U.S. patent application number 13/528399 was filed with the patent office on 2013-03-21 for microbubble complexes and methods of use.
This patent application is currently assigned to General Electric Company. The applicant listed for this patent is John Donald Burczak, Hae Won Lim, Lisa Anne Lowery, Praveena Mohan, James Edward Rothman, Anup Sood. Invention is credited to John Donald Burczak, Hae Won Lim, Lisa Anne Lowery, Praveena Mohan, James Edward Rothman, Anup Sood.
Application Number | 20130072854 13/528399 |
Document ID | / |
Family ID | 46852013 |
Filed Date | 2013-03-21 |
United States Patent
Application |
20130072854 |
Kind Code |
A1 |
Mohan; Praveena ; et
al. |
March 21, 2013 |
MICROBUBBLE COMPLEXES AND METHODS OF USE
Abstract
The present invention relates to a microbubble complex
comprising a microbubble having an outer shell comprising a mixture
of native and denatured albumin encapsulating a perfluorocarbon
gas, a therapeutic agent, a bifunctional linker having one end
attached to the therapeutic agent and the other attached to a
ligand and wherein the ligand is bound to the other shell of the
microbubble through hydrophobic interactions. Also included are
methods for delivering the aforementioned microbubble complex to a
tissue target.
Inventors: |
Mohan; Praveena; (Oakdale,
MN) ; Sood; Anup; (Clifton Park, NY) ;
Rothman; James Edward; (New York, NY) ; Burczak; John
Donald; (Voorheesville, NY) ; Lim; Hae Won;
(Niskayuna, NY) ; Lowery; Lisa Anne; (Niskayuna,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mohan; Praveena
Sood; Anup
Rothman; James Edward
Burczak; John Donald
Lim; Hae Won
Lowery; Lisa Anne |
Oakdale
Clifton Park
New York
Voorheesville
Niskayuna
Niskayuna |
MN
NY
NY
NY
NY
NY |
US
US
US
US
US
US |
|
|
Assignee: |
General Electric Company
Schenectady
NY
|
Family ID: |
46852013 |
Appl. No.: |
13/528399 |
Filed: |
June 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13235890 |
Sep 19, 2011 |
|
|
|
13528399 |
|
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|
Current U.S.
Class: |
604/22 ; 424/400;
514/1.1; 514/23; 514/44A; 514/44R |
Current CPC
Class: |
C12N 2320/32 20130101;
A61K 47/06 20130101; C12N 2310/14 20130101; A61K 41/0028 20130101;
C12N 15/87 20130101; C12N 15/1136 20130101; A61K 9/0019 20130101;
A61K 47/64 20170801; C12N 2310/3515 20130101; A61K 47/554 20170801;
A61M 2037/0007 20130101; A61K 47/6925 20170801; C12N 15/111
20130101; A61K 31/713 20130101; A61K 47/643 20170801; A61M 37/0092
20130101 |
Class at
Publication: |
604/22 ; 424/400;
514/1.1; 514/23; 514/44.R; 514/44.A |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/713 20060101 A61K031/713; A61K 31/70 20060101
A61K031/70; A61K 31/7088 20060101 A61K031/7088; A61M 37/00 20060101
A61M037/00; A61K 38/02 20060101 A61K038/02 |
Claims
1. A microbubble complex comprising: a microbubble having an outer
shell comprising a mixture of native and denatured albumin and a
hollow core encapsulating a perfluorocarbon gas; a therapeutic
agent selected from a group comprising a small molecule
chemotherapeutic agent, peptide, carbohydrate, oligonucleotide,
cytotoxin, protein synthesis inhibitor, or combination thereof; a
bifunctional linker having one end attached to the therapeutic
agent and the other attached to a ligand through reaction of a
reactive group on said ligand; and wherein the ligand is bound to
the outer shell of the microbubble through hydrophobic
interactions.
2. The complex of claim 1 wherein the therapeutic agent is an
oligonucleotide.
3. The complex of claim 2 wherein the oligonucleotide is a
naturally occurring or modified DNA or RNA.
4. The complex of claim 3 wherein the RNA is a small interfering
RNA.
5. The complex of claim 1 wherein the bifunctional linker comprises
a oligo- or poly-amino acid, peptide, saccharide, nucleotide,
organic moiety having approximately 1 to 250 carbon atoms, or a
combination thereof.
6. The complex of claim 1 wherein the bifunctional linker comprises
tetraethylene glycol (TEG) or polyethylene glycol.
7. The complex of claim 1 wherein the reactive group comprises an
activated ester, phosphoramidite, isocyanate, isothiocyanate,
aldehyde, acid chloride, sulfonyl chloride, maleimide, alkyl
halide, amine, phosphine, phosphate, alcohol or thiol.
8. The complex of claim 7 wherein the reactive group is an
amine.
9. The complex of claim 1 wherein the ligand is a fatty acid,
steroid, or a combination thereof.
10. The complex of claim 9 wherein the fatty acid is myristoyl,
lithocolic-oleyl, docosanyl, lauroyl, steoroyl, palmitoyl, oleoyl,
linoleoyl, or a combination thereof.
11. The complex of claim 9 wherein the steroid is cholesterol,
cholic acid, lithocholic acid, chenodeoxycholic acid or a
combination thereof.
12. The complex of claim 1 wherein the ligand is 4-iodophenyl
butyric acid or an analog or derivative thereof.
13. The complex of claim 1 wherein the amount of denature to native
albumin is in the range of approximately 0.5 to 30 wt %.
14. The complex of claim 13 wherein the range of denature to native
albumin is approximately 1 to 15 wt %.
15. The complex of claim 1 wherein the therapeutic agent comprises
siRNA, the linker comprises tetraethylene glycol, and the ligand
comprises cholesterol.
16. A method for delivering a microbubble complex to a tissue
target comprising the steps of: providing a microbubble complex,
said complex comprising; a microbubble having an outer shell
comprising a mixture of native and denatured albumin and a hollow
core encapsulating a perfluorocarbon gas; a therapeutic agent
selected from a group comprising a small molecule chemotherapeutic
agent, peptide, carbohydrate, oligonucleotide, cytotoxin, protein
synthesis inhibitor, or combination thereof; a bifunctional linker
having one end attached to the therapeutic agent and the other
attached to a ligand through reaction of a reactive group on said
ligand; and wherein the ligand is bound to the outer shell of the
microbubble through hydrophobic interactions; administrating the
microbubble complex to a subject wherein the subject is the source
of the tissue target; and administering ultrasonic energy to the
subject, wherein said energy is sufficient to cause cavitation of
the microbubble complex in the tissue target.
