U.S. patent application number 17/221867 was filed with the patent office on 2021-08-12 for short interfering rna templated lipoprotein particles (sirna-tlp).
This patent application is currently assigned to Northwestern University. The applicant listed for this patent is Northwestern University. Invention is credited to Kaylin M. McMahon, C. Shad Thaxton.
Application Number | 20210244826 17/221867 |
Document ID | / |
Family ID | 1000005540920 |
Filed Date | 2021-08-12 |
United States Patent
Application |
20210244826 |
Kind Code |
A1 |
Thaxton; C. Shad ; et
al. |
August 12, 2021 |
SHORT INTERFERING RNA TEMPLATED LIPOPROTEIN PARTICLES
(SIRNA-TLP)
Abstract
Nanostructures for the systemic delivery of nucleic acids, such
as RNA, are provided herein. The nanostructures include templated
lipoprotein nanoparticles (TLPs) composed of a core decorated with
proteins, a lipid bilayer and hydrophobic molecules that
self-assemble with nucleic acids, such as RNA. The nanostructures
are useful for research, therapeutic and diagnostic
applications.
Inventors: |
Thaxton; C. Shad; (Chicago,
IL) ; McMahon; Kaylin M.; (Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northwestern University |
Evanston |
IL |
US |
|
|
Assignee: |
Northwestern University
Evanston
IL
|
Family ID: |
1000005540920 |
Appl. No.: |
17/221867 |
Filed: |
April 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15499279 |
Apr 27, 2017 |
10967072 |
|
|
17221867 |
|
|
|
|
62328175 |
Apr 27, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/183 20130101;
A61K 47/6929 20170801; A61K 9/1273 20130101; A61K 9/1277
20130101 |
International
Class: |
A61K 47/69 20060101
A61K047/69; A61K 9/127 20060101 A61K009/127; A61K 47/18 20060101
A61K047/18 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No. R01CA167041 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. An anionic nanostructure aggregate, comprising: an aggregate of
cationic lipid-nucleic acid complexes and templated lipoprotein
particles (TLP), wherein the TLP comprises a core, and a lipid
shell surrounding the core; and the cationic lipid-nucleic acid
complex, comprised of nucleic acids, wherein each nucleic acid is
complexed with a cationic lipid, and wherein the aggregate of
cationic lipid-nucleic acid complexes and TLPs has a negative
.zeta.-potential and forms the anionic nanostructure aggregate.
2. The nanostructure of claim 1, wherein the core is a metal.
3. The nanostructure of claim 2, wherein the core is gold.
4. The nanostructure of claim 1, wherein the lipid shell comprises
phospholipids, and wherein the phospholipids are
1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC) and
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)pr-
opionate] (PDP-PE).
5. The nanostructure of claim 1, further comprising an
apolipoprotein functionalized to the core.
6. The nanostructure of claim 5, wherein the apolipoprotein is
apolipoprotein A-1 (apo A-1).
7. The nanostructure of claim 1, further comprising
cholesterol.
8. The nanostructure of claim 1, wherein the nucleic acids are
single-stranded.
9. The nanostructure of claim 1, wherein the nucleic acids are
RNAs.
10. The nanostructure of claim 9, wherein the RNAs are antisense
and sense RNAs of an siRNA duplex.
11. The nanostructure of claim 1, wherein the sense RNA and
antisense RNA are present in nearly equimolar amounts.
12. The nanostructure of claim 1, wherein the sense RNA and
antisense RNA are present in about a 1:2 ratio.
13. The nanostructure of claim 1, wherein the sense RNA and
antisense RNA are present in about a 1:1 ratio.
14. The nanostructure of claim 1, wherein the sense RNA and
antisense RNA are present in about a 2:1 ratio.
15. The nanostructure of claim 1, wherein the nucleic acid in the
nanostructure is more stable than free nucleic acid.
16. The nanostructure of claim 1, wherein the nanostructure
comprises alternating layers of
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and nucleic
acid.
17. The nanostructure of claim 1, wherein the nanostructure is
solid.
18. The nanostructure of claim 1, wherein the nucleic acid is not
chemically modified.
19. The nanostructure of claim 1, wherein the nucleic acid is mixed
with TLP in a molar ratio of 5:1, 15:1 or 25:1.
20. The nanostructure of claim 19, wherein the nucleic acid is
mixed with TLP in a molar ratio of 25:1.
21. The nanostructure of claim 1, wherein the cationic lipid is
DOTAP.
22. The nanostructure of claim 21, wherein the DOTAP is mixed with
nucleic acid in a molar ratio of 10:1, 20:1, 30:1 or 40:1.
23. The nanostructure of claim 1, wherein the TLP is a synthetic
HDL.
24. A method for delivering siRNA to a cell in a subject, the
method comprising: contacting the subject with an anionic
nanostructure aggregate, comprising an aggregate of cationic
lipid-nucleic acid complexes and templated lipoprotein particles
(TLP), wherein the cationic lipid-nucleic acid complex is comprised
of antisense and sense RNA of an siRNA duplex, each complexed with
a cationic lipid, and wherein the aggregate of cationic
lipid-nucleic acid complexes and TLPs has a negative
.zeta.-potential and forms the anionic nanostructure aggregate, to
deliver siRNA to the cell.
25. The method of claim 24, wherein the antisense RNA and/or the
sense RNA of the siRNA duplex is single-stranded.
26. The method of claim 24, wherein the antisense RNA and the sense
RNA of the siRNA duplex are single-stranded.
27. The method of claim 24, wherein the cell is a cancer cell.
28. The method of claim 27, wherein the cancer cell is a prostate
cancer cell, a breast cancer cell, a renal cancer cell or an
ovarian cancer cell.
29. The method of claim 24, wherein the cell is a LNCaP cell, an
enzalutamide resistant LNCaP cell, an MDA-MB-231 cell, a 786-O
cell, or a OvCar3 cell.
30. The method of claim 24, wherein the cell is contacted with the
nanostructure at a concentration of 1nM, 5 nM, 10 nM, or 20 nM.
31. The method of claim 24, wherein the cell is in contact with the
nanostructure for 24, 48, 72, and 96 hours.
32. The method of claim 24, wherein the cell expresses the androgen
receptor (AR) or the enhancer of zeste homolog 2 (EZH2)
proteins.
33. The method of claim 24, wherein the subject is a mammal.
34. The method of claim 33, wherein the subject is a human.
35. A method for treating a cancer, the method comprising:
systemically administering to a subject having a cancer an anionic
nanostructure aggregate, comprising an aggregate of cationic
lipid-nucleic acid complexes and templated lipoprotein particles
(TLP), wherein the cationic lipid-nucleic acid complex is comprised
of antisense and sense RNA of an siRNA duplex, each complexed with
a cationic lipid, and wherein the aggregate of cationic
lipid-nucleic acid complexes and TLPs has a negative
.zeta.-potential and forms the anionic nanostructure aggregate,
wherein the siRNA is an anti-cancer siRNA.
36. The method of claim 35, wherein the antisense RNA and/or the
sense RNA of the siRNA duplex is single-stranded.
37. The method of claim 35, wherein the antisense RNA and the sense
RNA of the siRNA duplex are single-stranded.
38. The method of claim 35, wherein the nanostructure is
administered in vivo.
39. The method of claim 35, wherein the subject is a mammal.
40. The method of claim 39, wherein the subject is a human.
41. The method of claim 35, wherein the nanostructure is
administered at a dose of about 0.7 mg siRNA/kg.
42. The method of claim 35, wherein the nanostructure is not toxic
to a surrounding non-cancerous cell or non-cancerous tissue.
43. A method of regulating gene expression in a cell with an
anionic nanostructure aggregate, comprising an aggregate of
cationic lipid-nucleic acid complexes and templated lipoprotein
particles (TLP), wherein the cationic lipid-nucleic acid complex is
comprised of antisense and sense RNA of an siRNA duplex, each
complexed with a cationic lipid, and wherein the aggregate of
cationic lipid-nucleic acid complexes and TLPs has a negative
.zeta.-potential and forms the anionic nanostructure aggregate.
44. The method of claim 43, wherein the antisense RNA and/or the
sense RNA of the siRNA duplex is single-stranded.
45. The method of claim 43, wherein the antisense RNA and the sense
RNA of the siRNA duplex are single-stranded.
46. The method of claim 43, wherein the nanostructure decreases the
expression of a gene.
47. The method of claim 46, wherein the nanostructure decreases the
expression of the gene that encodes for the AR protein.
48. The method of claim 46, wherein the nanostructure decreases the
expression of the gene that encodes for the EZH2 protein.
49. A method for synthesizing a templated lipoprotein particle
(TLP) comprising contacting gold nanoparticles with an
apolipoprotein to produce an apolipoprotein coated gold particle,
contacting the apolipoprotein coated gold particle with two
phospholipids and cholesterol to produce an anionic TLP.
50. The method of claim 49, further comprising mixing the anionic
TLP with a DOTAP RNA mixture.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/499,279, filed Apr. 27, 2017, which claims the benefit under
35 U.S.C. .sctn. 119(e) of U.S. provisional application Ser. No.
62/328,175, filed Apr. 27, 2016, the entire contents of each of
which are incorporated by reference herein.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 24, 2021, is named N049170017US02-SEQ-NTJ and is 2
kilobytes in size.
FIELD OF INVENTION
[0004] The present invention generally relates to products and
methods of using templated lipoprotein particles (TLPs) that
self-assemble with short interfering RNA (siRNA) for therapy.
BACKGROUND
[0005] Systemic administration of therapeutic short interfering RNA
(siRNA) is tremendously appealing due to the ability of siRNA to
potently silence any protein target and the opportunities that
exist for personalized medicine (Wu et al., Sci Transl Med, 2014
6(240):240ps7). Some progress has been made with recent clinical
trials demonstrating systemic delivery of therapeutic siRNA;
however, drugs mostly target protein expression in the liver and
none are approved by the U.S. Food and Drug Administration (FDA)
(Wu et al., Sci Transl Med, 2014; 6(240):240ps7; and Whitehead et
al., Nat Rev Drug Discov. 2009; 8(2):129-38). More specific to
oncology applications, only four systemically administered agents
have reached Phase I clinical trials, one was actively targeted,
one moved on to Phase II, and none have gained FDA approval
(Zuckerman et al., Nat Rev Drug Discov. 2015; 14(12):843-56).
Ultimately, targeted systemic delivery of therapeutic siRNA remains
difficult due to a number of well-known factors, such as: (1)
inherent siRNA instability, (2) inefficient active delivery to
target cells, (3) exclusion of siRNA from the target cell cytosol
where mRNA targets reside, (4) RNA target selection, (5) toxicity,
and (6) complexity, scalability, and cost (Choi et al., Mol Cell
Toxicol. 2014; 10:1-8; Nguyen et al., Curr Opin Mol Ther. 2008;
10:158-167; Singh et al., Curr Opin Biotechnol. 2016; 39:28-34; and
Cheng et al., Science. 2012; 338:903-910. In short, realizing the
full potential of systemically administered therapeutic siRNA,
especially for advanced solid tumors, requires the development of
new technology.
[0006] Individual and combinations of strategies have been employed
to enhance the efficacy of therapeutic siRNA. Notably, chemical
modifications to the siRNA phosphate backbone and/or ribose sugar
have improved RNA stability and can limit off-target effects..sup.8
However, chemical modifications can significantly increase cost,
may generate off-target effects, and can reduce siRNA efficacy
(McMahon et al., Expert Opin Drug Deliv. 2014; 11(2):231-47). In
addition, many siRNA delivery vehicles have been developed. Almost
exclusively, delivery vehicles are formed by self-assembling
cationic lipids (lipoplexes) or polymers (polyplexes) that
encapsulate siRNAs (Zhang et al., J Control Release. 2007;
123(1):1-10). Some of these cationic vehicles have been shown to
increase siRNA stability, improve circulating half-life, and
enhance cell uptake. However, toxicity, serum opsonization, and
lack of active targeting remain significant drawbacks to cationic
siRNA delivery vehicles (Lv, H. et al., J Control Release. 2006;
114(1):100-9; and Yang et al., Gene Ther. 1997; 4:950-960).
Ultimately, identifying simple strategies for formulating largely
unmodified siRNAs and alternatives to passively targeted cationic
delivery vehicles are important for developing the next generation
of therapeutic siRNAs.
SUMMARY OF THE INVENTION
[0007] According to one aspect, the invention is a nanostructure
which includes a templated lipoprotein particle (TLP) that includes
a core, a lipid shell surrounding the core, an apolipoprotein; and
single stranded antisense and sense RNA of an siRNA duplex
associated with the lipid.
[0008] In some embodiments, the core of the nanostructure is a
metal. Optionally, in another embodiment, the core of the
nanostructure is gold.
[0009] In some embodiments, the lipids in the lipid shell are
phospholipids. Optionally, in another embodiment, the phospholipids
are 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC) and
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)pr-
opionate] (PDP-PE).
[0010] In some embodiments, the apolipoprotein of the nanostructure
is apolipoprotein A-1 (apo A-1). Optionally, in some embodiments,
the nanostructure contains cholesterol.
[0011] In some embodiments, the RNA in the nanostructure is more
stable than free RNA.
[0012] In some embodiments, the nanostructure contains alternating
layers of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and RNA.
In another embodiment, optionally, the RNA is not chemically
modified. In another embodiment, the sense and antisense RNA are
present in equimolar amounts.
[0013] In some embodiments, the RNA is mixed with TLP in a molar
ratio of 5:1, 15:1 or 25:1. In another embodiment, optionally, the
RNA is mixed with TLP in a molar ratio of 25:1.
[0014] In some embodiments, the RNA is complexed to the cationic
lipid DOTAP. In another embodiment, optionally, the DOTAP is mixed
with RNA in a molar ratio of 10:1, 20:1, 30:1 or 40:1. In yet
another embodiment, optionally, DOTAP is mixed with RNA in a molar
ratio of 30:1 or 40:1. In an embodiment, optionally, DOTAP is mixed
with RNA in a molar ratio of 40:1. In yet another embodiment, DOTAP
is mixed with RNA in a charge ratio of about 1:4, 2:1, 3:4 or
1:1.
[0015] In some embodiments, DOTAP is mixed with RNA in a molar
ratio of 40:1 and the RNA is mixed with TLP in a molar ratio of
25:1.
[0016] According to another aspect, the invention is a synthetic
stable nanostructure that includes an anionic templated lipoprotein
particle (TLP) comprising a core, a lipid bilayer shell surrounding
the core, a cholesterol associated with the lipid bilayer and an
apolipoprotein, wherein the TLP has an anionic charge of about -35
to -50 mV.
[0017] In some embodiments, the core of the synthetic stable
nanostructure is a metal. In another embodiment, optionally, the
core of the nanostructure is gold.
[0018] In some embodiments, the lipids in the lipid bilayer shell
are phospholipids. In another embodiment, optionally, the
phospholipids are DOPC and PDP-PE.
[0019] According to another aspect, the invention is a method for
delivering siRNA to a cell, wherein in the method includes
contacting a cell with a nanostructure described herein to deliver
siRNA to the cell.
[0020] In some embodiments, the cell is in a subject. In yet
another embodiment, the cell is a cancer cell. In another
embodiment, optionally, the cancer cell is a prostate cancer cell,
a breast cancer cell, a renal cancer cell or an ovarian cancer
cell.
[0021] In some embodiments, the cell is a LNCaP cell, an
enzalutamide resistant LNCaP cell, a MDA-MB-231 cell, a 786-O cell,
or a OvCar3 cell.
[0022] In some embodiments, the cell is contacted with the
nanostructure at a concentration of 5 nM, 10 nM, or 20 nM.
[0023] In some embodiments, the cell is in contact with the
nanostructure for 24, 48, 72, and 96 hours.
[0024] In some embodiments, the cancer cell expresses the androgen
receptor (AR) or the enhancer of zeste homolog 2 (EZH2)
proteins.
[0025] In some embodiments, the subject is a mammal. In some
embodiments, the subject is human.
[0026] According to another aspect, the invention is a method for
treating a cancer, wherein the method comprises systemically
administering to a subject having a cancer a nanostructure
described herein to deliver siRNA to the subject and treat the
cancer, wherein the siRNA is an anti-cancer siRNA.
[0027] In some embodiments, the nanostructure is administered in
vivo.
[0028] In yet another embodiment, the subject is a mammal. In
another embodiment, optionally, the subject is a human.
[0029] In some embodiments, the nanostructure is administered at a
concentration of about 0.7 mg siRNA/kg.
[0030] In some embodiments, the nanostructure is not toxic to a
surrounding non-cancerous cell or non-cancerous tissue.
[0031] According to another aspect, the invention is a method of
regulating gene expression in a cell with a nanostructure described
herein.
[0032] In some embodiments, the nanostructure decreases the
expression of a gene. In another embodiment, optionally, the
nanostructure decreases the expression of the gene that encodes for
the AR protein. In yet another embodiment, the nanostructure
decreases the expression of the gene that encodes for the EZH2
protein.
[0033] According to another aspect, the invention is a method of
treating an autoimmune disorder, the method comprising
administering to a subject a nanostructure described herein.
[0034] In some embodiments, the nanostructure has a diameter of
about 110 nm.
[0035] According to another aspect, the invention is a
nanostructure described herein that further delivers a drug.
[0036] According to another aspect, the invention is a
nanostructure described herein that further delivers an
adjuvant.
[0037] According to another aspect, the invention is a
nanostructure described herein that further delivers a vaccine
adjuvant.
[0038] According to another aspect, the invention is a
nanostructure described herein that includes an antigen that
enhances antigen presentation in a cell.
[0039] According to another aspect, the invention is a method of
synthesizing an siRNA delivery vehicle that includes contacting a
TLP that includes a core, a lipid shell surrounding the core, and
an apolipoprotein with single stranded antisense and sense RNA of
an siRNA duplex formulated in a cationic acid.
