. In still another embodiment, the bioactive macromolecule
comprises a therapeutic plasmid, and the dose administered of the
plasmid is greater than or equal to about 10 mg/kg.
[0017] In one embodiment, the invention provides a pharmaceutical
composition comprising non-viral nanoparticles comprising a micelle
core comprising an antigen and a surfactant with an HLB value of
less than or equal to about 6.0, a shell adsorbed to the micelle
core and comprising a ligand, lithium, and an adjuvant, and having
a mean diameter of less than about 50 nanometers, wherein; (a) the
antigen is hydrophilic and comprises a DNA plasmid, a protein, or a
peptide; (b) the adjuvant comprises nickel; and (c) the ligand
comprises hyaluronan, wherein said hyaluronan ligand has an average
molecular weight of less than about 50,000 Daltons and is
non-covalently attached to the nanoparticles. In other embodiments,
the ligand is fibronectin, alpha V betaV, antiCV44, or antiDec205.
In still other embodiments, the ligand is a molecule such as a
carbohydrate, protein, peptide, or antibody that targets receptors
of both keratinocytes and dendritic cells.
[0018] It has been discovered that topically delivering
nickel-adjuvanted antigen in the inventive particles targeted to
both keratinocytes and dendritic cells generates improved and more
persistent mucosal IgA response over the formula targeted to
dendritic cells only. This is surprising because the view in the
art is that keratinocyte cells are only weakly immunogenic, and
that any antigen delivered to keratinocytes diverts from the more
immunogenic dendritic cells.
[0019] In another embodiment, the invention provides a method of
inducing an immune response in a subject, comprising administering
to the subject an effective amount of the composition, wherein the
composition is topically administered.
[0020] In one embodiment, the invention provides a composition for
imaging a subject, said composition comprising non-viral
nanoparticles comprising a micelle core comprising an imaging
agent, wherein the imaging agent is dysprosium, and a surfactant
with an HLB value of less than or equal to about 6.0, a shell
adsorbed to the micelle core and comprising a ligand and lithium,
and having a mean diameter of less than about 50 nanometers. In
another embodiment, the dysprosium is chelated to a DOTA-Dextran
conjugate. In still another embodiment, the subject is a human.
[0021] The foregoing has outlined rather broadly some of the
embodiments of the invention in order that the detailed description
of the invention that follows may be better understood. Other
aspects of the invention are described in or are obvious from the
following disclosure and are within the ambit of the invention.
[0022] The terms "nanocapsule" and "nanoparticle" are used
interchangeably herein.
[0023] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples described herein are illustrative only and are not
intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The following Detailed Description of the Invention, given
by way of Examples, but not intended to limit the invention to
specific embodiments described, may be understood in conjunction
with the accompanying figures, in which:
[0025] FIG. 1 shows a bar graph depicting the knockdown of Factor
VII (FVII) mRNA in mouse liver after a single treatment with
asialoorosomucoid (ASOR)-coated sub-50 nanometer (s50) capsules
bearing single- and double-stranded RNAi cargo. "D#" indicates
number of days after treatment. "ss" indicates FVII single-stranded
oligonucleotide. "ds" indicates FVII double-stranded siRNA. "sq"
indicates subcutaneous administration. "iv" indicates intravenous
administration. "NM" means not measured. There were n=5-13 animals
per group.
[0026] FIGS. 2A and 2B show bar graphs depicting the reduction of
FVII total protein in mouse liver (2A) and plasma (2B) after a
single treatment with ASOR-coated s50 capsules bearing single- and
double-stranded RNAi cargo. "D#" indicates number of days after
treatment. "ss" indicates FVII single-stranded oligonucleotide.
"ds" indicates FVII double-stranded siRNA. "sq" indicates
subcutaneous administration. "iv" indicates intravenous
administration. "NM" means not measured. There were n=3-8 animals
per group.
[0027] FIGS. 3A and 3B depict near infrared (NIR) imaging with ASOR
coated s50 particles bearing DyDex cargo, demonstrating capability
of s50 DyDex as an imaging agent to elucidate the specific sites of
capsule delivery. Mice were intravenously injected (right-side of
figure, 3B) or not (left-side of figure, 3A) with 100 nmol/kg of
s50 ASOR DyDex about 30 hours before imaging. Excised livers,
kidneys, or spleen were imaged on at Kodak Carestream FX pro
imaging station at 510 excitation and 700 nm emission. Results were
processed with Kodak Multispectral imaging analysis software to
extract Dy-specific signal. The checkerboard pattern denotes green
background signal (i.e. no Dy detection) in the original
pseudocolored data. Gray-to-white signal in liver denotes
increasing Dy concentration; a numerical representation of
intensity is illustrated in the calibration scale at the lower edge
of the figure.
[0028] FIG. 4 shows two gels demonstrating hepatocyte-targeted
subcutaneous (sq) delivery of ASOR-coated s50 capsules bearing
plasmid DNA in mice. SEAP-specific polymerase chain reaction (PCR)
products are visualized by electrophoresis on 1.25% agarose gels
using ethidium bromide. Input for the reactions was reverse
transcriptase product from total RNA isolated from animals treated
with ASOR sugar (3.times.10 mg/kg q3D intravenous) and ASOR
pCpGMarSEAP using different regimens: Group A=3.times.20 mg/kg
q3Day sq, Group C=6.times.10 mg/kg q3Day sq, and comparator Group
B=3.times.10 mg/kg q3Day intravenously. There were n=3 animals per
treatment group. Tissue was collected two days after the last
dose.
[0029] FIG. 5 shows a bar graph depicting site accumulation of s50
subcutaneously-delivered DyDex cargo targeted to liver in mice over
a 5-day period, demonstrating liver targeting specificity and
prolonged availability of the cargo. Mice were treated sq with
ASOR-ligand, s50 capsules having a mean diameter of 10-12
nanometers and bearing Dy-labeled Dextran at 100 nmol Dy/kg for
sensitive Dy-163 .mu.m neutron activation analysis (NAA) of
tissues. Tissues were collected daily for five days (n=1 mouse per
24 hour timepoint). Mice were housed in metabolism cages for urine
and feces collection for the last 24 hours of the respective
treatment periods. Tissues were weighed and neutron irradiated in a
fusion reactor facility for gamma counting of metastable Dy-163
spectra.
[0030] FIG. 6A shows a bar graph displaying 4-day
lymphoproliferation responses from lymphocyte cultures to challenge
by 50 .mu.g/ml of recombinant bacterial betagalactosidase.
Lymphocytes were either peripheral blood monocyte cells or isolated
from spleen, draining lymph nodes (mandibular), or distal lymph
nodes (pubescent) from week 7 terminal samples. For quantitation,
0.5 .mu.curie of tritium was added to cultures during the last
eighteen hours of culture to measure ongoing DNA synthesis and,
thus, proliferation.
[0031] FIG. 6B shows a bar graph displaying antigen-specific
mucosal IgA antibodies assayed in nasal lavage by Bgal ELISA and
normalized by lavage protein concentration. Nasal lavage was
collected from pigs under light isofluorane anesthesia by injecting
10 mL PBS up one nostril using a catheter as a syringe and
collecting outflow from the other nostril via a funnel. Samples
were collected for the last three weeks of the study. Very low
levels of normalized IgA antibodies were measured in week 7 lavage
from pig #863 receiving intradermal naked plasmid, providing an
estimate of background levels.
[0032] FIG. 6C shows a line graph depicting the development of
serum IgG antibodies against Bgal generated by ELISA. Background
from pre-immune serum is subtracted. Arrows denote treatment
schedule; sera were collected at weeks 2, 5, 6, and terminal week
7.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0033] The term "subject", as used herein, refers to a mammal. In
separate additional embodiments, the subject is a human or a
veterinary mammal. In the context of the vaccination/immune
response induction compositions and methods, in one embodiment, the
subject is a large mammal; in another embodiment, the subject is a
non-rodent mammal; in another embodiment, the subject is a
veterinary mammal; in another embodiment, the subject is a swine;
in another embodiment, the subject is a human; in another
embodiment, the subject weighs more than about five pounds. In the
context of imaging/diagnostic methods disclosed herein, the
"subject" includes, without limitation, a cell, a tissue, an organ,
and/or a mammal.
[0034] The term "extravascular", as used herein, refers to non-oral
methods of administration excluding intravenous methods. It
includes, without limitation, subcutaneous, topical, intramuscular,
intradermal, transdermal, transcutaneous, transmucosal, and
intracerebral administration.
[0035] The terms "comprises", "comprising", are intended to have
the broad meaning ascribed to them in U.S. Patent Law and can mean
"includes", "including" and the like. Moreover, the use of the term
"including", as well as other forms, such as "includes" and
"included", is not limiting.
[0036] The invention can be understood more fully by reference to
the following detailed description and illustrative examples, which
are intended to exemplify non-limiting embodiments of the
invention.
ADDITIONAL EMBODIMENTS OF THE INVENTION
[0037] In some embodiments, the nanoparticles described herein
comprise a core comprising a therapeutic or diagnostic agent; a
surfactant substantially surrounding the core to form a
surfactant-coated complex, wherein the surfactant has an HLB value
of less than about 6.0 units; and a shell that non-covalently
adheres to and substantially surrounds the surfactant-coated
complex, wherein the shell comprises a targeting moiety and
lithium. The mean diameter of the resulting nanoparticle is less
than about 50 nanometers (sub-50 nanometer capsules, "s50
capsules").
[0038] In a further embodiment of the invention, the mean size of
the nanoparticle is less than about 50 nanometers in diameter, as
measured by atomic force microscopy after drying of the
nanoparticles. In still a further embodiment of the invention, the
mean size of the nanoparticle is less than about 40 nanometers. In
still a further embodiment of the invention, the mean size of the
nanoparticle is less than about 30 nanometers. In still a further
embodiment of the invention, the mean size of the nanoparticle is
less than about 20 nanometers. In still a further embodiment of the
invention, the mean size of the nanoparticle is less than about 10
nanometers. In still a further embodiment of the invention, the
mean size of the nanoparticle is no less than about 8 nanometers.
In still a further embodiment of the invention, the mean size of
the nanoparticle is no less than about 5 nanometers. In yet another
embodiment of the invention, the mean size of the nanoparticle is
between about 5 and about 50 nanometers.
[0039] In additional embodiments comprising subcutaneous
administration of oligonucleotides, the mean diameter of the
nanoparticles is less than or equal to about 25 nm, less than or
equal to about 22 nm, between about 20 and about 25 nm, or between
about 10 and about 25 nm. Nanoparticle diameter is determined as
the mean of the minor and major particle axis of a dried-down
particle (for example, dried down on a mica sheet), and obtained by
atomic force microscopy, in one embodiment, using ImageJ
software.
[0040] In one embodiment of the invention, the core of the
nanoparticle comprises a drug/therapeutic agent, a vaccine agent,
or a diagnostic agent or a combination thereof. In another
embodiment, the agent comprises a bioactive macromolecule such as a
DNA plasmid, a single- or double-stranded RNAi molecule, an
antisense molecule, an antibody, a carbohydrate, a protein, or a
peptide, or a combination thereof. In still another embodiment, the
agent is hydrophilic. In still another embodiment, the agent is not
lipophilic.
[0041] In a further embodiment, the agent comprises an
oligonucleotide, which refers to a nucleic acid molecule of length
less than about 100, about 200, about 300, or about 400
nucleotides. Non-limiting examples of oligonucleotides include
antisense, aptamer(s), siRNA, miRNA, or single-stranded RNAi. As
used herein, the term "oligonucleotide" encompasses
polynucleotides. The active strand of the nucleic acid can, for
example, be a phosphodiester, with an oxygen atom linking
consecutive nucleotides. The phosphodiester can, in still a further
embodiment, be modified, for example, by replacing the oxygen-link
of the nucleotides with, for example, sulfur to provide a
phosphorothioate strand or with, for example, a nitrogen to provide
a morpholino.
[0042] In an embodiment of the invention, the targeting moiety of
the nanoparticle comprises a plurality of biocompatible polymers,
proteins, peptides, carbohydrates, antibodies, or small molecules.
In another embodiment, the targeting moiety does not comprise a
lipid derivative. As used herein, the terms "targeting moiety" and
"ligand" are used interchangeably.
[0043] In still another embodiment of the invention, the targeting
moiety is tenascin. Tenascin ("TN") is an extracellular matrix
molecule that is useful for nanoparticles as a biocompatible
polymer and/or as a targeting moiety. Tenascin is a branched, 225
KD fibronectin-like (FN) extracellular protein prominent in
specialized embryonic tissues, wound healing, and tumors. In the
adult, normal cells, aside from wound-activated keratinocytes, do
not migrate on tenascin. However, integrin receptors capable of
mediating migration on TN by carcinoma cells include
.alpha.2.beta.1, .alpha.v.beta.3 and .alpha.v.beta.6. In one
embodiment, TN nanoparticles deliver nucleic acids specifically via
receptor-mediated caveolar endocytosis. TN, or any subdomain(s)
thereof, is a suitable cell recognition polypeptide according to an
embodiment of the invention.
[0044] Tenascin has been implicated in cancer activities and, also,
as being specific for smooth muscle cells; furthermore, peptidic
domains of tenascin have been identified (e.g., as in U.S. Pat. No.
6,124,260) and are known in the art. In one embodiment, tenascin
suitable for the present invention is H. sapiens tenascin C,
Genbank Accession No. NM_002160. Moreover, tenascin peptides and
domains for adhesion with particular cell types, as well as
functional and structural aspects of tenascin, have been disclosed
and are known in the art (e.g., Aukhill, et al., 1993 J. Biol.
Chem. 268(4):2542-2553). Tenascin and/or any of its domains are, in
additional embodiments, suitable for the present invention. In one
embodiment, the ligand is the fibrinogen fragment of tenascin (also
referred to herein as Fbg-L domain of tenascin-C or tenfibgen or
TBG (nucleotide sequence of tenfibgen as disclosed in U.S. patent
application Ser. No. 13/071,067). is used as the biocompatible
polymer and/or the cell recognition polypeptide. Tenascin, its
subdomains, or any other biocompatible polymer may be expressed or
produced by methods provided herein, as well as by methods known in
the art.
[0045] In another embodiment of the invention, hyaluronan is
included as the targeting moiety for the nanoparticles of the
present invention. Hyaluronan is a negatively charged
glycosaminoglycan that can reach a molecular mass of several
million Daltons and is a ubiquitous component of extracellular
matrices. This carbohydrate is commercially available in a variety
of forms and has many known uses (e.g., U.S. Pat. No. 5,902,795).
In one embodiment of a method according to the invention, the
hyaluronan used is a low molecular weight hyaluronan. It is found
herein that such low molecular weight hyaluronan is surprisingly
effective for topical administration of vaccines.
[0046] In still another embodiment, the targeting moiety is
asialoorosomucoid (ASOR)(Stockert, et al. 1980 Lab. Invest.
43:556-63), which is capable of targeting hepatic cells of the
liver, for example. For example, subcutaneous administration of
therapeutic oligonucleotides at low dosages via nanoparticles may
be effective in the treatment of liver diseases or diseases
involving hepatocytes. As used herein, the term "therapeutic
oligonucleotides" describes oligonucleotides that can be used to
essentially silence genes and/or to inhibit a portion or all of the
activity of proteins that are responsible for a particular
disease.
[0047] In one embodiment of the invention, the nanoparticles
described herein can be used to treat or diagnose disease. In
another embodiment, the nanoparticles described herein can be used
to treat or diagnose diseases of the liver. For example, the
nanoparticles described herein can be used to treat hepatic
disease, where treatment comprises administering the nanoparticles
for the purpose of regulating, repairing, adding, inhibiting, or
deleting a genetic sequence in hepatic tissue and cells. In a
separate embodiment, the nanoparticles described herein can be used
to diagnose hepatic disease.
[0048] In another embodiment, the nanoparticles described herein
inhibit the expression of the Factor VII (FVII) gene in a cell or
mammal using an oligonucleotide, for example, for treating
pathological conditions and diseases caused by the expression of
the FVII gene, such as coagulation disorders.
[0049] In still another embodiment, the nanoparticles described
herein can be used to treat or diagnose diseases other than cancer.
For example, the nanoparticles described herein can be used to
treat tumors other than primary tumors. In one embodiment, the
nanoparticles described herein can be used to topically or
subcutaneously treat or diagnose diseases of the lymphatics or
lymph nodes, including via judicious selection of a targeting
moiety to enhance lymphatic or lymph node uptake.
[0050] The nanoparticles described herein are contemplated, in
separate embodiments, for use in in vitro, in vivo, and ex vivo
applications. In separate embodiments, the nanoparticles described
herein can be used to treat a subject in need of induction of an
immune response, prevention of disease, protection from effects of
infection, therapy of existing disease or symptoms, or combinations
thereof.
[0051] In another embodiment, the nanoparticles described herein
are used to treat, vaccinate, or diagnose a subject, wherein the
subject is a mammal. In additional embodiments, the subject is a
human or a veterinary mammal.
[0052] Nanoparticle sizing may be altered, in one embodiment, by
increasing or decreasing the weight of coating ligand applied to
the particle during synthesis, and/or increasing or decreasing the
length of crystallization time allowed during the precipitation and
hardening step. A less preferable but still viable method for
altering the size of the nanoparticles disclosed herein can be
achieved by increasing or decreasing the amount of condenser used
in mixing with, for example, nucleic acid cargo, to form the core
of the particle. In certain additional embodiments, reducing the
size of the nanoparticles described herein may increase the plasma
concentration of topically or subcutaneously administered
nanoparticles. In certain additional embodiments, increasing the
size of the nanoparticles described herein may increase the
lymphatic concentration of topically or subcutaneously administered
nanoparticles.
[0053] In some embodiments, a method of treating or diagnosing
disease is provided comprising administering the nanoparticles to a
host in a single dose once a day or more frequently. In some
embodiments, the nanoparticles are administered less than once a
day. In some embodiments, the nanoparticles are administered in a
depot that provides efficacious dosage without repeated injections.
In some embodiments, including plasmids coding for antigenic
sequences or therapeutic sequences, administration to a subject may
comprise single or repeat dosing.
[0054] As used herein, the term "therapeutic sequences" refers to
sequences intended to ultimately modulate cellular protein or
transcriptions levels, and the plasmids encoding for therapeutic
sequences are termed "therapeutic plasmids". Non-limiting examples
of therapeutic plasmids include plasmid DNA, including, for
example, shRNA plasmid DNA. Non-limiting examples of diseases that
can be treated with s50 particles bearing therapeutic plasmids
include cancer and haemophilia. For avoidance of doubt, unless
specified otherwise herein, therapeutic sequences do not include
antigenic sequences. As used herein, the term "repeat dosing"
refers to administering a drug or other substance to a given
subject more than once to treat a given disease or disorder, at
dosage levels that may or may not vary with each
administration.