17. The method of claim 16 wherein the tissue target is in vivo and
administrating the microbubble complex comprises intravenous or
intraperitoneal injection of the microbubble complex.
18. The method of claim 16 further comprising the step of
visualizing the microbubble complex at the tissue target prior to
administering the ultrasonic energy for cavitation of the
microbubble complex.
19. The method of claim 16 wherein the visualizing and
administering the ultrasonic energy are performed in real time.
20. The method of claim 16 wherein the tissue target is in
vitro.
21. The method of claim 16 wherein the therapeutic agent is an
oligonucleotide.
22. The method of claim 21 wherein the oligonucleotide is a
naturally occurring or modified DNA or RNA.
23. The method of claim 22 wherein the RNA is a small interfering
RNA.
24. The method of claim 16 wherein the bifunctional linker
comprises a oligo- or poly-amino acid, peptide, saccharide,
nucleotide, organic moiety having approximately 1 to 250 carbon
atoms, or a combination thereof.
25. The method of claim 16 wherein the bifunctional linker
comprises tetraethylene glycol (TEG) or polyethylene glycol.
26. The method of claim 16 wherein the reactive group comprises an
activated ester, phosphoramidite, isocyanate, isothiocyanate,
aldehyde, acid chloride, sulfonyl chloride, maleimide, alkyl
halide, amine, phosphine, phosphate, a alcohol or thiol.
27. The method of claim 26 wherein the reactive group is an
amine.
28. The method of claim 16 wherein the ligand is a fatty acid,
steroid, or a combination thereof.
29. The method of claim 28 wherein the fatty acid is myristoyl,
lithocolic-oleyl, docosanyl, lauroyl, steoroyl, palmitoyl, oleoyl,
linoleoyl, or a combination thereof.
30. The method of claim 28 wherein the steroid is cholesterol,
cholic acid, lithocholic acid, chenodeoxycholic acid or a
combination thereof.
31. The method of claim 16 wherein the ligand is 4-iodophenyl
butyric acid or an analog or derivative thereof.
32. The method of claim 16 wherein the amount of denature to native
albumin is in the range of approximately 0.5 to 30 wt %.
33. The method of claim 32 wherein the range of denature to native
albumin is approximately 1 to 15 wt %.
34. The method of claim 16 wherein the therapeutic agent comprises
siRNA, the linker comprises tetraethylene glycol, and the ligand
comprises cholesterol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part to U.S. patent
application Ser. No. 13/235,890 filed Sep. 19, 2011 the disclosure
of which is incorporated herein by reference in its entirety.
BACKGROUND
[0002] The invention relates generally to novel binding of
therapeutic agents to albumin microbubble pharmaceuticals using an
attachment of albumin affinity ligands to the agents. The binding
provides a method of microbubble-assisted delivery of therapeutic
agents to targeted cells or tissue of interest, either in vitro or
in vivo.
[0003] Ultrasound-mediated destruction of microbubbles carrying
drugs has been found to be useful as a noninvasive drug delivery
system. Drugs or other therapeutic agents can be incorporated into
the microbubbles in a number of different ways, including binding
of the drug to the microbubble shell and attachment of ligands. For
example, perfluorocarbon-filled microbubbles are sufficiently
stable for circulating in the vasculature as blood pool agents;
they act as carriers of these agents until the site of interest is
reached. Ultrasound applied over the skin surface can then be used
to burst the microbubbles at this site, causing localized release
of the drug or therapeutic agents on site specific locations.
[0004] More specifically, albumin microbubbles have been used and
delivered to a specific organ target by site-directed acoustic
ultrasound. Albumin is a major protein in blood, and its natural
physiological role is to bind and carry a wide variety of
lipophilic/poorly soluble ligands throughout the body. These
ligands, which may have an affinity to albumin, include fatty acids
and other biosynthetic and catabolic products that are hydrophobic
in nature. As such albumin microbubbles have been used to carry a
variety of therapeutic agents based on proteins and other biologics
including, oligonucleotides (ODN) and polynucleotides such as
antisense ODN, with sequences complementary to a specific targeted
messenger RNA (mRNA) sequence. These microbubble-nucleic acid
complexes may be generated from unmodified ODN that are mixed with
albumin or lipid components during microbubble shell formation or
alternatively, the complex formation can be performed by mixing
preformed microbubbles with an ODN of interest. In both cases, the
ODN acts as a mechanistic intervention in the processes of gene
translation or an earlier processing event. The advantage of this
approach is the potential for gene-specific actions which should be
reflected in a relatively low dose and minimal non-targeted side
effects.
[0005] However, a key barrier to translating the potent biology of
ODN into drugs is known to be at the level of drug delivery with
efficacy and safety. For example, ODN delivery with chemical
formulation, viral vectors, and particle delivery have been
hampered with clinical safety related problems before therapeutic
efficacy can be attained. Furthermore, the use of albumin
microbubbles as a carrier of ODNs such as siRNA is limited due to
the limited binding of the ODNs to the albumin microbubble as well
as the stability of the albumin-ODN complex. Due to negative shell
surface potential of albumin, the negatively charged shorter
nucleic acids do not bind very well to the microbubble and gene
transfection efficiencies using these complexes are generally
suboptimal.
[0006] Thus there is a need to improve the binding of the
therapeutic agents to the microbubble as well as improving the
stability and efficacy of the microbubble complex.
[0007] Furthermore there is a need to reduce toxicity in the
selective delivery of highly cytotoxic drugs. Non-targeted delivery
of these drugs can cause systemic toxicity and has prevented the
use of many of these drugs all together or at higher doses required
for good efficacy. Attempts to deliver these as pro-drugs in many
cases have reduced this problem, however, selective uptake in the
targeted tissue is not always easy to achieve as most of the uptake
mechanisms in the diseased tissue are also present in the normal
tissue. Enhancing the uptake of these drugs in selective tissues by
non-natural mechanisms as disclosed herein, therefore can add
considerable value.
BRIEF DESCRIPTION
[0008] Provided herein are novel compositions and methods for
increased binding of therapeutic drugs to microbubbles using the
affinity of ligand-therapeutic compositions towards the albumin
shell.