[0040] In some embodiments, the cancer comprises a cancer cell,
wherein the cancer cell overexpresses a scavenger receptor class B
type I (SR-BI), relative to a non-cancer cell or relative to a
cancer cell that does not overexpress SR-BI.
[0041] According to another aspect, the invention is a method for
synthesizing a TLP, wherein the method includes contacting gold
nanoparticles with an apolipoprotein to produce an apolipoprotein
coated gold particle, contacting the apolipoprotein coated gold
particle with two phospholipids to produce an anionic TLP.
[0042] In some embodiments, optionally, the method further includes
mixing the anionic TLP with a DOTAP RNA mixture.
[0043] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways. The
details of one or more embodiments of the invention are set forth
in the accompanying Detailed Description, Examples, Claims, and
Figures. Other features, objects, and advantages of the invention
will be apparent from the description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0045] FIGS. 1A-1F show a scheme for siRNA-TLP synthesis,
optimization, and function. (FIG. 1A) In Step 1, TLPs are
synthesized. In Step 2, TLPs are mixed with single-strand RNA
(ssRNA), complement strands of a siRNA duplex, complexed with
DOTAP. (FIG. 1B) UV-Vis spectroscopy measurement of nanoparticle
(.lamda..sub.max .about.520 nm) and RNA (.lamda..sub.max .about.260
nm) after centrifugation for purification from unreacted
components. (FIG. 1C) .zeta.-potential measurement of TLPs,
DOTAP-TLPs and siRNA-TLP particles synthesized with increasing
DOTAP:RNA molar ratios from 10:1-40:1. (FIG. 1D) Hydrodynamic
diameter (nm) measurement of 5 nm Au NP, TLP, and siRNA-TLP
particles synthesized with increasing DOTAP:RNA molar ratios from
10:1-40:1. (FIG. 1E) UV-Vis spectroscopy measuring purified
siRNA-TLPs synthesized with increasing DOTAP:RNA molar ratios.
(FIG. 1F) Western blot of siRNA-TLP function regulating target AR
expression in LNCaP cells (48 hrs, 20 nM siRNA-TLP). All UV-Vis
spectra were normalized according to .lamda..sub.max .about.520 nm.
AR=siRNA targeting the androgen receptor. Ctrl=scrambled control
siRNA sequence. Lipo=LIPOFECTAMINE.RTM. RNAiMAX.
[0046] FIGS. 2A-2D show siRNA-TLP characterization. (FIG. 2A) Table
depicting the molar ratio of each particle component in TLP and
siRNA-TLP following purification and quantification. (FIG. 2B)
Transmission electron microscopy of TLP and siRNA-TLP. Scale bar=50
nm. (FIG. 2C) Western blot of siRNA-TLPs function to regulate AR
expression using particles synthesized with either the sense (S),
antisense (AS), or both strands of the siRNA duplex pair (DS) in
LNCaP cells (48 hrs). (FIG. 2D) Western blot of siRNA-TLPs function
to regulate AR expression when formulated with either both RNA
complements (DS) on a single siRNA-TLP, or a mixed population of AS
and S siRNA-TLPs combined as a single treatment in LNCaP cells (48
hrs, 20 nM siRNA-TLP). AR=siRNA targeting the androgen receptor.
Ctrl=scrambled control siRNA sequence. Lipo=LIPOFECTAMINE.RTM.
RNAiMAX.
[0047] FIGS. 3A-3C show in vitro function of siRNA-TLP and cell
viability. (FIG. 3A) Western blot time course of siRNA-TLP function
regulating AR expression in LNCaP cells. (FIG. 3B) Cell viability
of LNCaP cells after treatment with siRNA-TLPs measured by MTS
assay (1, 5, 10, 20 nM siRNA-TLP). (FIG. 3C) LNCaP cell confluence
measured over time after treatment with siRNA-TLPs (20 nM), and
images taken 165 hours after siRNA-TLP treatment depicting cell
confluence. AR=siRNA targeting the androgen receptor.
Ctrl=scrambled control siRNA sequence. Lipo=LIPOFECTAMINE.RTM.
RNAiMAX.
[0048] FIGS. 4A-C show cell uptake of siRNA-TLP and functional
dependence on scavenger receptor type B-1 (SR-B1). (FIG. 4A)
Cellular uptake of siRNA-TLP by LNCaP cells measured by fluorescent
signal in cells over time using an Incucyte ZOOM (20 nM siRNA-TLP).
(FIG. 4B) Representative fluorescent images of siRNA-TLP uptake (72
hrs). (FIG. 4C) Western blot of LNCaP cells pre-treated with SR-B1
and Ctrl siRNA 48 hrs prior to siRNA-TLPs addition to determine if
function is dependent on cellular SR-B1 expression. The hours in
parenthesis indicate the total treatment time of siRNA sequences
added using LIPOFECTAMINE.RTM. RNAiMAX. LNCaP cells were treated
with siRNA-TLPs for 48 hrs. Quantification of western blot data.
AR=siRNA targeting the androgen receptor. Ctrl=scrambled control
siRNA sequence. Lipo=LIPOFECTAMINE.RTM. RNAiMAX.
[0049] FIGS. 5A-5C show the stability of RNA on siRNA-TLP and
siRNA-TLP function after incubation in human serum. (FIG. 5A) RNA
stability of siRNA-TLPs compared to free RNA sequences upon
exposure to RNase A. (FIG. 5B) RNA stability of siRNA-TLPs compared
to free RNA sequences after exposure to human plasma. (FIG. 5C)
Western blot of AR protein expression after treatment of LNCaP
cells with TLPs and siRNA-TLPs that have been incubated in human
serum (48 hrs, 20 nM siRNA-TLP), and serum supernatant (SN)
fraction containing only albumin and HDL separated from siRNA-TLPs.
The particle type added to human serum is indicated in parenthesis.
AR=siRNA targeting the androgen receptor. Ctrl=scrambled control
siRNA sequence. Lipo=LIPOFECTAMINE.RTM. RNAiMAX.
[0050] FIGS. 6A-6F show in vivo function of siRNA-TLP. (FIG. 6A)
Tumor volume measurements of LNCaP xenografts in athymic nude mice
over course of in vivo study. (FIG. 6B) The percent change in LNCaP
tumor volume over course of in vivo study. (FIG. 6C) Inductively
coupled plasmon mass spectrometry to measure Au NPs in tissues
after treatment with siRNA-TLP or water. (FIG. 6D) LNCaP tumor
uptake of sense (Cy3) and antisense (Cy5) labeled siRNA sequences
assembled with siRNA-TLPs following a single systemic
administration. Labeled RNA was visualized using confocal
fluorescence microscopy 24 hours after injection. Tumor tissues
were counterstained with DAPI. (FIG. 6E) Hematocrit (HCT),
hemoglobin (HGB), and platelet (PLT) count in whole blood collected
from study mice following treatment. (FIG. 6F) White blood cell
(WBC) and neutrophil count from study mice following treatment.
AR=siRNA targeting the androgen receptor. Ctrl=scrambled control
siRNA sequence.
[0051] FIGS. 7A-7F show RNA melting analysis, .zeta.-potential
measurement, and function of siRNA-TLP. (FIG. 7A) RNA melting
transition in water, PBS, and 9:1 water:ethanol (v/v) for AR+DOTAP
sample (40:1 DOTAP:RNA). (FIG. 7B) .zeta.-potential of DOTAP
liposomes, free RNA, and DOTAP-RNA mixtures of DOTAP:RNA ratios,
whereby 40, 30, 20, 10 represent molecules of DOTAP per RNA
phosphate (i.e. charge ratios of .about.1:1, 3:4, 1:2, 1:4). (FIG.
7C) Hydrodynamic diameter of particles formed with DOTAP-RNA
mixtures (FIG. 7D) Western blot of DOTAP-RNA particles targeting AR
in LNCaP cells (48 hrs). Total RNA concentration of DOTAP-RNA
mixtures to treat cells was equivalent to a 20 nM siRNA-TLP. (FIG.
7E) UV-Vis spectroscopy of purified siRNA-TLPs synthesized with
increasing RNA:TLP ratios, whereby 25, 15, and 5 represent the fold
molar excess of RNA molecules to TLP. UV-Vis spectra were
normalized according to nanoparticle peak (.lamda..sub.max
.about.520 nm), siRNA loading is shown by .lamda..sub.max
.about.260 nm. (FIG. 7F) Western blot of siRNA-TLP targeting AR in
LNCaP cells according to RNA:TLP ratios specified above (48
hrs).
[0052] FIG. 8 shows a Western blot of apolipoprotein A-I associated
with siRNA-TLP particles. (Lane 1) Purified human apo A-I. (Lanes
2-5) apo A-I associated with siRNA-TLPs.
[0053] FIGS. 9A-9D show in vitro function and modular loading.
(FIG. 9A) qRT-PCR of LNCaP cells after treatment with siRNA-TLPs to
measure AR mRNA expression (48 hrs). (FIG. 9B) Western blot of
siRNA-TLPs targeting AR in LNCaP enzultamide resistant cells (48
hrs). (FIG. 9C) UV-Vis spectroscopy of siRNA-TLP with siRNA
targeting AR (AR-TLP), EZH2 (EZH2-TLP) or Ctrl-TLP reveals equal
amounts of siRNA (.lamda..sub.max .about.260 nm). UV-Vis spectra
were normalized according to nanoparticle peak (.lamda..sub.max
.about.520 nm). (FIG. 9D) Western blot of siRNA-TLPs targeting EZH2
in multiple cancer cell lines, including prostate cancer (LNCaP)
and (LNCaP enzalutamide resistant), triple negative breast cancer
(MDA-MB-231), renal cell carcinoma (786-0), and ovarian cancer
(OvCar3) (48 hrs).
[0054] FIGS. 10A-10C show particle uptake of siRNA-TLP by cultured
LNCaP cells, SR-B1 expression by multiple target cell lines, and
SR-B1 knockdown in LNCaP cells. (FIG. 10A) Cy5-labeled siRNA-TLP
uptake in LNCaP cells represented by mock fluorescent images
obtained using an IncuCyte ZOOM at 6, 62, and 165 hours after
particle incubation. siRNA-TLP attachment to cell (6 hrs),
siRNA-TLP diffusion in cell (62 hrs), siRNA-TLP perinuclear
localization (165 hrs). Note reduced cell confluency secondary to
AR knockdown in AR-TLP samples (bottom). Scale bars=300 .mu.m.
(FIG. 10B) SR-B1 expression in cancer cells treated with
siRNA-TLPs, including prostate (LNCaP), LNCaP cells that are
resistant to enzalutamide (LNCaP MDV3100), triple negative breast
cancer (MDA-MB-231), renal cell carcinoma (786-0), and ovarian
cancer (OvCar3). (FIG. 10C) Western blot of SR-B1 knockdown in
LNCaP cells over time. SR-B1 and Ctrl siRNA were transfected using
LIPOFECTAMINE.RTM. RNAiMAX (Lipo).
[0055] FIG. 11 shows an experimental design for testing siRNA-TLP
function after prolonged incubation in human serum. (Top arrow) To
test siRNA-TLP function after serum incubation, siRNA-TLPs were
incubated in human serum for 1 hour prior to LNCaP cell treatment.
(Bottom arrow) To test RNA exchange to native HDL and/or albumin,
siRNA-TLP were incubated in human serum for 1 hour. After
incubation siRNA-TLPs were isolated from the serum fraction
containing native HDL and albumin. The isolated HDL and albumin was
used to treat LNCaP cells.
[0056] FIGS. 12A-12C show in vivo treatment scheme, mouse weight,
and tissue H&E sections following treatment. (FIG. 12A)
Treatment regimen for mice bearing LNCaP flank tumor xenografts.
(FIG. 12B) Mouse weight over the course of the study. (FIG. 12C)
H&E images of representative organs obtained from mice treated
with water, Ctrl-TLP, and AR-TLP at study conclusion. H&E
images were obtained 10.times. magnification.
[0057] FIGS. 13A-13B show serum chemistry and liver function
analysis after siRNA-TLP treatment regimen. (FIG. 13A) Serum
chemistry, kidney function, and cholesterol analysis. (FIG. 13B)
Liver function analysis. BUN=blood urea nitrogen, TP=total protein,
AST=aspartate aminotransferase, ALT=alanine aminotransferase,
ALK=alkaline phosphatase, ALB=albumin, TBIL=total bilirubin.
DETAILED DESCRIPTION
[0058] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
[0059] Efficient systemic administration of therapeutic short
interfering RNA (siRNA) is challenging. The present disclosure
presents a paradigm changing approach to systemic therapeutic siRNA
delivery by self-assembling single-stranded highly unmodified RNA
complements of an siRNA duplex pair in anionic delivery vehicles
that are inherently actively targeted. The approach was inspired
and designed according to properties of natural RNA delivery
vehicles, namely, high density lipoproteins (HDLs). The
siRNA-templated lipoprotein particles (siRNA-TLPs) presented herein
are a combination of synthetic bio-inspired lipoproteins and
cationic lipid-RNA assemblies. Some aspects of the present
disclosure detail the process of controlled self-assembly and the
exquisite functional tunability of siRNA-TLPs, the modular nature
allowing easy exchange of therapeutic siRNA cargo, active cell
targeting, potent target gene regulation, and in vivo efficacy
after systemic administration.
[0060] High-density lipoproteins (HDL) are natural in vivo RNA
delivery vehicles. The present disclosure uses the features of HDL
to develop templated lipoprotein particles (TLP) that self-assemble
with single-strand complements of, presumably, any highly
unmodified siRNA duplex pair after formulation with a cationic
lipid. Resulting siRNA templated lipoprotein particles (siRNA-TLP)
are anionic and tunable with regard to RNA assembly and function.
Quite surprisingly, siRNA-TLP are able to potently reduce gene
expression in vitro. Another surprising aspect of the disclosure,
as also shown herein, is that the systemic administration of
siRNA-TLPs in vivo significantly reduces the growth of cancer
xenografts and demonstrates no off-target toxicity. The present
disclosure presents a modular approach to siRNA delivery by
self-assembling single-strand complements of siRNA into actively
targeted anionic delivery vehicles that potently regulate target
gene expression in vitro and in vivo.
[0061] The present disclosure provides profound fundamental insight
into methods of synthesizing next generation siRNA delivery
vehicles for translation.
[0062] Nearly all of the technologies presently available for the
systemic delivery of siRNA are based upon cationic lipids or
cationic polymers. Most often, due to the cationic nature of these
vehicles and the synthetic properties, they can be highly toxic and
are not typically targeted to disease specific sites. The present
disclosure overcomes the barriers to systemic RNA therapy because
the nanostructure described herein is formulated such that it is
anionic and inherently targeted through specific receptors located
on the surface of cells.
[0063] Furthermore, many RNA therapies are designed around specific
disease targets. However, the formulation described herein is
highly modular, such that the siRNA-TLP can be tailored to
incorporate presumably any protein target of interest.
Additionally, most current techniques are not easily scaled and
have unknown biological composition(s), which can lead to in vivo
toxicity. The formulation described herein has been demonstrated in
vivo to have no inherent toxicity and it is formulated to mimic
natural RNA delivery vehicles to circumvent vehicle-related
toxicity.
[0064] In some aspects, the nanoparticles of the present disclosure
incorporate highly unmodified single-stranded complements of a
desired siRNA duplex in templated lipoprotein particles that mimic
the structure of natural HDL, which is an anionic delivery vehicle
for unmodified nucleic acids. In certain embodiments of the present
disclosure, the templated lipoprotein particle (TLP) inspired on
HDL are typically composed of a core, surrounded by
apolipoproteins, and a mixture of two phospholipids that form a
lipid bilayer; and a hydrophobic molecule (e.g., cholesterol). The
TLP associates with a cationic lipid complexed with a nucleic acid,
such as a single-strand of a duplex siRNA, to form a nanostructure
described herein.
[0065] The nanostructure of the present disclosure has several
useful applications, including but not limited to, cancer therapy,
autoimmune disease or disorder therapy, drug delivery,
antigen/adjuvant delivery vehicle, vaccine adjuvant, enhanced
antigen presentation, or as co-therapy with current cancer
therapies and immunomodulators.
[0066] The cancer may be a malignant or non-malignant cancer.
Cancers or tumors include, but are not limited to, biliary tract
cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver
cancer; lung cancer (e.g. small cell and non small cell); melanoma;
neuroblastomas; oral cancer; ovarian cancer; pancreas cancer;
prostate cancer; rectal cancer; sarcomas; skin cancer; testicular
cancer; thyroid cancer; and renal cancer, as well as other
carcinomas and sarcomas. In one embodiment, the cancer is hairy
cell leukemia, chronic myelogenous leukemia, cutaneous T-cell
leukemia, multiple myeloma, follicular lymphoma, malignant
melanoma, squamous cell carcinoma, renal cell carcinoma, prostate
carcinoma, bladder cell carcinoma, or colon carcinoma. In another
embodiment, the cancer is prostate cancer, breast cancer, renal
cancer or ovarian cancer.
[0067] In some embodiments, that nanostructures described herein
are useful for treating a cancer that overexpresses scavenger
receptor class B type I (SR-BI). Non-limiting examples of cancers
that overexpress SR-BI include human prostate cancer, breast
cancer, and renal cell carcinoma..sup.21,4244 Additional
non-limiting examples of cancers and cancer cell lines that
overexpress SR-BI are listed in Rajora et al. Front Pharmacol.
(2016) 7:326. As described herein, the term "overexpression" or
"increased expression," refers to an increased level of expression
of a given gene product in a given cell, cell type or cell state,
as compared to a reference cell, for example, a non-cancer cell or
a cancer cell that does not overexpress SR-BI.