[0055] Methods for identifying oligonucleotide chemistry and
sequence to inhibit a desired target are well known to those of
skill in the art. For example, conventional antisense design
typically optimizes for uniform hybridization energies across
sequences at sites of low target mRNA secondary structure, while
siRNA design is more focused on optimizing a hybridization profile
across a sequence within the context of sequence "rules".
Non-limiting examples of design algorithms such as Soligo for
antisense and SiRNA for siRNA are publicly available
(http:/sfold.wadsworth.org and websites for suppliers such as
Dharmacon). Sequence selection then consists of routine
optimization in the applicable oligonucleotide formats, followed by
nanoparticle formulation, in vitro assessment in target cells
plated on conventional 2D or, in a preferred embodiment, relevant
protein or 3-D matrices, and/or in vivo assessment in an animal
model.
[0056] Methods for high-throughput functional screening and
identification of potential candidate vaccine antigens from genomic
sequence data are well known to those of ordinary skill in the art.
In one non-limiting example, transcriptionally active PCR (TAP) and
TAP immunoscreening can be used (Regis, et al. 2007 Mol Biochem
Parasitol 158:32-45). TAP immunoscreening approaches for validation
of agents as potential vaccine candidates include for example
subcellular localization studies, gene knockout studies, and rodent
orthology protection studies.
[0057] In some embodiments, extravascular administration of the
nanoparticles described herein is achieved by, for example,
intramuscular, intradermal, transdermal, transcutaneous,
transmucosal, intracerebral, or subcutaneous injection or
device-based (e.g., infusion pumps or motor-driven devices)
application, or by topical application. It will be understood by
those of ordinary skill in the art that these methods of
administration refer to how the nanoparticles are applied to the
subject, and not where they remain or traverse to. In some
embodiments, administration according to the invention is performed
by extravascular administration other than topical administration,
for treating cancer and for treating other diseases.
[0058] As mentioned above, extravascular indicates administration
other than intravenous. In one embodiment, administration of the
nanoparticles is performed by subcutaneous administration by
needle, syringe, catheter, or similar device, with or without the
aid of an infusion pump or motor-driven device, and/or with or
without the aid of penetration enhancing agents. Penetration
enhancing agents for subcutaneous administration are well known to
those of ordinary skill in the art. Non-limiting examples of
penetration enhancing agents for subcutaneous treatment include
hyaluronidase or hyaluronidase glycoproteins.
[0059] As the skin is the largest and most accessible organ, it
provides an attractive target for drug and vaccine development.
However, an effective physical barrier in the epidermis protects
against the entry of environmental substances, including nucleic
acids. In particular, large hydrophilic molecules such as most
biopharmaceuticals normally do not pass through the skin. These
challenges emphasize the need for an easily applied system for
delivering drugs or vaccines that is able to transit the stratum
corneum.
[0060] Current strategies to overcome the challenges of transiting
the stratum corneum include physical methods that disrupt the
barrier properties of the stratum corneum by mechanical means, such
as electroporation, liquid jet device, biolistic, or abrasive tape
strip, or by microporation approaches that may comprise approaches
such as solid or hollow microneedles, thermal energy, or
radiofrequency. Other strategies include the use of skin
penetration enhancers, such as enzymatic penetration enhancers
and/or chemical penetration enhancers, such as solvents and
surfactants.
[0061] These strategies for crossing the stratum corneum have met
with little success because of associated cell damage, poor tissue
distribution, and/or cellular uptake, off-target delivery, high
treatment pain, and other factors. Therefore, in certain
embodiments of the present invention, the nanoparticles can be
co-administered with physical methods including mechanical means
and/or microporation means, and/or with external skin penetration
enhancers, to enable and/or increase the utility of these
strategies including less cell damage, improved tissue distribution
and/or cellular uptake, increased targeting, lower dosages, and
reduced treatment pain. For example, loading a microneedle array
patch with nanoparticles may, upon administration, increase the
tissue distribution of the cargo, increasing efficacy and
potentially reducing dose and/or number of required treatments (and
therefore, reducing the number of invasive treatments). Those of
ordinary skill in the art will appreciate that the nanoparticle
compositions described herein may be readily incorporated or
formulated with such physical methods and skin penetration
enhancers.
[0062] As disclosed herein, the nanoparticles can also cross the
stratum corneum and/or mucous membranes after topical application.
Thus, in certain embodiments, the nanoparticles described herein
may be topically administered to intact, unbroken skin. In further
embodiments, topical administration of the nanoparticles does not
comprise the use of physical methods and/or external enzymatic
penetration enhancers and/or external chemical penetration
enhancers, thus potentially reducing or avoiding cell damage,
treatment pain, and other adverse reactions at the site of
treatment.
[0063] In certain embodiments, the nanoparticles are topically
administered with facilitating agents, which are well known to one
skilled in the art and include, without limitation, lotions,
ointments, creams, pastes, sprays, gels, pipettes, and/or patches,
where such agents may or may not include enzymatic and/or chemical
skin penetration-enhancers. In certain embodiments, facilitating
agents may further improve bioavailability and/or uniformity of
delivery. As used herein, the term "bioavailability" refers to the
degree to which a drug or other substance becomes available to the
target tissue after administration. In one embodiment, hydration of
the skin before, during, or immediately after application of such a
formulation is desirable. For example, hydration may increase the
water content of the topmost layer of skin (e.g., stratum corneum
or superficial epidermis layer exposed by penetration enhancement
techniques) to above about 1%, about 5%, about 10%, about 25%,
about 50%, or about 75%. It is to be understood that uses of the
nanoparticles disclosed herein are contemplated to include standard
methods for cleaning, sterilizing, etc. the subject's area of
treatment before, during, or after administration of the
nanoparticles, as necessary. In certain embodiments, the surface
area to be treated is shaved before administration. In other
embodiments, the surface area to be treated is not shaved before
administration.
[0064] As used herein, "topical" administration refers to
administration of the nanoparticles or a composition comprising the
nanoparticles to a surface such as the skin or mucous membranes
such as the vagina, anus, throat, and ears. "Passive topical"
administration refers to topical administration without the use of
physical methods, such as mechanical and microporation methods,
that disrupt the barrier properties of the stratum corneum and/or
mucous membranes. The term "active topical" administration refers
to administration of the nanoparticles or a composition comprising
the nanoparticles with the use of physical methods, such as
mechanical and/or microporation methods, that disrupt the barrier
properties of the stratum corneum and/or mucous membranes.
[0065] The term "without external enhancers" refers to embodiments
and methods, such as topical delivery, that do not comprise the use
of external enzymatic penetration enhancers and/or external
chemical penetration enhancers for the skin and/or mucous
membranes. It is to be understood that the external enhancers
excluded by the term "without external enhancers" do not include
water used primarily for hydrating the skin and/or mucous membranes
during pre-treatment, co-treatment, or post-treatment.
[0066] In separate embodiments, the nanoparticles may be
administered extravascularly, including topically (passive and
active topical, in separate embodiments), to a subject for the
purpose of delivering DNA, protein, peptide, or carbohydrate
antigen cargos in the process of vaccination. As used herein, the
terms "DNA antigen" and "antigen comprising DNA" refers to a DNA
plasmid that encodes an antigenic sequence.
[0067] In certain embodiments, the extravascularly administered
nanoparticles provide co-stimulation of specific humoral and
cell-mediated responses. In one embodiment, an immune response,
subsequent to extravascular administration of the nanoparticles
described herein, is observed in primary sites, including blood,
skin and mucosal tissue. In another embodiment, an immune response,
after extravascular administration of the nanoparticles, is
observed in disseminated tissue, including draining and distal
lymph nodes.
[0068] The nanoparticles described herein may also be administered
in a non-clinical setting, such as in military field operations. In
certain embodiments, the nanoparticles may be stored in
ready-to-use formulations that do not require refrigeration for
periods of about 6 months, about 12 months, about 18 months, about
24 months, about 36 months, or about 60 months. In one embodiment,
nanoparticles encapsulating DNA antigen are administered by
topical, subcutaneous, or intradermal injection as a priming step,
followed by topical application in one or more boosting steps. In
an additional embodiment, the nanoparticles comprise a ligand for
spleen targeting to enhance specific IgG responses to the
encapsulated antigenic protein or DNA cargo.
[0069] In one embodiment, the nanoparticles described herein
comprise antigenic protein or DNA cargo and modulate IgA, IgD, IgE,
IgG, and/or IgM responses. In another embodiment, the nanoparticles
comprise a nickel adjuvant. In still another embodiment, the
nanoparticles are administered by an extravascular method, such as,
for example, a topical method, to treat a disease that requires
modulation of IgA, IgD, IgE, IgG, and/or IgM responses to be
effective. In another embodiment, the nanoparticles administered
extravascularly may be indicated to vaccinate subjects who cannot
tolerate treatment-site reactions. Determination of such subjects
can be made by a physician or other qualified individual based on,
for example, the subject's treatment history and/or medical or
physical condition.
[0070] An "immune response" is defined as a response of a cell of
the immune system, such as a B cell or a T cell, to a stimulus. An
immune response can be measured by several parameters, including,
but not limited to, cytokine secretion (IL-6, IL-10, IFN-.alpha,
etc.), immunoglobulin production, dendritic cell maturation, and
proliferation of a cell of the immune system. One of skill in the
art can readily determine an increase in any one of these
parameters, using known laboratory assays. In one specific
non-limiting example, to assess cell proliferation, incorporation
of (3)H-thymidine can be assessed.
[0071] A "substantial" increase in a parameter of the immune
response is a significant increase in this parameter as compared to
a control. Specific, non-limiting examples of a substantial
increase include at least about a 25% increase, at least about a
50% increase, at least about a 75% increase, at least about a 90%
increase, at least about a 100% increase, at least about a 200%
increase, at least about a 300% increase, and at least about a 500%
increase. One of skill in the art can readily identify a
significant increase using known statistical methods. For example,
to assess a substantial increase, a Z test can be used to compare
the percent of samples that respond to a formulated vaccine
contemplated for administration according to a method of the
invention as compared to the percent of samples that respond to a
control. A non-parametric ANOVA can be used to compare differences
in the magnitude of the response induced by the vaccine as compared
to the percent of samples that respond using a control. In this
example, p less than or equal to 0.05 is significant, and indicates
a substantial increase in the parameter of the immune response. One
of skill in the art can readily identify other statistical assays
of use.
[0072] In certain separate embodiments, the nanoparticles
contemplated for extravascular administration comprise a plurality
of low molecular weight hyaluronan ligands averaging less than
about 50,000 Daltons, less than about 40,000 Daltons, less than
about 30,000 Daltons, less than about 25,000 Daltons, less than
about 20,000 Daltons, less than about 15,000 Daltons, less than
about 10,000 Daltons, between about 5,000 and 30,000 Daltons, or
between about 10,000 and 30,000 Daltons. In a specific embodiment,
the hyaluronan ligand has a molecular weight of 22,480 Daltons. The
nanoparticles can be formulated for this range of hyaluronan
molecular weight, for example, by adjusting minor components
(dopants) in the crystallization bath.
[0073] In one embodiment, nanoparticles such as topical
hyaluronan-nickel nanoparticles provide a universal strategy for
engaging the immune system in the vaccination process. This
strategy improves efficacy and flexibility for addressing a range
of diseases by judicious design and selection of the DNA plasmid or
protein antigen. Thus, the nanoparticles described herein can be
used as vaccines for control of diseases such as infectious
diseases, including, without limitation, Chlamydia,
Cytomegalovirus, Helicobacter Pylori, Herpes Simplex,
Papillomavirus, Influenza, Meningococcus B, nvCreutzfeldt-Jakob
Disease, Respiratory Syncytial Virus, Severe Acute Respiratory
Syndrome, Shigella, Turberculosis, West Nile, and Zoster. In
another embodiment, hyaluronan-nickel nanoparticles are
administered extravascularly for tumor treatment.
[0074] In another embodiment, the targeted, antigen-bearing
nanoparticles of the present invention are used in topical or other
extravenously-administered treatments other than tumor
treatment.
[0075] In certain separate embodiments, vaccine nanoparticles
contemplated for extravascular, including topical, administration
for prophylactic and/or therapeutic use comprise an amount of
antigen in a unit dose anywhere in a range from about 0.001 .mu.g
to about 10 mg, from about 0.1 .mu.g to about 1 mg, from about 5
.mu.g to about 500 .mu.g, between about 1 .mu.g and about 10 .mu.g,
between about 10 .mu.g and about 50 .mu.g, between about 50 .mu.g
and about 200 .mu.g, and between about 1 mg and about 5 mg. In one
embodiment, the amount of antigen in the unit dose is about 100
.mu.g. In one embodiment, the amount of antigen in the unit dose is
about 500 .mu.g.
[0076] In certain embodiments, vaccine nanoparticles for topical
administration may be administered in an application that covers
from about 0.001% to about 100% of the subject's total body surface
area, or between about 0.001% and about 90% or about 80% or about
70% or about 60% or about 50% or about 40% or about 30% or about
25% or about 20% or about 15% or about 10% or about 5% of the
subject's total body surface area, or between about 5% and about
30% or between about 5% and about 20% of the subject's total body
surface area. In one embodiment, the topical vaccine preparation is
applied to cover about 5% or about 10% or about 20% or about 30% or
about 100% of the subject's total surface area. In a certain
embodiment, the topically-applied nanoparticle vaccine preparation
does not cover all or substantially all of the subject's total
surface area. In certain embodiments, the concentration of the
antigen applied in a unit dose is between about 0.001 and about 100
.mu.g/sq cm of treated surface area, between about 0.01 and about
50 .mu./sq cm of treated surface area, between about 0.01 and about
25 .mu.g/cm of treated surface area, less than about 10 .mu.g or
about 8 .mu.g or about 5 .mu.g or about 3 .mu.g or about 1 .mu.g/sq
cm of treated surface area, or about 0.44 .mu.g/sq cm of treated
surface area. The ratio of adjuvant to antigen in a unit dose will
vary depending on the strength of each, but non-limiting examples
include nanoparticles formulated with an adjuvant amounting to less
than about 5% of the antigen by weight, less than about 1% of the
antigen by weight, or less than about 0.1% by weight.
[0077] In some embodiments, the efficacy of the topical vaccine
nanoparticles can be enhanced or optimized by modifying the
concentration of antigen being applied. In applications where the
vaccine nanoparticles are to be sprayed on the surface of the
subject, non-limiting examples of the concentration of the antigen
to PBS or other acceptable diluents are between about 1 .mu.g/ml to
about 10 mg/ml, between about 25 .mu.g/ml to about 200 .mu.g/ml,
between about 25 .mu.g/ml to about 100 .mu.g/ml, about 50 .mu.g/ml.
For compounding into solid dosage forms, non-limiting examples of
concentration of the antigen to the solid dosage form are between
about 10 .mu.g/ml to about 100 mg/ml, between about 100 .mu.g/ml to
about 10 mg/ml, between about 0.5 mg/ml to about 10 mg/ml, between
about 0.5 mg/ml and 5 mg/ml, about 1 mg/ml.
[0078] With regard to formulations comprising other therapeutic
plasmids, the nanoparticles described herein can be administered
subcutaneously in a unit dose selected from a range of between
about 0.01 mg/kg and about 50 mg/kg, between about 0.1 mg/kg and
about 30 mg/kg, between about 1 mg/kg and 20 mg/kg, between about 5
mg/kg and about 20 mg/kg, between about 5 mg/kg and about 30 mg/kg,
about 1 mg/kg, about 10 mg/kg, about 20 mg/kg, less than or equal
to about 30 mg/kg, less than or equal to about 20 mg/kg, more than
or equal to about 1 mg/kg, about 5 mg/kg, and about 10 mg/kg. In
one embodiment, the dose is about 20 mg/kg.
[0079] In embodiments involving other therapeutic applications,
including formulations comprising oligonucleotides, the
nanoparticles described herein can be administered in a unit dose
of about 0.00001 mg drug/kg body weight, about 0.0001 mg/kg, about
0.001 mg/kg, about 0.01 mg/mg, about 0.1 mg/kg, about 1.0 mg/kg,
about 2.0 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, or about 5.0
mg/kg, less than or equal to about 1.0 mg/kg, less than or equal to
about 2.0 mg/kg, or less than or equal to about 5.0 mg/kg.
[0080] Non-limiting examples of methods for improving dosage
include optimizing sequence and medicinal chemistry. For example,
the incorporation of a 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
2'-O-allyl, 2'-O-aminoalkyl or 2'-deoxy-2'-fluoro group in
nucleosides of an oligonucleotide confers enhanced hybridization
properties to the oligonucleotide. Further, oligonucleotides
containing phosphorothioate backbones have enhanced nuclease
stability. Thus, in one embodiment, functionalized, linked
nucleosides can be augmented to include either or both a
phosphorothioate backbone or a 2'-O-methyl, 2'-O-ethyl,
2'-O-propyl, 2'-O-aminoalkyl, 2'-O-allyl or 2'-deoxy-2'-fluoro
group.
[0081] The improved bioavailability and targeting of the
nanoparticles contemplated for extravascular administration,
combined with flexibility in incorporating various cargoes and
ligands, provides for the potential treatment of a wide range of
diseases. Non-limiting examples of diseases the nanoparticle
formulations can be used to treat include hepatitis, cancer,
atherosclerosis, tuberculosis, and asthma.
[0082] In some applications, the nanoparticles described herein are
administered to a subject who has cells that are expressing or
over-expressing a particular receptor that is indicative of a
disease state, and which can be targeted by judicious selection of
nanoparticle ligand. For example, a subject with cells that express
or over-express tenascin receptors may have cancer. Methods for
diagnosing or detecting tenascin or tenfibgen receptors are
provided herein.
[0083] A variety of cancers may be diagnosed or detected by
measuring tenascin or tenfibgen expression levels, where the
over-expression level of tenascin or tenfibgen indicates a cancer
diagnosis, and/or where the expression level of tenascin or
tenfibgen indicates a target for tumor-targeted nanoparticles
according to certain embodiments of the invention. By
"over-expression" is meant an increase in, for example, expression
of a particular molecule, e.g., tenascin or tenfibgen, relative to
a control, e.g., relative to the level of expression that is
normally produced by non-cancerous cells. The exact amount of
over-expression or increase is not necessarily critical, as long as
the over-expression or increase is statistically significant.
Diagnostic methods for cancer and the clinical delineation of
cancer diagnoses are known to those of ordinary skill in the
art.