[0009] Systemic circulation of the microbubbles carrying the
therapeutic composition can be easily visualized through ultrasound
imaging. Therapeutic agent is released from the microbubbles using
a trigger of high energy pulsed ultrasound specific to the site of
treatment. The cavitation of microbubbles causes sonoporation of
the neighboring cells/tissue.
[0010] In one embodiment a microbubble complex is disclosed
comprising a microbubble having an outer shell comprising a mixture
of native and denatured albumin and a hollow core encapsulating a
perfluorocarbon gas, a therapeutic agent selected from the group
comprising a small molecule chemotherapeutic agent, a peptide, a
carbohydrate, or an oligonucleotide, and a bifunctional linker
having one end attached to the therapeutic agent and the other
attached to the ligand through reaction of a reactive group on the
ligand. The ligand is bound to the outer shell of the microbubble
through hydrophobic interactions.
[0011] In another embodiment a method is for delivering the
aforementioned microbubble complex to a tissue target is disclosed.
The method comprising the steps of providing the microbubble
complex, administrating the microbubble complex to a subject
wherein the subject is the source of the tissue target, and
administering ultrasonic energy to the subject, wherein the
ultrasound energy is sufficient to cause cavitation of the
microbubble complex in the tissue target.
BRIEF DESCRIPTION OF THE FIGURES
[0012] These and other features, aspects, and advantages of the
present invention will become better understood when the following
detailed description is read with reference to the accompanying
figures wherein:
[0013] FIG. 1 is a representation of non-covalent binding of
siRNA-ligand to albumin microbubbles.
[0014] FIG. 2 is representative micrograph of a gel shift assay for
a mixture of cholesterol conjugated siRNA (2 pmoles) and varying
amounts of Optison (0, 9, 22 and 46 pmoles for i, ii, iii and iv
respectively).
[0015] FIG. 3 is a. representative micrograph of a gel shift assay
for Cy3-siRNA (4 pmoles) mixed with varying concentrations of
either Optison or native HSA which shows no shift in gel assay.
[0016] FIG. 4 is a. representative micrograph of a gel assay for
Cy3-cholesterol-siRNA (2 pmoles) mixed with varying concentrations
of either Optison, native HSA or denatured HSA.
[0017] FIG. 5 is a graphical representation of binding properties
of cholesterol-siRNA to Optison and native HSA; (A) Relative
fluorescence of cholesterol-siRNA bands is measured from the gel
shift assay (B) fraction bound of siRNA calculated from relative
fluorescence and plotted against the albumin concentration.
[0018] FIG. 6 is a graphical representation of the uptake of siRNA
by U-87 tumor cells in opticell is quantified by measuring cell
cy3-fluorescence.
[0019] FIG. 7 are micrographs of fluorescence images showing a
comparison of siRNA transfection between a lipid transfection
reagent (RNAifect) and Optison.
[0020] FIG. 8 is a graphical representation of the mean cell
fluorescence values and standard error of siRNA transfection
between a lipid transfection reagent (RNAifect) and Optison.
[0021] FIG. 9 is a graphical representation of the fraction bound
of fluorescein-myristate to Optison and native HSA as calculated
from anisotropy values.
[0022] FIGS. 10 A and B are the fluorescein bound to Optison (0, 8,
40 and 200 pmoles for i, ii, iii and iv respectively) and
visualized on the gel as dark bands for fluorescein-myristate (63
pmoles) and fluorescein-stearate (180 pmoles) respectively.
DETAILED DESCRIPTION
[0023] The following detailed description is exemplary and not
intended to limit the invention of the application and uses of the
invention. Furthermore, there is no intention to be limited by any
theory presented in the preceding background of the invention or
descriptions of the drawings.
[0024] The invention relates generally to microbubble-assisted
delivery of a therapeutic agent to cells or tissue of interest,
either in vitro or in vivo.
[0025] In certain embodiment the therapeutic agent may be comprised
of a small molecule chemotherapeutic agent, a peptide, a
carbohydrate, or an oligonucleotide, and a bifunctional linker
having one end attached to the therapeutic agent and the other
attached to the ligand through reaction of a reactive group on the
ligand. In certain embodiment the therapeutic agent may be an
oligonucleotide (ODN). Oligonucleotides refers to nucleic acid
polymers that are formed by bond cleavage of longer nucleic acids
or are synthesized using building blocks, protected
phosphoramidites of natural or chemically modified nucleosides or,
to a lesser extent, of non-nucleosidic compounds. The length of the
oligonucleotide may vary from a short nucleic acid polymer of fifty
or fewer base pairs to more than 200 base pairs. As used herein,
ODN also refers to polynucleotides having more than 200 base pairs.
Also included are antisense ODN which refer to single strands of
DNA or RNA that are complementary to a chosen sequence. In the case
of antisense RNA, antisense RNA prevents protein translation of
certain messenger RNA strands by binding to them. Antisense DNA can
be used to target a specific, complementary (coding or non-coding)
RNA. Also included are small interfering RNA (siRNA), sometimes
known as short interfering RNA or silencing RNA, is a class of
double-stranded RNA molecules, typically 20-25 nucleotides in
length, that play a variety of roles in biology including the RNA
interference (RNAi) pathway, where it interferes with the
expression of a specific gene, as an antiviral mechanism, or in
shaping the chromatin structure of a genome.
[0026] In certain embodiments, the therapeutic agent may be a
cytotoxin. As used herein cytotoxin refers to a substance that has
a toxic effect on cells. For example a cytotoxin may cause undergo
necrosis, in which they lose membrane integrity and die as a result
of cell lysis. In other examples a cytotoxin may be associated with
antibody-dependent cell mediated cytotoxicity wherein a cell is
marked by an antibody and acted upon by certain lymphocytes.
