U.S. patent application number 16/470506 was filed with the patent office on 2020-03-19 for co-delivery of nucleic acids for simultaneous suppression and expression of target genes.
The applicant listed for this patent is The Brigham and Women's Hospital, Inc.. Invention is credited to Omid Farokhzad, Jinjun Shi, Xiaoding Xu.
Application Number | 20200085758 16/470506 |
Document ID | / |
Family ID | 61148465 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200085758 |
Kind Code |
A1 |
Farokhzad; Omid ; et
al. |
March 19, 2020 |
CO-DELIVERY OF NUCLEIC ACIDS FOR SIMULTANEOUS SUPPRESSION AND
EXPRESSION OF TARGET GENES
Abstract
Nanoparticulate pharmaceutical formulations and methods for
co-delivery of two or more species of nucleic acids for
simultaneous suppression and expression of target genes in a cell,
are provided. The nanoparticles encapsulate two or more nucleic
acid species. The first nucleic acid suppresses expression of a
gene or product thereof, e.g., inhibitory nucleic acid, such as
antisense, siRNA, miRNA, Dicer siRNA, piRNA, etc. The second
nucleic acid increases expression of, or encodes, an endogenous or
exogenous protein or polypeptide, e.g., an mRNA. The first and
second nucleic acid species simultaneously target or affect the
same or different cellular processes within a cell including
communication, senescence, DNA repair, gene expression, metabolism,
necrosis, and apoptosis.
Inventors: |
Farokhzad; Omid; (Waban,
MA) ; Xu; Xiaoding; (Malden, MA) ; Shi;
Jinjun; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Brigham and Women's Hospital, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
61148465 |
Appl. No.: |
16/470506 |
Filed: |
December 18, 2017 |
PCT Filed: |
December 18, 2017 |
PCT NO: |
PCT/US2017/067090 |
371 Date: |
June 17, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62435171 |
Dec 16, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/7088 20130101; C12N 2310/351 20130101; A61K 9/5146
20130101; A61K 38/465 20130101; C12N 2320/31 20130101; C12N
2310/141 20130101; A61K 47/60 20170801; C12N 15/113 20130101; C12Y
301/03048 20130101; A61K 47/6935 20170801; A61K 48/0041 20130101;
C12N 15/111 20130101; A61K 47/62 20170801; C12N 2320/32 20130101;
A61K 45/06 20130101; C12N 2310/14 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; C12N 15/113 20060101 C12N015/113; A61K 48/00 20060101
A61K048/00; A61P 35/00 20060101 A61P035/00; A61K 47/69 20060101
A61K047/69; A61K 45/06 20060101 A61K045/06; A61K 31/7088 20060101
A61K031/7088; A61K 47/62 20060101 A61K047/62; A61K 47/60 20060101
A61K047/60; A61K 38/46 20060101 A61K038/46 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. R01HL127464 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A nanoparticle formulation comprising polymeric or inorganic
nanoparticles, liposomes or micelles encapsulating an inhibitory
functional nucleic acid or an expression construct encoding an
inhibitory functional nucleic acid, wherein the inhibitory
functional nucleic acid inhibits an activity in a cell, and a
stimulatory nucleic acid species which enhances or increases an
activity in a cell or encodes a protein or peptide.
2. The nanoparticle formulation of claim 1, wherein the inhibitory
functional nucleic acid specifically inhibits or reduces expression
of a target gene or product thereof in a cell, and the stimulatory
functional nucleic acid increases or induces expression of an
endogenous or heterologous protein or polypeptide.
3. The nanoparticle formulation of claim 1 wherein the inhibitory
functional nucleic acid is an inhibitory RNA.
4. The nanoparticle formulation of claim 3, wherein the inhibitory
RNA is antisense, siRNA, miRNA, piRNA, Dicer siRNA or shRNA.
5. The nanoparticle formulation of claim 1, wherein the stimulatory
functional nucleic acid is an mRNA or a deoxyribonucleic acid
(DNA).
6. The nanoparticle formulation of claim 4, wherein the inhibitory
functional nucleic acid reduces or suppresses the expression of an
immune costimulatory molecule or signal.
7. The nanoparticle formulation of claim 6, wherein the
costimulatory molecule or signal is selected from the group
consisting of B7/CD28 family members, Butyrophilins, LAIR Family
members, Nectin and Nectin-like Ligand/Receptor co-signaling
molecules, ILT/CD85 family proteins, TNF superfamily members, SLAM
family members, and TIM family co-Signaling molecules.