[0084] Assays according to some embodiments of the invention may be
carried out in vivo, in vitro, or ex vivo using samples obtained
from standard sources and by standard procedures. A "sample" can be
any organ, tissue, cell, or cell extract isolated from a subject,
such as a sample isolated from a mammal having a cancer. For
example, a sample can include, without limitation, cells or tissue
(e.g., from a biopsy or autopsy) from plasma, organ or tumor, or
any other specimen, or any extract thereof, obtained from a patient
(human or animal), test subject, or experimental animal. In some
embodiments, it may be desirable to separate cancerous cells from
non-cancerous cells in a sample.
[0085] A "control" includes a sample obtained for use in
determining base-line expression or activity. Accordingly, a
control sample may be obtained by a number of means including from
non-cancerous cells or tissue, e.g., from cells surrounding a tumor
or cancerous cells of a subject; from subjects not having a cancer;
from subjects not suspected of being at risk for a cancer; or from
cells or cell lines derived from such subjects. A control also
includes a previously established standard. Accordingly, any test
or assay conducted according to an embodiment of the invention may
be compared with the established standard, and it may not be
necessary to obtain a control sample for comparison each time.
[0086] Tenfibgen or tenfibgen expression or activity can be assayed
using a variety of techniques, including immunohistochemistry
(IHC), in situ hybridization (ISH), Northern blotting, polymerase
chain reaction (e.g., real time quantitative PCR or RT-PCR),
antibody-based assays, such as immunoprecipitation,
immunofluorescence, Western blotting, nucleic acid sequencing,
etc.
[0087] In some embodiments, cells in a subject may be exposed in
vivo to an antibody (e.g., a tenfibgen or tenascin antibody, or a
tenfibgen-coated nanoparticle at about 10 .mu.g/.mu.l of
oligonucleotide concentration), which is optionally detectably
labeled, e.g., radioactive isotope; and binding of the antibody to
the cells may be evaluated by, e.g., external scanning for
radioactivity or analysis of a biopsy.
[0088] The assays may be conducted using detectably labelled
molecules, i.e., any means for marking and identifying the presence
of a molecule, e.g., an oligonucleotide probe or primer, a gene or
fragment thereof, a peptide, or a cDNA molecule. Methods for
detectably labelling a molecule are known in the art and include,
without limitation, radioactive labelling (e.g., with an isotope
such as .sup.32P or .sup.35S) and non-radioactive labelling such as
enzymatic labelling (for example, using horseradish peroxidase or
alkaline phosphatase), chemiluminescent labeling, fluorescent
labeling (for example, using fluorescein), bioluminescent labeling,
or antibody detection of a ligand attached to the probe. Also
included in this definition is a molecule that is detectably
labelled by indirect means, for example, a molecule that is bound
with a first moiety (such as biotin) that is, in turn, bound to a
second moiety that may be observed or assayed (such as
fluorescein-labeled streptavidin). Labels also include digoxigenin,
luciferases, and aequorin.
[0089] The term "detecting", as used herein, is intended to include
determining the presence or absence of a substance and/or
quantifying the amount of the substance. The term, thus, refers to
the use of the materials, compositions, and methods according to
certain embodiments of the invention for qualitative and
quantitative determinations. In general, the particular technique
used for detection is not critical for practice of the invention.
Detecting may include quantifying a change (increase or decrease)
of any value between about 10% and about 90%, or of any value
between about 30% and about 60%, or over about 100%, when compared
to a control. Detecting may include quantifying a change of any
value between about 2-fold to about 10-fold, inclusive, or more,
e.g., about 100-fold.
[0090] As used herein, an "effective amount" or "therapeutically
effective amount" is defined as an amount effective, at dosages and
for periods of time necessary, to produce the intended
pharmacological, therapeutic or preventive result. For example, if
a given clinical treatment is considered effective when there is at
least about a 25% reduction in a measurable parameter associated
with a disease or disorder, a therapeutically effective amount of a
drug for the treatment of that disease or disorder is the amount
necessary to effect at least about a 25% reduction in that
parameter. The effective amount of the formulated drug, vaccine, or
imaging agent of the invention may vary according to factors such
as the disease state, age, sex, and weight of the subject. The
precise amount and number of doses and timing can be readily
determined by those skilled in the art. With regard to topically
applied vaccines, factors influencing dosing include, without
limitation, the chemical structure and biological activity of the
antigen and adjuvant, the concentration of the application, the
body site to be treated, the surface area, age, weight, and
treatment history of the subject, and the precise condition
requiring treatment and its severity.
[0091] Dosage regimens may be adjusted to provide the optimum
response. For example, doses may be one-time or multi-dose, or
several divided doses may be administered daily, or the dose may be
proportionally reduced as indicated by the exigencies of the
medical situation. An effective amount of antigen sufficient to
induce an immune response may be delivered, for example, at a
single cutaneous and/or mucous membrane location, or at multiple
cutaneous and/or mucous locations, or, in a separate embodiment,
over an area of skin covering multiple draining lymph node fields
(e.g., cervical, axillary, inguinal, epitrochelear, popliteal,
mandibular, those of the abdomen and thorax). Such locations close
to numerous different lymphatic nodes at locations all over the
body may provide a more widespread stimulus to the immune system
than when a small amount of antigen is injected at a single
location intradermally, subcutaneously, or intramuscularly.
[0092] Example 7 describes a study using three courses of 500 .mu.g
plasmid DNA antigen encapsulated in sub-50 nanometer nanoparticles
comprising a low-molecular weight hyaluronan shell spiked with
nickel for adjuvant effect, administered in a weanling pig model
via passive topical application. IgG titers generated with this
formulation were within one log of standard intradermal protein
treatment (3.times. at 500 g), and were two-fold the titer level
generated by intradermal naked DNA treatment (3.times. at 500 g).
Specific IgA response was persistent and 2.5.times. vs. naked
intradermal DNA by grand average while specific lymphocyte response
was 37.times. for draining nodacytes and 12.1.times. for distal
nodacytes (p<0.05) over standard intradermal protein
inoculation. No treatment site reactions were found after topical
administration at the end of the 7-week study.
[0093] The artisan will recognize the methods and results for
weanling pigs are relevant to other mammal models, including
humans. For example, with regards to the relevance to humans, the
physiology of the skin is known to be very similar between humans
and pigs, and the development of the immune system is also similar
between humans and pigs.
[0094] In one embodiment, a method according to the invention
includes evaluating the subject for lymph node concentration and/or
plasma concentration of the administered drug or diagnostic agent.
Such an evaluation can, for example, be performed before, during,
and/or after the administration of the agent. For example, the
evaluation can be performed at least about 1 day, about 2 days,
about 4, about 7, about 14, about 21, or about 30 or more days
before and/or after the administration.
[0095] Treatment site reactions can occur when a therapeutic is
deposited, for example, in the subcutaneous tissue or topically on
the tissue of a subject. In one embodiment, the extravascular
administration of the nanoparticles described herein avoids or
diminishes such reactions, due to low dosage, effective tissue
clearance, absence of prolonged treatment-site retention due in
part to the crystal nature and/or essentially neutral charge of the
nanoparticles, and/or other factors.
[0096] Furthermore, the volume of subcutaneous injection is limited
(e.g., in humans, subcutaneous injection is limited to about 1 mL)
because of the pain and tissue distortion, irritation, and redness
(erythema) that accompany large volumes. This limitation prevents
subcutaneous delivery of drugs which typically require high
dosages. Thus, in an embodiment of the present invention, the
nanoparticles described herein enable subcutaneous delivery of such
high-dose drugs, due to more effective biodistribution, less
aggregation at or near the injection site, and, in comparison to
other nucleic-acid delivery technologies, more efficient packaging
of the drug in the nanoparticle (e.g., where the encapsulated drug
accounts for more than about 60%, more than about 70%, more than
about 80%, more than about 85%, or more than about 90% of the
weight of the nanoparticle). In separate embodiments, the
formulations described herein are suitable for subcutaneous
administration volumes of about 2 mL, about 5 mL, about 10 mL,
about 20 mL, and between about 2 and about 20 mL.
[0097] Also provided herein, in additional embodiments, are
diagnostic agents and methods of use to improve the utility and
accuracy of diagnostic imaging techniques in research and medical
applications. Generally, diagnostic agents are agents that can be
detected or observed following administration into a cell, tissue,
organ, or mammal. Diagnostic agents include contrast agents, which
are used to improve the visualization of cells, tissues, organs,
and physiologic processes by increasing the relative difference of
imaging signal intensities in adjacent regions of the body.
Diagnostic imaging techniques of the nanoparticles described herein
can be used, for example, in the identification, planning, staging,
treatment, and monitoring of, for example, lesions or other areas
of abnormal tissue. For example, diagnostic imaging techniques may
be used in the treatment of patients with tumor lesions, including
where the treatment consists of chemotherapy, surgery, and/or
radiation therapy.
[0098] Diagnostic techniques can also be used in molecular imaging,
which is broadly defined as characterization and measurement of the
biologic processes of disease by assessing functional (e.g.,
vascularization, blood flow and lymphatic flow), metabolic (e.g.,
glycolysis and tumor viability), or molecular phenomena (e.g.,
proliferation, epidermal growth factor receptor expression and
apoptosis, at the cellular and molecular level).
[0099] In some embodiments, the nanoparticle diagnostic agent
comprises a core comprised of dysprosium or dysprosium derivatives
associated with a functionalized chelating ligand complex, wherein
the functionalized chelating ligand complex is comprised of a
covalently-bound functionalized chelating ligand and a polymer
comprised of dextran or derivatives of dextran; a surfactant
substantially surrounding the core to form a surfactant coated
complex, wherein the surfactant has an HLB value of less than about
6.0 units; and a shell which non-covalently adheres to and
substantially surrounds the surfactant-coated complex, wherein the
shell comprises a precipitate comprising a targeting moiety and a
cationic precipitating agent, wherein the cationic precipitating
agent is comprised of lithium.
[0100] In some embodiments, a water-soluble polysaccharide other
than dextran can be used. Non-exhaustive examples of such a
polysaccharide would include mannose and sucrose.
[0101] In another embodiment, the chelating ligand is chosen from
the group of diethylenetriamine tetraacetate (DTTA),
diethylenetriamine pentaacetate (DTPA),
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(2-propionic acid)
(DOTMA), and 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic
acid (TETA).
[0102] In further embodiments, methods of using the nanoparticle
diagnostic agents include analyzing a cell, a tissue, or a subject,
the methods comprising administering to a cell, a tissue, or a
subject a nanoparticle diagnostic agent and subjecting the cell,
tissue, or subject to fluorescent imaging, neutron activation
analysis (NAA), and/or related applications, and analyzing the
resulting information to detect, diagnose, monitor, or report on a
parameter of a cell, a tissue, or a subject. The use of the
nanoparticle diagnostic agents disclosed herein as fluorescent
(optical) agents is surprising, as dysprosium is not known to
possess useful fluorescent properties. Relative to other
water-quenched lanthanides, dysprosium luminescence is considered
the least stable, with a time constant of only 9-11 .mu.sec in
aqueous media. Without wishing to be bound by theory, it is
believed the nanoparticle diagnostic agents described herein are
not subject to the some or all of the complications of
photobleaching, photodegradation, and diffusion that are typically
associated with the use of optical imaging dyes and protocols.
[0103] In one embodiment, the nanoparticle diagnostic agents of the
present invention are used as a tissue marker to mark a position in
a body, such as a specific position in a tissue or organ, in order
to allow re-visiting of the position to check for progress or
developments of an ailment or a treatment, or to allow re-treatment
at the same site. For example, tissue marking can be used during a
biopsy or other tissue removal procedure to accurately mark the
site of the tissue removal or biopsy, thus allowing later return to
the same site if desired, in order, for example, to monitor the
status of the tissue in question or to carry out a further biopsy.
A tissue marker may be viewed broadly as a type of imaging agent
that does not move or stays substantially in the same position once
it has been administered or implanted. It is often desirable that
the tissue marker is biodegradable over a period of time and is
resorbed safely by the body.
[0104] In another embodiment, the nanoparticle diagnostic agents
disclosed herein are used in and/or administered to an aqueous
environment, such as a mammal including, for example, a human. In
additional embodiments, agents are used in imaging and/or
diagnosis, wherein a sample is subjected to imaging outside of the
human or animal body. This includes, for example, uses wherein a
sample has been removed from the human or animal body or generated
outside of the human or animal body.
[0105] In certain embodiments, the nanoparticle diagnostic agents
are used in optical imaging techniques such as, for example, near
infrared (NIR) imaging. This technique involves excitation of a
fluorophore that emits light at a wavelength in the red or far red
end of the light spectrum (longer than 600 nm). This is a desirable
range, as red light passes through living entities better by
avoiding absorption by common substances such as hemoglobin.
Equipment suitable for optical imaging is well-known in the art and
generally consists of a light source, filters, detector, and
appropriate electronics for signal processing. Commercial systems
such as the Kodak Carestream FXpro, MAESTRO and Xenogen systems are
readily available. Key operating parameters for the inventive
nanocapsule-based Dy composition include optimal excitation in
living creatures at 510 nm and emission at 700. Multispectral
analysis to extract the Dy-specific signal is typically supplied
with the imaging system and is very beneficial to use. Following
dosing which may range from about 10 nmol Dy/kg to about 1 mmol
Dy/kg of body weight and a desired amount of time for capsule
binding to occur which may last from about 1 minute to about 5
days, image capture is executed for an exposure period that may
last from about 1 second to about 15 minutes.
[0106] The s50 nanoparticle chemistry together with Dy-chelated
cargo provides the important advantage and benefit of facile
preparation methods, incorporating proteins, peptides, antibodies,
carbohydrates, or small molecules as ligands in the shell. Ligands
may be readily exchanged for diagnostic purposes in s50
ligand-targeted particles, e.g., tenfibgen or ASOR may be
substituted for an anti-PSA antibody as a ligand to enable a
diagnostic imaging agent or theranostic capable of identifying
patient populations for treatment guidance.
[0107] In another embodiment, methods are described for bulk
quantitation of target cells (e.g., tumor cells) in tissue by
analytical methods (e.g., ICP-AES, Neutron Activation Analysis
(NAA)). It has been discovered that the s50 capsule together with
Dy-chelated cargo such as DyDex also provides high sensitivity in
NAA (Dy detectable to 0.1 ppb). Without wishing to be bound by
theory, this physical characteristic derives, in part, from the
large size of the Dy nucleus, enabling it to function as a
favorable target for neutron bombardment and activation. This
feature enables measurement of Dy cargo as an isotopic tracer by
quantitative method. Such bulk measurement would improve
sensitivity of detection over spot sampling methods such as tissue
sectioning and microscopic observation.
[0108] Results presented herein demonstrate the quantitative nature
of Dy cargo as a tracer and suggest methods for bulk quantitation.
Tissue samples may be ashed, dissolved in dilute nitric acid, and
detected for Dy sensitively using established ICP-AES methods. For
neutron activation analysis, tissues are simply weighed and shipped
to a fusion reactor facility for neutron bombardment and
measurement of metastable Dy-163 gamma spectra. Additionally, in
line with the multi-modal nature of Dy detection, these bulk
measurements can be readily correlated with current
microscopy-based methods of detection, due to the long-term
retained fluorescence of Dy within the hydrophobic environment
within the s50 capsule within formalin-fixed tissue. For example,
the use of tumor-targeted capsules bearing fluorescent Dy cargo has
been administered in tumor margin analysis post-resection. Dy has
excitation peaks at 320 nm and 510 nm with emission peaks at 547 nm
and 720 nm. Therefore, both conventional fluorescence microscopy
based on mercury-arc illumination with a broad UV excitation
emission filtering in the range of 547, e.g., Cy3, Texas Red or
confocal microscopy based on yellow laser excitation (514 nm) and
image capture at either 547 nm or about 720 nm is feasible.
[0109] The utility of the Dy-bearing s50 particle as a diagnostic
agent for detecting and quantitating cargo at specific organ
locations by extravascular routes is demonstrated in Example 5,
where Dy was transferred into the body of mice by both topical and
subcutaneous routes, as well as intravenous. Example 5 further
demonstrates the utility of s50 Dy agents (here, delivered i.v.)
for optical (NIR) imaging of internal organs such as liver and gall
bladder in a mouse model. Taken together, those of ordinary skill
in the art will appreciate from these results that the s50 Dy
particle can be used for optical imaging methods such as, for
example, NIR, as well as for detection methods such as NAA, by a
variety of routes of administration, and that the s50 Dy particle
provides utility for as a diagnostic agent in extravascular, as
well as intravenous, applications.
[0110] In another embodiment, the nanoparticles described herein
are used to identify and measure the disease-fighting capacity of
the organ or tissue or cell of interest by delivering a diagnostic
payload to a target that corresponds with the disease-fighting
capacity of the organ or tissue or cell of interest.
[0111] In additional separate embodiments, the nanoparticles
contemplated for extravascular administration are used for uptake
into cells by lipid rafts, for avoidance of endosomal entrapment,
and/or for delivery to the nucleus and/or the cytoplasm of the
cell.
[0112] In one embodiment, the nanoparticles described herein are
administered extravascularly to treat chronic diseases such as
cardiovascular or metabolic (e.g., diabetes) diseases, where
intravenous delivery is not desirable due, for example, to cost,
convenience, or other factors.
[0113] The following is a brief description of methods that can be
used to make the nanoparticles as disclosed herein. The following
description is meant to be representative only and is not meant to
be limiting. Briefly, a negatively-charged cargo moiety such as
nucleic acid that is to be targeted and delivered to a cell can be
complexed with a polycationic polymer to condense or reduce its
size to about 50 nm or less. A number of different polycationic
polymers (also known as "condensing" agents or proteins) can be
used and are well-known in the art. See, for example, Rolland
(1998, Crit. Rev. Therapeutic Drug Carr. Syst., 15:143-198). For
example, enough polycationic condensing protein can be complexed
with the negatively-charged cargo moiety to neutralize at least
about 75% (e.g., about 80%, about 85%, about 90%, about 95%, about
99%, or about 100%) of the negatively-charged cargo moiety, which,
for nucleic acids, can be measured by ethidium dye exclusion (1998
J. Controlled Release 53:289-99). Non-limiting examples of
condensers include spermine, polyornithine, and protamine. Simply
by way of non-limiting example, 125 .mu.g of 10 kD polyornithine
can be used to condense 500 .mu.g of a 20-mer oligonucleotide or
87.5 .mu.g of spermine may be used to condense 250 .mu.g of a 14 kD
siRNA oligo. For cargo moieties lacking a negative charge or
bearing a positive charge, a condensing polycationic polymer may
not be necessary.