[0027] Examples of cytotoxic agents are listed in Goodman and
Gilman's "The Pharmacological Basis of Therapeutics," Tenth
Edition, McGraw-Hill, New York, 2001. These include taxol; nitrogen
mustards, such as mechlorethamine, cyclophosphamide, melphalan,
uracil mustard and chlorambucil; ethylenimine derivatives, such as
thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as
carmustine, lomustine, semustine and streptozocin; triazenes, such
as dacarbazine; folic acid analogs, such as methotrexate;
pyrimidine analogs, such as fluorouracil, cytarabine and azaribine;
purine analogs, such as mercaptopurine and thioguanine; vinca
alkaloids, such as vinblastine and vincristine; antibiotics, such
as dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin
and mitomycin; enzymes, such as L-asparaginase; platinum
coordination complexes, such as cisplatin; substituted urea, such
as hydroxyurea; methyl hydrazine derivatives, such as procarbazine;
adrenocortical suppressants, such as mitotane; hormones and
antagonists, such as adrenocortisteroids (prednisone), progestins
(hydroxyprogesterone caproate, medroprogesterone acetate and
megestrol acetate), estrogens (diethylstilbestrol and ethinyl
estradiol), antiestrogens (tamoxifen), and androgens (testosterone
propionate and fluoxymesterone).
[0028] Drugs that interfere with intracellular protein synthesis,
protein synthesis inhibitors, can also be coupled to the ligand;
such drugs are known to these skilled in the art and include
puromycin, cycloheximide, and ribonuclease.
[0029] In one embodiment, the protein includes, but is not limited
to, enzymes, soluble and serum proteins, proteins expressed on a
surface of a cell, non-immunoglobulin proteins, intracellular
proteins, and segment of proteins that are or can be made
water-soluble, either individually or in combinations thereof as
well as any derivatives of the proteins.
[0030] In a particular embodiment, the protein includes such as,
but not limited to, cysteine proteases, glutathione S transferase,
epoxide hydrolase (EH), thiolase, NAD/NADP-dependent
oxidoreductase, enoyl coA hydratase, aldehyde dehydrogenase,
hydroxypyruvate reductase, tissue transglutaminase (tTG),
formiminotransferase cyclodeaminase (FTCD),
aminolevulinate-dehydratase (ADD), creatin kinase, carboxylesterase
(LCE), monoacylglycerol (MAG) lipase, metalloproteases (MP),
phosphotases (protein tyrosine phosphotases, PTP), proteosome,
FK506 binding protein (FKBP12), mammalian target of Rapamycin
(mTOR; alternatively known as FKBP-rapamycin binding domain (FRB)),
serine hydrolase (superfamily), ubiquitin-binding protein,
-galactosidase, nucleotide binding enzymes, protein kinases,
GTP-binding proteins, cutinase, adenylo succinate synthase, adenylo
succinate lyase, glutamate dehydrogenase, dihydrofolate reductase,
fatty acid synthase, aspartate transcarbamylase,
acetylcholinesterase, HMG cholate reductase, and cyclo-oxygenase
(COX-1 and COX-2), either individually or in combinations thereof.
Also included are any derivatives of any of the proteins.
[0031] In another example, the protein is substantially free of a
cofactor. "Substantially free of a cofactor" includes proteins that
do not require any additional cofactor, chemical, chemical
modification, or physical modification to be naturally stable under
physiological conditions and room temperature and pressure in
solution or as a solid, and can bind its corresponding ligand in
vivo.
[0032] In one embodiment, an albumin microbubble may be utilized to
carry a therapeutic agent in systemic delivery. Tissue targeted
ultrasound acoustic energy may then be used to cavitate the albumin
microbubble and deliver the therapeutic agent into the
intracellular environment. For example the microbubble complex may
be administered intravenously or into the peritoneum
(intraperitoneally) of a subject whose cells or tissues are to be
targeted. Once the microbubble complex is carried through the
subject to the targeted cell, the ultrasound acoustic energy is
delivered. In certain embodiments, visualization of the targeted
cells may occur prior to delivering the ultrasound while in still
other embodiments visualization may be performed in real time and
the cavitation monitored.
[0033] In certain embodiments, the albumin outer shell of the
microbubble is comprised of both native and denatured albumin held
together by mostly cysteine to cysteine bonds. In certain
embodiments, the primary composition of the albumin shell is mostly
in the native form wherein the denatured portion allows for
increased cysteine bond attachments. In certain embodiments the
relative amount of denatured albumin to native albumin ranges from
approximately 0.5 to 30 wt %. In other embodiments the relative
amount is in the range of approximately 1% to 15 wt %. The mixture
of native and denatured albumin provides a balance of shell
elasticity needed for cavitation, with increased reactive binding
sites on the microbubble surface. The microbubbles are formed by
sonication of perfluorocarbon gas in the presence of pre-heated
albumin solution. A small part of the albumin molecules rearrange
during sonication of pre-heated albumin solution and crosslinking
occurs through disulfide linkages between albumin molecules. These
albumin molecules are believed to be similar in structure to an F
form of albumin which has more hydrophobic residues exposed. This
allows increased binding sites for hydrophobic interactions.
[0034] In certain embodiments, the microbubble may be filled with
an insoluble perfluorocarbon gas, such as but not limited to,
perfluoromethane, perfluoroethane, perfluoropropane,
perfluorobutane, perfluoropentane, or a combination thereof. In
certain embodiments, the microbubbles are about 1 to about 5
microns in diameter, the size being optimized to allow circulation
through the blood stream.
[0035] In certain embodiments, the therapeutic-microbubble
complexes comprise a therapeutic agent modified with a linker
having a reactive group capable of binding with a ligand having
affinity towards albumin. As such, the therapeutic agent may be
coupled to albumin though the ligand.
[0036] The linker includes any linking moiety that attaches the
ligand to the therapeutic agent through a first moiety. The linker
can be as short as one carbon or a long polymeric species such as
polyethylene glycol, tetraethylene glycol (TEG), polylysine or
other polymeric species normally used in the pharmaceutical
industry for modulating pharmacokinetic and biodistribution
characteristics of therapeutic agents. Other linkers of varying
length include C1-C250 length with one or more heteroatoms selected
from O, S, N, P, and optionally substituted with halogen atoms. In
a particular embodiment, the linker comprises at least one of an
oligomeric or polymeric species made of natural or synthetic
monomers, oligomeric or polymeric moiety selected from a
pharmacologically acceptable oligomer or polymer composition, an
oligo- or poly-amino acid, peptide, saccharide, a nucleotide, and
an organic moiety with 1-250 carbon atoms, either individually or
in combination thereof. The organic moiety with 1-250 carbon may
contain one or more heteroatoms such as O, S, N or P and optionally
substituted with halogen atoms at one or more places.