[0068] The nanostructures are also useful for treating and
preventing autoimmune disease or disorder. Autoimmune disease or
disorder is a class of diseases in which an subject's own
antibodies react with host tissue or in which immune effector T
cells are autoreactive to endogenous self peptides and cause
destruction of tissue. Thus, an immune response is mounted against
a subject's own antigens, referred to as self antigens. Autoimmune
diseases or disorders include, but are not limited to, rheumatoid
arthritis, Crohn's disease, multiple sclerosis, systemic lupus
erythematosus (SLE), autoimmune encephalomyelitis, myasthenia
gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome,
pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune
hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma
with anti-collagen antibodies, mixed connective tissue disease,
polymyositis, pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus.
[0069] The nanostructure of the present disclosure includes a core.
The core may be a solid or a hollow core, such as a liposomal core.
A solid core is a spherical shaped material that does not have a
hollow center. The term spherical as used herein refers to a
general shape and does not imply or is not limited to a perfect
sphere or round shape. It may include imperfections.
[0070] The core of the nanostructure whether being a solid core or
a hollow core, may have any suitable shape and/or size. For
instance, the core may be substantially spherical, non-spherical,
oval, rod-shaped, pyramidal, cube-like, disk-shaped, wire-like, or
irregularly shaped. The core (e.g., a nanostructure core or a
hollow core) may have a largest cross-sectional dimension (or,
sometimes, a smallest cross-section dimension) of, for example,
less than or equal to about 500 nm, less than or equal to about 250
nm, less than or equal to about 100 nm, less than or equal to about
75 nm, less than or equal to about 50 nm, less than or equal to
about 40 nm, less than or equal to about 35 nm, less than or equal
to about 30 nm, less than or equal to about 25 nm, less than or
equal to about 20 nm, less than or equal to about 15 nm, or less
than or equal to about 5 nm. In some cases, the core has an aspect
ratio of greater than about 1:1, greater than 3:1, or greater than
5:1.
[0071] The core may be formed of an inorganic material. The
inorganic material may include, for example, a metal (e.g., Ag, Au,
Pt, Fe, Cr, Co, Ni, Cu, Zn, and other transition metals), a
semiconductor (e.g., silicon, silicon compounds and alloys, cadmium
selenide, cadmium sulfide, indium arsenide, and indium phosphide),
or an insulator (e.g., ceramics such as silicon oxide). The
inorganic material may be present in the core in any suitable
amount, e.g., at least 1 wt %, 5 wt %, 10 wt %, 25 wt %, 50 wt %,
75 wt %, 90 wt %, or 99 wt %. In one embodiment, the core is formed
of 100 wt % inorganic material. The core may, in some cases, be in
the form of a quantum dot, a carbon nanotube, a carbon nanowire, or
a carbon nanorod. In some cases, the core comprises, or is formed
of, a material that is not of biological origin. In some
embodiments, a nanostructure includes or may be formed of one or
more organic materials such as a synthetic polymer and/or a natural
polymer. Examples of synthetic polymers include non-degradable
polymers such as polymethacrylate and degradable polymers such as
polylactic acid, polyglycolic acid and copolymers thereof. Examples
of natural polymers include hyaluronic acid, chitosan, and
collagen. In addition, these cores may be inert, paramagnetic, or
supramagnetic. These solid cores can be constructed from either
pure compositions of described materials, or in combinations of
mixtures of any number of materials, or in layered compositions of
materials. In addition, solid cores can be composed of a polymeric
core such as amphiphilic block copolymers, hydrophobic polymers
such as polystyrene, poly(lactic acid), poly(lactic co-glycolic
acid), poly(glycolic acid), poly(caprolactone) and other
biocompatible polymers known to those skilled in the art.
[0072] Furthermore, a shell of a structure can have any suitable
thickness. For example, the thickness of a shell may be at least 10
Angstroms, at least 0.1 nm, at least 1 nm, at least 2 nm, at least
4 nm, at least 5 nm, at least 7 nm, at least 10 nm, at least 15 nm,
at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, or
at least 200 nm (e.g., from the inner surface to the outer surface
of the shell). In some cases, the thickness of a shell is less than
200 nm, less than 100 nm, less than 50 nm, less than 30 nm, less
than 20 nm, less than 15 nm, less than 10 nm, less than 7 nm, less
than 5 nm, less than 3 nm, less than 2 nm, or less than 1 nm (e.g.,
from the inner surface to the outer surface of the shell).
[0073] The shell of a structure described herein may comprise any
suitable material, such as a hydrophobic material, a hydrophilic
material, and/or an amphiphilic material. Although the shell may
include one or more inorganic materials such as those listed above
for the nanostructure core, in many embodiments the shell includes
an organic material such as a lipid or certain polymers. The
components of the shell may be chosen, in some embodiments, to
facilitate the binding capacity.
[0074] In one set of embodiments, a structure described herein or a
portion thereof, such as a shell of a structure, includes one or
more natural or synthetic lipids or lipid analogs (i.e., lipophilic
molecules). One or more lipids and/or lipid analogues may form a
single layer or a multi-layer (e.g., a bilayer) of a structure. In
some instances where multi-layers are formed, the natural or
synthetic lipids or lipid analogs interdigitate (e.g., between
different layers). Non-limiting examples of natural or synthetic
lipids or lipid analogs include fatty acyls, glycerolipids,
glycerophospholipids, sphingolipids, saccharolipids and polyketides
(derived from condensation of ketoacyl subunits), and sterol lipids
and prenol lipids (derived from condensation of isoprene
subunits).
[0075] In one particular set of embodiments, a structure described
herein includes one or more phospholipids. The one or more
phospholipids may include, for example, phosphatidylcholine,
phosphatidylglycerol, lecithin,
.beta.,.gamma.-dipalmitoyl-.alpha.-lecithin, sphingomyelin,
phosphatidylserine, phosphatidic acid,
N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium
chloride, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylinositol, cephalin,
cardiolipin, cerebrosides, dicetylphosphate,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,
palmitoyl-oleoyl-phosphatidylcholine,
di-stearoyl-phosphatidylcholine,
stearoyl-palmitoyl-phosphatidylcholine,
di-palmitoyl-phosphatidylethanolamine,
di-stearoyl-phosphatidylethanolamine,
di-myrstoyl-phosphatidylserine, di-oleyl-phosphatidylcholine,
1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, and combinations
thereof. In some cases, a shell (e.g., a bilayer) of a structure
includes 50-200 natural or synthetic lipids or lipid analogs (e.g.,
phospholipids). For example, the shell may include less than about
500, less than about 400, less than about 300, less than about 200,
or less than about 100 natural or synthetic lipids or lipid analogs
(e.g., phospholipids), e.g., depending on the size of the
structure.
[0076] Non-phosphorus containing lipids may also be used such as
stearylamine, docecylamine, acetyl palmitate, and fatty acid
amides. In other embodiments, other lipids such as fats, oils,
waxes, cholesterol, sterols, fat-soluble vitamins (e.g., vitamins
A, D, E and K), glycerides (e.g., monoglycerides, diglycerides,
triglycerides) can be used to form portions of a structure
described herein.
[0077] A portion of a structure described herein such as a shell or
a surface of a nanostructure may optionally include one or more
alkyl groups, e.g., an alkane-, alkene-, or alkyne-containing
species, that optionally imparts hydrophobicity to the structure.
An "alkyl" group refers to a saturated aliphatic group, including a
straight-chain alkyl group, branched-chain alkyl group, cycloalkyl
(alicyclic) group, alkyl substituted cycloalkyl group, and
cycloalkyl substituted alkyl group. The alkyl group may have
various carbon numbers, e.g., between C.sub.2 and C.sub.40, and in
some embodiments may be greater than C.sub.5, C.sub.10, C.sub.15,
C.sub.20, C.sub.25, C.sub.30, or C.sub.35. In some embodiments, a
straight chain or branched chain alkyl may have 30 or fewer carbon
atoms in its backbone, and, in some cases, 20 or fewer. In some
embodiments, a straight chain or branched chain alkyl may have 12
or fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.12 for
straight chain, C.sub.3-C.sub.12 for branched chain), 6 or fewer,
or 4 or fewer Likewise, cycloalkyls may have from 3-10 carbon atoms
in their ring structure, or 5, 6 or 7 carbons in the ring
structure. Examples of alkyl groups include, but are not limited
to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,
tert-butyl, cyclobutyl, hexyl, cyclochexyl, and the like.
[0078] The alkyl group may include any suitable end group, e.g., a
thiol group, an amino group (e.g., an unsubstituted or substituted
amine), an amide group, an imine group, a carboxyl group, or a
sulfate group, which may, for example, allow attachment of a ligand
to a nanostructure core directly or via a linker. For example,
where inert metals are used to form a nanostructure core, the alkyl
species may include a thiol group to form a metal-thiol bond. In
some instances, the alkyl species includes at least a second end
group. For example, the species may be bound to a hydrophilic
moiety such as polyethylene glycol. In other embodiments, the
second end group may be a reactive group that can covalently attach
to another functional group. In some instances, the second end
group can participate in a ligand/receptor interaction (e.g.,
biotin/streptavidin).
[0079] Where a shell includes an amphiphilic material, the material
can be arranged in any suitable manner with respect to the
nanostructure core and/or with each other. For instance, the
amphiphilic material may include a hydrophilic group that points
towards the core and a hydrophobic group that extends away from the
core, or, the amphiphilic material may include a hydrophobic group
that points towards the core and a hydrophilic group that extends
away from the core. Bilayers of each configuration can also be
formed.
[0080] The lipid bilayer is composed of two layers of lipid
molecules. Each lipid molecule in a layer is oriented substantially
parallel to adjacent lipid bilayers, and two layers that form a
bilayer have the polar ends of their molecules exposed to the
aqueous phase and the non-polar ends adjacent to each other.
[0081] "Lipid" refers to its conventional sense as a generic term
encompassing fats, lipids, alcohol-ether-soluble constituents of
protoplasm, which are insoluble in water. Lipids usually consist of
a hydrophilic and a hydrophobic moiety. In water, lipids can
self-organize to form bilayers membranes, where the hydrophilic
moieties (head groups) are oriented towards the aqueous phase, and
the lipophilic moieties (acyl chains) are embedded in the bilayers
core. Lipids can comprise as well two hydrophilic moieties
(bolaamphiphiles). In that case, membranes may be formed from a
single lipid layer, and not a bilayer. Typical examples for lipids
in the current context are fats, fatty oils, essential oils, waxes,
steroid, sterols, phospholipids, glycolipids, sulpholipids,
aminolipids, chromolipids, and fatty acids. The term encompasses
both naturally occurring and synthetic lipids. Preferred lipids in
connection with the present invention are: steroids and sterol,
particularly cholesterol, phospholipids, including phosphatidyl,
phosphatidylcholines and phosphatidylethanolamines and
sphingomyelins. Where there are fatty acids, they could be about
12-24 carbon chains in length, containing up to 6 double bonds. The
fatty acids are linked to the backbone, which may be derived from
glycerol. The fatty acids within one lipid can be different
(asymmetric), or there may be only 1 fatty acid chain present, e.g.
lysolecithins. Mixed formulations are also possible, particularly
when the non-cationic lipids are derived from natural sources, such
as lecithins (phosphatidylcholines) purified from egg yolk, bovine
heart, brain, liver or soybean.
[0082] The nanostructures described herein may also include one or
more proteins, polypeptides and/or peptides (e.g., synthetic
peptides, amphiphilic peptides). In one set of embodiments, the
structures include proteins, polypeptides and/or peptides that can
increase the rate of cholesterol transfer or the
cholesterol-carrying capacity of the structures. The one or more
proteins or peptides may be associated with the core (e.g., a
surface of the core or embedded in the core), the shell (e.g., an
inner and/or outer surface of the shell, and/or embedded in the
shell), or both. Associations may include covalent or non-covalent
interactions (e.g., hydrophobic and/or hydrophilic interactions,
electrostatic interactions, van der Waals interactions).
[0083] The nanostructure is composed of a core, which may be an
inorganic material, surrounded by a shell of a lipid layer. The
nanostructure also includes a protein, such as an apolipoprotein.
The apolipoprotein can be apolipoprotein A (e.g., apo A-I, apo
A-II, apo A-IV, and apo A-V), apolipoprotein B (e.g., apo B48 and
apo B100), apolipoprotein C (e.g., apo C-I, apo C-II, apo C-III,
and apo C-IV), and apolipoproteins D, E, and H. Specifically, apo
A1, apo A2, and apo E promote transfer of cholesterol and
cholesteryl esters to the liver for metabolism and may be useful to
include in structures described herein. Additionally or
alternatively, a structure described herein may include one or more
peptide analogues of an apolipoprotein, such as one described
above. Of course, other proteins (e.g., non-apolipoproteins) can
also be included in the nanostructures described herein.
[0084] It should be understood that the components described
herein, such as the lipids, phospholipids, alkyl groups, polymers,
proteins, polypeptides, peptides, enzymes, bioactive agents,
nucleic acids, and species for targeting described above (which may
be optional), may be associated with a nano structure in any
suitable manner and with any suitable portion of the nanostructure,
e.g., the core, the shell, or both. For example, one or more such
components may be associated with a surface of a core, an interior
of a core, an inner surface of a shell, an outer surface of a
shell, and/or embedded in a shell.
[0085] A variety of methods can be used to fabricate the
nanostructures described herein. Examples of methods are provided
in International Patent Publication No. WO/2009/131704, filed Apr.
24, 2009 and entitled, "Nanostructures Suitable for Sequestering
Cholesterol and Other Molecules", which is incorporated herein by
reference in its entirety for all purposes.
[0086] The shell may have an inner surface (facing the core) and an
outer surface (facing the surroundings), such that the
apolipoprotein may be adsorbed on the outer shell and/or
incorporated between the inner surface and outer surface of the
shell. The shell is comprised of lipids and may be a lipid
monolayer or bilayer, for instance.
[0087] It should be understood that a shell which surrounds a core
need not completely surround the core, although such embodiments
may be possible. For example, the shell may surround at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
99% of the surface area of a core. In some cases, the shell
substantially surrounds a core. In other cases, the shell
completely (100%) surrounds a core. The components of the shell may
be distributed evenly across a surface of the core in some cases,
and unevenly in other cases. For example, the shell may include
portions (e.g., holes) that do not include any material in some
cases. If desired, the shell may be designed to allow penetration
and/or transport of certain molecules and components into or out of
the shell, but may prevent penetration and/or transport of other
molecules and components into or out of the shell. The ability of
certain molecules to penetrate and/or be transported into and/or
across a shell may depend on, for example, the packing density of
the components forming the shell and the chemical and physical
properties of the components forming the shell. The shell may
include one layer of material, or multilayers of materials in some
embodiments.
[0088] In some embodiments, the nanostructure includes a cationic
lipid. The cationic lipid may be, for example,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.C1), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.C1), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),
1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA) or analogs thereof,
(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-
- -3 aH-cyclopenta[d][1,3]dioxol-5-amine,
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)b-
utanoate, or a mixture thereof.
[0089] Other cationic lipids, which carry a net positive charge at
about physiological pH, in addition to those specifically described
above, may also be included in the lipid nanoparticle. Such
cationic lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride ("DOTMA");
N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt
("DOTAP.Cl");
3.beta.-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
-ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"),
N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"),
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"), and 1,2-dioleoyl-sn-glycero-3-phosphocholine
("DOPC").
[0090] In some aspects of the disclosure, the nanostructure
comprises a cationic lipid (e.g., DOTAP) is mixed with a nucleic
acid (e.g., RNA) in a molar ratio of about 1:1, of about 2:1, of
about 3:1, of about 4:1, of about 5:1, of about 6:1, of about 7:1,
of about 8:1, of about 9:1, of about 10:1, of about 11:1, of about
12:1, of about 13:1, of about 14:1, of about 15:1, of about 16:1,
of about 17:1, of about 18:1, of about 19:1, of about 20:1, of
about 21:1, of about 22:1, of about 23:1, of about 24:1, of about
25:1, of about 26:1, of about 27:1, of about 28:1, of about 29:1,
of about 30:1, of about 31:1, of about 32:1, of about 33:1, of
about 34:1, of about 35:1, of about 36:1, of about 37:1, of about
38:1, of about 39:1, of about 40:1, of about 41:1, of about 42:1,
of about 43:1, of about 44:1, of about 45:1, of about 46:1, of
about 47:1, of about 48:1, of about 49:1, of about 50:1, of about
60:1, of about 70:1, of about 80:1, of about 90:1, or of about
100:1. In some embodiments, the cationic lipid (e.g. DOTAP) is
mixed with the nucleic acid (e.g., RNA) in a molar ratio of 10:1,
20:1, 30:1 or 40:1.
[0091] "Amphipathic lipids" refer to any suitable material, wherein
the hydrophobic portion of the lipid material orients into a
hydrophobic phase, while the hydrophilic portion orients toward the
aqueous phase. Such compounds include, but are not limited to,
phospholipids, aminolipids, and sphingolipids. Representative
phospholipids include sphingomyelin, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, palmitoyloleoyl phosphatdylcholine,
lysophosphatidylcholine, lysophosphatidylethanolamine,
dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
distearoylphosphatidylcholine, or dilinoleylphosphatidylcholine,
monophosphoryl lipid A (MPLA), or glycopyranoside lipid A
(GLA).
[0092] In some aspects, the nucleic acid or oligonucleotide
regulate the expression of a gene. As used herein, "regulating gene
expression" or "gene regulation" are used interchangeably and
includes a wide range of mechanisms that are used by cells to
increase or decrease the production of specific gene products
(e.g., protein, RNA, etc.).