8. The nanoparticle formulation of claim 6, wherein the
costimulatory molecule or signal is selected from the group
consisting of B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H4, B7-H6,
B7-H7/HHLA2, BTLA, CD28, CD30L, CTLA-4, ICOS, PD-1, PD-L1/B7-H1,
PD-L2/B7-DC, PDCD6, TMIGD2/CD28H, VISTA/B7-H5/PD-1H,
BTN1A1/Butyrophilin, NTB-A/SLAMF6, and SLAM/CD150,
TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD160, CD200, CD300a/LMIR1,
CD300d/LMIR4, CLECL1/DCAL-1, DAP12, Dectin-1/CLEC7A, DPPIV/CD26,
EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1,
LAG-3, and TSLP R.
9. The nanoparticle formulation of claim 1, wherein the stimulatory
nucleic acid encodes an antigen, and wherein the inhibitory
functional nucleic acid inhibits one or more co-stimulatory
molecules of an antigen presenting cell to induce immunological
tolerance to the antigen encoded by the stimulatory nucleic
acid.
10. The nanoparticle formulation of claim 9, wherein the
stimulatory nucleic acid expresses an antigen selected from the
group consisting of an antigen to which tolerance is desired.
11. The nanoparticle formulation of claim 4, wherein the inhibitory
nucleic acid reduces or suppresses the expression of or inhibits
the function of a tumorigenic driver or oncogene, and wherein the
stimulatory nucleic acid encodes or enhances the function of a
tumor repressor.
12. The nanoparticle formulation of claim 10, wherein the antigen
is a viral capsid protein from a virus selected from the group
consisting of an Adeno-Associated Virus (AAV), a Herpesvirus, a
retrovirus and a lentivirus.
13. The nanoparticle formulation of claim 12, wherein the viral
capsid protein is a VP1, VP2, or VP3 capsid protein from an
Adeno-Associated Virus (AAV) subtype selected from the group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
and AAV10.
14. The nanoparticle formulation of claim 1, wherein the inhibitory
functional nucleic acid, and/or the stimulatory functional nucleic
acid is modified to acquire one or more properties selected from
the group consisting of increase nuclease resistance, enhanced
membrane permeability, and reduced immunogenicity.
15. The nanoparticle formulation of claim 1, wherein the first and
stimulatory functional nucleic acids can affect the same, or
different cellular process(es).
16. The nanoparticle formulation of claim 15, wherein the cellular
process is selected from the group consisting of cell
communication, cellular senescence, DNA repair, gene expression,
metabolism, necrosis, and programmed cell death (apoptosis), immune
stimulatory, or immune co-stimulatory signal.
17. The nanoparticle formulation of claim 1 comprising an excipient
for administration to an individual in need thereof.
18. The nanoparticle formulation of claim 17, wherein the
nanoparticles have a diameter of between about 10 nm and about 500
nm, inclusive.
19. The nanoparticle formulation of claim 17, wherein the
nanoparticles have a diameter of between 20 nm and about 500 nm,
inclusive, between about 25 nm and about 250 nm, between about 40
nm and about 150 nm, between about 50 nm and about 150 nm, or
between about 50 nm and about 100 nm, inclusive.
20. The nanoparticle formulation of claim 17, wherein the
nanoparticles are in a form selected from the group consisting of
polymeric nanoparticles, lipid nanoparticles, metallic or ceramic
nanoparticles, and combinations thereof.
21. The nanoparticle formulation of claim 1 wherein the
nanoparticles comprises one or more polymer.
22. The nanoparticle formulation of claim 21, wherein the
nanoparticles comprise a polymer selected from the group consisting
of polyesters, polyanhydrides, polycaprolactone, polyorthoesters,
polyhydroxyalkanoates, polyalkylene oxides, copolymers thereof, and
blends thereof.
23. The nanoparticle formulation of claim 22, comprising a blend of
polyesters selected from the group consisting of polyglycolic acid,
polylactic acid, polyglycolic-lactic acid, copolymers of
polyglycolic acid, polylactic acid, polyglycolic-lactic acid and
polyalkylene glycol or copolymers thereof.
24. The nanoparticle formulation of claim 1, wherein the
nanoparticles comprise one or more lipids, alone or in combination
with polymer.
25. The nanoparticle formulation of claim 24, wherein the
nanoparticles are in a form selected from the group consisting of
liposomes, micelles, and combinations thereof.