[0037] The first moiety may simply be an extension of the linker,
formed by the reaction of a reactive species on the linker with a
reactive group on the therapeutic agent. Examples of reactive
species and the reactive group include, but are not limited to,
activated esters (such as N-hydroxysuccinimide ester,
pentafluorophenyl ester), a phosphoramidite, an isocyanate, an
isothiocyanate, an aldehyde, an acid chloride, a sulfonyl chloride,
a maleimide an alkyl halide, an amine, a phosphine, a phosphate, an
alcohol or a thiol with the proviso that the reactive species and
reactive group are matched to undergo a reaction yielding
covalently linked conjugates.
[0038] In certain embodiments, the reactive moiety may be a primary
amine functionality, and as such, the amine modified therapeutic
agent may be conjugated to the affinity ligand through reaction of
a carboxyl moiety of the ligand. In certain other embodiments, the
reactive moiety may be an alcohol attached to the ligand through a
phosphate group.
[0039] The affinity ligand includes fatty acids, steroids, small
aromatic compounds or a combination thereof. Examples of albumin
binding molecules may be found in US patent application publication
number 2010/0172844, published Jul. 8, 2010.
[0040] For example in certain embodiments the affinity ligand is a
fatty acid, including but not limited to myristoyl,
lithocolic-oleyl, docosanyl, lauroyl, steoroyl, palmitoyl, oleoyl,
or linoleoyl. In other embodiments, the lipophilic molecule is a
steroid or modified steroid including, cholesterol, cholic acid,
lithocholic acid, or chenodeoxycholic acid. In other embodiment,
the high affinity molecule is selected from 4-p-iodophenyl-butyric
acid and analogs or derivatives thereof. In still other
embodiments, the therapeutic agent comprises siRNA, the linker
comprises tetraethylene glycol, and the ligand comprises
cholesterol.
[0041] In certain embodiments, the therapeutic-albumin complexes
may be prepared either by sonicating ligand-modified therapeutic
agent with albumin or lipid in the presence of perfluorocarbon or
by mixing preformed bubbles with ligand modified therapeutic
agents. In certain embodiments, these molecules may be attached to
therapeutic agents of interest during therapeutic agent synthesis.
For example the phosphoramidites of cholesterol may be used to
incorporate cholesterol during DNA or RNA synthesis on a nucleic
acid synthesizer, or post synthesis by incorporating a reacting
moiety.
[0042] In certain embodiments, the therapeutic agent is a modified
ODN which may be prepared enzymatically by using modified
nucleoside triphosphates; modified either with the ligand itself or
with a reactive functionality for post synthesis modification with
the ligand. Ligand attachment may be at one or both termini,
internal to the nucleic acid sequence or at multiple positions
depending upon the ODN use. In certain embodiments, where siRNA is
the ODN, attachment may be through the 3' OH position.
[0043] In certain embodiments, in addition to the ligand, the
therapeutic agent, including where the therapeutic agent is ODN,
may be selectively modified to protect from nucleases. In certain
embodiments, stabilizing modification may include phosphorothioate
modification, or 2'-OMe modification.
[0044] In certain embodiments, the microbubble complexes may be
incubated with cells or the tissue of interest or injected into the
body, preferably intravenously, and then cavitated with ultrasound
energy at desired site and at a predetermined time or during live
imaging.
[0045] In certain embodiments, the microbubble complex may be
viewed during systemic travel in the blood circulation under normal
ultrasound diagnostic imaging. When the bubble arrives on tissue
target, in this case the tumor, a series of pulsed acoustic energy
waves are sent to the tumor. This creates inertial cavitation on
the microbubble, which collapses the microbubble. Cavitation of the
microbubble occurs where the acoustic energy is maximally located.
This direction is achieved on the ultrasound probe by parameters
related to mechanical index force, optimal ultrasound acoustic
distance, and dimensions of the ultrasound acoustic sweep. The
force generated can then potentially form micropores within the
cellular plasma membrane. Typically the pulsed energy is
administered at a frequency of about 0.5 to about 5 MHz.
[0046] These micropores, along with the microjetting force created
under inertial cavitation, may facilitate the entrance of ODN into
the cellular cytoplasmic environment. For example when the ODN is
siRNA, siRNA in the intracellular environment will utilize the host
machinery to silence mRNA and later protein synthesis. Similarly,
where mRNA message acts as an angiogenesis promoting proteins
including vascular endothelial growth factor (VEGF), the reduction
of VEGF expression in a tumor may halt or slow tumor growth. After
microbubble cavitation the dense gas of the microbubble center is
exhaled out of the body and the albumin shell is metabolized and
excreted via the liver elimination pathway.
[0047] In an exemplary embodiment, a bolus of the microbubble
complex may be mixed to an optimal ratio from previous therapeutic
investigations. Once the mixture of the complex is established, the
bolus drug is injected systemically by venous route.
[0048] For example in the use of a siRNA-microbubble bolus, the
bolus may be monitored in the first pass blood kinetics. The
microbubble resonance and thus enhanced ultrasound contrast may be
monitored with an ultrasound probe using low diagnostic levels of
acoustic energy. During circulation the bolus arrives on organ
target. Cardiovascular tissue perfusion may assist in delivering
the bolus into deep microvessels with small lumen diameters. By
supplying pulsed acoustic energy, sufficient energy may be provided
for the microbubble to undergo inertial cavitation. Once the
microbubble cavitation is complete the siRNA contents may be
delivered across the plasma membrane and into the diseased cell.
While in the cytoplasmic intracellular environment the siRNA can
have a therapeutic effect.
[0049] During microbubble cavitation siRNA entrance into the cell
may occur by various mechanisms. For example the siRNA may enter
the cell by a: a microjetting force from the collapsing microbubble
which can push siRNA into the cytoplasm. Alternatively the
mechanism may include microjetting energy or sonoluminescence
energy which creates temporary micropores within the plasma
membrane to allow for passive diffusion of the siRNA into the cell,
or during microbubble resonance before actual cavitation the
microbubble bumping into the plasma membrane may push the siRNA in
the cell.
[0050] As such, the mechanism of microbubble delivery has potential
applications in the treatment of a wide variety of diseases, which
can include cancer, inflammatory, infectious, cardiovascular,
metabolic, autoimmune, and central nervous system diseases. Many of
these diseases cannot currently be effectively treated by virtue of
targeting molecular mechanisms not accessible to conventional small
molecule drugs and monoclonal antibodies.