[0114] An aqueous solution of the complexed or uncomplexed cargo
moiety can be encapsulated by first dispersing the cargo moiety
into a biocompatible, water-miscible solvent using a biocompatible,
water-insoluble surfactant system suitable for preparation of an
inverted or reverse micelle. Suitable surfactant systems are
well-known in the formulation arts as amphiphilic materials that
are essentially hydrophobic and characterized by a
hydrophile-lipophile balance (HLB) of about 6.0 or less, a critical
micelle concentration (CMC) of less than about 200 .mu.M, or a
critical packing diameter greater than about 1.
[0115] Hydrophobic surfactants and hydrophobic, water-miscible
solvents suitable for preparing reverse micelles are described in
Pashley & Karaman (2004, In Applied Colloid and Surface
Chemistry, John Wiley, pgs 60-85), Rosen (2004, In Surfactants and
Interfacial Phenomena, John Wiley), The Handbook of Industrial
Surfactants (1993, Ash, ed., Gower Pub), and Perry's Chemical
Engineer's Handbook (1997, Perry & Green, 7th Ed., McGraw-Hill
Professional). In one embodiment, a hydrophobic surfactant can be
2,4,7,9-tetramethyl-5-decyn-4,7-diol (TM-diol) used in a
concentration of up to 0.5% by weight of surfactant micelle volume,
and a water-miscible solvent can be dimethylsulfoxide (DMSO). In
other embodiments, the water-miscible solvent can be acetone,
acetonitrile, ethanol, dimethyl acetamide (DMA), tetrahydrofuran
(THF), dioxane, and dimethylformamide (DMF).
[0116] The concentration of surfactant selected should, in one
embodiment, be sufficient to prepare an optically clear
nanoemulsion, but not so much as to induce aggregation, since
aggregation can lead to overly large nanoparticles.
[0117] In some embodiments, the surfactant includes at least one of
cetyl alcohol, 2, 4, 7, 9-tetramethyl-5-decyn-4, 7-diol, molecules
containing an acetylenic diol portion, and blends of 2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol. In some embodiments, the HLB of
the surfactant is less than about 5.0. In some embodiments, the
hydrophobic surfactant at least partially surrounds or encloses the
diagnostic or therapeutic-containing core. In some embodiments, the
surfactant complex is at least partially surrounded by the
targeting moiety and cationic precipitating agent.
[0118] The micelles carrying the cargo moieties can be coated by
adsorption with cell-targeting moieties (e.g., proteins, peptides,
carbohydrates, or antibodies) by mixing one or more targeting
moieties with an aqueous suspension of the micelles. In some
non-limiting embodiments, targeting moieties can be mixed with
nanocapsules in a ratio (by weight) of about 1 part ligand to about
100 parts drug, to about 1 parts ligand to about 10 parts drug,
depending upon the rate at which the nanocapsule is desired to
dissolve or disassemble. In one embodiment, the coating weight
ratio is about 1:16 of nanocapsules to targeting moieties. In
another embodiment, the ratio is about 1:20.
[0119] Targeting moieties may also, in some embodiments, be
modified by processes designed to enhance final nanoparticle
function, e.g., tenascin polypeptides may be precipitated from cell
culture supernatants using metal-containing ammonium sulfate such
that metals known to promote oxidative stress are adsorbed onto
coating ligands preceding nanoparticles preparation. Coating
ligands may be readily modified with pharmaceutically acceptable
heavy metals by re-precipitating protein in saturated ammonium
sulfate solutions prepared with known levels of heavy metals.
Incubation of an about 0.1-about 1 mg/ml solution of protein about
1:2 with a saturated ammonium sulfate solution is most
expeditiously executed for about 4-36 hours before recovering
metal-modified coating ligand by centrifugation. Metal
concentrations in the ultrapure ammonium sulfate may range from
about 1 part per thousand-about 1 part per trillion.
[0120] To stabilize the targeting moiety-adsorbed nanocapsule, the
aqueous suspension of nanocapsules coated with one or more
targeting moieties can be mixed into an aqueous solution of metal
ions (i.e., a "stabilization solution") capable of precipitating,
crystallizing, or iontophoretic exchange with the coated
nanocapsules. Representative, non-limiting examples of solutes that
can be used to precipitate the coated nanocapsules include ionic
species derived from elements listed in the periodic table. Ions
may be included in the aqueous stabilization composition in a range
from about 0.1 part per trillion to about 1 Molar (M). An adequate
amount of ion should be included such that the coated nanocapsules
are sufficiently contacted with ions, but not so much that
aggregation occurs, which can lead to overly large capsules. In one
embodiment, a stabilization solution can include about 10
millimolar (mM) Ca2+ and about 200 mM Li+. If ultrapure reagents
are used in the stabilization solution, addition of very small
amounts (e.g., less than 1 mM) of ions such as Ba, Fe, Mg, Sr, Pb
and Zn, normally found in sufficient quantities in preparations of
lithium and calcium salts that are not ultrapure, may be added to
optimize stabilization of the coated nanocapsules. Addition of
cations such as, without limitation, Ni, Bi, Pb, Ur, Al, Ba, and
Dy, in the receiving bath can also be useful for manipulating
nanoparticle properties. Suitable counterions include chloride,
acetate, sulfate, and similar anions. In a certain embodiment for
vaccine nanoparticles with 250 .mu.g of plasmid DNA, the cations in
the receiving bath are comprised of 135 mM Li+, 9 mM Ca2+, 1.25 nM
Sr2+, 10.0 nM Mg2+ and 380 aM Ni2+(all ultrapure), wherein the
counterion for each is chloride.
[0121] In some embodiments, the nanoparticles are prepared in a
receiving solution for subsequent precipitation and
crystallization, wherein the solution comprises Li.sup.+ which may
range from about 10-about 250 mM, Ca.sup.2+ which may range from
about 1-about 20 mM, Sr.sup.2+ which may range from about 0-about 1
.mu.M, and Mg.sup.2+ which may range from about 0-about 1 uM.
Suitable counterions for these cations include chloride, acetate,
sulfate, and similar anions.
[0122] In some embodiments, the nanoparticles described herein do
not comprise polyethylene glycol (PEG) or derivatives thereof. In
some embodiments, the nanoparticles do not comprise
polyethylenimine (PEI) or derivatives thereof.
[0123] Any configuration of the nanoparticles that gives rise to an
overall compact and/or globular shape having an appropriate
morphology is acceptable, including where there is substructure to
the nanoparticle, as long as the overall compact shape occurs. When
the overall morphology of the nanoparticle comprises a compact
shape, the final surface charge approaches about neutral or
slightly negative charge. In one embodiment, a final surface charge
of neutral or slightly negative is a preferred surface charge for
the nanoparticles contemplated herein for extravascular
administration. In another embodiment, particles with a charge of
between about +2 and about -15 milli electron volts (mev) are
desirable. Additionally, any other components that are capable of
increasing the stability of the nanoparticles can be included as
part of the stabilization solution, such that the final dry average
diameter of the nanoparticles is between a range of about 5-about
50 nm by AFM.
[0124] For a more consistent size of nanoparticles, the
nanoparticles can, in one embodiment, be atomized through a nozzle.
Atomization should be sufficient to apply a shear force capable of
breaking up flocculated aggregates without so much force as to
induce hard aggregates. Those skilled in the art will understand
that a particular nozzle diameter will lead to range of feed
pressures suitable for atomizing the nanoparticles to a suitable
and consistent size. In one embodiment, a nozzle diameter of less
than about 250 microns with feed pressures of less than about 10
psi produces suitable nanoparticles. In another embodiment, the
nanoparticles can be atomized into a stabilization solution.
[0125] The incubation time and temperature may be varied from about
8 hrs to about 7 days to vary the amount of time required for
particle dissolution or disassembly in end use. After
precipitating, atomizing, and/or incubating the nanocapsules in a
stabilization solution, the nanocapsules can be filtered,
centrifuged, and/or dried to obtain separate and discrete sub-50 nm
nanocapsules. In one embodiment, nanocapsules are incubated for
about 2 days at about 4.degree. C., with nominal rotation in 50-ml
round-bottomed tubes with salt solution. The resultant nanocapsules
can be frozen or dried and reconstituted for later use. In some
embodiments, the particles are subjected to one freeze-thaw before
sizing analysis. Encapsulation yields can be determined by methods
known in the art, including methods of Burton and ICP.
[0126] Methods for synthesizing the nanoparticle diagnostic agents
disclosed herein include mixing dysprosium or derivatives of
dysprosium with amide-terminated dextran or dextran derivatives
covalently linked to a functionalized chelating ligand to create a
core mixture, subjecting the mixture to purification by a
column-based method to create a purified core mixture, disposing
into the purified mixture a hydrophobic surfactant to create a
surfactant complex mixture, wherein the hydrophobic surfactant has
an HLB value of less than about 6.0 units, and mixing the
surfactant complex mixture with a precipitating solution comprising
a targeting moiety and a cationic precipitating agent comprised of
lithium to create a precipitated nanoparticle mixture, resulting in
a nanoparticle diagnostic agent comprising the
dysprosium-containing core substantially surrounded by the
hydrophobic surfactant, which is substantially surrounded by the
targeting moiety and cationic precipitating agent.
[0127] The disclosed diagnostic agents provide specific targeting,
high resolution, and high sensitivity. In addition, the disclosed
diagnostic agents are compatible with a range of imaging
modalities. Further, the disclosed diagnostic agents provide a
modular targeting component that can be readily synthesized for a
given biomarker target. The disclosed diagnostic agents also can be
used in both an imaging application and in an analytic application,
such as histological diagnoses of collected cells for biochemical
tests of blood, to validate that the image observed at a given site
after administration of the agent consistently and accurately
represents accumulation of the agent at that site.
[0128] As described above, the nanoparticles of the present
invention may be administered topically with facilitating agents
that include, without limitation, patches, pipettes, lotions,
ointments, creams, sprays, and/or gels. Those of ordinary skill in
the art will appreciate that the inventive nanoparticle
compositions may be readily incorporated or formulated with such
agents. For example, a variety of transdermal patch structures are
known in the art. In an additional embodiment of the present
invention, a transdermal patch includes an adhesive. Some examples
of adhesive patches are well known (for example, U.S. Pat. Nos.
6,010,715; 5,591,767; 5,008,110; 5,683,712; 5,948,433; and
5,965,154; all of which are incorporated herein by reference).
Adhesive patches are generally characterized as having an adhesive
layer, which will be applied to a person's skin, a depot or
reservoir for holding a pharmaceutical agent, and an exterior
surface that prevents leakage of the pharmaceutical from the depot.
The exterior surface of a patch is typically non-adhesive.
[0129] In another embodiment, the nanoparticles are prepared as a
pack or kit, including as an outpatient pack or a kit for
self-administration.
[0130] Methods of synthesizing nanoparticle vehicles are described
in, for example, the following U.S. patents and patent applications
and are incorporated herein by reference in their entireties, with
modifications as described herein: Unger, U.S. Pat. No. 6,632,671,
issued Oct. 14, 2003; as well as U.S. Patent Publication Nos.
20090238883, 20080113932, 20070098713, 20060018826, 20040137071,
20040038303, and 20040023855, U.S. patent application Ser. Nos.
12/525,652, and 12/644,610; and PCT/US2008/052863. Examples of such
nanoparticle manufacture are also disclosed herein in the Examples
section, below.
[0131] Various embodiments of the disclosure could also include
permutations of the various elements recited in the claims as if
each dependent claim was a multiple dependent claim incorporating
the limitations of each of the preceding dependent claims as well
as the independent claims. Such permutations are expressly within
the scope of this disclosure.
[0132] While the invention has been particularly shown and
described with reference to a number of embodiments, it would be
understood by those skilled in the art that changes in the form and
details may be made to the various embodiments disclosed herein
without departing from the spirit and scope of the invention and
that the various embodiments disclosed herein are not intended to
act as limitations on the scope of the claims. All references cited
herein are incorporated in their entirety by reference.
EXAMPLES
[0133] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1--Preparation of Dysprosium-Chelated Dextran (DyDex)
[0134] This method is based on a modification of Armitage, F. E. et
al., Bioconjugate Chem 1990 1(6):365-374. Using a 10.times. molar
excess of p-SCN-Bn-Dota (Macrocyclics B-205, MW 688, 0.1M in DMSO)
to dextran amino groups, react p-SCN-Bn-DOTA with amino-terminated
dextran (Invitrogen D1861, 40KD) in 0.1 M Sodium bicarbonate pH
9.0. Incubate overnight at 25.degree. C. Optionally, in place of
p-SCN-Bn-Dota, p-NH.sub.2-Bn-DTPA, p-NH.sub.2-Bn-DOTA,
p-NH.sub.2--SCN-DTPA, p-NH.sub.2-Bn-NOTA, p-SCN-Bn-oxo-DO3A,
p-NH.sub.2-Bn-oxo-DO3A, p-NO.sub.2-Bn-DTPA, p-NO.sub.2-Bn-DOTA,
p-NH.sub.2-Bn-DOTA (t-Bu-ester), p-NH.sub.2-Bn-DTPA(t-Bu-ester)
p-SCN-Bn-NOTA, p-SCN-PCTA p-NH.sub.2-PCTA, or other similar
compositions may be suitable. Calculation of the activated chelate
as 90.times. molar excess on dextran is also acceptable. Dialyze,
using a 3500 MWCO cartridge, against 0.1 M Sodium bicarbonate pH
9.0 for 4 buffer exchanges, then against ddH.sub.2O for 4
additional exchanges. Following lyophilization, incubate dextran
intermediate in deionized water with a 10.times. molar excess of
0.1M DyCL3 (Sigma 203173) overnight at 25.degree. C. Dialyze, using
a 3500 MWCO cartridge, against deionized water for 4 to 6
exchanges. Lyophilize and weigh product, measuring Dy incorporation
by ICP-AES. Yields in this process are approximately 70% by dextran
with a 4-4.5% incorporation of Dy by weight.
Example 2--Preparation of Liver-Targeted Nanoparticles for
Subcutaneous Administration
[0135] This example describes how colloidal formulations of diverse
cargos and biocompatible polymers may be generated, for subsequent
subcutaneous administration. Nanoparticles were prepared by the
"dispersion atomization" method described in U.S. Pat. No.
6,632,671, which is incorporated herein by reference in its
entirety, with some modifications. Oligonucleotide preparations
were synthesized by Dharmacon (Boulder, Colo.; siFVII and siRFP)
and Trilink Biotechnologies (San Diego, Calif.; single strand FVII
and siAboB).
[0136] Briefly, to prepare each formula below, the following
procedures were used:
[0137] Formula A, 250 g of 21 mer, unmodified, double-stranded RNA
oligonucleotide (siFVII, Akinc, et. al, 2009 Mol Ther
17(5)872-879), was first complexed with 87.5 .mu.g of 300 MW
spermine (Sigma), then was dispersed into 100 .mu.l of sterile
water using a water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE-30 (Air Products),6.25
.mu.g in 50% DMSO. Following emulsification with 150 .mu.l of a
water-miscible solvent (DMSO), the complexes were then inverted and
diluted by the addition of 750 .mu.l of PBS.
[0138] The resultant hydrophobic micelles were coated
(non-covalently) by the addition of 12.5 .mu.g of asialoorosomucoid
(ASOR; prepared by the method of Stockert, et al. 1980 Lab. Invest.
43:556-63), then atomized into a modified LiCl salt receiving
solution (135 mM Li.sup.+, 9 mM Ca.sup.2+, 31.25 nM Sr.sup.2+, 12.5
nM Mg.sup.2+ (all ultrapure, Sigma)). Following cold-room
incubation (4.degree. C.) with nominal rotation in 50 ml
round-bottomed tubes for 48 hours, which stabilizes the coated
micelles in the salt solution, the sub-50 nm nanocapsules were
recovered by centrifugation at 20,000.times.g for 2 hrs and
resuspended in PBS+10% lactitol (at a concentration of 0.5
.mu.g/.mu.l) for filter sterilization through a 0.2 .mu.m filter.
In all formulations described, a small amount (1% of coating
weight) of Syrian Hamster Fab fragment was "spiked" into the ligand
coat to enable immunodetection of nanocapsules uptake by
anti-syrian hamster IgG antibodies. Average capsule size with
standard error was about 22.5.+-.about 1 nm, as measured by tapping
mode atomic force microscopy using average of minor and major
elliptical diameters of a 0.5 ng/ml sample dried down on a mica
sheet. Average zeta potential was between about +2 and -15 mev
(-5.5.+-.5.4 mev) when measured on Zetasizer 4 dynamic light
scattering device at a potential of 20 volts with a 2-second pause
between measurements in 1 mM KCl at 2 .mu.g/ml. Encapsulation yield
was 100% by the modified method of Burton, Kren, et al. 2009 JCl
119(7):2086-99.
[0139] Formula B: sub-50 nm nanocapsules coated with
asialoorosomucoid (ASOR; prepared by the method of Stockert, et al.
1980 Lab. Invest., 43:556-63, incorporated herein by reference in
its entirety) bearing single-stranded DNA/RNA chimeric guide strand
against murine coagulation Factor VII (rniFVII, 5'-gta aga ctt gag
a [none] 2o'ME[uga ucc]-(propyl)-3', antisense strand sequence from
Akinc, et al. 2009 Mol Ther 17(5)872-879) were generated as
described in Formula A except that chimeric guide strand was
complexed in 63 .mu.g of 10 kD polyornthine (Sigma) before
dispersion and a modified LiCl salt receiving solution (135 mM
Li.sup.+, 9 mM Ca.sup.2+, 14.06 nM Sr.sup.2+, 6.25 nM Mg.sup.2+
(all ultrapure)) was used for generating nanocapsules. Average
particle size was 17.8.+-.1.5 nm (mean.+-.SE), and surface charge
was -1.+-.3.5 mev for the instant preparation.
[0140] Formula C: sub-50 nm nanocapsules coated with ASOR and
bearing a modified, double-stranded RNA oligonucleotide against
murine ApoB (siApoB-1, sequence as described in Rozema, et al. 2007
PNAS 104(32):12982-87) were prepared as described in Formula A
except that 25 mcg of ASOR was added to the hydrophobic micelles.
When generating these nanocapsules, the ASOR-coated micelles were
atomized into a modified LiCl salt receiving solution (135 mM
Li.sup.+, 9 mM Ca.sup.2+, 26.3 nM Sr.sup.2+, 15 nM Mg.sup.2+ (all
ultrapure)).