[0093] In some embodiments the nucleic acid or oligonucleotide is
an inhibitory nucleic acid. The inhibitory nucleic acid may be, for
instance, an siRNA or an antisense molecule that inhibits
expression of a protein that will have a therapeutic effect. The
inhibitory nucleic acids may be designed using routine methods in
the art.
[0094] An inhibitory nucleic acid typically causes specific gene
knockdown, while avoiding off-target effects. Various strategies
for gene knockdown known in the art can be used to inhibit gene
expression. For example, gene knockdown strategies may be used that
make use of RNA interference (RNAi) and/or microRNA (miRNA)
pathways including small interfering RNA (siRNA), short hairpin RNA
(shRNA), double-stranded RNA (dsRNA), miRNAs, and other small
interfering nucleic acid-based molecules known in the art. In one
embodiment, vector-based RNAi modalities (e.g., shRNA expression
constructs) are used to reduce expression of a gene in a cell. In
some embodiments, therapeutic compositions of the invention
comprise an isolated plasmid vector (e.g., any isolated plasmid
vector known in the art or disclosed herein) that expresses a small
interfering nucleic acid such as an shRNA. The isolated plasmid may
comprise a specific promoter operably linked to a gene encoding the
small interfering nucleic acid. In some cases, the isolated plasmid
vector is packaged in a virus capable of infecting the individual.
Exemplary viruses include adenovirus, retrovirus, lentivirus,
adeno-associated virus, and others that are known in the art and
disclosed herein.
[0095] A broad range of RNAi-based modalities could be employed to
inhibit expression of a gene in a cell, such as siRNA-based
oligonucleotides and/or altered siRNA-based oligonucleotides.
Altered siRNA based oligonucleotides are those modified to alter
potency, target affinity, safety profile and/or stability, for
example, to render them resistant or partially resistant to
intracellular degradation. Modifications, such as
phosphorothioates, for example, can be made to nucleic acids or
oligonucleotides to increase resistance to nuclease degradation,
binding affinity and/or uptake. In addition, hydrophobization and
bioconjugation enhances siRNA delivery and targeting (De Paula et
al., RNA. 13(4):431-56, 2007) and siRNAs with ribo-difluorotoluyl
nucleotides maintain gene silencing activity (Xia et al., ASC Chem.
Biol. 1(3):176-83, (2006)). siRNAs with amide-linked
oligoribonucleosides have been generated that are more resistant to
S1 nuclease degradation than unmodified siRNAs (Iwase R et al. 2006
Nucleic Acids Symp Ser 50: 175-176). In addition, modification of
siRNAs at the 2'-sugar position and phosphodiester linkage confers
improved serum stability without loss of efficacy (Choung et al.,
Biochem. Biophys. Res. Commun. 342(3):919-26, 2006).
[0096] Other molecules that can be used to inhibit expression of a
gene include antisense nucleic acids (single or double stranded),
ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple
helix forming oligonucleotides, antibodies, and aptamers and
modified form(s) thereof directed to sequences in gene(s), RNA
transcripts, or proteins. Antisense and ribozyme suppression
strategies have led to the reversal of a tumor phenotype by
reducing expression of a gene product or by cleaving a mutant
transcript at the site of the mutation (Carter and Lemoine Br. J.
Cancer. 67(5):869-76, 1993; Lange et al., Leukemia. 6(11):1786-94,
1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994;
Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng
et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer
Res. 55(1):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998).
Ribozymes have also been proposed as a means of both inhibiting
gene expression of a mutant gene and of correcting the mutant by
targeted trans-splicing (Sullenger and Cech Nature
371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8,
1996).
[0097] Triple helix approaches have also been investigated for
sequence-specific gene suppression. Triple helix forming
oligonucleotides have been found in some cases to bind in a
sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci.
U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl.
Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc.
Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer
Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have
been shown to inhibit gene expression (Hanvey et al., Antisense
Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res.
24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83,
1997). Minor-groove binding polyamides can bind in a
sequence-specific manner to DNA targets and hence may represent
useful small molecules for suppression at the DNA level (Trauger et
al., Chem. Biol. 3(5):369-77, 1996). In addition, suppression has
been obtained by interference at the protein level using dominant
negative mutant peptides and antibodies (Herskowitz Nature
329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6,
1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203,
1989). The diverse array of suppression strategies that can be
employed includes the use of DNA and/or RNA aptamers that can be
selected to target a protein of interest.
[0098] Other inhibitor molecules that can be used include antisense
nucleic acids (single or double stranded). Antisense nucleic acids
include modified or unmodified RNA, DNA, or mixed polymer nucleic
acids, and primarily function by specifically binding to matching
sequences resulting in modulation of peptide synthesis (Wu-Pong,
November 1994, BioPharm, 20-33). Antisense nucleic acid binds to
target RNA by Watson Crick base-pairing and blocks gene expression
by preventing ribosomal translation of the bound sequences either
by steric blocking or by activating RNase H enzyme. Antisense
molecules may also alter protein synthesis by interfering with RNA
processing or transport from the nucleus into the cytoplasm
(Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7,
151-190).
[0099] As used herein, the term "antisense nucleic acid" describes
a nucleic acid that is an oligoribonucleotide,
oligodeoxyribonucleotide, modified oligoribonucleotide, or modified
oligodeoxyribonucleotide which hybridizes under physiological
conditions to DNA comprising a particular gene or to an mRNA
transcript of that gene and, thereby, inhibits the transcription of
that gene and/or the translation of that mRNA. The antisense
molecules are designed so as to interfere with transcription or
translation of a target gene upon hybridization with the target
gene or transcript. Those skilled in the art will recognize that
the exact length of the antisense oligonucleotide and its degree of
complementarity with its target will depend upon the specific
target selected, including the sequence of the target and the
particular bases which comprise that sequence.
[0100] An inhibitory nucleic acid useful in the invention will
generally be designed to have partial or complete complementarity
with one or more target genes. The target gene may be a gene
derived from the cell, an endogenous gene, a transgene, or a gene
of a pathogen which is present in the cell after infection thereof.
Depending on the particular target gene, the nature of the
inhibitory nucleic acid and the level of expression of inhibitory
nucleic acid (e.g. depending on copy number, promoter strength) the
procedure may provide partial or complete loss of function for the
target gene. Quantitation of gene expression in a cell may show
similar amounts of inhibition at the level of accumulation of
target mRNA or translation of target protein.
[0101] "Inhibition of gene expression" refers to the absence or
observable decrease in the level of protein and/or mRNA product
from a target gene. "Specificity" refers to the ability to inhibit
the target gene without manifest effects on other genes of the
cell. The consequences of inhibition can be confirmed by
examination of the outward properties of the cell or organism or by
biochemical techniques such as RNA solution hybridization, nuclease
protection, Northern hybridization, reverse transcription, gene
expression monitoring with a microarray, antibody binding, enzyme
linked immunosorbent assay (ELISA), Western blotting,
radioimmunoassay (RIA), other immunoassays, and fluorescence
activated cell analysis (FACS). For RNA-mediated inhibition in a
cell line or whole organism, gene expression is conveniently
assayed by use of a reporter or drug resistance gene whose protein
product is easily assayed. Such reporter genes include
acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta
galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol
acetyltransferase (CAT), green fluorescent protein (GFP),
horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase
(NOS), octopine synthase (OCS), and derivatives thereof. Multiple
selectable markers are available that confer resistance to
ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,
kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin,
and tetracyclin.
[0102] Depending on the assay, quantitation of the amount of gene
expression allows one to determine a degree of inhibition which is
greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell
not treated according to the present invention. As an example, the
efficiency of inhibition may be determined by assessing the amount
of gene product in the cell: mRNA may be detected with a
hybridization probe having a nucleotide sequence outside the region
used for the inhibitory nucleic acid, or translated polypeptide may
be detected with an antibody raised against the polypeptide
sequence of that region.
[0103] An expression enhancing nucleic acid or oligonucleotide as
used herein is a synthetic oligonucleotide that encodes a protein.
The synthetic oligonucleotide may be delivered to a cell such that
it is used by a cells machinery to produce a protein based on the
sequence of the synthetic oligonucleotide. The synthetic
oligonucleotide may be, for instance, synthetic DNA or synthetic
RNA. "Synthetic RNA" refers to a RNA produced through an in vitro
transcription reaction or through artificial (non-natural) chemical
synthesis. In some embodiments, a synthetic RNA is an RNA
transcript. In some embodiments, a synthetic RNA encodes a protein.
In some embodiments, the synthetic RNA is a functional RNA. In some
embodiments, a synthetic RNA comprises one or more modified
nucleotides. In some embodiments, a synthetic RNA is up to 0.5
kilobases (kb), 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 4 kb, 5 kb, 6 kb,
7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb or more in
length. In some embodiments, a synthetic RNA is in a range of 0.1
kb to 1 kb, 0.5 kb to 2 kb, 0.5 kb to 10 kb, 1 kb to 5 kb, 2 kb to
5 kb, 1 kb to 10 kb, 3 kb to 10 kb, 5 kb to 15 kb, or 1 kb to 30 kb
in length.
[0104] A diagnostic nucleic acid or oligonucleotide is an nucleic
acid or oligonucleotide that interacts with a cellular marker to
identify the presence of the marker in a cell or subject.
Diagnostic oligonucleotides are well known in the art and typically
include a label or are otherwise detectable.
[0105] The terms "oligonucleotide" and "nucleic acid" are used
interchangeably to mean multiple nucleotides (i.e., molecules
comprising a sugar (e.g., ribose or deoxyribose) linked to a
phosphate group and to an exchangeable organic base, which is
either a substituted pyrimidine (e.g., cytosine (C), thymidine (T)
or uracil (U)) or a substituted purine (e.g., adenine (A) or
guanine (G)). Thus, the term embraces both DNA and RNA
oligonucleotides. The terms shall also include polynucleosides
(i.e., a polynucleotide minus the phosphate) and any other organic
base containing polymer. Oligonucleotides can be obtained from
existing nucleic acid sources (e.g., genomic or cDNA), but are
preferably synthetic (e.g., produced by nucleic acid
synthesis).
[0106] An oligonucleotide of the nanostructure can be single
stranded or double stranded. A double stranded oligonucleotide is
also referred to herein as a duplex. Double-stranded
oligonucleotides of the invention can comprise two separate
complementary nucleic acid strands.
[0107] The nucleic acids useful in the nanostructures of the
invention are synthetic or isolated nucleic acids.
[0108] As used herein, "duplex" includes a double-stranded nucleic
acid molecule(s) in which complementary sequences are hydrogen
bonded to each other. The complementary sequences can include a
sense strand and an antisense strand. The antisense nucleotide
sequence can be identical or sufficiently identical to the target
gene to mediate effective target gene inhibition (e.g., at least
about 98% identical, 96% identical, 94%, 90% identical, 85%
identical, or 80% identical) to the target gene sequence.
[0109] A double-stranded nucleic acid or oligonucleotide can be
double-stranded over its entire length, meaning it has no
overhanging single-stranded sequences and is thus blunt-ended. In
other embodiments, the two strands of the double-stranded
polynucleotide can have different lengths producing one or more
single-stranded overhangs. A double-stranded polynucleotide of the
invention can contain mismatches and/or loops or bulges. In some
embodiments, it is double-stranded over at least about 70%, 80%,
90%, 95%, 96%, 97%, 98% or 99% of the length of the
oligonucleotide. In some embodiments, the double-stranded
oligonucleotide of the invention contains at least or up to 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
[0110] Nucleic acids or oligonucleotides associated with the
invention can be modified such as at the sugar moiety, the
phosphodiester linkage, and/or the base. As used herein, "sugar
moieties" includes natural, unmodified sugars, including pentose,
ribose and deoxyribose, modified sugars and sugar analogs.
Modifications of sugar moieties can include replacement of a
hydroxyl group with a halogen, a heteroatom, or an aliphatic group,
and can include functionalization of the hydroxyl group as, for
example, an ether, amine or thiol.
[0111] Modification of sugar moieties can include 2'-O-methyl
nucleotides, which are referred to as "methylated." In some
instances, polynucleotides associated with the invention may only
contain modified or unmodified sugar moieties, while in other
instances, polynucleotides contain some sugar moieties that are
modified and some that are not.
[0112] In some instances, modified nucleomonomers include sugar- or
backbone-modified ribonucleotides. Modified ribonucleotides can
contain a non-naturally occurring base such as uridines or
cytidines modified at the 5'-position, e.g., 5'-(2-amino)propyl
uridine and 5'-bromo uridine; adenosines and guanosines modified at
the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g.,
7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyl
adenosine. Also, sugar-modified ribonucleotides can have the 2'-OH
group replaced by an H, alkoxy (or OR), R or alkyl, halogen, SH,
SR, amino (such as NH2, NHR, NR2,), or CN group, wherein R is lower
alkyl, alkenyl, or alkynyl. In some embodiments, modified
ribonucleotides can have the phosphodiester group connecting to
adjacent ribonucleotides replaced by a modified group, such as a
phosphorothioate group.
[0113] In some aspects, 2'-O-methyl modifications can be beneficial
for reducing undesirable cellular stress responses, such as the
interferon response to double-stranded nucleic acids. Modified
sugars can include D-ribose, 2'-O-alkyl (including 2'-O-methyl and
2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo
(including 2'-fluoro), 2'-methoxyethoxy, 2'-allyloxy
(--OCH.sub.2CH.dbd.CH.sub.2), 2'-propargyl, 2'-propyl, ethynyl,
ethenyl, propenyl, and cyano and the like. The sugar moiety can
also be a hexose.
[0114] The term "base" includes the known purine and pyrimidine
heterocyclic bases, deazapurines, and analogs (including
heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine),
derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and
1-alkynyl derivatives) and tautomers thereof. Examples of purines
include adenine, guanine, inosine, diaminopurine, and xanthine and
analogs (e.g., 8-oxo-N6-methyladenine or 7-diazaxanthine) and
derivatives thereof. Pyrimidines include, for example, thymine,
uracil, and cytosine, and their analogs (e.g., 5-methylcytosine,
5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and
4,4-ethanocytosine). Other examples of suitable bases include
non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and
triazines.
[0115] In some aspects, polynucleotides of the invention comprise
3' and 5' termini (except for circular oligonucleotides). The 3'
and 5' termini of a polynucleotide can be substantially protected
from nucleases, for example, by modifying the 3' or 5' linkages
(e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). Oligonucleotides
can be made resistant by the inclusion of a "blocking group." The
term "blocking group" as used herein refers to substituents (e.g.,
other than OH groups) that can be attached to oligonucleotides or
nucleomonomers, either as protecting groups or coupling groups for
synthesis (e.g., FITC, propyl (CH.sub.2--CH.sub.2--CH.sub.3),
glycol (--O--CH.sub.2--CH.sub.2--O--) phosphate (PO32-), hydrogen
phosphonate, or phosphoramidite). "Blocking groups" also include
"end blocking groups" or "exonuclease blocking groups" which
protect the 5' and 3' termini of the oligonucleotide, including
modified nucleotides and non-nucleotide exonuclease resistant
structures.
[0116] Exemplary end-blocking groups include cap structures (e.g.,
a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3'-3'
or 5'-5' end inversions (see, e.g., Ortiagao et al. 1992. Antisense
Res. Dev. 2:129), methylphosphonate, phosphoramidite,
non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers,
conjugates) and the like. The 3' terminal nucleomonomer can
comprise a modified sugar moiety. The 3' terminal nucleomonomer
comprises a 3'-O that can optionally be substituted by a blocking
group that prevents 3'-exonuclease degradation of the
oligonucleotide. For example, the 3'-hydroxyl can be esterified to
a nucleotide through a 3'.fwdarw.3' internucleotide linkage. For
example, the alkyloxy radical can be methoxy, ethoxy, or
isopropoxy, and preferably, ethoxy. Optionally, the 3'.fwdarw.3'
linked nucleotide at the 3' terminus can be linked by a substitute
linkage. To reduce nuclease degradation, the 5' most 3'.fwdarw.5'
linkage can be a modified linkage, e.g., a phosphorothioate or a
P-alkyloxyphosphotriester linkage. Preferably, the two 5' most
3'.fwdarw.5' linkages are modified linkages. Optionally, the 5'
terminal hydroxy moiety can be esterified with a phosphorus
containing moiety, e.g., phosphate, phosphorothioate, or
P-ethoxyphosphate.
[0117] The term "nucleoside" includes bases which are covalently
attached to a sugar moiety, preferably ribose or deoxyribose.
Examples of preferred nucleosides include ribonucleosides and
deoxyribonucleosides. Nucleosides also include bases linked to
amino acids or amino acid analogs which may comprise free carboxyl
groups, free amino groups, or protecting groups. Suitable
protecting groups are well known in the art (see P. G. M. Wuts and
T. W. Greene, "Protective Groups in Organic Synthesis", 2nd Ed.,
Wiley-Interscience, New York, 1999).