EXPERIMENTAL
Example 1
[0051] FIG. 1 is a representation illustration of binding of siRNA
to albumin encapsulated microbubbles to form a microbubble-siRNA
complex.
[0052] The target siRNA for VEGF silencing (vascular endothelial
growth factor) were synthesized by IDTDNA technologies. IDTDNA
provided lipid modifications such as cholesteryl TEG on the siRNA
(Chol-siRNA) as well as dye conjugation.
TABLE-US-00001 Sense strand:
5'-Cy3/GCAUUUGUUUGUCCAAGAUmUmU/3'-Lipid Antisense strand; 5'/mAmArA
rUrCrU rUrGrG rArCrA rArArC rArArA rUrGrC/3'
[0053] Cyanine dye, Cy3 on the siRNA has an excitation wavelength
of 550 nm and a peak emission of 580 nm. The siRNA has been labeled
with a cy3 dye for easy visualization of siRNA during binding
assays and other characterization techniques. Optison.TM. (GE
Healthcare, Chalfont St. Giles, United Kingdom, 10 mg/ml albumin)
was centrifuged; the top layer was discarded and the excess albumin
solution in the bottom was used for the binding studies.
Lyophilized human serum albumin (HSA) powder (Sigma Aldrich, St.
Louis Mo.) was dissolved in 1.times. phosphate buffered saline
(PBS) to make a stock solution of 10 mg/mL. Both Optison and native
albumin solution dilutions were prepared with 1.times.PBS.
Denatured HSA solution was prepared by heating native HSA solution
to 80.degree. C. for 20 minutes.
Binding Reaction:
[0054] The stock solutions of cy3-siRNA and cy3-siRNA-cholesterol,
20 .mu.M were prepared in RNAse free water and stored at
-20.degree. C. 4 pmoles of cy3-siRNA and 2 pmoles of
cholesterol-siRNA solutions were mixed with varying amounts of
Optison solution, native HSA and denatured HSA solution, ranging
from 0 to 50 pmoles. The reaction buffer was 1.times.PBS, pH 7.4.
The reaction mixture was incubated under dark at 25.degree. C. for
45 minutes. After incubation, ten .mu.L of siRNA mixtures was mixed
with 2 .mu.L of Novex.RTM. Hi-Density TBE Sample Buffer (5.times.)
(Invitrogen, Carlsbad, Calif., USA).
Gel Electrophoresis:
[0055] All the reactions were loaded onto precast 6% nondenaturing
polyacrylamide gels (Invitrogen, Carlsbad, Calif., USA). The gel
was run at 100 V for 45 min in 0.5.times. Novex TBE Running buffer
(Invitrogen, Carlsbad, Calif., USA). The gels containing either
DNA, protein or both were imaged for cy3 fluorescence using a
typhoon scanner (Typhoon.TM. 9410, GE Healthcare).
Results:
[0056] The fluorescence of Cy3 attached to siRNA can be visualized
as distinct siRNA bands on the gel. When a mixture of siRNA and
albumin solution was run on the gel, the mobility of albumin
bound-siRNA is slower than free-siRNA resulting in two bands on the
gel. In preliminary trials, sypro ruby stain from EMSA kit
(Molecular Probes, Eugene, Oreg., USA) was used to observe the
albumin bands in the gel. An example is shown in FIG. 2 which is a
gel shift assay for a mixture of cholesterol conjugated siRNA (2
pmoles) and varying amounts of Optison (0, 9, 22 and 46 pmoles for
i, ii, iii and iv respectively). Fluorescence imaging of the gel
shows distinct bands for siRNA (lower band sections) and albumin
(upper band sections).
Cy3-siRNA
[0057] When the mixture of cy3-siRNA and native HSA/Optison was run
on the gel, there was no bound-siRNA visualized for increasing
concentrations of albumin. There was no significant binding of
cy3-siRNA with either native HSA or Optison solution. This is shown
in FIG. 3 which is a fluorescence image of a gel for Cy3-siRNA (4
pmoles) mixed with varying concentrations of either Optison or
native HSA shows no shift in gel assay. The dark bands on the gel
are the cy3-fluorescence on siRNA. There is no significant binding
of cy3-siRNA to both Optison and native HSA.
Chol-siRNA
[0058] FIG. 4 shows the gel images for binding of chol-siRNA with
Optison, native HSA and denatured HSA. Chol-siRNA bound to both
native HSA and Optison solution, while the binding significantly
decreased for the same amount of denatured HSA. The fluorescence
intensity of siRNA in each lane was estimated manually by drawing a
box around the bands. Background, equivalent to the average
intensity value of the gel, was subtracted from the intensity value
of each siRNA band. Fluorescence intensities of bound siRNA over a
wide range of albumin concentrations were calculated. Relative
fluorescence, R was calculated as:
R=(Fbound-Ffree)/Ffree (1)
Fbound is fluorescence intensity of bound-siRNA band and Ffree is
fluorescence intensity of free-siRNA band. Relative fluorescence
was plotted against albumin concentration. This is shown in FIG. 5,
which is a graphical comparison of binding properties of
cholesterol-siRNA to Optison and native HSA as described below
[0059] At low albumin concentration ranging from 0 to 15 .mu.M,
linear dependence of bound fluorescence on albumin concentration
was visualized. FIG. 5, graph A shows that at this concentration
range, the amount of chol-siRNA bound to Optison solution was
higher than the binding to native HSA. To estimate binding
constants, higher concentration of albumin was used to allow
saturation of amount of siRNA bound to albumin. Fraction bound, x
is determined as:
x=(Fbound-Ffree)/(Fsat-Ffree) (2)
Fsat is the fluorescence intensity of maximum bound-siRNA under
saturation conditions.
[0060] Fraction bound was plotted against increasing albumin
concentrations, as shown in FIG. 5 graph B, and the data points
were fitted to the following equilibrium equation;
x=n*[Albumin]/(kd+[Albumin]) (3)
kd is the dissociation constant, n is the number of binding sites
and [Albumin] is the total albumin concentration for the respective
samples. Equation 3 was solved using a non-linear fit to determine
kd and n for binding of chol-siRNA to both Optison and native HSA
(Table 1). Microsoft Excel's solver tool was utilized for this
non-linear fit, and the sum of squared errors (SSE) was found to be
0.07 and 0.06 for Optison and native HSA respectively. The binding
constant of chol-siRNA was similar for both Optison and native
HSA.