26. The nanoparticle formulation of claim 24, wherein the
lipid-conjugated polymer is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-terminated
polyethylene glycol (PEG).
27. The nanoparticle formulation of claim 1, wherein the
nanoparticles further comprise a ligand.
28. The nanoparticle formulation of claim 27, wherein the ligand is
selected from the group consisting of a targeting ligand, an
adhesion ligand, a cell-penetrating ligand, an
endosomal-penetrating ligand, and combinations thereof.
29. The nanoparticle formulation of claim 30, wherein the ligand is
covalently, or non-covalently attached to the surface of the
nanoparticle.
30. The nanoparticle formulation of claim 1 formed by emulsion with
a non-aqueous solvent, solvent extraction, or
nanoprecipitation.
31. The nanoparticle formulation of claim 30, wherein the
nanoparticle is formed by self-assembly of amphiphilic polymer
optionally in combination with hydrophobic polymer.
32. The nanoparticle formulation of claim 31 comprising a blend of
hydrophobic polymer and amphiphilic polymer.
33. The nanoparticle formulation of claim 31 wherein the
hydrophilic portion of the amphiphilic polymers is a polyalkylene
oxide or derivative thereof.
34. The nanoparticle formulation of claim 1 comprising an
additional therapeutic, prophylactic or diagnostic agent selected
from the group consisting of proteins or peptides, nucleic acids,
lipids, sugars or polysaccharides, small molecules, or combinations
thereof.
35. The nanoparticle formulation of claim 34, wherein the
additional agent is a chemotherapeutic or antiinfective for
treatment of a disorder characterized by a stimuli effecting
release or which can be exposed to a stimuli.
36. The nanoparticle formulation of claim 1 comprising between
about 1% and about 70% weight/weight, between about 5% and about
50% weight/weight, or between about 10% and about 30% weight/weight
of the first and stimulatory functional nucleic acid, alone or in
combination with an additional therapeutic agent, prophylactic
agent, diagnostic agent, or combination thereof.
37. The nanoparticle formulation of claim 1, wherein the particles
release the inhibitory and stimulatory functional nucleic acid
primarily within certain target cells.
38. A method of simultaneously delivering two nucleic acid species
to cells comprising administering the nanoparticle formulation of
claim 1 to an individual in need thereof or claims therefrom.
39. The method of claim 38, wherein the subject has a disease or
disorder, or is at risk of developing a disease or disorder
selected from the group consisting of cancer, infection,
inflammation, and autoimmune disease or disorder.
40. The method of claim 39 for treating cancer comprising
administering to a subject with cancer the nanoparticles of claim
1, wherein the inhibitory and stimulatory nucleic acids affect the
same or different cellular processes in cancer cells in an
effective amount to reduce one or more symptoms of the cancer.
41. The method of claim 40, wherein the cellular process or
processes is selected from the group consisting of apoptosis, cell
survival signaling, proliferation, sensitivity to anticancer
agents, a DNA damage and repair pathway or signaling.
42. The method of claim 40 for modulating an immunological response
towards an antigen comprising administering to a subject in need
thereof the nanoparticle formulation of claim 1, wherein the
inhibitory nucleic acid inhibits or reduces a target gene or
product thereof which is directly or indirectly involved in the
immunological response, and the stimulatory nucleic acid encodes
the antigen.
43. The method of claim 42, wherein the target gene or product
thereof is a molecule associated with the mammalian target of
rapamycin (mTOR) pathway.
44. The method of claim 43, wherein the target gene or product
thereof inhibits the mTOR complex 1 (mTORC1), and/or any of the
downstream effector molecules thereof.
45. The method of claim 42, wherein the target gene or product
thereof is a costimulatory molecule of a professional antigen
presenting cell.
46. The method of claim 45, wherein the costimulatory molecule is
selected from the group consisting of B7-1, B7-2, B7-H3, B7-H4,
CD40, OX40L, ICOS-L, PD-L1, PD-L2, LIGHT, CD70, 4-1BBL, CD30L,
SLAM, and combinations thereof.
47. The method of claim 38, wherein the effect of the inhibitory
nucleic acid in the cells is greater when delivered by the
nanoparticles, than when the inhibitory nucleic acid is delivered
in the absence of the stimulatory nucleic acid.
48. The method of claim 38, wherein the effect of the stimulatory
nucleic acid in the cells is greater when delivered by the
nanoparticles, than when the stimulatory nucleic acid is delivered
in the absence of the inhibitory nucleic acid.