[0118] As used herein, the nanostructure is a construct having an
average diameter on the order of nanometers (i.e., between about 1
nm and about 1 micrometer. For example, in some instances, the
diameter of the nanoparticle is from about 1 nm to about 250 nm in
mean diameter, about 1 nm to about 240 nm in mean diameter, about 1
nm to about 230 nm in mean diameter, about 1 nm to about 220 nm in
mean diameter, about 1 nm to about 210 nm in mean diameter, about 1
nm to about 200 nm in mean diameter, about 1 nm to about 190 nm in
mean diameter, about 1 nm to about 180 nm in mean diameter, about 1
nm to about 170 ran in mean diameter, about 1 nm to about 160 nm in
mean diameter, about 1 nm to about 150 nm in mean diameter, about 1
nm to about 140 nm in mean diameter, about 1 nm to about 130 nm in
mean diameter, about 1 nm to about 120 nm in mean diameter, about 1
nm to about 110 nm in mean diameter, about 1 nm to about 100 nm in
mean diameter, about 1 nm to about 90 nm in mean diameter, about 1
nm to about 80 nm in mean diameter, about 1 nm to about 70 nm in
mean diameter, about 1 nm to about 60 nm in mean diameter, about 1
nm to about 50 nm in mean diameter, about 1 nm to about 40 nm in
mean diameter, about 1 nm to about 30 nm in mean diameter, about 1
nm to about 20 nm in mean diameter, about 1 nm to about 10 nm in
mean diameter, about 5 nm to about 150 nm in mean diameter, about 5
to about 50 nm in mean diameter, about 10 to about 30 nm in mean
diameter, about 10 to 150 nm in mean diameter, about 10 to about
100 nm in mean diameter, about 10 to about 50 nm in mean diameter,
about 30 to about 100 nm in mean diameter, or about 40 to about 80
nm in mean diameter. In a set of embodiments, the nanostructure is
about 110 nm in diameter.
[0119] In some embodiments, the nanostructures may be used at a
concentration of about 1 nM to about 1000 nM, of about 1 nM to
about 900 nM, of about 1 nM to about 800 nM, of about 1 nM to about
700 nM, of about 1 nM to about 600 nM, of about 1 nM to about 500
nM, of about 1 nM to about 400 nM, of about 1 nM to about 300 nM,
of about 1 nM to about 200 nM, of about 1 nM to about 100 nM, of
about 1 nM to about 50 nM, of about 1 nM to about 40 nM, of about 1
nM to about 30 nM, of about 1 nM to about 20 nM, or of about 1 nM
to about 10 nM. In a set of embodiments, the nanostructure is used
at a concentration of 5 nM, 10 nM or 20 nM.
[0120] In some aspects of the disclosure, the nanostructure
comprises a nucleic acid (e.g., RNA) mixed with TLP in a molar
ratio of about 1:1, of about 2:1, of about 3:1, of about 4:1, of
about 5:1, of about 6:1, of about 7:1, of about 8:1, of about 9:1,
of about 10:1, of about 11:1, of about 12:1, of about 13:1, of
about 14:1, of about 15:1, of about 16:1, of about 17:1, of about
18:1, of about 19:1, of about 20:1, of about 21:1, of about 22:1,
of about 23:1, of about 24:1, of about 25:1, of about 26:1, of
about 27:1, of about 28:1, of about 29:1, of about 30:1, of about
31:1, of about 32:1, of about 33:1, of about 34:1, of about 35:1,
of about 36:1, of about 37:1, of about 38:1, of about 39:1, of
about 40:1, of about 41:1, of about 42:1, of about 43:1, of about
44:1, of about 45:1, of about 46:1, of about 47:1, of about 48:1,
of about 49:1, of about 50:1, of about 60:1, of about 70:1, of
about 80:1, of about 90:1, or of about 100:1. In some embodiments
the nucleic acid, such as RNA, is mixed with TLP in a molar ratio
of 5:1, 15:1 or 25:1. In a set of embodiments, the nanostructure
comprises a nucleic acid (e.g., RNA) mixed with TLP at a molar
ratio of 25:1 and a cationic lipid (e.g., DOTAP) mixed with a
nucleic acid (e.g., RNA) in a molar ratio of 40:1.
[0121] The nanostructures may be used in "pharmaceutical
compositions" or "pharmaceutically acceptable" compositions, which
comprise a therapeutically effective amount of one or more of the
structures described herein, formulated together with one or more
pharmaceutically acceptable carriers, additives, and/or diluents.
The pharmaceutical compositions described herein may be useful for
treating cancer or autoimmune diseases or disorders, or other
related diseases. It should be understood that any suitable
structures described herein can be used in such pharmaceutical
compositions, including those described in connection with the
figures.
[0122] The pharmaceutical compositions may be specially formulated
for administration in solid or liquid form, including those adapted
for the following: oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets, e.g.,
those targeted for buccal, sublingual, and systemic absorption,
boluses, powders, granules, pastes for application to the tongue;
parenteral administration, for example, by subcutaneous,
intramuscular, intravenous or epidural injection as, for example, a
sterile solution or suspension, or sustained-release formulation;
topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin, lungs, or
oral cavity; intravaginally or intrarectally, for example, as a
pessary, cream or foam; sublingually; ocularly; transdermally; or
nasally, pulmonary and to other mucosal surfaces.
[0123] The phrase "pharmaceutically acceptable" is employed herein
to refer to those structures, materials, compositions, and/or
dosage forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0124] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient, or
solvent encapsulating material, involved in carrying or
transporting the subject compound from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; pH buffered
solutions; polyesters, polycarbonates and/or polyanhydrides; and
other non-toxic compatible substances employed in pharmaceutical
formulations.
[0125] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0126] Examples of pharmaceutically-acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0127] The structures described herein may be orally administered,
parenterally administered, subcutaneously administered, and/or
intravenously administered. In certain embodiments, a structure or
pharmaceutical preparation is administered orally. In other
embodiments, the structure or pharmaceutical preparation is
administered intravenously. Alternative routes of administration
include sublingual, intramuscular, and transdermal
administrations.
[0128] Pharmaceutical compositions described herein include those
suitable for oral, nasal, topical (including buccal and
sublingual), rectal, vaginal and/or parenteral administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the host being treated, and the particular mode of
administration. The amount of active ingredient that can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, this amount will range from about 1%
to about 99% of active ingredient, from about 5% to about 70%, or
from about 10% to about 30%.
[0129] The inventive compositions suitable for oral administration
may be in the form of capsules, cachets, pills, tablets, lozenges
(using a flavored basis, usually sucrose and acacia or tragacanth),
powders, granules, or as a solution or a suspension in an aqueous
or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of a structure described herein as an active ingredient. The
HDL-NP may also be administered as a bolus, electuary or paste.
[0130] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol, glycerol monostearate,
and non-ionic surfactants; absorbents, such as kaolin and bentonite
clay; lubricants, such as talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, and
mixtures thereof; and coloring agents. In the case of capsules,
tablets and pills, the pharmaceutical compositions may also
comprise buffering agents. Solid compositions of a similar type may
also be employed as fillers in soft and hard-shelled gelatin
capsules using such excipients as lactose or milk sugars, as well
as high molecular weight polyethylene glycols and the like.
[0131] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made in a suitable machine in which a mixture
of the powdered structure is moistened with an inert liquid
diluent.
[0132] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be formulated for rapid release, e.g.,
freeze-dried. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions that
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or in a certain
portion of the gastrointestinal tract, optionally, in a delayed
manner. Examples of embedding compositions that can be used include
polymeric substances and waxes. The active ingredient can also be
in micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0133] Liquid dosage forms for oral administration of the
structures described herein include pharmaceutically acceptable
emulsions, microemulsions, solutions, dispersions, suspensions,
syrups and elixirs. In addition to the inventive structures, the
liquid dosage forms may contain inert diluents commonly used in the
art, such as, for example, water or other solvents, solubilizing
agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0134] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0135] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0136] Formulations of the pharmaceutical compositions described
herein (e.g., for rectal or vaginal administration) may be
presented as a suppository, which may be prepared by mixing one or
more compounds of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the body and release the
structures.
[0137] Dosage forms for the topical or transdermal administration
of a structure described herein include powders, sprays, ointments,
pastes, foams, creams, lotions, gels, solutions, patches and
inhalants. The active compound may be mixed under sterile
conditions with a pharmaceutically-acceptable carrier, and with any
preservatives, buffers, or propellants which may be required.
[0138] The ointments, pastes, creams and gels may contain, in
addition to the inventive structures, excipients, such as animal
and vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0139] Powders and sprays can contain, in addition to the
structures described herein, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates and polyamide
powder, or mixtures of these substances. Sprays can additionally
contain customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0140] Transdermal patches have the added advantage of providing
controlled delivery of a structure described herein to the body.
Dissolving or dispersing the structure in the proper medium can
make such dosage forms. Absorption enhancers can also be used to
increase the flux of the structure across the skin. Either
providing a rate controlling membrane or dispersing the structure
in a polymer matrix or gel can control the rate of such flux.
[0141] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0142] Pharmaceutical compositions described herein suitable for
parenteral administration comprise one or more inventive structures
in combination with one or more pharmaceutically-acceptable sterile
isotonic aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain sugars, alcohols, antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0143] Examples of suitable aqueous and nonaqueous carriers, which
may be employed in the pharmaceutical compositions described herein
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0144] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. It may also be desirable to include isotonic agents, such
as sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0145] Delivery systems suitable for use with structures and
compositions described herein include time-release, delayed
release, sustained release, or controlled release delivery systems,
as described herein. Such systems may avoid repeated
administrations of the structures in many cases, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include, for example, polymer based systems such
as polylactic and/or polyglycolic acid, polyanhydrides, and
polycaprolactone; nonpolymer systems that are lipid-based including
sterols such as cholesterol, cholesterol esters, and fatty acids or
neutral fats such as mono-, di- and triglycerides; hydrogel release
systems; silastic systems; peptide based systems; wax coatings;
compressed tablets using conventional binders and excipients; or
partially fused implants. Specific examples include, but are not
limited to, erosional systems in which the composition is contained
in a form within a matrix, or diffusional systems in which an
active component controls the release rate. The compositions may be
as, for example, microspheres, hydrogels, polymeric reservoirs,
cholesterol matrices, or polymeric systems. In some embodiments,
the system may allow sustained or controlled release of the active
compound to occur, for example, through control of the diffusion or
erosion/degradation rate of the formulation. In addition, a
pump-based hardware delivery system may be used in some
embodiments. The structures and compositions described herein can
also be combined (e.g., contained) with delivery devices such as
syringes, pads, patches, tubes, films, MEMS-based devices, and
implantable devices.
[0146] Use of a long-term release implant may be particularly
suitable in some cases. "Long-term release," as used herein, means
that the implant is constructed and arranged to deliver therapeutic
levels of the composition for at least about 30 or about 45 days,
for at least about 60 or about 90 days, or even longer in some
cases. Long-term release implants are well known to those of
ordinary skill in the art, and include some of the release systems
described above.
[0147] Injectable depot forms can be made by forming microencapsule
matrices of the structures described herein in biodegradable
polymers such as polylactide-polyglycolide. Depending on the ratio
of structure to polymer, and the nature of the particular polymer
employed, the rate of release of the structure can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides).
[0148] When the structures described herein are administered as
pharmaceuticals, to humans and animals, they can be given per se or
as a pharmaceutical composition containing, for example, about 0.1%
to about 99.5%, about 0.5% to about 90%, or the like, of structures
in combination with a pharmaceutically acceptable carrier.
[0149] The administration may be localized (e.g., to a particular
region, physiological system, tissue, organ, or cell type) or
systemic, depending on the condition to be treated. For example,
the composition may be administered through parental injection,
implantation, orally, vaginally, rectally, buccally, pulmonary,
topically, nasally, transdermally, surgical administration, or any
other method of administration where access to the target by the
composition is achieved. Examples of parental modalities that can
be used with the invention include intravenous, intradermal,
subcutaneous, intracavity, intramuscular, intraperitoneal,
epidural, or intrathecal. Examples of implantation modalities
include any implantable or injectable drug delivery system. Oral
administration may be useful for some treatments because of the
convenience to the patient as well as the dosing schedule.
[0150] Regardless of the route of administration selected, the
structures described herein, which may be used in a suitable
hydrated form, and/or the inventive pharmaceutical compositions,
are formulated into pharmaceutically-acceptable dosage forms by
conventional methods known to those of skill in the art.
[0151] The compositions described herein may be given in dosages,
e.g., at the maximum amount while avoiding or minimizing any
potentially detrimental side effects. The compositions can be
administered in effective amounts, alone or in a combinations with
other compounds. For example, when treating cancer, a composition
may include the structures described herein and a cocktail of other
compounds that can be used to treat cancer. When treating
conditions associated with abnormal lipid levels, a composition may
include the structures described herein and other compounds that
can be used to reduce lipid levels (e.g., cholesterol lowering
agents).
[0152] The phrase "therapeutically effective amount" as used herein
means that amount of a material or composition comprising an
inventive structure which is effective for producing some desired
therapeutic effect in a subject at a reasonable benefit/risk ratio
applicable to any medical treatment. Accordingly, a therapeutically
effective amount may, for example, prevent, minimize, or reverse
disease progression associated with cancer or an autoimmune
disorder. Disease progression can be monitored by clinical
observations, laboratory and imaging investigations apparent to a
person skilled in the art. A therapeutically effective amount can
be an amount that is effective in a single dose or an amount that
is effective as part of a multi-dose therapy, for example an amount
that is administered in two or more doses or an amount that is
administered chronically.
[0153] The effective amount of any one or more structures described
herein may be from about 10 ng/kg of body weight to about 1000
mg/kg of body weight, and the frequency of administration may range
from once a day to once a month. However, other dosage amounts and
frequencies also may be used as the invention is not limited in
this respect. A subject may be administered one or more structure
described herein in an amount effective to treat one or more
diseases or bodily conditions described herein.
[0154] An effective amount may depend on the particular condition
to be treated. The effective amounts will depend, of course, on
factors such as the severity of the condition being treated;
individual patient parameters including age, physical condition,
size and weight; concurrent treatments; the frequency of treatment;
or the mode of administration. These factors are well known to
those of ordinary skill in the art and can be addressed with no
more than routine experimentation. In some cases, a maximum dose be
used, that is, the highest safe dose according to sound medical
judgment.
[0155] Actual dosage levels of the active ingredients in the
pharmaceutical compositions described herein may be varied so as to
obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0156] The selected dosage level will depend upon a variety of
factors including the activity of the particular inventive
structure employed, the route of administration, the time of
administration, the rate of excretion or metabolism of the
particular structure being employed, the duration of the treatment,
other drugs, compounds and/or materials used in combination with
the particular structure employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0157] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the structures described herein
employed in the pharmaceutical composition at levels lower than
that required to achieve the desired therapeutic effect and then
gradually increasing the dosage until the desired effect is
achieved.
[0158] In some embodiments, a structure or pharmaceutical
composition described herein is provided to a subject chronically.
Chronic treatments include any form of repeated administration for
an extended period of time, such as repeated administrations for
one or more months, between a month and a year, one or more years,
or longer. In many embodiments, a chronic treatment involves
administering a structure or pharmaceutical composition repeatedly
over the life of the subject. For example, chronic treatments may
involve regular administrations, for example one or more times a
day, one or more times a week, or one or more times a month. In
general, a suitable dose such as a daily dose of a structure
described herein will be that amount of the structure that is the
lowest dose effective to produce a therapeutic effect. Such an
effective dose will generally depend upon the factors described
above. Generally doses of the structures described herein for a
patient, when used for the indicated effects, will range from about
0.0001 to about 100 mg per kg of body weight per day. The daily
dosage may range from 0.001 to 50 mg of compound per kg of body
weight, or from 0.01 to about 10 mg of compound per kg of body
weight. In some embodiments, the nanostructure is administered at a
dose of about 1000 mg/kg, of about 500 mg/kg, of about 100 mg/kg,
of about 50 mg/kg, of about 25 mg/kg, of about 10 mg/kg, of about 5
mg/kg, of about 4 mg/kg, of about 3 mg/kg, of about 2 mg/kg, of
about 1 mg/kg, of about 0.7 mg/kg, of about 0.5 mg/kg, 0.1 mg/kg.
In certain embodiments, the nanostructure is administered at a dose
of 0.7 mg/kg. However, lower or higher doses can be used. In some
embodiments, the dose administered to a subject may be modified as
the physiology of the subject changes due to age, disease
progression, weight, or other factors.
[0159] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. For example,
instructions and methods may include dosing regimens wherein
specific doses of compositions, especially those including
structures described herein having a particular size range, are
administered at specific time intervals and specific doses to
achieve reduction of cholesterol (or other lipids) and/or treatment
of disease while reducing or avoiding adverse effects or unwanted
effects.
[0160] While it is possible for a structure described herein to be
administered alone, it may be administered as a pharmaceutical
composition as described above. The present invention also provides
any of the above-mentioned compositions useful for diagnosing,
preventing, treating, or managing a disease or bodily condition
packaged in kits, optionally including instructions for use of the
composition. That is, the kit can include a description of use of
the composition for participation in any disease or bodily
condition, including those associated with abnormal lipid levels.
The kits can further include a description of use of the
compositions as discussed herein. The kit also can include
instructions for use of a combination of two or more compositions
described herein. Instructions also may be provided for
administering the composition by any suitable technique, such as
orally, intravenously, or via another known route of drug
delivery.
[0161] The kits described herein may also contain one or more
containers, which can contain components such as the structures,
signaling entities, and/or biomolecules as described. The kits also
may contain instructions for mixing, diluting, and/or
administrating the compounds. The kits also can include other
containers with one or more solvents, surfactants, preservatives,
and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose)
as well as containers for mixing, diluting or administering the
components to the sample or to the patient in need of such
treatment.
[0162] The compositions of the kit may be provided as any suitable
form, for example, as liquid solutions or as dried powders. When
the composition provided is a dry powder, the powder may be
reconstituted by the addition of a suitable solvent, which may also
be provided. In embodiments where liquid forms of the composition
are used, the liquid form may be concentrated or ready to use. The
solvent will depend on the particular inventive structure and the
mode of use or administration. Suitable solvents for compositions
are well known and are available in the literature.
[0163] The kit, in one set of embodiments, may comprise one or more
containers such as vials, tubes, and the like, each of the
containers comprising one of the separate elements to be used in
the method. For example, one of the containers may comprise a
positive control in the assay. Additionally, the kit may include
containers for other components, for example, buffers useful in the
assay.