Example 2
Delivery of siRNA to Tumor Cells
Cell Culture:
[0061] MATBIII rat mammary carcinoma and U-87 human glioblastoma
cells were cultured in McCoy's 5A Medium (modified) (1.times.)
(Invitrogen, Carlsbad, Calif., USA) and Eagle's Minimum Essential
Medium (EMEM) (ATCC, Manassas, Va.) respectively. Both the media
solutions were supplemented with 10% heat deactivated fetal bovine
serum (FBS) (Fisher Scientific, Springfield, N.J.) and 1%
penicillin-streptomycin (Sigma Aldrich, St Louis, Mo.). The cells
were maintained at 37.degree. C. in a humidified atmosphere with 5%
CO2.
Sonication of Substrate-Attached Cells:
[0062] MATB-III and U-87 cells were grown in 10 mL capacity
Opticell units (Nalge Nunc International, Rochester, N.Y.) to 90%
confluence. The media in OptiCell was replaced with 10 mL fresh
media containing 40 pmoles of either cy3-siRNA or
cholesterol-siRNA. The opticell was left in the incubator for 24
hours at 37.degree. C. Separately, the cells were either treated
with siRNA solution mixed with Optison microbubbles (300 .mu.L) or
a lipid transfection reagent (90 .mu.L) (RNAifect, Qiagen,
Valencia, Calif.). For siRNA/Optison mixtures, Vivid i imagers with
a cardiac sector probe (3S) was used to rupture the microbubbles
and deliver the siRNA drug from microbubbles. The opticell was
immobilized in a water bath, and the ultrasound probe was attached
to a motion arm that spanned the entire length of the opticell. The
tip of the probe was immersed in water, and the distance between
the probe and opticell surface was 3 cm that allowed sonication of
the entire width of the opticell. The microbubbles in opticell were
treated with a mechanical index, MI>1.3 continuous sonication.
The probe moved at a speed of 1 m/s over the entire length of the
opticell. After sonication, the cells were incubated for 24 hours
at 37.degree. C. Similarly, the cells treated with RNAifect were
also kept in the incubator for 24 hours. After incubation, the
cells were imaged using a fluorescence microscope (Zeiss Axio
Imager.Z1, Carl Zeiss). The filter used for cy3 was DsRed/Cy3 (546
ex/620 em). In the region of interest (ROI) in the fluorescent
images, cell fluorescence was measured and the mean values of cell
fluorescence were calculated. ImageJ was used to process the images
and calculate fluorescence intensities.
[0063] The data are reported as mean+1.0 standard error (SE) for
N=4. The statistical significance of the differences between the
groups was evaluated using two-sample t-test and the statistical
analyses were carried out using Minitab.RTM. 12 (Minitab Inc, State
College, Pa. USA).
Results:
[0064] The effect of the delivery system is illustrated in FIG. 6
for U-87 cells incubated with either cy3-siRNA or chol-siRNA. The
delivery of siRNA into the tumor cells is represented by average
cell cy3-fluorescence. For each group, mean fluorescence values and
standard errors are reported in. Cell sonication substantially
enhanced cy3-siRNA penetration into the cell. Due to the effects of
sonoporation, average cell fluorescence for Optison/ultrasound
treated cells was 39% more than untreated cells. For
cholesterol-siRNA, there was a 53% increase in average cell
fluorescence after treatment with Optison/ultrasound. Significant
differences between the groups were evaluated using two sample
t-tests (p=0.032 for cy3-siRNA and p=0.059 for
cholesterol-siRNA).
[0065] Similarly for MATBIII cells, the effect of
Optison/ultrasound treatment was compared to a commercially
available lipid transfection reagent. The cells were treated with
either Cy3-siRNA or chol-siRNA in combination with either RNAifect
or Optison/ultrasound delivery agents and the results are shown in
FIGS. 7 and 8. FIG. 7 shows representative images of the cells
after treatment. FIG. 8 reports the mean cell fluorescence for all
groups with standard errors represented as error bars. For
cy3-siRNA, the average cell fluorescence was higher for
Optison/ultrasound treatment (two sample t-test, p=0.007). This is
primarily due to sonoporation of the cells in the presence of
microbubbles. There was no significant difference between RNAifect
and Optison/ultrasound delivery of chol-siRNA into cells. Although
the average cell fluorescence was similar, the lipid transfection
reagent was found to be toxic to the tumor cells as evidenced by
the irregular shape of the cells in FIG. 7. It should be noted that
the same amount of transfection reagent and siRNA was used in both
unmodified and cholesterol-siRNA. While the transfection reagent
was toxic in both the cases, it was higher for chol-siRNA. Example
3
[0066] Preliminary binding studies of therapeutic-fatty acid
conjugates to microbubbles were evaluated using fluorescein-fatty
acid conjugates.
Conjugation Method;
[0067] Fatty acid NHS ester (2 equivalents, 5.37 mg Myristic acid
NHS ester or 6.38 mg Stearic acid NHS ester was taken in a 50:50
mixture of DMSO and dichloromethane (100 ul) and mixed with a
solution of Fluorescein cadaverine (FL-Cadaverine, 5 mg, 1
equivalent, in 50 ul DMSO). To this diisopropylethyl amine (3.8
equivalents) was added and mixture was vortexed to give a clear
solution. Samples were kept in the dark at room temperature. After
4.5 h, reaction was checked by HPLC and was found to be complete. A
large shift in retention time was observed for both conjugates
(Retention times FL-Cadaverine 4.7 min, FL-Cadaverine stearate 12.1
minute and FL-Cadaverine Myristate 9.9 min, column X-Bridge Shield
RP 18, 4.6.times.50 mm column, particle size 5 um, gradient method
0-100% B in 15 min and 100% B for 5 min, solvent A 0.1M TEAA, pH
7.0 and solvent B 100% acetonitrile, flow rate 1 ml/min) as
expected. Crude product was diluted with DMSO to .about.2 ml and
purified on AKTA purifier using Xterra MS C18, 19.times.100 mm
column and a gradient of 0-100% B in 18.75 column volumes at a flow
rate of 10 ml/min. Solvent A and B were as described above for the
analytical method. Product was collected in multiple fractions and
each fraction was analyzed by analytical HPLC. Only the purest
fraction in each case (.about.90% purity) was used for binding
studies (starting material itself was .about.86% pure, remaining
likely a regioisomer with same spectral properties). This fraction
was concentrated to dryness at room temperature. Residue was
suspended in water (.about.2 ml) and extracted with dichloromethane
(3.times.2 ml). Organic extracts were combined, dried over
anhydrous sodium sulfate and concentrated to dryness.