49. A nanoparticular formulation for inducing immunological
tolerance to an antigen in a subject, comprising nanoparticles
having an average diameter of between 40 and 100 nm, the
nanoparticles having encapsulated therein one or more
tolerance-inducing nucleic acids that silence or down-regulate one
or more costimulatory molecules, wherein the nanoparticles are
preferentially taken up by dendritic cells, subcapsular
macrophages, or antigen-presenting cells, and the nucleic acid is
expressed in the cells in an amount effective to induce tolerance
to a co-administered antigen.
50. The nanoparticular formulation of claim 49 for inducing
tolerance wherein the nanoparticles are selected from the group
consisting of polymeric nanoparticles, metal or ceramic
nanoparticles, liposomes, lipid micelles, and polymeric-lipid
nanoparticles.
51. The nanoparticular formulation of claim 49 for inducing
tolerance wherein the nanoparticles are formulated in a suspension
with antigen to which tolerance is to be induced, the antigen in
solution or in particles.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/435,171, filed on Dec. 16, 2016, which is
incorporated by reference herein in its entirety.
REFERENCE TO SEQUENCE LISTING
[0003] The Sequence Listing submitted Dec. 18, 2017 as a text file
named "BWH_23932_ST25.txt," created on Dec. 14, 2017, and having a
size of 2,000 bytes, is hereby incorporated by reference pursuant
to 37 C.F.R. .sctn. 1.52(e)(5).
FIELD OF THE INVENTION
[0004] This invention is generally in the field of pharmaceutical
compositions including one or more inhibitory nucleic acids and one
or more nucleic acids encoding a protein or polypeptide or
stimulating expression of a protein or peptide, and methods of use
thereof.
BACKGROUND OF THE INVENTION
[0005] Use of nucleic acid in therapeutic and vaccine applications
is on the rise. For example, inhibitory RNAs such as siRNA can be
used to reduce gene expression, while mRNA can be used to increase
expression of an endogenous or exogenous protein. Efficient
delivery of RNA (e.g., siRNA or mRNA) or DNA remains the key
challenge for broad applications of nucleic acid-based
pharmaceuticals in vivo (Whitehead K A et al., Nat. Rev. Drug
Discovery, 8(2), 129-138 (2009); Islam M A et al., Biomater. Sci.
3:1519-1533 (2015)).
[0006] Drug delivery systems have become an important tool in many
industries including healthcare. For example, lipid and polymer
nanoparticles have been used to deliver siRNA or mRNA (Akinc, et
al., Nat. Biotechnol., 26 (5), 561-9 (2008), Semple, et al., Nat.
BiotechnoL, 28 (2), 172-6 (2010), Cheng and Bryers, Biomaterials,
33(28), 6868-76 (2012)). Some nanoparticles for siRNA delivery have
advanced to clinical trials to treat diverse diseases (Pascolo,
Handb. Exp. Pharmacol., 183, 221-235 (2008)), Burnett, Biotechnol.
J., 6:(9), 1130-46 (2011)), de Fougerolles, Nat. Rev. Drug
Discovery, 6(6):443-53 (2007), and Weide, J. Immunother.,
31(2):180-8 (2008)).
[0007] It is therefore an object of the present invention to
provide pharmaceutical compositions and methods of use thereof for
simultaneous stimulation of production of a protein or gene and
inhibition of expression of gene.
[0008] It is a further object of the invention to provide
nanoparticles with increased efficacy in getting to the targeted
tissue and deliver one or more inhibitory nucleic acids in
combination with one or more nucleic acids encoding proteins or
increasing expression thereof.
SUMMARY OF THE INVENTION
[0009] Pharmaceutical formulations and methods of use thereof for
co-delivery of two or more species of nucleic acids for suppression
of target genes and increasing expression of genes or proteins in
the same cell are provided. In preferred embodiments, the
pharmaceutical formulations are in the form of nanoparticles.
Nanoparticles can be polymeric particles, non-polymeric particles,
liposomes, micelles, hybrids thereof, and/or combinations thereof,
optionally with at least one moiety responsive to an environmental
stimulus, and/or at least one targeting moiety. The nanoparticle is
not a virus, or a virus-like particle.
[0010] The nanoparticles include at least two nucleic acid species.