[0164] As used herein, a "subject" or a "patient" refers to any
mammal (e.g., a human), for example, a mammal that may be
susceptible to a disease or bodily condition such as a disease or
bodily condition associated with abnormal gene expression. Examples
of subjects or patients include a human, a non-human primate, a
cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such
as a mouse, a rat, a hamster, or a guinea pig. Generally, the
invention is directed toward use with humans. A subject may be a
subject diagnosed with a certain disease or bodily condition or
otherwise known to have a disease or bodily condition such as
cancer or an autoimmune disorder. In some embodiments, a subject
may be diagnosed as, or known to be, at risk of developing a
disease or bodily condition.
[0165] In some embodiments, the nanostructure may be administered
to a subject systemically. Systemic routes of administration,
include but are not limited to, enteral or parenteral routes.
Examples of enteral routes of administration include oral,
sublingual or rectal administration. Parenteral routes of
administration include inhalational, transdermal, or injections,
such as intravenous, intramuscular, subcutaneous, intra-arterial,
intra-articular, intra-thecal injections.
[0166] In some embodiments, a subject may be diagnosed with, or
otherwise known to have, a disease or bodily condition associated
with cancer or an autoimmune disorder.
[0167] A subject having a cancer is a subject that has detectable
cancerous cells. The cancer may be a malignant or non-malignant
cancer. Cancers or tumors include, but are not limited to, biliary
tract cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver
cancer; lung cancer (e.g. small cell and non small cell);
melanoma;
[0168] neuroblastomas; oral cancer; ovarian cancer; pancreas
cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;
testicular cancer; thyroid cancer; and renal cancer, as well as
other carcinomas and sarcomas. In one embodiment, the cancer is
hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell
leukemia, multiple myeloma, follicular lymphoma, malignant
melanoma, squamous cell carcinoma, renal cell carcinoma, prostate
carcinoma, bladder cell carcinoma, or colon carcinoma. In another
embodiment, the cancer is prostate cancer, breast cancer, renal
cancer or ovarian cancer.
[0169] The nanostructures are also useful for treating and
preventing autoimmune disease or disorder in a subject. Autoimmune
disease or disorder is a class of diseases in which an subject's
own antibodies react with host tissue or in which immune effector T
cells are autoreactive to endogenous self peptides and cause
destruction of tissue. Thus, an immune response is mounted against
a subject's own antigens, referred to as self antigens. Autoimmune
diseases or disorders include, but are not limited to, rheumatoid
arthritis, Crohn's disease, multiple sclerosis, systemic lupus
erythematosus (SLE), autoimmune encephalomyelitis, myasthenia
gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome,
pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune
hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma
with anti-collagen antibodies, mixed connective tissue disease,
polymyositis, pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus.
[0170] Major groups of vaccine adjuvants include, but are not
limited to, mineral salt adjuvants, such as alum, calcium, iron and
zirconium-based adjuvants; tensoactive adjuvants; bacteria derived
adjuvants; adjuvant emulsions; liposome adjuvants; polymeric
microsphere adjuvants; cytokines; carbohydrate adjuvants, such as
inulin-derived adjuvants, polysaccharides based on glucose and
mannose with adjuvant action, such as glucans, dextrans, lentinans,
glucomannans, levans, xylans; adjuvant formulations; mucosal
adjuvants, such as bacterial derivatives, synthetic or inactivate
antigen delivery systems, living antigen mucosal delivery systems;
adjuvants for DNA immunization; or DNA vaccines and particulate
adjuvant systems. (See e.g., Petrovsky et al., Immunol Cell Biol
(2004) 82, 488-496).
[0171] An antigen as used herein is a molecule capable of provoking
an immune response. Antigens include but are not limited to cells,
cell extracts, proteins, polypeptides, peptides, polysaccharides,
polysaccharide conjugates, peptide and non-peptide mimics of
polysaccharides and other molecules, small molecules, lipids,
glycolipids, carbohydrates, viruses and viral extracts and
muticellular organisms such as parasites and allergens. The term
antigen broadly includes any type of molecule which is recognized
by a host immune system as being foreign. Antigens include but are
not limited to cancer antigens, microbial antigens, and
allergens.
[0172] A cancer antigen as used herein is a compound, such as a
peptide or protein, associated with a tumor or cancer cell surface
and which is capable of provoking an immune response when expressed
on the surface of an antigen presenting cell in the context of an
MHC molecule. Cancer antigens can be prepared from cancer cells
either by preparing crude extracts of cancer cells, for example, as
described in Cohen, et al., 1994, Cancer Research, 54:1055, by
partially purifying the antigens, by recombinant technology, or by
de novo synthesis of known antigens. Cancer antigens include but
are not limited to antigens that are recombinantly expressed, an
immunogenic portion of, or a whole tumor or cancer. Such antigens
can be isolated or prepared recombinantly or by any other means
known in the art.
[0173] The function and advantage of these and other embodiments
will be more fully understood from the examples below. The
following examples are intended to illustrate the benefits of the
present invention, but do not exemplify the full scope of the
invention. Accordingly, it will be understood that the example
section is not meant to limit the scope of the invention.
EXAMPLES
Example 1
Self-Assembly of Single-Strand Complements of siRNA, Lipids, and
Bio-Inspired Nanoparticles Yields Anionic Vehicles for Active siRNA
Delivery
[0174] There is significant interest in developing synthetic mimics
of natural RNA delivery vehicles..sup.8 In particular, high-density
lipoproteins (HDL) are appealing because they naturally bind
endogenous RNAs, like microRNA, stabilize the single-stranded RNA
(ssRNA) to nuclease degradation, and deliver them to target cells
to regulate gene expression..sup.12,13 HDL-mediated delivery of RNA
is dependent upon target cell expression of scavenger receptor type
B-1 (SR-B1), the high-affinity receptor for mature spherical HDLs,
such as the mature HDLs that have apolipoprotein A-I (apoA-I) on
their surface..sup.12,14,15In addition to HDL, SR-B1 binds anionic
particulate ligands in a wide variety of sizes..sup.16-18 HDLs
appear to overcome hurdles to successful systemic delivery of RNA
to target cells that express SR-B1. These data have motivated the
development of synthetic mimics of HDLs that efficiently load,
stabilize, and deliver therapeutic RNAs, like siRNA..sup.19-23
[0175] In the present example, the Applicant focused on the
following properties of natural HDL that appear to enable targeted
systemic delivery of RNA, including: 1) the ability to bind and
stabilize single-stranded RNA in a scalable and modular fashion, 2)
charge reconciliation between HDL and RNA that enables nucleic acid
binding and efficient RNA delivery, and 3) active targeting of
SR-B1 for RNA delivery..sup.12 According to these design elements,
the Applicant synthesized templated lipoprotein particles (TLP)
that initiate a self-assembly process that incorporates and
stabilizes ssRNA complements of siRNA duplexes after complexation
with a cationic lipid. The use of a cationic lipid reconciles the
negative charge of ssRNAs and TLPs enabling efficient and tunable
siRNA-TLP self-assembly and function. The particles actively target
SR-B1 to potently regulate target gene expression in multiple
cancer cell lines in vitro and in an in vivo xenograft model
without inherent toxicity. Finally, with an eye toward translation
to human patients, siRNA-TLPs are modular such that specific siRNAs
targeting different disease-relevant proteins can be formulated
with pre-fabricated TLPs demonstrating the potential to
manufacture, scale-up, and provide on-demand patient-specific siRNA
therapy.
Results
Templated Lipoprotein Nanoparticles (TLP) Synthesis
[0176] Natural HDL is an anionic delivery vehicle for unmodified
nucleic acids..sup.12 As such, one goal of this work was to
fundamentally understand and develop synthetic particles that
incorporate highly unmodified single-stranded complements of a
desired siRNA duplex. The Applicant started by generating a
lipoprotein inspired particle by surface-functionalizing a 5 nm
diameter gold nanoparticle (Au NP) template with apolipoprotein A-I
(apoA-I), the defining HDL protein,.sup.24 a mixture of two
phospholipids, and cholesterol (FIG. 1A). The phospholipid
containing a di-sulfide headgroup binds to the Au NP providing a
hydrophobic surface for the assembly of the outer phospholipid and
cholesterol..sup.25-27 The outer phospholipid and cholesterol were
chosen because they favorably associate with nucleic acids and have
been shown to enhance nucleic acid delivery..sup.28
Characterization of the TLPs reveals similar shape (spherical),
size (13.+-.2 nm), and anionic charge (-42.+-.1 mV), comparable to
natural HDL..sup.27 Large batches of TLPs were synthesized,
purified, and stored for several months at 4.degree. C., providing
a platform for investigating modular addition of RNA.
DOTAP Provides Charge Reconciliation and Enables Optimal
Self-Assembly of Single-Stranded RNA to Form siRNA-TLPs
[0177] Next, conditions supporting the self-assembly of RNA with
TLPs were identified. As an initial proof-of-concept, siRNA
sequences were designed to target the androgen receptor (AR), a
well-established target for prostate cancer (Table 1)..sup.29 The
AR, and the AR signaling axis, are well-known targets in patients
suffering from advanced prostate cancer even after treatment
failure due to castration, which is the gold standard therapy for
systemic disease..sup.49 Due to the negative charge of TLPs and
RNA, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a cationic
lipid known to complex RNA through electrostatic (head group-RNA
phosphate) and hydrophobic (lipid tail-RNA nucleobase)
interactions,.sup.30 was added to mixtures of RNA in water or
1.times. phosphate buffered saline (PBS). A DOTAP:RNA molar ratio
of 40:1 provided approximately one DOTAP molecule per phosphate of
the ssRNA sequences (charge ratio .about.1:1). RNA alone or
DOTAP-RNA mixtures were added to TLPs. After overnight incubation,
TLPs mixed with DOTAP-RNA in PBS were irreversibly aggregated and
precipitated. Purified solutions of the remaining particles were
subjected to ultraviolet-visible (UV-Vis) spectroscopy, which
revealed a strong absorption band at .about.520 nm, characteristic
of disperse 5 nm diameter Au NPs..sup.31 However, only TLPs mixed
with DOTAP-RNA in water demonstrated the presence of RNA by UV-Vis
spectroscopy as indicated by a strong absorption at .about.260 nm
(FIG. 1B). These data suggest that charge reconciliation by DOTAP
is required for the self-assembly of RNA with TLPs. In addition,
because self-assembly only occurred in water, these data suggest
that RNA bound to TLPs are single-stranded.
TABLE-US-00001 TABLE 1 Individual siRNA sequences. Control, AR,
EZH2 sequences were designed using NCBI software. Uppercase letters
indicate RNA bases. Two deoxyribose bases reside on the 3' end of
each sequence and are denoted by a lower case "d" followed by a
capital letter. The 5' end of each antisense sequence contains a
5'phosphate. Fluorescent cyanine dyes are denoted by "Cy" followed
by the specific cyanine fluorophore. The sequences below correspond
to SEQ ID NOs: 1-10 from top to bottom, respectively. Sequence Name
Sequence Control Sense 5'-GCAUUCUUAAACUCGUAAAdTdT-3' Control
Antisense 5-Phosphate UUUACGAGUUUAAGAAUGCdAdA-3' Control Sense
5'-GCAUUCUUAAACUCGUAAAdTdT (Cyanine-labeled) (Cy3 or 5)-3' Control
Antisense 5-Phosphate (Cyanine-labeled) UUUACGAGUUUAAGAAUGCdAdA
(Cy5)-3' AR Sense 5'-GCCCAUUGACUAUUACUUUdTdT-3' AR Antisense
5'-Phosphate AAAGUAAUAGUCAAUGGGCdAdA-3' AR Sense (Cyanine-
5'-GCCCAUUGACUAUUACUUUdTdT labeled) (Cy3 or 5)-3' AR Antisense
5'-Phosphate (Cyanine-labeled) AAAGUAAUAGUCAAUGGGCdAdA (Cy5)-3'
EZH2 Sense 5'-GAGGUUCAGACGAGCUGAUdTdT-3' EZH2 Antisense
5'-Phosphate AUCAGCUCGTCUGAACCUCdAdA-3'
[0178] To directly test if RNA bound to TLPs were single strands or
if DOTAP facilitated siRNA duplex formation in water, thermal
denaturation experiments were performed. RNA melting transitions
were measured in water and in PBS and compared to the DOTAP-RNA
mixture. A clear melting transition was observed for the siRNA
duplex in PBS. No melting transition was observed for the RNA
sequences in water, which was similar to the data collected for the
DOTAP-RNA mixture (FIG. 7A). Thus, data demonstrate that DOTAP
facilitates the assembly of ssRNA with TLPs.
[0179] DOTAP is required for the formation of stable siRNA-TLPs.
Thus, the Applicant hypothesized that DOTAP neutralizes the
negative charge of RNA for assembly with anionic TLPs. To this end,
the zeta potential (.zeta.-potential) of solutions containing
DOTAP, RNA, and DOTAP-RNA mixtures were measured in water.
DOTAP:RNA molar ratios of 10:1, 20:1, 30:1, and 40:1 (charge ratios
.about.1:4, 2:1, 3:4, 1:1) were analyzed. Data show that,
regardless of the molar ratio, DOTAP had a positive
.zeta.-potential and free RNA had a negative .zeta.-potential.
Surprisingly, all DOTAP-RNA mixtures were negative regardless of
the DOTAP:RNA molar ratio (FIG. 7B). Collectively, these data
suggested that siRNA-TLP self-assembly results from DOTAP binding
with TLPs that enables subsequent binding of DOTAP-RNA. To validate
these assumptions, TLPs were formulated with DOTAP alone at
concentrations consistent with the previously tested DOTAP-RNA
molar ratios. Only TLPs mixed with DOTAP at the appropriate 30:1 or
40:1 concentration were stable to irreversible aggregation.
.zeta.-potential measurements show that stable DOTAP-TLPs were
positively charged (+31 and +34 mV, respectively), while all of the
DOTAP-RNA mixtures, when added to TLPs, yielded negatively charged
siRNA-TLPs (FIG. 1C). Ultimately, the collective data showed that
self-assembled siRNA-TLPs are stabilized, at least in part, by
negatively charged RNA molecules at the particle surface. Dynamic
light scattering measurements of each of the siRNA-TLPs
demonstrated a progressive increase in size with increasing
DOTAP:RNA molar ratio (FIG. 1D). UV-Vis data supported that the
progressive increase in siRNA-TLP size was due, at least in part,
to increasing amounts of RNA (FIG. 1E).
siRNA-TLPs Require TLPs to Function In Vitro
[0180] To measure siRNA-TLP function, the Applicant treated lymph
node derived prostate cancer cells (LNCaP), known to express AR,
with siRNA-TLPs made using each DOTAP:RNA molar ratio used above.
Western blotting demonstrated that the most potent reduction in AR
expression was achieved with the 40:1 siRNA-TLPs followed in
step-wise order by the 30:1, 20:1, and 10:1 molar ratios (FIG. 1F).
As a critical control, because DOTAP has been employed as a nucleic
acid transfection reagent,.sup.32,33 the Applicant explored if TLPs
are required for siRNA-TLP function. Importantly, data showed that
mixtures of DOTAP-RNA particles without TLPs (FIG. 7C) did not
function to knockdown target AR expression (FIG. 7D). Next, the
optimal RNA:TLP molar ratio was explored using the 40:1 and 30:1
DOTAP:RNA siRNA-TLPs. In all appropriate pairwise comparisons,
UV-Vis data showed that the 40:1 DOTAP:RNA and the 25:1 RNA:TLP
molar ratios were optimal for RNA assembly to siRNA-TLPs (FIG. 7E).
Treatment of LNCaP cells with each of the particles demonstrated
dose-dependent reductions in AR expression consistent with the
amount of RNA associated with TLPs (FIG. 7F). In short, the
self-assembly of siRNA-TLPs can be tailored to potently regulate
target gene expression, and the optimal siRNA-TLP resulted from the
use of 40:1 (DOTAP:RNA) and 25:1 (RNA:TLP) molar ratios,
respectively.
siRNA-TLPs Characterization
[0181] Multiple modalities were used to characterize optimized
siRNA-TLPs. Fluorescently labeled apo A-I, cholesterol, DOPC,
DOTAP, and RNA were used to quantify the amount of each of these
molecules in siRNA-TLPs (FIG. 2A). In addition, western blotting
was performed to confirm apo A-I presence on the TLP and siRNA-TLP
(FIG. 8). Transmission electron microscopy (TEM) was used to obtain
images of TLPs and siRNA-TLPs. As shown in FIG. 2B, there is clear
indication of TLP surface functionalization. TEM images of the
siRNA-TLPs revealed spherical particles that show TLP self-assembly
with solid alternating layers of DOTAP and RNA. Further, because
siRNA-TLPs are formulated with a mixture of ssRNAs, the Applicant
sought to conclusively demonstrate that siRNA-TLPs incorporate and
require each complement of the siRNA duplex for optimal
function..sup.34,35 Particles were synthesized with only the sense
(S) or antisense (AS) RNA sequence of the siRNA pair and their
function was compared to siRNA-TLPs synthesized with both sequences
(DS). Only siRNA-TLPs synthesized with both sequences of the siRNA
pair functioned to reduce AR expression (FIG. 2C). In addition, the
Applicant determined if function could be achieved by mixing
siRNA-TLPs synthesized with only the S or AS RNA sequence.