Fluorescence Polarization Assay
[0068] The stock solution of fluorescein-myristate was prepared in
1.times.PBS. The concentration of fluorescein was kept low for the
fluorescence polarization assay, at 126 nM. Varying concentrations
of either Optison or HSA solutions, ranging from 0 to 15 .mu.M
albumin concentrations, were added to the fluorescein myristate
solution. The reaction buffer was 1.times.PBS, pH 7.4. The reaction
mixture was incubated under dark at 25.degree. C. for 15 minutes.
After incubation, the changes in raw anisotropy values of
fluorescein were measured using a microplate reader (SpectraMax 5,
Molecular Devices, Sunnyvale, Calif.).
[0069] The samples were measured in Corning 96-well plates (black
plate with a clear bottom) (Sigma Aldrich, St Louis, Mo.).
Fluorescein was excited at 470 nm, and emission was measured at 540
nm. Fraction bound (x) was calculated using the same equation as
before (Equation 2), but replacing fluorescence values with
anisotropy values. The fraction bound calculated was then plotted
against albumin concentration as shown in FIG. 9. The data are
reported as mean+1.0 standard error (SE) for N=3. Equation 3 was
used to determine kd and n for fluorescein-myristate binding to
both Optison and native HSA (Table 2). This is represented also in
FIG. 10 which shows the fluorescein bound to Optison (0, 8, 40 and
200 pmoles for i, ii, iii and iv respectively) is visualized on the
gel as dark bands for fluorescein-myristate (FIG. 10A) (63 pmoles)
and fluorescein-stearate (FIG. 10B) (180 pmoles).
[0070] When the fluorescein without the myristate conjugation was
tested for its binding properties to albumin, no significant
changes in anisotropy was observed. It is well known that the fatty
acids have stronger binding properties than cholesterol, and is
also confirmed here with the lower dissociation constants, kd,
observed for fluorescein-myristate conjugate (Table 1 and Table
2).
TABLE-US-00002 TABLE 1 Number of binding sites and dissociation
constants for binding of cholesterol-siRNA to Optison and native
HSA Optison Native HSA Number of binding sites, n 1.16 1.13
Dissociation constant, k.sub.d (.mu.M) 7.16 6.4
TABLE-US-00003 TABLE 2 Number of binding sites and dissociation
constants for binding of fluorescein-myristate to Optison and
native HSA Optison Native HSA Number of binding sites, n 1.03 1.02
Dissociation constant, k.sub.d (.mu.M) 0.238 0.378
[0071] Therefore, conjugating a fatty acid such as myristate to a
therapeutic compound can increase the binding of such therapeutic
compounds to the albumin shell microbubbles. The dissociation
constant of fluorescein-myristate binding to Optison was lower than
that of binding to native HSA. This suggests better hydrophobic
binding properties of microbubble shell that has both native and
partially denatured albumin.
Example 4
Stability of siRNA In Vivo
[0072] The stability of therapeutic compounds such as siRNA is very
low once injected into the body. A comparison between subcutaneous
and tail-vein injection of a mixture of albumin microbubbles and
native siRNA (without conjugates) was studied.
[0073] Eleven to fourteen weeks (body weight .about.30 g) old nu/nu
mice were obtained from Charles River Laboratories (Wilmington,
Mass.). Animals were housed in accordance with the Guide for the
Care and Use of Laboratory Animals as adopted by the National
Institutes of Health. Lewis lung carcinoma cells (LLC) were
inoculated subcutaneously to the right flank of anaesthetized mice
(3.5.times.106 cells/100 .mu.l/mouse).
[0074] On the fourth day after inoculation, the mice were treated
with anti-VEGF siRNA (Sigma Life Sciences, St. Louis,
Mo.)--microbubble mixtures, a siRNA dose of 1.0 mg/kg for
subcutaneous injections and a dose of 2.0 mg/kg for tail-vein
injections. The injection mixture contained 100 .mu.L of
microbubble solution and 100 .mu.L of siRNA in RNAse free water.
After injection, the tumors were sonicated using Vivid i imagers
with a cardiac sector probe (3S). The energies were delivered in a
pulsatile form with the peak MI at 1.3. Control group did not
receive any treatments.
[0075] After 24 hours from the treatment day, the mice were
euthanized and the tumors were extracted. The tumors were frozen
immediately and stored at -20.degree. C. The tumors were thawed out
at room temperature on the day of VEGF measurement. The tumors were
then lysed in RIPA buffer (protease inhibitors added) using a
lysing matrix tube (Lysing matrix tube A, RP Biomedical). The
lysate collected from the samples were then diluted, and measured
for total protein using a protein kit (Pierce BCA reagent protein
assay kit) and for VEGF using an ELISA kit (Mouse VEGF ELISA kit,
RayBiotech, Norcross, Ga.).
[0076] The results are reported in Table 3 as mean pg VEGF/mg
protein for control and different treatment groups. The
subcutaneous injection of 1.0 mg/kg siRNA-microbubble mixture
resulted in an approximately 39% decrease in VEGF when compared to
the control group (two-sample t-test; p=0.0096). While there was
only a minor difference between the control group and 2.0 mg/kg
tail-vein injection of siRNA-microbubble mixtures, this may be due
to lack or less efficient binding of unmodified siRNA to the
microbubble.
TABLE-US-00004 TABLE 3 The effect of siRNA delivery to tumors; mean
pg VEGF/mg total protein. Mean pg VEGF/ Std Conditions mg protein
error 95% CI n control 296.19 24.89 48.79 5 Optison/siRNA SQ 1
mg/kg 181.97 24.38 47.78 7 Optison/siRNA TV 2 mg/kg 255.1 51.06
100.07 3
[0077] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects as illustrative rather than limiting on the
invention described herein. The scope of the invention is thus
indicated by the appended claims rather than by the foregoing
description, and all changes that come within the meaning and range
of equivalency of the claims are therefore intended to be embraced
therein.
Sequence CWU 1
1
2121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gcauuuguuu guccaagauu u
21221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2aaaucuugga caaacaaaug c 21
* * * * *