[0141] Formula D: 1000 .mu.g of Dysprosium-chelated Dextran (from
Example 1) was dispersed into 100 .mu.l of sterile water using a
water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE-30 (Air Products),
6.25 .mu.g in 50% DMSO). Following emulsification with 150 .mu.l of
a water-miscible solvent (DMSO), the complexes were then inverted
and diluted by the addition of 750 .mu.l of PBS.
[0142] The resultant hydrophobic micelles were coated
(non-covalently) by the addition of 50 .mu.g of ASOR, then atomized
into a modified LiCl salt receiving solution (135 mM Li.sup.+, 9 mM
Ca.sup.2+, 27.5 nM Sr.sup.2+, 13.75 nM Mg.sup.2+ (all ultrapure)).
Following cold-room incubation (4.degree. C.) with nominal rotation
in 50 ml round-bottomed tubes for 48 hours, which stabilizes the
coated micelles in the salt solution, the sub-50 nm nanocapsules
were recovered by centrifugal filtration with a Millipore
Centricon-70 10KD device and resuspended in PBS+10% lactitol for
filter sterilization through a 0.2 .mu.m filter. In all
formulations described, a small amount (1% of coating weight) of
Syrian Hamster Fab fragment was "spiked" into the ligand coat to
enable immunodetection of nanocapsules uptake by anti-syrian
hamster IgG antibodies. Average capsule size was between about 8-20
nm as measured by tapping mode atomic force microscopy using
average of minor and major elliptical diameters of a 0.5 ng/ml
sample dried down on a mica sheet. Formulations were subjected to
one freeze-thaw before sizing analysis. Average zeta potential was
between about +2 and -15 mev when measured on Zetasizer 4 dynamic
light scattering device at a potential of 20 volts with a 2-second
pause between measurements in 1 mM KCl at 2 .mu.g/ml. Encapsulation
yield for Dy content was determined by ICP.
[0143] Formula E: sub-50 nm nanocapsules coated with TBG were
generated as described in Formula A except that 31.25 mcg of
recombinant fibrinogen fragment of tenascin (TBG; prepared by the
modified method of Aukhill, et al. (1993 J. Biol Chem. 268:2542-53)
see below for method, precipitated in ultra-pure 40% ammonium
sulfate containing 250 ppb As.sup.+3, 25 ppm Se.sup.+4, 2.5 ppm
Hg.sup.+2 and 25 ppm Mo.sup.+5 for about 16 hours) was added to 500
mcg of an antisense oligo to CK2 (phosphodiester 3' and
propylendblocked-2OME RNA chimeric, "LCK-6", (PCT/US 2005/045820,
incorporated herein by reference) and condensed with 125 mcg of 10
kD polyornthine (Sigma). When generating these nanocapsules, the
Tbg-coated micelles were atomized into a modified LiCl salt
receiving solution (135 mM Li.sup.+, 9 mM Ca.sup.2+, 7.5 nM
Sr.sup.2+, 2.3 nM Mg.sup.2+ (all ultrapure, Sigma)) and capsules
were incubated for 48 hours before centrifugation. Capsules were
resuspended following centrifugation in PBS+10% Lactitol. Average
capsule size was about 26.2.+-.about 1 nm as measured by tapping
mode atomic force microscopy using average of minor and major
elliptical diameters of a 1 ng/ml sample dried down on a mica sheet
and a surface charge of -8.+-.4 mev was measured on Zetasizer 4
dynamic light scattering device at a potential of 20 volts with a
2-second pause between measurements in 1 mM KCl at 2 .mu.g/ml.
[0144] The nanocapsules were administered in a pilot study to
athymic nude mice bearing palpable flank xenograft tumors of FaDu
hypopharangeal tumor. Growth inhibition at 24 hours after a third
10 .mu.g/kg dose every third day was significant for tumors treated
by a variety of treatment routes vs. controls (pooled treated vs.
untreated 65% inhibition, 83.+-.19 cu.cm vs. 236.+-.33 cu. cm).
Within the treatment group, growth inhibition was comparable or
better to intravenous for subcutaneous via the back of the neck
(tumor volume: (sq vs. iv): 92 vs. 126 cu. cm.; (topical by ear vs.
i.v.): 36 vs. 126 cu. cm.), further supporting the view that s50
capsules can be administered subcutaneously with a variety of
targeting ligands and cargo, and provide efficacious results.
[0145] Tenfibgen (TBG) Preparation:
[0146] TBG was prepared by the method of Aukhill with
modifications, i.e., TBG was isolated and refolded from bacterial
lysate by washing the insoluble pellet once with lysis buffer (50
mM Tris-HCl, 1.0 mM EDTA, 0.1 M NaCl, 0.2 mg/ml lysozyme, 0.1%
Triton X-100, 0.1 mM PMSF, pH 8.0), containing 2 M urea and
resuspending in 4M GuCL, 5 mM DTT in 0.02 M Tris-HCl, pH 8.0. After
additional centrifugation, the clarified TBG solution was diluted
with 2 M Guanidine-HCl, 20 mM Tris-HCl, pH 8.0 to make a final
OD280 of about 1 and diluted dropwise about 10-fold into 20 mM
Tris-HCl, 0.2 M NaCl, 0.5 M Arginine-HCl, 10 .mu.M CuCl.sub.2 pH
8.0 for overnight stirred incubation (4.degree. C.). After
diafiltration against 20 mM Tris-HCl, pH 8.0 with an approximate
4-5 fold reduction in concentration and 0.45 uM filtration, a final
purification was performed on heparan sepharose in 20 mM Tris-HCl,
pH 8.0, with elution by bringing the NaCl concentration to 0.6
M.
[0147] Formula F, 250 .mu.g of 21 mer, sistable.TM.-modified,
double-stranded RNA (Dharmacon) oligonucleotide (siFVII sequence,
Akinc, et al. 2009 Mol Ther 17(5)872-879), was first complexed with
87.5 .mu.g of 300 MW spermine (Sigma), then was dispersed into 100
.mu.l of sterile water using a water-insoluble surfactant system
(2, 4, 7, 9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE-30 (Air
Products), 6.25 .mu.g in 50% DMSO). Following emulsification with
150 .mu.l of a water-miscible solvent (DMSO), the complexes were
then inverted and diluted by the addition of 750 .mu.l of PBS.
[0148] The resultant hydrophobic micelles were coated
(non-covalently) by the addition of 12.5 .mu.g of asialoorosomucoid
(ASOR; prepared by the method of Stockert, et al. (1980 Lab.
Invest. 43:556-63)), then atomized into a modified LiCl salt
receiving solution (135 mM Li.sup.+, 9 mM Ca.sup.2+, 14.1 nM
Sr.sup.2+, 11.25 nM Mg.sup.2+ (all ultrapure, Sigma)). Following
cold-room incubation (4.degree. C.) with nominal rotation in 50 ml
round-bottomed tubes for 48 hours, which stabilizes the coated
micelles in the salt solution, the sub-50 nm nanocapsules were
recovered by centrifugation at 20,000.times.g for 2 hrs and
resuspended in PBS+10% lactitol (at a concentration of 0.5
.mu.g/.mu.l) for filter sterilization through a 0.2 .mu.m filter.
In all formulations described, a small amount (1% of coating
weight) of Syrian Hamster Fab fragment was "spiked" into the ligand
coat to enable immunodetection of nanocapsules uptake by
anti-syrian hamster IgG antibodies. Average capsule size was about
20.2.+-.0.6 nm as measured by tapping mode atomic force microscopy
using average of minor and major elliptical diameters of a 1 ng/ml
sample dried down on a mica sheet. Formulations were subjected to
one freeze-thaw before sizing analysis. Average zeta potential was
between about +2 and -15 mev (-3.+-.4.5 mev) when measured on
Zetasizer 4 dynamic light scattering device at a potential of 20
volts with a 2-second pause between measurements in 1 mM KCl at 2
.mu.g/ml. Encapsulation yield was 100% by the modified method of
Burton, Kren, et al. 200, JCl 119(7):2086-99.
[0149] Formula G, 500 .mu.g of plasmid DNA was first complexed with
73 .mu.g of 25 kDa polyethyleneimine (PEI; Sigma Chemical Co., St.
Louis, Mo.), a branched cationic polymer, and dispersed into 150
.mu.l of sterile water using a water-insoluble surfactant system
(TM-diol; SE-30 (Air Products) in 50% DMSO). The DNA used in these
experiments was a commercially available 4.5 kb reporter plasmid
containing a Secreted Alkaline Phosphatase (SEAP) expression
cassette (pCpg-marSEAP, Invivogen). Following emulsification with a
water-miscible solvent (DMSO), the complexes were then inverted and
diluted by the addition of 700 .mu.l of PBS.
[0150] The resultant hydrophobic micelles were coated
(non-covalently) by the addition of 25 .mu.g of asialoorosomucoid
(ASOR; prepared by the method of Stockert et al. (1980, Lab.
Invest., 43:556-63)) then atomized into a modified LiCl salt
receiving solution (135 mM Li.sup.+, 9 mM Ca.sup.2+, 15 nM
Sr.sup.2+, 7.5 nM Mg.sup.2+ (all ultrapure, Sigma)). Following
cold-room incubation (4.degree. C.) with nominal rotation in 50 ml
round-bottomed tubes for 48 hours, which stabilizes the coated
micelles in the salt solution, the sub-50 nm nanocapsules were
recovered by centrifugation at 20,000.times.g for 2 hrs and
resuspended in PBS+10% lactitol (at a concentration of 0.5
.mu.g/.mu.l) for filter sterilization through a 0.2 .mu.m filter.
In all formulations described, a small amount (1% of coating
weight) of Syrian Hamster Fab fragment was "spiked" into the ligand
coat to enable immunodetection of nanocapsules uptake by
anti-syrian hamster IgG antibodies. Average capsule size was about
8.8.+-.0.3 nm as measured by tapping mode atomic force microscopy
using average of minor and major elliptical diameters of a 1 ng/ml
sample dried down on a mica sheet. Formulations were subjected to
one freeze-thaw cycle before sizing analysis. Zeta potential was
between about +2 and -15 mev (-8.+-.5 mev) when measured on
Zetasizer 4 dynamic light scattering device at a potential of 20
volts with a 2-second pause between measurements in 1 mM KCl at 2
.mu.g/ml. Encapsulation yield was 100% by the modified method of
Burton, Kren, et al. 2009 JCl 119(7):2086-99.
[0151] Formula H1, 250 .mu.g of plasmid DNA (pVivo2-sahIRF7/3-LacZ,
Invivogen) was first complexed with 38.7 .mu.g of 25 kDa
polyethyleneimine (PEI; Sigma Chemical Co., St. Louis, Mo.), a
branched cationic polymer, and dispersed into 150 .mu.l of sterile
water using a water-insoluble surfactant system (TM-diol; SE-30
(Air Products), 7.5 .mu.g in 50% DMSO). The DNA used in these
experiments was a 10.5 kb commercially-available vaccine plasmid
containing a LacZ expression cassette to produce betagalactosidase
as a model antigen. Following emulsification with a water-miscible
solvent (DMSO), the complexes were then inverted and diluted by the
addition of 750 .mu.l of PBS.
[0152] The resultant hydrophobic micelles were coated
(non-covalently) by the addition of 6.3 .mu.g of 22,480 Daltons
hyaluronan (Sodium Hylarunonate Powder, GSP223-20, resuspended in
water; Lifecore Biomedical); Formula H then was atomized into a
LiCl salt receiving solution (135 mM Li+, 9 mM Ca2+, 1.25 nM Sr2+,
10.0 nM Mg2+ and 380 uM Ni2+(all ultrapure)). Following cold-room
incubation (4.degree. C.) with nominal rotation for 14.5 hours in
50 ml round-bottomed tubes with the salt solution, which stabilizes
the coated micelles, the sub-50 nm nanocapsules were recovered by
centrifugation at 20,000.times.g for 2 hrs and resuspended in
PBS+10% lactitol (at a concentration of 0.5 .mu.g/.mu.l) for filter
sterilization through a 0.2 .mu.m filter. In all formulations
described, a small amount (1% of coating weight) of Syrian hamster
Fab Fragment was "spiked" into the ligand coat to enable
immunodetection of nanocapsules uptake by anti-syrian hamster IgG
antibodies.
[0153] For formulas H2, H3, H4 and H5, the following substitutions
from H1 were made. For H2 (LhaAl pVivo), 827 .mu.M AL.sup.3+ was
substituted for nickel ion in the salt receiving solution in which
10 nM Sr2+, 27.5 Mg.sup.2+ were used. For H3 (HaNi pVivo), 6.3
.mu.g of 1MM MW hyaluronan (Lifecore Biomedical) was substituted
for low MW hyaluronan fragment with 1.75 nM Sr.sup.2+ and 21 nM
Mg.sup.2+ (i.e., the quantities of Sr.sup.2+ and Mg.sup.2+ used for
Formula H1 were changed to 1.75 nM and 21 nM, respectively, for
Formula H2) in the receiving bath. For H4 (WJ6Ni pVivo), 6.3 .mu.g
of mouse anti-bovine Dec205 antibody (clone WJ6, MCA1651G, Serotec)
was substituted for hyaluronan fragment as the coating ligand and
capsule shell with 1.5 nM Sr.sup.2+ and 27 nM Mg.sup.2+ in the
receiving bath. Dec205 is a surface receptor present on dendritic
cell creating a nanoparticle targeting dendritic cells but not
keratinocytes. For H5, a commercial betagal-expressing sequence
(pcDNA3.1/LacZ, Invitrogen) based on the CMV promoter that was used
in Formula H1, was condensed with 36.5 .mu.g of 25KD PEI, with 2 nM
of Mg.sup.2++1.75 nM of Sr.sup.2+ used in the receiver bath.
[0154] For all capsules, average capsule diameter was less than 50
nm as measured by tapping mode atomic force microscopy using
elliptical diameters of a 0.5 ng/ml sample dried down on a mica
sheet. Typical average capsule size was about 30-40 nm diameter.
Zeta potential was between about +2 and -15 mev when measured on
Zetasizer 4 dynamic light scattering device at a potential of 20
volts with a 2-second pause between measurements in 1 mM KCl at 2
.mu.g/ml.
Example 3--Knockdown of FVII mRNA and Total Protein in Mice after
Single Treatment with ASOR-Coated s50 Capsules Bearing RNAi
Cargo
[0155] In an effort to identify administrative routes more amenable
than intravenous to chronic administration of nucleic acid and
other pharmaceutical agents, C57B/6 mice of a minimum of 11 weeks
were treated with bolus subcutaneous injection of s50 ASOR capsules
bearing RNAi oligos in the back of the neck. In order to administer
equal amounts of active agent, i.e., antisense guide strand, mice
were treated with 10-25 nm s50 ASOR capsules bearing either 1 mg/kg
siFVII (Formula A, example 2) or 0.5 mg/kg RNAi FVII, as
single-stranded chimeric oligo displaying the antisense guide
strand sequence for siFVII (Formula B, example 2).
[0156] Injection volumes were approximately 200 .mu.l. Control
formulations consisted of subcutaneous PBS (phosphate-buffered
saline, 200 al), intravenous 1 mg/kg s50 ASOR siApoB (Formula C,
example 2) and subcutaneous s50 ASOR capsules bearing Dysprosium
Dextran chelate, an experimental contrast agent @ 1 .mu.g/kg or 300
nmol Dy/kg in 200 .mu.l (.about.3.times. an imaging dose, Formulas
D, example 2).
[0157] Animals were bled and euthanized in groups over a 5-day
timecourse to follow the relationship between changes in
coagulation FVII mRNA and protein levels. A significant 50%
reduction in mRNA transcript (fold-HPRT, n=7-9) was measured at 48
hours post-treatment, the first timepoint (FIG. 1). This
significant reduction in FVII liver mRNA preceded by 24 hours a
significant 50% reduction in liver FVII total protein (by ELISA,
n=6-8, FIG. 2) for double-stranded RNAi cargo and coincided for
single-stranded RNAi cargo. The more rapid kinetics of change
continued for single-stranded cargo with a significant nadir of
plasma FVII protein knockdown of 93% on Day 3 and of 87% on Day 4
post-treatment for double-stranded RNAi cargo (n=6-8). As a
comparison, mice treated intravenously with s50 ASOR capsules
bearing double-stranded siFVII showed a significant 84% reduction
in plasma FVII protein on Day 4 post-treatment (n=3), indicating
comparable performance by subcutaneous and intravenous routes by
the s50 capsule delivery carrier.
[0158] These effects were not attributable to the capsule delivery
carrier itself, as mice treated with either the ASOR s50 capsules
bearing a non-nucleic acid cargo (dysprosium) or with the s50 ASOR
capsules bearing an irrelevant siRNA (siApoB, Example 2, Formula
C), sacrificed over the course of the study, showed no significant
change in FVII mRNA or protein levels (FIG. 1, 2). A third control
s50 ASOR capsule, bearing an siRNA against a non-mammalian target,
Red Fluorescent Protein, exhibited a pattern not unexpected with
introducing siRNA against non-endogenous targets, in that a
significant 30% knockdown in FVII liver mRNA was observed, coupled
with non-significant inhibition of plasma and liver protein (2010
Nucl Acids Res 38 (1):1-16). Thus, the s50 capsule technology
showed significant utility for subcutaneous delivery of charged
macromolecules.
[0159] Plasma and liver protein levels were determined by a direct
antibody ELISA using the manufacturer's recommended protocol (MAB
3305 clone 406722; R&D Systems, Mpls, MN). Liver mRNA levels of
Factor VII were determined by qPCR using standard primers for mouse
FVII and HPRT from SAB Biosciences per the manufacturer's
recommended protocols and reagents (SAB Biosciences, Bethesda,
Md.).
Example 4--Inhibition of Clotting Activity by s50 Encapsulated
siFVII by Decreased Subcutaneous Dosing
[0160] In an effort to further identify methodologies for reduced
invasiveness in treatment administration of nucleic acids and other
diagnostic and therapeutic cargos, dose response was investigated
for anti-coagulation in female C57/B6 mice of at least 11 weeks,
using an anti-FVII sequence (sistFVII, sequence same as Example 3,
prepared as Formula F of example 2) with a stabilized backbone
chemistry (Dharmacon, Lafayette, Co. sistable chemistry). Mice were
treated with bolus subcutaneous injection of s50 ASOR capsules
bearing siRNA oligos in the back of the neck. Injection volumes
were approximately 200 .mu.l. Control formulation consisted of
subcutaneous PBS (phosphate-buffered saline, 200 .mu.l).