Ultimately, mixed siRNA-TLPs functioned similarly to siRNA-TLPs
synthesized with both sequences (FIG. 2D). These data support that
optimal gene regulation requires delivery of both sequences of the
siRNA pair, and that siRNA can be delivered as component single
strands on a single or mixed population of siRNA-TLPs.
siRNA-TLPs In Vitro Function and Modular Design
[0182] In vitro siRNA-TLP efficacy was determined over time. LNCaP
cells were treated with siRNA-TLPs for 24, 48, 72, and 96 hours at
Au NP concentrations of 20, 10, and 5 nM. Western blot data
demonstrated that siRNA-TLPs reduced AR protein expression in a
time and dose-dependent fashion (FIG. 3A). In addition, siRNA-TLPs
reduced AR mRNA expression measured using qRT-PCR (FIG. 9A). In
addition to LNCaP cells, AR targeted siRNA-TLPs were tested in a
cell culture model of advanced prostate cancer known to be
resistant to a common AR blocker, enzalutamide (MDV3100)..sup.36 As
in LNCaP cells, siRNA-TLPs reduced AR expression in dose response
(FIG. 9B).
[0183] The AR is important for prostate cancer cell
survival..sup.37 Thus, LNCaP cell viability was tested after
treatment with siRNA-TLPs. The AR siRNA-TLPs reduced LNCaP cell
viability over time and with a clear dose-response (FIG. 3B).
Control siRNA-TLPs did not reduce cell viability demonstrating no
apparent toxicity and siRNA specificity. In addition, serial images
were collected over six days after cell treatment. Using imaging
software and analysis, data showed that cell confluence was reduced
in the presence of AR-TLPs over time, and images taken at 165 hours
confirmed reduced cell confluence (FIG. 3C).
[0184] Modular addition of desired siRNA sequences with TLPs would
enable rapid and facile targeting of different proteins and protein
variants relevant to cancer in individual patients. Thus, in
addition to targeting AR, siRNA-TLPs targeting EZH2, a histone
lysine N-methyltransferase enzyme, known for its oncogenic
relevance in prostate cancer and other malignancies such as breast,
renal, and ovarian cancers, were synthesized..sup.38-41 UV-Vis data
demonstrated equivalent RNA assembly to EZH2-, AR-, and Ctrl-TLPs
(FIG. 9C). Data showed that siRNA-TLPs reduce EZH2 expression in
LNCaP, enzalutamide resistant LNCaP cells, MDA-MB-231 (breast
cancer), 786-O (renal cell carcinoma), and OvCar3 (ovarian cancer)
in a dose-dependent fashion (FIG. 9D).
siRNA-TLPs Taken Up by Cell and Targeted to SR-B1
[0185] Next, to study cellular internalization of siRNA-TLPs, LNCaP
cells were treated with siRNA-TLPs synthesized with
fluorophore-labeled RNA. Ctrl- and AR-TLP uptake and cell
confluence were captured with imaging software after treating cells
for 165 hours. Data showed an apparent two-phase uptake of
siRNA-TLPs (FIG. 4A and FIG. 10A). Representative images of the
second uptake phase are presented in FIG. 4B.
[0186] To explore if the high-affinity HDL receptor, SR-B1, was
required for siRNA-TLP function, western blotting was performed to
confirm SR-B1 expression in all of the cultured cells used in this
study (FIG. 10B). Of note, human prostate cancer, breast cancer,
and renal cell carcinoma, among others, have been shown to
overexpress SR-B1..sup.21,42-44 In LNCaP cells, optimal SR-B1
knockdown using conventional LIPOFECTAMINE.RTM. RNAiMAX was tested
in order to ensure maximal SR-B1 reduction at the time of siRNA-TLP
addition (48 hours), and that SR-B1 knockdown was maintained for
the duration of the experiment to subsequently test siRNA-TLP
function (96 hours) (FIG. 10C). Thus, conventional SR-B1 knockdown
was performed in LNCaP cells followed by addition of siRNA-TLPs, or
controls, targeting AR. Following treatments, data showed that
conventional knockdown of SR-B1 partially reduces AR expression,
which is a finding supported by the published literature but
requires further study..sup.45,46 Quantitative analysis showed that
AR-TLPs added after SR-B1 knockdown do not function to reduce AR
expression. On the other hand, conventional delivery of siRNA
targeting AR functioned to reduce AR expression in the presence of
SR-B1 knockdown (FIG. 4C). These data clearly show that active
targeting of SR-B1 is required for siRNA-TLP function.
Nuclease Stability of siRNA-TLPs Natural HDLs stabilize RNA from
nuclease degradation in blood and actively deliver RNA to target
cells.sup.12,15. To demonstrate the stability of RNA assembled in
siRNA-TLPs, free siRNA and siRNA-TLPs were exposed to RNase A and
human plasma. Gel electrophoresis was used to determine RNA
stability. RNA assembled in siRNA-TLP was protected from
degradation after exposure to RNase A (FIG. 5A) and human plasma
(FIG. 5B). Further, the Applicant tested the function of siRNA-TLPs
after incubation in human serum, and if siRNA-TLPs directly deliver
RNA to target cells or if RNA is exchanged with native HDL in serum
and indirectly delivered. See FIG. 11 for experimental design.
Treatment of LNCaP cells with siRNA-TLPs after incubation in human
serum demonstrated reduced AR expression to the same level as
siRNA-TLPs directly added to cultured cells. Native HDL isolated
from serum after incubation with siRNA-TLPs had no effect on AR
expression (FIG. 5C). In addition, siRNA-TLPs were incubated in
human serum for 2, 4, 6, 10, and 24 h at 37.degree. C. The
siRNA-TLP that had been incubated in serum were then added to
cultured LNCaP cells and, following 48 hours of incubation, AR
expression was measured. ARTLPs continue to reduce AR expression,
even after incubation in serum for 10 hours (Data not shown). These
data show that siRNA is stable to degradation and is directly
delivered to target cells by siRNA-TLPs. Efficacy of siRNA-TLPs In
Vivo
[0187] Next, the in vivo efficacy of siRNA-TLPs was investigated.
Subcutaneous LNCaP xenografts were established in male nude mice. A
total of thirteen treatments were administered via tail vein and
tumor volumes were recorded over a 26-day period (FIG. 12A). Mice
treated with AR-TLPs (0.7 mg siRNA/kg) showed a significant
reduction in tumor volume (FIG. 6A) and percent change in tumor
volume over time (FIG. 6B). Targeted siRNA-TLP delivery to tumor
tissue was assessed using inductively coupled plasmon mass
spectrometry (ICP-MS) to quantify Au NPs, and confocal fluorescent
microscopy to visualize RNA. ICP-MS data showed the presence of
gold in tumor tissue after the treatment regimen (FIG. 6C).
Following a single dose of siRNA-TLPs synthesized with
fluorescently labeled RNA [sense (Cy3) and antisense (Cy5)], tumor
tissue was obtained 24 hours after the injection and confocal
fluorescent microscopy confirmed RNA in tumor tissue (FIG. 6D).
Known consequences of systemic AR knockdown in mice include a
reduction in hematocrit and neutrophils..sup.47 Indeed, a
significant reduction in hematocrit (HCT) (FIG. 6E), white blood
cells (WBC) (FIG. 6F), and neutrophils (FIG. 6F) was measured in
AR-TLP treated mice. Consistent with published literature,.sup.47
no change in hemoglobin (HGB) or platelets (PLT) was measured (FIG.
6E).
[0188] Body weights were maintained and consistent across all study
groups (FIG. 12B). To determine any off-target side effects,
tissues obtained from representative organs were analyzed by
hematoxylin and eosin (H&E) staining followed by microscopic
examination, and standard serum parameters were measured. Data show
no histopathologic changes in the examined organs (FIG. 12C). No
untoward alterations were observed in serum electrolyte or
cholesterol levels and kidney function was normal (FIG. 13A). In
addition, markers of liver function were normal (FIG. 13B).
Ultimately, data collected in mice treated with water and Ctrl-TLPs
revealed no side effects confirming a lack of toxicity of the
vehicle and control RNA.
Discussion
[0189] With a goal of synthesizing efficient in vitro and in vivo
siRNA delivery vehicles, the Applicant adhered to a set of design
rules that appear critical for natural HDL to deliver nucleic
acids, including: 1) preference for ssRNA, 2) charge
reconciliation, and 3) active targeting. A pre-synthesized TLP was
added to a mixture of DOTAP and ssRNA. Resulting self-assembled
siRNA-TLPs are a hybrid between bio-inspired lipoprotein
nanoparticles and lipid-RNA structures. Uniquely, delineation of a
synthetic method that produces highly efficient siRNA delivery
vehicles with a preference for ssRNA complements of a siRNA duplex
pair has not been reported. siRNA-TLPs are highly uniform and their
synthesis and function are tailorable based upon the appropriate
addition of TLP, RNA, and DOTAP. The Applicant clearly shows that
the TLPs are absolutely required for siRNA-TLP self-assembly (FIG.
1D and FIG. 7C), active targeting (FIG. 4C), and function (FIG. 1F
and FIG. 7D). Presumably, any siRNA duplex pair can be used for
self-assembly of siRNA-TLPs, and the particles work to regulate
target gene expression in multiple cancer cell types that express
SR-B1 (FIG. 10B). In vivo efficacy was achieved in a prostate
cancer xenograft model suggesting translational potential.
Proof-of-concept in vivo data was gathered using an every-other-day
dosing regimen. Significant AR knockdown was observed for up to 96
h in the in vitro model. Also, since gold is biocompatible and the
siRNA-TLPs contain a gold core, our data demonstrate no off-target
toxicity after 13 intravenous doses of the siRNA-TLPS.
[0190] Approaches to develop synthetic versions of HDL are variable
and design rules that enable RNA delivery by native or synthetic
HDLs are not well understood. Importantly, native HDLs bind and
deliver ssRNA, which is not in accordance.sup.12 with the delivery
of duplex siRNAs, which is where most delivery strategies are
focused. The data described herein demonstrate that formulating
ssRNAs of a siRNA pair, either in individual or separate
siRNA-TLPs, significantly reduce target gene expression. The
findings described herein provide evidence that ssRNA complements
of a siRNA duplex can be formulated for systemic siRNA delivery.
The data described herein also shows that miRNAs adsorbed to native
HDLs localize to the particle surface..sup.12 Similarly, the
siRNA-TLP is formed through a self-assembly process that localizes
the RNA to the surface of the particle, and does not encapsulate
the RNA inside of a lipid or polymer particle. RNA that is
localized to the surface of the siRNA-TLP remains stable and is
efficiently delivered to cells for target gene knockdown. RNA
stability may result from the anionic nature of the particle and/or
because of the solid nature of the particle. The function of
siRNA-TLPs may also be enhanced because the RNA is more freely
available to the host cell, not encapsulated. Finally, siRNA-TLPs
are negatively charged and require the TLP for efficient siRNA
delivery and knockdown of target gene expression. Although
particles made without the TLP have a charge similar to particles
made with the TLP, the particles do not regulate target gene
expression. Thus, anionic particles are, actually, poor delivery
vehicles for RNA unless the particle is inherently targeted or
contains a moiety that enables target cell binding and RNA uptake
into the cytoplasm.
[0191] In short, the Applicant reports an siRNA delivery vehicle
that delivers highly unmodified single strand RNA self-assembled in
an anionic particle that is actively targeted. These findings,
inspired by native HDL, may enable new approaches for the
development of potent and modular siRNA delivery vehicles for
personalized medicine.
Methods
[0192] Synthesis of Templated Lipoprotein Particles (TLP) and
siRNA-TLPs
[0193] For TLP synthesis, an aqueous solution of citrate stabilized
gold nanoparticles (Au NP) (80 nM, 5.+-.0.75 nm, Ted Pella, Inc.)
was mixed with a 5-fold molar excess of purified human apoA-I (400
nM, Meridian Life Sciences, >95% pure by SDS PAGE) in a glass
vial. The Au NP/apo A-I mixture was incubated overnight at room
temperature (RT) on a flat bottom shaker at low speed. Next, a 1:1
ratio of two phospholipids:
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)pr-
opionate] (PDP-PE) and 1,2-dioleoyl-sn-glycero-3-phophocholine
(DOPC) (Avanti Polar Lipids), each dissolved in chloroform
(CHCl.sub.3, 1 mM), are added to the Au NP/apo A-I solution in
250-fold molar excess to the Au NP. PDP-PE was added first and the
solution was vortexed prior to adding DOPC. Next, cholesterol
dissolved in CHCl.sub.3 (1 mM, Sigma Aldrich) was added in 25-fold
molar excess to the Au NP. The mixture was vortexed and briefly
sonicated (.about.2 mins) causing the solution to become opaque and
pink in color. The resulting mixture was gradually heated to
.about.65.degree. C. with constant stirring to evaporate CHCl.sub.3
and to transfer the phospholipids onto the particle surface and
into the aqueous phase (.about.20 minutes). The reaction was
complete when the solution returned to a transparent red color. The
resultant TLPs were incubated overnight at RT and then purified via
centrifugation (15,870.times.g, 50 min). The supernatant was
removed and the resulting purified and concentrated TLPs were
combined into a single vial. TLPs were stored at 4.degree. C. until
use. The concentration of the TLPs was measured using UV-Vis
spectroscopy (Agilent 8453) where Au NPs have a characteristic
absorption at .lamda..sub.max=520 nm, and the extinction
coefficient for 5 nm Au NPs is 9.696.times.10.sup.6
M.sup.-1cm.sup.-1.
[0194] To synthesize siRNA-TLP, RNA and
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were first mixed.
Individual sense and antisense RNA sequences of the AR, control
(Ctrl), or EZH2 siRNA (Integrated DNA Technologies) were
re-suspended in nuclease free water (500 .mu.M, final). Complement
pairs were then mixed in nuclease free water at a concentration
enabling direct addition to TLPs (100 nM) at 25-fold molar excess
of each RNA sequence (2.5 .mu.M, final per RNA sequence). An
ethanolic (EtOH) solution of DOTAP was then added to the RNA
mixture to desired DOTAP:RNA molar ratios. In each case the
resulting solvent ratio was 9:1, EtOH:water (v/v). The mixture of
DOTAP and RNA was briefly sonicated and vortexed (.times.3) and
then incubated at RT for 15 minutes prior to addition to a solution
of TLPs in water. After the DOTAP-RNA mixture was added to the
TLPs, the solvent mixture was 9:1, water:EtOH (v/v). This solution
was incubated overnight at RT with gentle shaking on a flat bottom
shaker at low speed. Resulting siRNA-TLPs were purified via
centrifugation (15,870.times.g, 50 min), the supernatant with
unbound starting materials was removed, and the pellets were
combined in a single tube to concentrate the siRNA-TLPs. The
concentration of the siRNA-TLPs was calculated as described for
TLP. For siRNA-TLPs, a strong absorption at .lamda..sub.max=260 nm
confirmed the presence of RNA. For particles synthesized with only
one strand of the siRNA duplex pair, the synthetic procedure
proceeded similarly; however, twice the amount of RNA was added to
the TLPs (5 .mu.M, final).
Single vs. Double Strand RNA Assembly with DOTAP and TLPs
[0195] To investigate if single stranded RNA complements or double
stranded siRNA duplexes assembled with TLPs, and the requirement
for DOTAP, siRNA-TLPs were synthesized per the above protocol.
However, the synthesis was carried out with and without DOTAP and
using either water or 1.times. PBS as the aqueous solvent for
siRNA-TLP assembly.
Thermal Denaturation Experiments
[0196] Thermal denaturation experiments were performed to measure
RNA melting transition temperatures (T.sub.m) between
25.degree.-90.degree. C. using an Agilent 8453 UV-Vis
spectrophotometer equipped with a Peltier temperature controller.
Solutions without RNA, but only with water, EtOH, water:EtOH (v/v),
and/or DOTAP had no appreciable absorbance at 260 nm (data not
shown).
Quantification of RNA, apo A-I, DOTAP, DOPC and Cholesterol
[0197] To confirm the presence of each of the molecules used to
synthesize TLPs and siRNA-TLPs, and to quantify the amount present,
the Applicant used molecules labeled with molecular fluorophores to
synthesize TLPs and siRNA-TLPs according to the previously
described synthetic method. After purification, the amount of each
of the fluorescent molecules with reference to standard titration
curves developed with each of the fluorescently labeled molecules
was measured. More specifically, the number of RNA strands per
siRNA-TLP was quantified by incorporating 3' end-labeled (Cy5) RNA
sequences. For Cy5, measurements were obtained using a Biotek
Synergy 2 fluorescent plate reader using Ex=620/40 nm and Em=680/30
nm. Apo A-I on the particle surface was confirmed by western
blotting. For quantification, apoA-I was labeled with Alexa-488
using a commercially available protein labeling kit (Invitrogen)
according to the instruction provided by the manufacturer.
Measurements were taken using a Biotek Synergy 2 fluorescent plate
reader using Ex=485/20 nm and Em=528/20 nm. DOTAP, DOPC, and
cholesterol were quantified by incorporating nitrobenzoxadiazole
(NBD)-fluorescent analogs of each of the molecules (Avanti Polar
Lipids) into the particle synthesis at a 10% dilution. All samples
were measured in a 1:1 mixture of EtOH:water (v/v), including the
standards. Measurements were taken using a Biotek Synergy 2
fluorescent plate reader using the same settings as for apo A-I
measurements.
Dynamic Light Scattering and .zeta.-Potential Measurements
[0198] Hydrodynamic diameter and 4-potential measurements were
performed using TLP or siRNA-TLP in water (10 nM). Triplicate
measurements were made under 173.degree. backscatter setting with
10 runs, 30 sec/run/measurement. RNA and RNA-DOTAP mixtures were
measured using a concentration of 5.mu.M RNA. Mixtures containing
DOTAP were made where the final concentration of DOTAP=100 .mu.M,
75 .mu.M, 50 .mu.M, and 25 .mu.M to achieve the 40, 30, 20, 10 fold
excesses to RNA, respectively. Particle free measurements were
taken in 9:1 water:ethanol (v/v) solutions. Measurements were made
using a Zetasizer Nano ZS (Malvern). The hydrodynamic diameter data
are represented using the number function.