[0161] Animals were bled and euthanized in groups after 3 days to
follow the relationship between changes in coagulation and drug
administration. Animals were treated in groups of three at the
following doses; 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.25 mg/kg and 0.1
mg/kg. Surprisingly, anti-clotting activity, as assessed by
examination for clots in the lung and carcass, and the ability of
blood to flow easily from a hematocrit collection tube after 10-15
minutes of rest, were present in all mice in the first four dosage
groups as well as 2/3 of the mice in the lowest dose group,
indicating a capacity for 4-10-fold or more dose reduction and an
accompanying reduction in dose volume (and injection pain) by
subcutaneous injection relative to our comparator dose from Example
3. For the 0.25 mg/kg group, active protein as measured by Factor
VII activity ELISA was reduced (0.69.+-.0.02 vs. control, n=3)
corresponding to a parallel 25% reduction in mRNA transcript of
0.76.+-.0.01 vs. control, n=3. Further significant utility was thus
found for subcutaneous delivery of charged macromolecules via s50
capsules.
Example 5--Sub-50 nm Capsules Bearing Dysprosium-Dota-Dextran
(DyDex) for Near IR (NIR) Imaging, Fluorescence Microscopy and
Quantitative Neutron Activation Analysis (NAA)
[0162] Results herein demonstrate DyDex within the s50 capsule
chemistry functions as a long-term, non-bleaching fluorescent probe
in a water environment. This is highly surprising, as lanthanide
luminescence, derived from electron mobility between outer F
orbitals, is rapidly quenched by water contact. Relative to other
water-quenched lanthanides, Dysprosium luminescence is considered
the least stable with a time constant of only 9-11 .mu.sec in
aqueous media (Mudring, et al. 2006 J Alloys Cmpd 418:204-208),
e.g., for lanthanide label series, fluorescent lifetimes were
Tb(III), 695 us; Eu(III) 618 us; Sm(III) 89 us; Dy(III) not
detectable (Huhtinen, et al. 2005 Anal. Chem. 77:2643-2648).
[0163] FIG. 3 shows excised organs (spleen, kidney, liver) from two
mice, one of which (right hand side) was administered 1 .mu.mol/kg
(expressed as Dy amount) of s50 ASOR DyDex i.v. 30 hours before
imaging (Formula D from Example 2). The other mouse (left-hand
side) was not treated. Excised livers, kidneys, or spleen were
imaged on at Kodak Carestream FX pro imaging station at 510
excitation and 700 nm emission with 20 nm bandpass filtering using
a Kodak Carestream Multispectral imaging system with a xenon lamp
light source. Results were processed with Kodak Multispectral
imaging analysis software to extract Dy-specific signal and
presented as grayscale (white as positive signal, for a
pseudocolour scale (red-orange-yellow-white, 1750-2450 au),
pseudocolor not shown. Specific Dy emission was observed as 50% of
scale signal uniformly throughout two major liver lobes and minor
lobe (depicted as medium white, identified with three narrow arrows
or red-orange on a pseudocolor scale). A 75% scale signal was
detected in the gall bladder (depicted as bright white with broad
arrow or orange-yellow on a pseudocolor scale), indicating a higher
accumulation in this organ consistent with hepatobilliary excretion
of intact s50 ASOR capsule eventually into the feces. The spleen
and kidney of the treated mouse, and the spleen, kidney, gall
bladder, and liver of the untreated mouse were completely negative
for signal (depicted as dark grey for grayscale or a green on a
pseudocolor scale), establishing by s50 DyDex optimal imaging the
liver-targeting capability of ASOR-targeted capsules administered
intravenously.
[0164] The s50 nanoparticle chemistry together with Dy-chelated
cargo provides the important advantage and benefit of facile
preparation methods, incorporating proteins, peptides, antibodies,
carbohydrates or small molecules as ligands in the shell. Ligands
may be readily exchanged for diagnostic purposes in s50
ligand-targeted particles, e.g., tenfibgen or ASOR may be
substituted for an anti-PSA antibody as a ligand to enable a
diagnostic imaging agent or theranostic capable of identifying
patient populations for treatment guidance.
[0165] The s50 capsule together with Dy-chelated cargo such as
DyDex also provides high sensitivity in NAA (Dy detectable to 0.1
ppb). This physical characteristic derives in part from the large
size of the Dy nucleus enabling it to function as a favorable
target for neutron bombardment and activation. This feature enables
measurement of Dy cargo as an isotopic tracer by quantitative
method. For example, Table 1, below, shows quantitation of Dy metal
in organs and fluids collected during a 24 hour period from mice
injected by either intravenous, intraperitoneal, subcutaneous, or
topical routes (one mouse each) with 250 nmol/kg (expressed as Dy
amount) s50 ASOR DyDex. Fluids were collected using metabolism
cages and tissue samples were carefully weighed before shipment to
a nuclear reactor facility (Univ. Missouri) for neutron bombardment
and gamma counting.
TABLE-US-00001 TABLE 1 NAA measurement after 250 nmol/ kg s50 ASOR
DyDex @ 24 hours Organ accumulation passive (% injected dose/g
intra- intra- sub- topical by tissue) venous peritoneal cutaneous
ear Blood 0.00 0.00 0 0 Spleen 3.14 0.00 0 0 Liver 38.50 3.10 1.39
0 Lung 0.83 ND ND ND Kidney 0.52 0.00 0.10 0 Jejunum 0.16 0.00 ND
ND Urine 16.61 4.15 1.58 1.3 Feces 7.54 11.64 23.8 61.3 Injection
site (tail) 0.99 ND ND ND TOTAL IDENTIFIED 68.29 18.89 26.87
62.60
[0166] Examination of the table shows that consistent with NIR
imaging described in FIG. 3 where Dy signal was highest in the gall
bladder, significant amounts of Dy were found in the feces relative
to urine supporting hepatobiliary excretion. Subcutaneous delivery
showed a small amount of Dy delivery to the target organ, liver.
Topical delivery did not result in delivery to the liver but was as
effective as intravenous in terms of delivering Dy across the skin
barrier and into the body (iv vs. to; 68.29 vs. 62.60 ng Dy, Table
1). As the majority of topically-delivered Dy was found in the
feces, this is consistent with direct delivery to the colon by
lymphatics, however, it still demonstrates quantitatively very
efficient transport of cargo by s50 nanoparticles across the
skin.
[0167] It appears the ASOR ligands, when applied topically, is
directed to the colon and feces, but topical work with another s50
ligand indicated desired organ (tumor) targeting was achieved
(Example 2, Formula E, tenfibgen ligand). Additionally, the lower
amount of DyDex measured for subcutaneous delivery in Table 1
(26.87) is consistent with the likely lymphatic transport of
subcutaneously administered s50 ASOR particles (lymphatics were not
assayed in this study; see also Example 4).
[0168] These data demonstrate the quantitative nature of Dy cargo
as a tracer and suggest methods for bulk quantitation of target
cells (e.g., tumor cells) in tissue by analytical methods (e.g.,
ICP-AES, NAA). Such bulk measurement would improve sensitivity of
detection over spot sampling methods such as tissue sectioning and
microscopic observation. Also advantageously, in line with the
multi-modal nature of Dy detection, these bulk measurements can be
readily correlated with current microscopy-based methods of
detection due to the long-term retained fluorescence of Dy within
the hydrophobic environment within the s50 capsule within
formalin-fixed tissue. For example, the use of tumor-targeted
capsules bearing fluorescent Dy cargo has been administered in
tumor margin analysis post-resection.
Example 6--Subcutaneous Delivery of Plasmid DNA by s50
Nanocapsule
[0169] In an effort to identify feasible strategies for
patient-friendly, chronic dosing in gene therapy protocols, two
different regimens for subcutaneous (sq) administration and
intravenous (iv) administration were compared. Outbred,
immunocompetent mice (Swiss Webster, groups of three each) were
treated with hepatocyte-targeting ASOR s50 capsules bearing the
non-integrating reporter plasmid pCpgfreeSEAP (Formula G of Example
2) with the following regimens; 1) 3.times.10 mg/kg q3Day
intravenous, 2) 3.times.20 mg/kg q3Day sq or 3) 6.times.10 mg/kg
q3Day sq. Comparator/control mice were treated with saline or
3.times.10 mg/kg q3Day intravenous ASOR s50 capsules bearing
trehalose sugar. Mice were sacrificed and livers collected for
microscopy and RNA analysis two days after the last dose. For
analysis of SEAP transcript, total RNA was isolated from .about.50
mg of snap frozen liver tissue using the Qiagen RNeasy Lipid Tissue
Mini Kit including the on column DNase digestion step according to
the manufacturers protocol. RT-PCR was performed using the QIAGEN
OneStep RT-PCR kit using forward primer and backward primer as
disclosed in Example 6 of U.S. patent application Ser. No.
13/071,067,` to amplify a 207 bp fragment of SEAP corresponding to
nt 332 to 539 of the coding sequence (CDS).
[0170] The 50 .mu.l reactions used 1 .mu.g of total liver RNA as
template, and the concentrations of all the other components
including the Q buffer used were those specified by the
manufacturer. The reverse transcriptase step was performed at
50.degree. C. for 30 min, followed by a 15 min denaturation step at
95.degree. C., followed by 35 cycles of (94.degree. C. 45 sec,
56.degree. C. 30 sec, 72.degree. C. 1 min), followed by a 10 min
extension step at 72.degree. C. Band analysis was performed by
electrophoresis of 20 .mu.l of the reactions on 1.25% agarose gels
and the material visualized using ethidium bromide and UVlight.
Gels, depicted in FIG. 4A, indicate that 2/3 animals for each group
of treated animals (intravenous and two subcutaneous protocols,
Groups A and C) exhibited positive PCR bands for alkaline
phosphatase transcript (6.times.10 mg/kg: 40% of iv, 3.times.20
mg/kg: 30% of iv by mean band density). No animals in the control
groups (water and sugar s50) exhibited positive PCR bands for
alkaline phosphatase transcript. Expression at the protein level
was confirmed by microscopy (data not shown). These results are
surprising, as repeat dosing has been highly problematic for
plasmid delivery strategies, which have typically been based on
particles larger than 50 nm in diameter and/or protein or
carbohydrate targeting. For viral-based delivery strategies,
antibodies against the vehicle/vector generally develop after one
administration.
[0171] To study sq administration of s50 capsules independently of
cargo transgene expression we used ASOR capsules carrying
Dysprosium (Dy)-chelated dextran (Formula D of Example 2). Dy is a
large nuclei, fluorescent lanthanide enabling sensitive isotopic
measurements of tissues by neutron activation analysis of Dy
content. A cohort of mice (5 groups, one mouse per group) were
injected with 100 nmol/kg (expressed as Dy amount) sq over a 5 day
period and held in metabolism cages for the final 24 hrs before
sacrifice and tissue collection for Dysprosium content analysis.
Over 5 days, .about.4.3% of the injected dose accumulated in the
liver (based on summation of 24-hr collection points for the 5
days) peaking at 3 days post-injection (see FIG. 5). In this
analysis, 23% of the injected dose (summing all data points over
the five day period) is accounted for. In other s50 studies (not
shown), much of the non-organ distribution at late timepoints was
found in the carcass (e.g., lymphatics); potentially, this would
continue to distribute to the s50-targeted organ over a period of
time as a depot effect.
[0172] Of note, no significant uptake was observed in spleen,
kidney, lung, heart or testes (kidney data shown in FIG. 5, other
data not shown), nor was there any evidence of injection site
reaction from repeat injection. These data support the view that sq
delivery of ASOR targeted s50 capsules to hepatocytes is specific
and provides a depot effect, with prolonged availability of cargo
and possible avoidance of duration-adverse effects such as
saturation of cellular uptake, intracellular enzymatic degradation
of cargo, etc.
[0173] Taken together, these results are very surprising, as
targeted delivery of therapeutic plasmid DNA by subcutaneous
delivery has not been demonstrated in vivo by non-viral or fully
synthetic means and has not been believed to be possible due to the
large size of the plasmid molecule.
Example 7--Enhanced Treatment Outcomes for s50 Capsule-Mediated DNA
Vaccine Delivery in a Weanling Pig Model
[0174] A 6 weanling pig pilot study was initiated comparing the
immunostimulation from standard i.d. treatments (naked DNA
expressing antigen or protein and protein antigen adjuvanted with
incomplete Freund's) to candidate topical nanocapsule DNA vaccines
directed against the model antigen .beta.-galactosidase. The pilot
study was designed to allow comparison of i) nickel vs. aluminum
ion as adjuvant, ii) antigen vector designs, iii) the relative
value of targeting keratinocytes+Langerhans cells/dendritic cells
(LC/DC) vs. LC/DC alone and iv) specific antibody and cellular
responses relative to standard i.d. inoculation of adjuvanted
protein antigen and naked DNA. Preparation of important
formulations is described in Example 2 under Formula H. A regimen
of 3.times.500 .mu.g DNA @ weeks 0, 3, and 5 with termination at
week 7 was used. Specific cellular targeting for either dendritic
cells alone (Formula H4) or combination DC and keratinocyte was
confirmed for capsule designs by transduction studies in pig biopsy
skin organ culture.
[0175] After cleaning the skin with cloth and water, s50 hyaluronan
capsules encapsulating DNA sequence for antigen and targeted to
keratinocytes and DC (LHaNi, Formula H1) were sprayed on back of
neck, thorax, and throat (500 .mu.g DNA in 10 ml PBS plus 1 drop
blue food coloring, equivalent to a dose of 0.44 .mu.g/sq.cm based
on treated surface area and approximately 10% of total body surface
area, pig #866)), dosed at 3.times.500 .mu.g (week 0, 3, 5). This
formulation generated by ELISA specific IgG within about 1 log of
the IgG level generated by i.d. protein antigen combined with
incomplete Freund's adjuvant (3.times. at 500 .mu.g, pig #864).
Functional protein antigen production by DNA vaccine plasmid in
skin was confirmed by polymerase chain reaction for Bgal transcript
for Formula H1 (administered to pig #866, data not shown). No
treatment site complications were observed at week 7 study
termination in the topical delivery of nickel-adjuvant capsules
bearing DNA, while complications including granuloma and redness
and irritation were observed with the protein antigen i.d.
treatment. s50 capsule IgG titer was two-fold that of naked i.d.
DNA inoculation (3.times. at 500 .mu.g, pig #863) and s50 capsules
targeted to DC exclusively using anti-Dec205 binding (FIG. 6C) A
second betagal-expressing vector was formulated (H5) in the optimal
capsule design and produced a specific immune response (pig #865),
demonstrating capability of the capsules to effectively deliver
different antigenic loads.
[0176] Combined s50 targeting to keratinocytes and dendritic cells
together with nickel adjuvant induced equivalent or better specific
IgA responses as compared to standard i.d. inoculation of protein
antigen (naked DNA from nasal lavage, demonstrating enhancement in
mucosal humoral response (see FIG. 6B). Formula H1, s50 LhaNi,
administered to pig #866, showed improved and more persistent
mucosal IgA response over pig#867, treated with Formula H4,
targeted at DC only. These results were particularly surprising, as
the view in the art has been that keratinocyte cells are only
weakly immunogenic and do not productively contribute to vaccine
response, thus creating a weaker response for those vaccine
formulations with any ancillary targeting to keratinocytes.
Consistent with this observation, both the nanoparticle targeting
dendritic cells, but not keratinocytes (Formula H4 of Example 2),
and the low molecular weight hyaluronan utilizing aluminum ion as
an adjuvant (Formula H2 of Example 2) showed no increase in IgA
response vs. intradermal treatments.
[0177] Regional biasing of responses was suggested with i.d.
inoculation showing strong splenic cellular responses but poorer
nodal responses. In contrast, splenic cellular responses were
completely absent following topical DNA nanocapsule application
with statistically significant stronger nodal responses in both
distal and draining lymph nodes over standard intradermal
adjuvanted protein antigen (See FIG. 6A).
[0178] The candidate combo-targeted capsule design of LHaNi pVivo
(administered to Pig #866, Formula H1 of Example 2) appeared to be
optimal amongst the s50 capsule options tested. Specific IgA
response was persistent and 2.5.times. naked i.d. DNA by grand
average while specific lymphocyte response was 37.times. for
draining nodacytes and 12.1.times. for distal nodacytes (p<0.05)
over standard i.d. protein inoculation.
[0179] These results were surprising, as low molecular weight
hyaluronan-nickel particles exhibited combination targeting to
dendritic cells and keratinocytes relative to dendritic cells alone
(DC targeting executed by Formula H4 of Example 2). Low molecular
weight hyaluronan as a ligand relative to high MW (1MM Da)
hyaluronan was also surprising as an optimal ligand. Western blot
analysis showed the low molecular weight treatment
(LhaNi-p.beta.gal, Formula H1, 5 .mu.g plasmid DNA) induced twice
as much betagal production in pigskin biopsy organ culture three
days after treatment, as the high molecular weight treatment (high
MW hyaluronan-Ni p.beta.gal, Formula H3, 5 .mu.g) (measured
relative to lactate dehydrogenase, data not shown).
LhaNi-p.beta.gal treatment stimulated 1.6.times. more IL-1B
cytokine than high MW hyaluronan-Ni p.beta.gal (data not shown)
into supernatants from pigskin biopsy organ culture three days
after treatment. IL-1B is a cytokine associated with keratinocytes
in the skin, and keratinocytes are believed in the art to be weakly
immunogenic and not contributory to vaccine responses. Formula H4,
the DC-targeting nanocapsule, did not stimulate any increased IL-1B
into supernatants. Further, keratinocytes are surrounded by a
glycocalyx of high MW hyaluronan and not known to respond to low
molecular weight hyaluronan at all.
[0180] The results are likewise surprising, because they are
produced in a large-animal model. Indeed, it is known in the art
that, despite the success of non-viral DNA-based vaccines in small
animal models, studies in large animals have not yielded similar
success.
Example 8--Comparison of Factor VII (FVII) Activity Levels in Mice
Treated with Oligonucleotides Via Subcutaneous and Intravenous
Routes, with Nanoparticles of Different Size
[0181] Studies were conducted in C57/B6 female mice to evaluate
FVII activity levels for different routes and formulations of
ASOR-coated sub-50 nanometer (s50) particles encapsulating siRNA
cargo targeting FVII (siFVII). The siRNA medicinal chemistry
included modifications for the 20 and 28 nm particle formulations,
and no modifications for the 22 nm formulations (22 nm: Example 2,
Formula A, 20 nm: Example 2, Formula F). A common siFVII sequence
for all formulations was used as described in Akinc, et al. 2009
Mol Ther 17(5)872-879 for siFVII sense and siFVII antisense, as
disclosed in U.S. patent application Ser. No. 13/071,067. Surface
charge measured by zeta potential was equivalent between
formulations, leaving particle size as the major statistical
significant parameter different between formulations.
[0182] All mice were treated 1.times. at 1 mg/kg of body weight.
FVII activity levels were determined by FVII activity ELISA
(Biophen FVII; Aniara, Mason, Ohio) for the 20 and 28 nm particles,
and by direct protein ELISA (MAB 3305 clone 406722; R&D
Systems, Minneapolis, Minn.) for the 22 nm particles. FVII activity
levels were measured on Days 2, 3, and 4 post-treatment for the
mice treated subcutaneously (sq) with 20 and 28 nm particles, on
Days 2, 3, 4, and 5 for mice treated sq with 22 nm particles, on
Days 2 and 3 for mice treated i.v. with 20 nm and 28 nm particles,
and on Day 4 for mice treated i.v. with 22 nm particles.
[0183] Table 2, below, shows nadir FVII activity levels for each
formulation and route cohort. Only minor differences were found in
FVII activity levels between sq and intravenous (i.v.) routes for
the smallest (20 and 22 nm) particles, but substantial differences
between routes for the largest (28 nm) particles.
TABLE-US-00002 TABLE 2 s50 nanoparticles with s50 nanoparticles
with siRNA FVII plasma activity levels, vs. PBS control siRNA cargo
(diameter*, cargo (surface charge**, mev, (% activity, measurement
day, no. mice) nm, mean .+-. SE) mean .+-. SE) Subcutaneous
Intravenous 28 .+-. 0.6 nm, (n = 25) -5 .+-. 3.8 74% .+-. 0 (D 3, n
= 2) 19% .+-. 7 (D 3, n = 2) 22 .+-. 1 nm, (n = 10).sup. -5.5 .+-.
5.4.sup. 13% .+-. 2 (D 4, n = 8) 16% .+-. 5 (D 4, n = 3) 20 .+-.
0.6 nm, (n = 25) -3 .+-. 4.5 32% .+-. 10 (D 2, n = 4) 37% .+-. 10
(D 2, n = 5) *Particle diameter measured as average of major and
minor elliptical axes by atomic force microscopy after drying of
the suspension onto mica at 0.5 ng/ml or less of oligo. **Zeta
Potential measured by DLS across a 20 volt potential in 1 mM KCl at
2 .mu.g/ml. of oligo.
[0184] The findings that i.v. formulations were observed to
substantially inhibit FVII activity for all of the modified and
unmodified cargoes indicates the s50 particle is compatible and
effective with a range of oligo chemistries. Those observations,
and the findings that the sq oligo formulations were most effective
for the smallest particles (with modified and unmodified cargoes),
show the diminished sq efficacy at 28 nm is attributable to
particle size and not to oligo-related factors (e.g., intracellular
nuclease resistance or target binding efficiency).
[0185] The description of the present invention has been presented
for purposes of illustration and description, but is not intended
to be exhaustive or limiting of the invention to the form
disclosed. The scope of the present invention is limited only by
the scope of the following claims. Many modifications and
variations will be apparent to those of ordinary skill in the art.
The embodiment described and shown in the figures was chosen and
described in order to best explain the principles of the invention,
the practical application, and to enable others of ordinary skill
in the art to understand the invention for various embodiments with
various modifications as are suited to the particular use
contemplated.
Sequence CWU 1
1
817616DNAHomo sapiens 1attacagagg aaggagctcg ctatataagc cagccaaagt
tggctgcacc ggccacagcc 60tgcctactgt cacccgcctc tcccgcgcgc agatacacgc
ccccgcctcc gtgggcacaa 120aggcagcgct gctggggaac tcgggggaac
gcgcacgtgg gaaccgccgc agctccacac 180tccaggtact tcttccaagg
acctaggtct ctcgcccatc ggaaagaaaa taattctttc 240aagaagatca
gggacaactg atttgaagtc tactctgtgc ttctaaatcc ccaattctgc
300tgaaagtgag ataccctaga gccctagagc cccagcagca cccagccaaa
cccacctcca 360ccatgggggc catgactcag ctgttggcag gtgtctttct
tgctttcctt gccctcgcta 420ccgaaggtgg ggtcctcaag aaagtcatcc
ggcacaagcg acagagtggg gtgaacgcca 480ccctgccaga agagaaccag
ccagtggtgt ttaaccacgt ttacaacatc aagctgccag 540tgggatccca
gtgttcggtg gatctggagt cagccagtgg ggagaaagac ctggcaccgc
600cttcagagcc cagcgaaagc tttcaggagc acacagtgga tggggaaaac
cagattgtct 660tcacacatcg catcaacatc ccccgccggg cctgtggctg
tgccgcagcc cctgatgtta 720aggagctgct gagcagactg gaggagctgg
agaacctggt gtcttccctg agggagcaat 780gtactgcagg agcaggctgc
tgtctccagc ctgccacagg ccgcttggac accaggccct 840tctgtagcgg
tcggggcaac ttcagcactg aaggatgtgg ctgtgtctgc gaacctggct
900ggaaaggccc caactgctct gagcccgaat gtccaggcaa ctgtcacctt
cgaggccggt 960gcattgatgg gcagtgcatc tgtgacgacg gcttcacggg
cgaggactgc agccagctgg 1020cttgccccag cgactgcaat gaccagggca
agtgcgtaaa tggagtctgc atctgtttcg 1080aaggctacgc cggggctgac
tgcagccgtg aaatctgccc agtgccctgc agtgaggagc 1140acggcacatg
tgtagatggc ttgtgtgtgt gccacgatgg ctttgcaggc gatgactgca
1200acaagcctct gtgtctcaac aattgctaca accgtggacg atgcgtggag
aatgagtgcg 1260tgtgtgatga gggtttcacg ggcgaagact gcagtgagct
catctgcccc aatgactgct 1320tcgaccgggg ccgctgcatc aatggcacct
gctactgcga agaaggcttc acaggtgaag 1380actgcgggaa acccacctgc
ccacatgcct gccacaccca gggccggtgt gaggaggggc 1440agtgtgtatg
tgatgagggc tttgccggtg tggactgcag cgagaagagg tgtcctgctg
1500actgtcacaa tcgtggccgc tgtgtagacg ggcggtgtga gtgtgatgat
ggtttcactg 1560gagctgactg tggggagctc aagtgtccca atggctgcag
tggccatggc cgctgtgtca 1620atgggcagtg tgtgtgtgat gagggctata
ctggggagga ctgcagccag ctacggtgcc 1680ccaatgactg tcacagtcgg
ggccgctgtg tcgagggcaa atgtgtatgt gagcaaggct 1740tcaagggcta
tgactgcagt gacatgagct gccctaatga ctgtcaccag cacggccgct
1800gtgtgaatgg catgtgtgtt tgtgatgacg gctacacagg ggaagactgc
cgggatcgcc 1860aatgccccag ggactgcagc aacaggggcc tctgtgtgga
cggacagtgc gtctgtgagg 1920acggcttcac cggccctgac tgtgcagaac
tctcctgtcc aaatgactgc catggccagg 1980gtcgctgtgt gaatgggcag
tgcgtgtgcc atgaaggatt tatgggcaaa gactgcaagg 2040agcaaagatg
tcccagtgac tgtcatggcc agggccgctg cgtggacggc cagtgcatct
2100gccacgaggg cttcacaggc ctggactgtg gccagcactc ctgccccagt
gactgcaaca 2160acttaggaca atgcgtctcg ggccgctgca tctgcaacga
gggctacagc ggagaagact 2220gctcagaggt gtctcctccc aaagacctcg
ttgtgacaga agtgacggaa gagacggtca 2280acctggcctg ggacaatgag
atgcgggtca cagagtacct tgtcgtgtac acgcccaccc 2340acgagggtgg
tctggaaatg cagttccgtg tgcctgggga ccagacgtcc accatcatcc
2400aggagctgga gcctggtgtg gagtacttta tccgtgtatt tgccatcctg
gagaacaaga 2460agagcattcc tgtcagcgcc agggtggcca cgtacttacc
tgcacctgaa ggcctgaaat 2520tcaagtccat caaggagaca tctgtggaag
tggagtggga tcctctagac attgcttttg 2580aaacctggga gatcatcttc
cggaatatga ataaagaaga tgagggagag atcaccaaaa 2640gcctgaggag
gccagagacc tcttaccggc aaactggtct agctcctggg caagagtatg
2700agatatctct gcacatagtg aaaaacaata cccggggccc tggcctgaag
agggtgacca 2760ccacacgctt ggatgccccc agccagatcg aggtgaaaga
tgtcacagac accactgcct 2820tgatcacctg gttcaagccc ctggctgaga
tcgatggcat tgagctgacc tacggcatca 2880aagacgtgcc aggagaccgt
accaccatcg atctcacaga ggacgagaac cagtactcca 2940tcgggaacct
gaagcctgac actgagtacg aggtgtccct catctcccgc agaggtgaca
3000tgtcaagcaa cccagccaaa gagaccttca caacaggcct cgatgctccc
aggaatcttc 3060gacgtgtttc ccagacagat aacagcatca ccctggaatg
gaggaatggc aaggcagcta 3120ttgacagtta cagaattaag tatgccccca
tctctggagg ggaccacgct gaggttgatg 3180ttccaaagag ccaacaagcc
acaaccaaaa ccacactcac aggtctgagg ccgggaactg 3240aatatgggat
tggagtttct gctgtgaagg aagacaagga gagcaatcca gcgaccatca
3300acgcagccac agagttggac acgcccaagg accttcaggt ttctgaaact
gcagagacca 3360gcctgaccct gctctggaag acaccgttgg ccaaatttga
ccgctaccgc ctcaattaca 3420gtctccccac aggccagtgg gtgggagtgc
agcttccaag aaacaccact tcctatgtcc 3480tgagaggcct ggaaccagga
caggagtaca atgtcctcct gacagccgag aaaggcagac 3540acaagagcaa
gcccgcacgt gtgaaggcat ccactgaaca agcccctgag ctggaaaacc
3600tcaccgtgac tgaggttggc tgggatggcc tcagactcaa ctggaccgca
gctgaccagg 3660cctatgagca ctttatcatt caggtgcagg aggccaacaa
ggtggaggca gctcggaacc 3720tcaccgtgcc tggcagcctt cgggctgtgg
acataccggg cctcaaggct gctacgcctt 3780atacagtctc catctatggg
gtgatccagg gctatagaac accagtgctc tctgctgagg 3840cctccacagg
ggaaactccc aatttgggag aggtcgtggt ggccgaggtg ggctgggatg
3900ccctcaaact caactggact gctccagaag gggcctatga gtactttttc
attcaggtgc 3960aggaggctga cacagtagag gcagcccaga acctcaccgt
cccaggagga ctgaggtcca 4020cagacctgcc tgggctcaaa gcagccactc
attataccat caccatccgc ggggtcactc 4080aggacttcag cacaacccct
ctctctgttg aagtcttgac agaggaggtt ccagatatgg 4140gaaacctcac
agtgaccgag gttagctggg atgctctcag actgaactgg accacgccag
4200atggaaccta tgaccagttt actattcagg tccaggaggc tgaccaggtg
gaagaggctc 4260acaatctcac ggttcctggc agcctgcgtt ccatggaaat
cccaggcctc agggctggca 4320ctccttacac agtcaccctg cacggcgagg
tcaggggcca cagcactcga ccccttgctg 4380tagaggtcgt cacagaggat
ctcccacagc tgggagattt agccgtgtct gaggttggct 4440gggatggcct
cagactcaac tggaccgcag ctgacaatgc ctatgagcac tttgtcattc
4500aggtgcagga ggtcaacaaa gtggaggcag cccagaacct cacgttgcct
ggcagcctca 4560gggctgtgga catcccgggc ctcgaggctg ccacgcctta
tagagtctcc atctatgggg 4620tgatccgggg ctatagaaca ccagtactct
ctgctgaggc ctccacagcc aaagaacctg 4680aaattggaaa cttaaatgtt
tctgacataa ctcccgagag cttcaatctc tcctggatgg 4740ctaccgatgg
gatcttcgag acctttacca ttgaaattat tgattccaat aggttgctgg
4800agactgtgga atataatatc tctggtgctg aacgaactgc ccatatctca
gggctacccc 4860ctagtactga ttttattgtc tacctctctg gacttgctcc
cagcatccgg accaaaacca 4920tcagtgccac agccacgaca gaggccctgc
cccttctgga aaacctaacc atttccgaca 4980ttaatcccta cgggttcaca
gtttcctgga tggcatcgga gaatgccttt gacagctttc 5040tagtaacggt
ggtggattct gggaagctgc tggaccccca ggaattcaca ctttcaggaa
5100cccagaggaa gctggagctt agaggcctca taactggcat tggctatgag
gttatggtct 5160ctggcttcac ccaagggcat caaaccaagc ccttgagggc
tgagattgtt acagaagccg 5220aaccggaagt tgacaacctt ctggtttcag
atgccacccc agacggtttc cgtctgtcct 5280ggacagctga tgaaggggtc
ttcgacaatt ttgttctcaa aatcagagat accaaaaagc 5340agtctgagcc
actggaaata accctacttg cccccgaacg taccagggac ataacaggtc
5400tcagagaggc tactgaatac gaaattgaac tctatggaat aagcaaagga
aggcgatccc 5460agacagtcag tgctatagca acaacagcca tgggctcccc
aaaggaagtc attttctcag 5520acatcactga aaattcggct actgtcagct
ggagggcacc cacagcccaa gtggagagct 5580tccggattac ctatgtgccc
attacaggag gtacaccctc catggtaact gtggacggaa 5640ccaagactca
gaccaggctg gtgaaactca tacctggcgt ggagtacctt gtcagcatca
5700tcgccatgaa gggctttgag gaaagtgaac ctgtctcagg gtcattcacc
acagctctgg 5760atggcccatc tggcctggtg acagccaaca tcactgactc
agaagccttg gccaggtggc 5820agccagccat tgccactgtg gacagttatg
tcatctccta cacaggcgag aaagtgccag 5880aaattacacg cacggtgtcc
gggaacacag tggagtatgc tctgaccgac ctcgagcctg 5940ccacggaata
cacactgaga atctttgcag agaaagggcc ccagaagagc tcaaccatca
6000ctgccaagtt cacaacagac ctcgattctc caagagactt gactgctact
gaggttcagt 6060cggaaactgc cctccttacc tggcgacccc cccgggcatc
agtcaccggt tacctgctgg 6120tctatgaatc agtggatggc acagtcaagg
aagtcattgt gggtccagat accacctcct 6180acagcctggc agacctgagc
ccatccaccc actacacagc caagatccag gcactcaatg 6240ggcccctgag
gagcaatatg atccagacca tcttcaccac aattggactc ctgtacccct
6300tccccaagga ctgctcccaa gcaatgctga atggagacac gacctctggc
ctctacacca 6360tttatctgaa tggtgataag gctgaggcgc tggaagtctt
ctgtgacatg acctctgatg 6420ggggtggatg gattgtgttc ctgagacgca
aaaacggacg cgagaacttc taccaaaact 6480ggaaggcata tgctgctgga
tttggggacc gcagagaaga attctggctt gggctggaca 6540acctgaacaa
aatcacagcc caggggcagt acgagctccg ggtggacctg cgggaccatg
6600gggagacagc ctttgctgtc tatgacaagt tcagcgtggg agatgccaag
actcgctaca 6660agctgaaggt ggaggggtac agtgggacag caggtgactc
catggcctac cacaatggca 6720gatccttctc cacctttgac aaggacacag
attcagccat caccaactgt gctctgtcct 6780acaaaggggc tttctggtac
aggaactgtc accgtgtcaa cctgatgggg agatatgggg 6840acaataacca
cagtcagggc gttaactggt tccactggaa gggccacgaa cactcaatcc
6900agtttgctga gatgaagctg agaccaagca acttcagaaa tcttgaaggc
aggcgcaaac 6960gggcataaat tccagggacc actgggtgag agaggaataa
ggcccagagc gaggaaagga 7020ttttaccaaa gcatcaatac aaccagccca
accatcggtc cacacctggg catttggtga 7080gagtcaaagc tgaccatgga
tccctggggc caacggcaac agcatgggcc tcacctcctc 7140tgtgatttct
ttctttgcac caaagacatc agtctccaac atgtttctgt tttgttgttt
7200gattcagcaa aaatctccca gtgacaacat cgcaatagtt ttttacttct
cttaggtggc 7260tctgggaatg ggagaggggt aggatgtaca ggggtagttt
gttttagaac cagccgtatt 7320ttacatgaag ctgtataatt aattgtcatt
atttttgtta gcaaagatta aatgtgtcat 7380tggaagccat cccttttttt
acatttcata caacagaaac cagaaaagca atactgtttc 7440cattttaagg
atatgattaa tattattaat ataataatga tgatgatgat gatgaaaact
7500aaggattttt caagagatct ttctttccaa aacatttctg gacagtacct
gattgtattt 7560tttttttaaa taaaagcaca agtacttttg agtttgttaa
aaaaaaaaaa aaaaaa 761622201PRTHomo sapiens 2Met Gly Ala Met Thr Gln
Leu Leu Ala Gly Val Phe Leu Ala Phe Leu 1 5 10 15 Ala Leu Ala Thr
Glu Gly Gly Val Leu Lys Lys Val Ile Arg His Lys 20 25 30 Arg Gln