UV-Vis Spectroscopy
[0199] A UV-Vis spectrophotometer (Agilent 8453) was used to
measure the concentration and stability of NPs to aggregation, The
concentration of solutions of Au NPs was determined by measuring
the absorbance at .about.520 nm (extinction coefficient
9.696.times.10.sup.6 M.sup.-1cm.sup.-1; Ted Pella). Disperse
colloidal gold nanoparticles strongly absorb and scatter light at
.about.520 nm (A.sub..lamda.max). The molar concentration of NPs in
the preparations was calculated using the formula:
(A.sub..lamda.max.times.dilution factor)/9.696.times.10.sup.6
M.sup.-1cm.sup.-1. UV-Vis spectrophotometry was also used to
determine RNA loading using the strong absorbance of RNA at
.about.260 nm.
Cell Culture
[0200] The human lymph node derived prostate cancer LNCaP clone FGC
(fast growing colony), androgen receptor positive, androgen
sensitive, was obtained from American Type Cell Culture (ATCC).
LNCaP cells were grown in RPMI 1640 medium (Invitrogen),
supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin (Invitrogen). The enzalutamide resistant
LNCaP cell line (MDV3100) was a generous gift from Dr. Donald
Vander Griend's laboratory (University of Chicago). LNCaP MDV3100
resistant cells were cultured using the same conditions as LNCaP
cells; however, 10 .mu.M (final) MDV3100 was added to the growth
medium. A375 cells (human malignant melanoma) and MDA-MB-231 cells
(human triple negative breast cancer) were obtained from ATCC. Both
cell lines were cultured in DMEM (Invitrogen), supplemented with
10% fetal bovine serum (FBS), 1% penicillin/streptomycin
(Invitrogen), and 1.times. Glutamax. 786-O (human renal cell
carcinoma) cells were obtained from ATCC and grown under the same
conditions as the LNCaP cells. OvCar3 (human ovarian
adenocarcinoma) cells were obtained from ATCC and cultured in RPMI
1640 medium (Invitrogen), supplemented with 10% FBS and 1%
penicillin/streptomycin (Invitrogen), and 1% insulin. In general,
cells were cultured in T75 flasks and plated into appropriate
dishes (e.g. 6-well, 24-well, 96-well) 24-48 hours prior to
experiments. All cells were incubated at 37.degree. C. in a
humidified 5% CO.sub.2 incubator.
Conventional siRNA Transfection
[0201] Prior to all cell transfections, the cell culture media was
changed to fresh growth medium. TLPs or RNA-TLPs were directly
added to the cultured cells. For comparisons against conventional
transfection reagents, LIPOFECTAMINE.RTM. RNAiMax transfections
were used to treat cells with Ctrl, AR, EZH2, or SR-B1 siRNAs
according to the protocol provided by the manufacturer
(Invitrogen). Briefly, siRNA was mixed with RNAiMax in OpitMEM
media to achieve final concentrations of siRNA ranging from 6-12
nM. siRNA RNAiMAX transfections were optimized to achieve maximal
target gene knockdown at 48 hours.
Western Blotting
[0202] Cells were plated at 1.5.times.10.sup.5 cells/well for all
western blotting experiments. Cells were harvested 48 hours
following treatment unless otherwise specified. To harvest protein
lysate, cells were washed in ice cold 1.times. PBS and lysed in
M-PER (Mammalian Protein Extraction Reagent) supplemented with
1.times. protease and phosphatase inhibitors (Thermo Scientific).
After protein isolation, the bicinchoninic acid assay (BCA) assay
was used to quantify total protein. Protein absorbance was measured
at 562 nm (BioTek, Synergy 2). Protein concentrations were
normalized and then mixed with 4.times. Laemmli loading buffer
containing .beta.-mercaptoethanol (Bio-Rad) and boiled for 10
minutes at 100.degree. C. prior to gel loading. Cellular proteins
were resolved by 4-20% SDS-PAGE (200 volts, 32 minutes) and
transferred to a 0.2 .mu.m PVDF membrane (65 volts, 1 hour)
(Bio-Rad). Membranes were blocked in 5% milk in Tris buffered
saline (TBS) and Tween-20 (0.1%) for 1 hour prior to antibody (Ab)
addition. Membranes were incubated overnight at 4.degree. C. using
rabbit polyclonal antibodies directed against androgen receptor
(1:1000, Santa Cruz), beta actin (1:2000, Cell Signaling), EZH2
(1:1000, BD Biosciences), apo A-I (1:1000, Abcam), or SR-BI
(1:2000, Abcam). Goat anti-rabbit or goat anti-mouse IgG-HRP
(1:2000, Bio-Rad) were used as secondary antibodies. The secondary
Ab was applied at RT for 30-60 minutes. Blots were washed
(3.times.) in TBST (0.1% Tween-20) for 10 minutes/wash prior to
protein detection. Proteins were detected using enhanced
chemiluminescence (ECL) detection (GE Healthcare Life Sciences) on
x-ray film using Konica SRX101A X-Ray Film Processor (MXR Source
One Healthcare). Densitometry measurements for western blot
analysis were made using ImageJ software.
Transmission Electron Microscopy
[0203] A pair of tweezers was used to hold a 200 mesh carbon coated
copper grid (Electron Microscopy Sciences) while a 5 .mu.L drop of
particles (250 nM) was pipetted onto the grid. The drop was allowed
to adsorb to the grid for 10 minutes and the excess solution was
wicked away with filter paper. Grids were stained with 5 .mu.L of
4% uranyl acetate (UA) for three minutes. Excess UA was wicked off
and the staining was repeated two times. The remaining UA was
wicked off and the samples were allowed to dry for 10 minutes. For
some transmission electron microscopy experiments, a pair of
tweezers was used to hold a 200 mesh carbon coated copper grid
(Electron Microscopy Sciences). An equal volume of particles
(150.times.10-9 m) and a 2% uranyl acetate solution were mixed. 10
.mu.L of this solution was added to the grid and allowed to sit for
20 s. The excess volume was removed with a piece of filter paper
and the grid was allowed to dry. TEM images were taken with a FEI
Tecnai Spirit G2 transmission electron microscope operating at 80
kV. In UA-stained samples, phospholipids are visible as white rings
around the electron dense NP. Nikon Elements Imaging Software was
used to analyze transmission electron microscopy images to measure
the size of TLP, Ctrl-TLP, and AR-TLP. The measurements were taken
from three TEM images for each sample, combined, and plotted as
histograms using GraphPad Prism.
Cell Viability Assay
[0204] Cells were plated at 3.times.10.sup.4 cells/well in 96-well
plates 48 hours prior to particle treatment. LNCaP cells were
treated with increasing concentrations of particles 1, 5, 10, 20
nM. Cell viability was measured at 24, 48, 72, 96 hours using
CELLTITER 96.RTM. AQ.sub.ueous One Solution Cell Proliferation
Assay (Promega) according to the instructions provided by the
manufacturer. Absorbance was measured at 490 nm (Biotek Synergy
2).
Real-Time Measurements of Particle Uptake and Confluence
[0205] LNCaP cells were plated at 6.times.10.sup.4 cells/well in
96-well plates 48 hours prior to treatment. LNCaP cells were
treated with 20 nM siRNA-TLPs (Ctrl and AR) where the RNA was
labeled with Cy5 per the above protocol. Cell confluence and
particle uptake were captured for 165 hours using an INCUCYTE.RTM.
Zoom system and software. Cy5 fluorescence was captured using
light-emitting diodes with Ex=585/20 nm and Em=524/20 nm. The
entire experiment was performed at 37.degree. C. in a 5% CO.sub.2
humidified incubator. siRNA-TLP uptake was measured by fluorescent
labeled (Cy5) RNA bound to the siRNA-TLPs.
Real-Time qRT-PCR
[0206] Total RNA was isolated from LNCaP cells using RNeasy mini
kit (Qiagen). Reverse transcription was preformed using 0.2m of RNA
and TaqMan Reverse Transcription Kit following the protocol
provided by Life Technologies. Real-time qRT-PCR was preformed
using TaqMan PCR Master Mix and TaqMan androgen receptor (Catalog
#Hs0171172_m1) and .beta.-actin primers/probes for relative mRNA
quantification (Life Technologies). qRT-PCR analysis was carried
out using an ABI Prism Model 7900HT. Data was analyzed using the
comparative C.sub.t method using .beta.-actin as an endogenous
control.
Human Serum and Plasma Isolation and Lipoprotein Depletion
[0207] Following IRB approval and informed consent, blood samples
were collected via venipuncture from an antecubital vein from a
healthy donor into a serum separator tube (Becton Dickinson). Serum
was isolated by centrifugation (1,000.times.g for 10 minutes) at
4.degree. C. Serum was aliquoted (250 .mu.L) and stored at
-20.degree. C. prior to use. To generate serum depleted of
lipoproteins other than HDL, serum was first mixed with a solution
of polyethylene glycol [PEG8000, 20% (v/v) solution in 200 mM
glycine, pH 7.4] at a 10:4 serum:PEG solution ratio. The sample was
gently mixed and incubated at RT for 20 minutes. Next, the samples
were centrifuged for 30 minutes (12,700.times.g) at 4.degree. C.
The supernatant, consisting of albumin and HDL was set aside and
the pellet was discarded. Human plasma samples were collected
similarly to serum; however, blood was collected into heparinized
tubes (Becton Dickinson) and then centrifuged for 15 minutes
(2,000.times.g) at 4.degree. C. Resulting plasma was aliquoted (250
.mu.L), used immediately, or stored at -20.degree. C.
siRNA-TLP Function and Stability Post Serum Incubation
[0208] TLPs and siRNA-TLPs were incubated with human serum for 1
hour at 37.degree. C. To determine if siRNA exchanged to natural
HDL after incubation, human HDL was separated from particles using
the isolation assay described above. The albumin/human HDL mixture
was directly added to plated LNCaP cells. In addition, experiments
were conducted by adding siRNA-TLPs to human serum, incubating for
1 hour, and then directly adding the mixture to LNCaP cells. See
FIG. 11 for experimental design.
Nuclease Protection Assay and siRNA-TLP Stability
[0209] The stability of RNA in siRNA-TLPs was compared to free RNA
in the presence of RNase A (Bio-Rad). siRNA-TLPs (.about.1 .mu.M
RNA) and free RNA sequences (1 .mu.M) were exposed to 2.0 ng/.mu.L
RNase A for 0, 5, 15, 30, and 60 minutes at 37.degree. C. Reactions
were quenched by addition of 2.times. RNA loading buffer [90%
formamide, 10% glycerol, 1% SDS (w/v), and bromophenol blue] and
heated to 65.degree. C. for 3 minutes. Samples were transferred to
a pre-run polyacrylamide gel (25% polyacrylamide with 5% stacking
layer) and subjected to electrophoretic separation (400 volts for
30 minutes). The gel was stained with ethidium bromide and imaged
using ChemiDoc System (Bio-Rad).
siRNA-TLP Stability in Human Plasma
[0210] A Cy3 labeled RNA was used to measure the physiologic
stability of the siRNA-TLP with comparison to free RNA sequences.
siRNA-TLPs (400 nM siRNA-TLP, 1.6 .mu.M RNA of each strand) and
free RNA (1.6 .mu.M of each sequence) were exposed to 50% human
plasma for 0, 5, 15, 30, and 60 minutes at 37.degree. C. Reactions
were quenched by the addition of 2.times. RNA loading buffer [90%
formamide, 10% glycerol, 1% (SDS w/v), and bromophenol blue], and
heated to 65.degree. C. for 3 minutes. The samples were transferred
to a pre-run polyacrylamide gel and underwent electrophoresis. The
gel consisted of 25% polyacrylamide with a 5% stacking layer and
ran at room temperature at 400 V for 30 minutes. The Cy3-RNA was
detected using a G:BOX Chemi XT4 Imager (Synoptics).
In Vivo Efficacy Studies
[0211] All animal experiments were approved by the Institutional
Animal Care and Use Committee (IACUC) of Northwestern University,
and the studies were performed in accordance with institutional and
national guidelines and regulations. LNCaP flank tumor xenografts
were established in 6 to 8 week male athymic nude mice by
subcutaneous implantation of 1.times.10.sup.6 cells. When tumors
reached .about.500 mm.sup.3, mice were randomized to three
treatment groups (i.e. water, Ctrl-TLP, and AR-TLP), n=8
mice/group. Body weights were measured over the course of the study
on a standard laboratory scale. Mice were treated every other day
with Ctrl-TLPs or AR-TLPs (100 .mu.L, 2 .mu.M siRNA-TLP, .about.0.7
mg siRNA/kg) or 100 .mu.L of water for a total of 13 treatments.
Treatments were administered via tail vein. When tumors reached
.about.2000 mm.sup.3 in the control groups the study was
terminated. Whole blood was obtained by cardiac puncture and
collected in heparinized blood collection tubes. Blood samples were
separated and subjected to complete blood count and serum chemistry
analysis. Tissues (liver, lung, kidney, spleen, heart, brain,
adrenal, testes, small intestine, and tumor) were harvested for
inductively coupled plasmon mass spectrometry (ICP-MS) and
hematoxylin and eosin (H&E) analysis. Fresh tissue sections for
ICP-MS were stored immediately at -80.degree. C. until prepped for
ICP-MS analysis. ICP-MS analysis was conducted at the Chemistry of
Life Processes Core Facility at Northwestern University after
digestion of the tissues and Au NPs using strong acid. The amount
of Au NPs was quantified with reference to calibrated additional
standards. Tissues harvested for H&E were immediately fixed in
10% formalin in PBS. Within 48 hours the tissues were prepped for
paraffin embedding and sectioning. Tissue sectioning and H&E
staining was performed by the Mouse Histology and Phenotyping
Laboratory (MHPL) at Northwestern University. Images of the H&E
stained tissues were obtained using a Nikon Eclipse TE2000-U and
SPOT imaging software. All images were obtained at 10.times.
magnification.
Tumor Volume Measurements, Blood Analysis, and Serum Chemistry
[0212] Tumors were measured using digital calipers over the course
of the experiment. The volume was calculated using the equation
[Volume=length.times.width/2)]..sup.48 Whole blood collected at
time of sacrifice was analyzed for complete blood cell counts using
a Hemavet 950FS (Drew Scientific). Plasma was obtained from an
aliquot of whole blood and a complete chemistry panel, cholesterol,
and liver function analysis was performed by Charles River.
Confocal Fluorescence Microscopy of Tumor Tissues
[0213] AR-TLP and Ctrl-TLP were synthesized with Cy3-labeled sense
RNA and Cy5-labeled antisense RNA according to the protocol
described above. Mice with established LNCaP xenografts (2000
mm.sup.3) (see above) were treated with a single tail vein
injection of 100 .mu.L of 2 .mu.M (siRNA-TLP) AR-TLP-Cy3/5 or
Ctrl-TLP-Cy3/5, or 100 .mu.L of water. Mice were sacrificed 24
hours following the injection and LNCaP xenograft tumors were
harvested and then embedded in optimal cutting temperature (O.C.T)
matrix and immediately frozen on a block of dry ice. Tissues were
sectioned (10 .mu.m), mounted on glass slides, and counterstained
with DAPI diluted (1:50,000) in 1.times. PBS. Fluoromount-G
(Southern Biotech) mounting media and coverslips were applied prior
to imaging. Images were acquired using a Nikon C2+ laser scanning
confocal microscope (Northwestern University Center for Advanced
Microscopy) and analyzed by Nikon Elements software and ImageJ.
Images were taken at 60.times. magnification. Laser settings were
consistent across samples.
Statistical Analysis
[0214] Data are expressed as means.+-.standard deviation. Blood
cell count comparison analyses were performed using unpaired
two-tailed t-test with Welch's correction using GraphPad Prism
software. An effects model was used to compare changes in tumor
volume over time within groups and changes between groups. Group
and time were fixed effects and animal was a random effect. The
model took into account the repeated measures across animals.
Post-hoc comparisons were done using Tukey's method. Statistical
significance was considered significant for P.ltoreq.0.05; *
denotes P.ltoreq.0.05, ** P.ltoreq.0.01, ** P.ltoreq.0.001, ****
P.ltoreq.0.0001.
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[0257] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used.
[0258] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0259] Furthermore, the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the invention,
or aspects of the invention, is/are referred to as comprising
particular elements and/or features, certain embodiments of the
invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein. It is also noted that the terms "comprising"
and "containing" are intended to be open and permits the inclusion
of additional elements or steps. Where ranges are given, endpoints
are included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0260] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0261] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0262] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0263] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
10121DNAArtificial SequenceSynthetic Polynucleotide 1gcauucuuaa
acucguaaat t 21221DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(1)..(1)Modified by phosphate 2uuuacgaguu
uaagaaugca a 21321DNAArtificial SequenceSynthetic
Polynucleotidemisc_featureModified by Cy3 or Cy5 3gcauucuuaa
acucguaaat t 21421DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(1)..(1)Modified by
phosphatemisc_feature(21)..(21)Modified by Cy5 4uuuacgaguu
uaagaaugca a 21521DNAArtificial SequenceSynthetic Polynucleotide
5gcccauugac uauuacuuut t 21621DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(1)..(1)Modified by phosphate 6aaaguaauag
ucaaugggca a 21721DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(21)..(21)Modified by Cy3 or Cy5
7gcccauugac uauuacuuut t 21821DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(1)..(1)Modified by
phosphatemisc_feature(21)..(21)Modified by Cy5 8aaaguaauag
ucaaugggca a 21921DNAArtificial SequenceSynthetic Polynucleotide
9gagguucaga cgagcugaut t 211021DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(1)..(1)Modified by phosphate
10aucagcucgt cugaaccuca a 21
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