Ser Gly Val Asn Ala Thr Leu Pro Glu Glu Asn Gln Pro Val 35 40 45
Val Phe Asn His Val Tyr Asn Ile Lys Leu Pro Val Gly Ser Gln Cys 50
55 60 Ser Val Asp Leu Glu Ser Ala Ser Gly Glu Lys Asp Leu Ala Pro
Pro 65 70 75 80 Ser Glu Pro Ser Glu Ser Phe Gln Glu His Thr Val Asp
Gly Glu Asn 85 90 95 Gln Ile Val Phe Thr His Arg Ile Asn Ile Pro
Arg Arg Ala Cys Gly 100 105 110 Cys Ala Ala Ala Pro Asp Val Lys Glu
Leu Leu Ser Arg Leu Glu Glu 115 120 125 Leu Glu Asn Leu Val Ser Ser
Leu Arg Glu Gln Cys Thr Ala Gly Ala 130 135 140 Gly Cys Cys Leu Gln
Pro Ala Thr Gly Arg Leu Asp Thr Arg Pro Phe 145 150 155 160 Cys Ser
Gly Arg Gly Asn Phe Ser Thr Glu Gly Cys Gly Cys Val Cys 165 170 175
Glu Pro Gly Trp Lys Gly Pro Asn Cys Ser Glu Pro Glu Cys Pro Gly 180
185 190 Asn Cys His Leu Arg Gly Arg Cys Ile Asp Gly Gln Cys Ile Cys
Asp 195 200 205 Asp Gly Phe Thr Gly Glu Asp Cys Ser Gln Leu Ala Cys
Pro Ser Asp 210 215 220 Cys Asn Asp Gln Gly Lys Cys Val Asn Gly Val
Cys Ile Cys Phe Glu 225 230 235 240 Gly Tyr Ala Gly Ala Asp Cys Ser
Arg Glu Ile Cys Pro Val Pro Cys 245 250 255 Ser Glu Glu His Gly Thr
Cys Val Asp Gly Leu Cys Val Cys His Asp 260 265 270 Gly Phe Ala Gly
Asp Asp Cys Asn Lys Pro Leu Cys Leu Asn Asn Cys 275 280 285 Tyr Asn
Arg Gly Arg Cys Val Glu Asn Glu Cys Val Cys Asp Glu Gly 290 295 300
Phe Thr Gly Glu Asp Cys Ser Glu Leu Ile Cys Pro Asn Asp Cys Phe 305
310 315 320 Asp Arg Gly Arg Cys Ile Asn Gly Thr Cys Tyr Cys Glu Glu
Gly Phe 325 330 335 Thr Gly Glu Asp Cys Gly Lys Pro Thr Cys Pro His
Ala Cys His Thr 340 345 350 Gln Gly Arg Cys Glu Glu Gly Gln Cys Val
Cys Asp Glu Gly Phe Ala 355 360 365 Gly Val Asp Cys Ser Glu Lys Arg
Cys Pro Ala Asp Cys His Asn Arg 370 375 380 Gly Arg Cys Val Asp Gly
Arg Cys Glu Cys Asp Asp Gly Phe Thr Gly 385 390 395 400 Ala Asp Cys
Gly Glu Leu Lys Cys Pro Asn Gly Cys Ser Gly His Gly 405 410 415 Arg
Cys Val Asn Gly Gln Cys Val Cys Asp Glu Gly Tyr Thr Gly Glu 420 425
430 Asp Cys Ser Gln Leu Arg Cys Pro Asn Asp Cys His Ser Arg Gly Arg
435 440 445 Cys Val Glu Gly Lys Cys Val Cys Glu Gln Gly Phe Lys Gly
Tyr Asp 450 455 460 Cys Ser Asp Met Ser Cys Pro Asn Asp Cys His Gln
His Gly Arg Cys 465 470 475 480 Val Asn Gly Met Cys Val Cys Asp Asp
Gly Tyr Thr Gly Glu Asp Cys 485 490 495 Arg Asp Arg Gln Cys Pro Arg
Asp Cys Ser Asn Arg Gly Leu Cys Val 500 505 510 Asp Gly Gln Cys Val
Cys Glu Asp Gly Phe Thr Gly Pro Asp Cys Ala 515 520 525 Glu Leu Ser
Cys Pro Asn Asp Cys His Gly Gln Gly Arg Cys Val Asn 530 535 540 Gly
Gln Cys Val Cys His Glu Gly Phe Met Gly Lys Asp Cys Lys Glu 545 550
555 560 Gln Arg Cys Pro Ser Asp Cys His Gly Gln Gly Arg Cys Val Asp
Gly 565 570 575 Gln Cys Ile Cys His Glu Gly Phe Thr Gly Leu Asp Cys
Gly Gln His 580 585 590 Ser Cys Pro Ser Asp Cys Asn Asn Leu Gly Gln
Cys Val Ser Gly Arg 595 600 605 Cys Ile Cys Asn Glu Gly Tyr Ser Gly
Glu Asp Cys Ser Glu Val Ser 610 615 620 Pro Pro Lys Asp Leu Val Val
Thr Glu Val Thr Glu Glu Thr Val Asn 625 630 635 640 Leu Ala Trp Asp
Asn Glu Met Arg Val Thr Glu Tyr Leu Val Val Tyr 645 650 655 Thr Pro
Thr His Glu Gly Gly Leu Glu Met Gln Phe Arg Val Pro Gly 660 665 670
Asp Gln Thr Ser Thr Ile Ile Gln Glu Leu Glu Pro Gly Val Glu Tyr 675
680 685 Phe Ile Arg Val Phe Ala Ile Leu Glu Asn Lys Lys Ser Ile Pro
Val 690 695 700 Ser Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro Glu Gly
Leu Lys Phe 705 710 715 720 Lys Ser Ile Lys Glu Thr Ser Val Glu Val
Glu Trp Asp Pro Leu Asp 725 730 735 Ile Ala Phe Glu Thr Trp Glu Ile
Ile Phe Arg Asn Met Asn Lys Glu 740 745 750 Asp Glu Gly Glu Ile Thr
Lys Ser Leu Arg Arg Pro Glu Thr Ser Tyr 755 760 765 Arg Gln Thr Gly
Leu Ala Pro Gly Gln Glu Tyr Glu Ile Ser Leu His 770 775 780 Ile Val
Lys Asn Asn Thr Arg Gly Pro Gly Leu Lys Arg Val Thr Thr 785 790 795
800 Thr Arg Leu Asp Ala Pro Ser Gln Ile Glu Val Lys Asp Val Thr Asp
805 810 815 Thr Thr Ala Leu Ile Thr Trp Phe Lys Pro Leu Ala Glu Ile
Asp Gly 820 825 830 Ile Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly
Asp Arg Thr Thr 835 840 845 Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr
Ser Ile Gly Asn Leu Lys 850 855 860 Pro Asp Thr Glu Tyr Glu Val Ser
Leu Ile Ser Arg Arg Gly Asp Met 865 870 875 880 Ser Ser Asn Pro Ala
Lys Glu Thr Phe Thr Thr Gly Leu Asp Ala Pro 885 890 895 Arg Asn Leu
Arg Arg Val Ser Gln Thr Asp Asn Ser Ile Thr Leu Glu 900 905 910 Trp
Arg Asn Gly Lys Ala Ala Ile Asp Ser Tyr Arg Ile Lys Tyr Ala 915 920
925 Pro Ile Ser Gly Gly Asp His Ala Glu Val Asp Val Pro Lys Ser Gln
930 935 940 Gln Ala Thr Thr Lys Thr Thr Leu Thr Gly Leu Arg Pro Gly
Thr Glu 945 950 955 960 Tyr Gly Ile Gly Val Ser Ala Val Lys Glu Asp
Lys Glu Ser Asn Pro 965 970 975 Ala Thr Ile Asn Ala Ala Thr Glu Leu
Asp Thr Pro Lys Asp Leu Gln 980 985 990 Val Ser Glu Thr Ala Glu Thr
Ser Leu Thr Leu Leu Trp Lys Thr Pro 995 1000 1005 Leu Ala Lys Phe
Asp Arg Tyr Arg Leu Asn Tyr Ser Leu Pro Thr 1010 1015 1020 Gly Gln
Trp Val Gly Val Gln Leu Pro Arg Asn Thr Thr Ser Tyr 1025 1030 1035
Val Leu Arg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val Leu Leu 1040
1045 1050 Thr Ala Glu Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val
Lys 1055 1060 1065 Ala Ser Thr Glu Gln Ala Pro Glu Leu Glu Asn Leu
Thr Val Thr 1070 1075 1080 Glu Val Gly Trp Asp Gly Leu Arg Leu Asn
Trp Thr Ala Ala Asp 1085 1090 1095 Gln Ala Tyr Glu His Phe Ile Ile
Gln Val Gln Glu Ala Asn Lys 1100 1105 1110 Val Glu Ala Ala Arg Asn
Leu Thr Val Pro Gly Ser Leu Arg Ala 1115 1120 1125
Val Asp Ile Pro Gly Leu Lys Ala Ala Thr Pro Tyr Thr Val Ser 1130
1135 1140 Ile Tyr Gly Val Ile Gln Gly Tyr Arg Thr Pro Val Leu Ser
Ala 1145 1150 1155 Glu Ala Ser Thr Gly Glu Thr Pro Asn Leu Gly Glu
Val Val Val 1160 1165 1170 Ala Glu Val Gly Trp Asp Ala Leu Lys Leu
Asn Trp Thr Ala Pro 1175 1180 1185 Glu Gly Ala Tyr Glu Tyr Phe Phe
Ile Gln Val Gln Glu Ala Asp 1190 1195 1200 Thr Val Glu Ala Ala Gln
Asn Leu Thr Val Pro Gly Gly Leu Arg 1205 1210 1215 Ser Thr Asp Leu
Pro Gly Leu Lys Ala Ala Thr His Tyr Thr Ile 1220 1225 1230 Thr Ile
Arg Gly Val Thr Gln Asp Phe Ser Thr Thr Pro Leu Ser 1235 1240 1245
Val Glu Val Leu Thr Glu Glu Val Pro Asp Met Gly Asn Leu Thr 1250
1255 1260 Val Thr Glu Val Ser Trp Asp Ala Leu Arg Leu Asn Trp Thr
Thr 1265 1270 1275 Pro Asp Gly Thr Tyr Asp Gln Phe Thr Ile Gln Val
Gln Glu Ala 1280 1285 1290 Asp Gln Val Glu Glu Ala His Asn Leu Thr
Val Pro Gly Ser Leu 1295 1300 1305 Arg Ser Met Glu Ile Pro Gly Leu
Arg Ala Gly Thr Pro Tyr Thr 1310 1315 1320 Val Thr Leu His Gly Glu
Val Arg Gly His Ser Thr Arg Pro Leu 1325 1330 1335 Ala Val Glu Val
Val Thr Glu Asp Leu Pro Gln Leu Gly Asp Leu 1340 1345 1350 Ala Val
Ser Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr 1355 1360 1365
Ala Ala Asp Asn Ala Tyr Glu His Phe Val Ile Gln Val Gln Glu 1370
1375 1380 Val Asn Lys Val Glu Ala Ala Gln Asn Leu Thr Leu Pro Gly
Ser 1385 1390 1395 Leu Arg Ala Val Asp Ile Pro Gly Leu Glu Ala Ala
Thr Pro Tyr 1400 1405 1410 Arg Val Ser Ile Tyr Gly Val Ile Arg Gly
Tyr Arg Thr Pro Val 1415 1420 1425 Leu Ser Ala Glu Ala Ser Thr Ala
Lys Glu Pro Glu Ile Gly Asn 1430 1435 1440 Leu Asn Val Ser Asp Ile
Thr Pro Glu Ser Phe Asn Leu Ser Trp 1445 1450 1455 Met Ala Thr Asp
Gly Ile Phe Glu Thr Phe Thr Ile Glu Ile Ile 1460 1465 1470 Asp Ser
Asn Arg Leu Leu Glu Thr Val Glu Tyr Asn Ile Ser Gly 1475 1480 1485
Ala Glu Arg Thr Ala His Ile Ser Gly Leu Pro Pro Ser Thr Asp 1490
1495 1500 Phe Ile Val Tyr Leu Ser Gly Leu Ala Pro Ser Ile Arg Thr
Lys 1505 1510 1515 Thr Ile Ser Ala Thr Ala Thr Thr Glu Ala Leu Pro
Leu Leu Glu 1520 1525 1530 Asn Leu Thr Ile Ser Asp Ile Asn Pro Tyr
Gly Phe Thr Val Ser 1535 1540 1545 Trp Met Ala Ser Glu Asn Ala Phe
Asp Ser Phe Leu Val Thr Val 1550 1555 1560 Val Asp Ser Gly Lys Leu
Leu Asp Pro Gln Glu Phe Thr Leu Ser 1565 1570 1575 Gly Thr Gln Arg
Lys Leu Glu Leu Arg Gly Leu Ile Thr Gly Ile 1580 1585 1590 Gly Tyr
Glu Val Met Val Ser Gly Phe Thr Gln Gly His Gln Thr 1595 1600 1605
Lys Pro Leu Arg Ala Glu Ile Val Thr Glu Ala Glu Pro Glu Val 1610
1615 1620 Asp Asn Leu Leu Val Ser Asp Ala Thr Pro Asp Gly Phe Arg
Leu 1625 1630 1635 Ser Trp Thr Ala Asp Glu Gly Val Phe Asp Asn Phe
Val Leu Lys 1640 1645 1650 Ile Arg Asp Thr Lys Lys Gln Ser Glu Pro
Leu Glu Ile Thr Leu 1655 1660 1665 Leu Ala Pro Glu Arg Thr Arg Asp
Ile Thr Gly Leu Arg Glu Ala 1670 1675 1680 Thr Glu Tyr Glu Ile Glu
Leu Tyr Gly Ile Ser Lys Gly Arg Arg 1685 1690 1695 Ser Gln Thr Val
Ser Ala Ile Ala Thr Thr Ala Met Gly Ser Pro 1700 1705 1710 Lys Glu
Val Ile Phe Ser Asp Ile Thr Glu Asn Ser Ala Thr Val 1715 1720 1725
Ser Trp Arg Ala Pro Thr Ala Gln Val Glu Ser Phe Arg Ile Thr 1730
1735 1740 Tyr Val Pro Ile Thr Gly Gly Thr Pro Ser Met Val Thr Val
Asp 1745 1750 1755 Gly Thr Lys Thr Gln Thr Arg Leu Val Lys Leu Ile
Pro Gly Val 1760 1765 1770 Glu Tyr Leu Val Ser Ile Ile Ala Met Lys
Gly Phe Glu Glu Ser 1775 1780 1785 Glu Pro Val Ser Gly Ser Phe Thr
Thr Ala Leu Asp Gly Pro Ser 1790 1795 1800 Gly Leu Val Thr Ala Asn
Ile Thr Asp Ser Glu Ala Leu Ala Arg 1805 1810 1815 Trp Gln Pro Ala
Ile Ala Thr Val Asp Ser Tyr Val Ile Ser Tyr 1820 1825 1830 Thr Gly
Glu Lys Val Pro Glu Ile Thr Arg Thr Val Ser Gly Asn 1835 1840 1845
Thr Val Glu Tyr Ala Leu Thr Asp Leu Glu Pro Ala Thr Glu Tyr 1850
1855 1860 Thr Leu Arg Ile Phe Ala Glu Lys Gly Pro Gln Lys Ser Ser
Thr 1865 1870 1875 Ile Thr Ala Lys Phe Thr Thr Asp Leu Asp Ser Pro
Arg Asp Leu 1880 1885 1890 Thr Ala Thr Glu Val Gln Ser Glu Thr Ala
Leu Leu Thr Trp Arg 1895 1900 1905 Pro Pro Arg Ala Ser Val Thr Gly
Tyr Leu Leu Val Tyr Glu Ser 1910 1915 1920 Val Asp Gly Thr Val Lys
Glu Val Ile Val Gly Pro Asp Thr Thr 1925 1930 1935 Ser Tyr Ser Leu
Ala Asp Leu Ser Pro Ser Thr His Tyr Thr Ala 1940 1945 1950 Lys Ile
Gln Ala Leu Asn Gly Pro Leu Arg Ser Asn Met Ile Gln 1955 1960 1965
Thr Ile Phe Thr Thr Ile Gly Leu Leu Tyr Pro Phe Pro Lys Asp 1970
1975 1980 Cys Ser Gln Ala Met Leu Asn Gly Asp Thr Thr Ser Gly Leu
Tyr 1985 1990 1995 Thr Ile Tyr Leu Asn Gly Asp Lys Ala Glu Ala Leu
Glu Val Phe 2000 2005 2010 Cys Asp Met Thr Ser Asp Gly Gly Gly Trp
Ile Val Phe Leu Arg 2015 2020 2025 Arg Lys Asn Gly Arg Glu Asn Phe
Tyr Gln Asn Trp Lys Ala Tyr 2030 2035 2040 Ala Ala Gly Phe Gly Asp
Arg Arg Glu Glu Phe Trp Leu Gly Leu 2045 2050 2055 Asp Asn Leu Asn
Lys Ile Thr Ala Gln Gly Gln Tyr Glu Leu Arg 2060 2065 2070 Val Asp
Leu Arg Asp His Gly Glu Thr Ala Phe Ala Val Tyr Asp 2075 2080 2085
Lys Phe Ser Val Gly Asp Ala Lys Thr Arg Tyr Lys Leu Lys Val 2090
2095 2100 Glu Gly Tyr Ser Gly Thr Ala Gly Asp Ser Met Ala Tyr His
Asn 2105 2110 2115 Gly Arg Ser Phe Ser Thr Phe Asp Lys Asp Thr Asp
Ser Ala Ile 2120 2125 2130 Thr Asn Cys Ala Leu Ser Tyr Lys Gly Ala
Phe Trp Tyr Arg Asn 2135 2140 2145 Cys His Arg Val Asn Leu Met Gly
Arg Tyr Gly Asp Asn Asn His 2150 2155 2160 Ser Gln Gly Val Asn Trp
Phe His Trp Lys Gly His Glu His Ser 2165 2170 2175 Ile Gln Phe Ala
Glu Met Lys Leu Arg Pro Ser Asn Phe Arg Asn 2180 2185 2190 Leu Glu
Gly Arg Arg Lys Arg Ala 2195 2200 3687DNAHomo sapiens 3attggactcc
tgtacccctt ccccaaggac tgctcccaag caatgctgaa tggagacacg 60acctctggcc
tctacaccat ttatctgaat ggtgataagg ctcaggcgct ggaagtcttc
120tgtgacatga cctctgatgg gggtggatgg attgtgttcc tgagacgcaa
aaacggacgc 180gagaacttct accaaaactg gaaggcatat gctgctggat
ttggggaccg cagagaagaa 240ttctggcttg ggctggacaa cctgaacaaa
atcacagccc aggggcagta cgagctccgg 300gtggacctgc gggaccatgg
ggagacagcc tttgctgtct atgacaagtt cagcgtggga 360gatgccaaga
ctcgctacaa gctgaaggtg gaggggtaca gtgggacagc aggtgactcc
420atggcctacc acaatggcag atccttctcc acctttgaca aggacacaga
ttcagccatc 480accaactgtg ctctgtccta caaaggggct ttctggtaca
ggaactgtca ccgtgtcaac 540ctgatgggga gatatgggga caataaccac
agtcagggcg ttaactggtt ccactggaag 600ggccacgaac actcaatcca
gtttgctgag atgaagctga gaccaagcaa cttcagaaat 660cttgaaggca
ggcgcaaacg ggcataa 687419DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined Molecule Synthetic oligonucleotide 4gtaagacttg agaugaucc
19522DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5tgggacaggc acagcattct tg 22622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6tgggcataag ttccagcagg ag 22721DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined Molecule Synthetic oligonucleotide 7ggaucaucuc aagucuuact
t 21821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined Molecule Synthetic
oligonucleotide 8guaagacuug agaugaucct t 21
* * * * *
References