The first nucleic acid species directly suppresses expression of
one or more genes, or a gene product thereof such as mRNA, within a
target cell. The first nucleic acid species can be, for example, a
functional nucleic acid or an expression construct, such as a
vector, encoding a functional nucleic acid. In preferred
embodiments, the first nucleic acid species is an inhibitory
nucleic acid, for example, antisense, siRNA, miRNA, shRNA, Dicer
siRNA or piRNA. The functional nucleic acid can specifically target
the gene or gene product.
[0011] The second nucleic acid species can induce or increase
expression of an endogenous or exogenous protein or polypeptide, or
a functional non-coding RNA molecule such as transfer RNA,
ribosomal RNA, or regulatory RNA. For example, the second nucleic
acid species can be mRNA or a functional non-coding RNA molecule.
In preferred embodiments, the second nucleic acid species is an
mRNA encoding a protein or polypeptide, although the nucleic acid
species can also be a molecule that turns on or increases gene
expression. The protein or polypeptide can be endogenous or
exogenous to the target cell. The protein or polypeptide can be
heterologous.
[0012] As a result of co-delivery to the same cell by the
nanoparticles, the desired effect from the biological activity of
the first nucleic acid species in the cells is greater when
delivered by the co-loaded nanoparticles, than when the first
nucleic acid species is delivered in the absence of the second
nucleic acid species. The desired effect from the biological
activity of the second nucleic acid species in the cells is greater
when delivered by the co-loaded nanoparticles, than when the second
nucleic acid species is delivered in the absence of the first
nucleic acid species.
[0013] The first and second nucleic acid species can target or
affect the same or different cellular processes within a cell.
Cellular processes include, but are not limited to, cell
communication, cellular senescence, DNA repair, gene expression,
metabolism, necrosis, and programmed cell death (apoptosis). In
some embodiments the first and second nucleic acid species target
or affect the biological functions of immune cells, such as
leukocytes. Exemplary leukocytes include neutrophils, basophils,
eosinophils, lymphocytes, monocytes, and macrophages. Exemplary
biological activities of immune effector cells include activation,
differentiation, proliferation, suppression and apoptosis of the
immune cell. In some embodiments, the first and second nucleic acid
species are selected to target one or more cellular pathways that
contribute to one or more symptoms of a disease. The compositions
can be used to treat a disease or disorder by delivering to a cell
two nucleic acid species that affect the same or different cellular
processes in target cells in an effective amount to reduce one or
more symptoms of the disease or disorder. For example, if the
disease or disorder is cancer, target cellular processes can be,
but are not limited to, apoptosis, cell survival signaling,
proliferation, sensitivity to anticancer agents, a DNA damage and
repair pathway or signaling cascade, cellular metabolism, and
combinations thereof. In this example, the inhibitory nucleic acid
inhibits the proliferation of the cancer cells while at the same
time as enhancing the expression of cytolytic proteins.
[0014] Methods of using the nanoparticles are also provided. For
example, a method of simultaneously delivering two nucleic acid
species (siRNA and mRNA) to cells can include contacting target
cells with nanoparticles encapsulating the two nucleic acid
species. The contacting can occur in vitro or in vivo. In some
embodiments, the contacting occurs in vivo following administration
of the nanoparticles to a subject in need thereof. The subject can
have a disease or disorder, for example, cancer, an infection,
inflammation, or an autoimmune disease or disorder. In some
embodiments, the target cells for the nanoparticles are
antigen-presenting cells such as dendritic cells, B-lymphocytes, or
macrophages, including subcapsular macrophages.
[0015] Methods of using nanoparticles encapsulating or otherwise
associated with two or more species of nucleic acids for modulating
an immunological response towards a target antigen in a subject are
also provided. In some embodiments, one nucleic acid species
carries the genetic material encoding the target antigen while the
second or further nucleic acid species upregulates and/or
downregulates a target gene or product thereof. Exemplary target
molecules on antigen presenting cells include those associated with
the mammalian target of rapamycin (mTOR) pathway, and costimulatory
molecules such as B7-1, B7-2, B7-H3, B7-H4, CD40, OX40L, ICOS-L (or
B7-H2), PD-L1 (or B7-H1), PD-L2 (or B7-DC), LIGHT, CD70, 4-1BBL,
CD30L, RelB, and SLAM, or others such as TLR3, TLR4, IL-6, and
IL-23.
[0016] siRNAs can be used to reduce or prevent the expression or
function of one or more co-stimulatory proteins, and thereby
mediate developmental and/or immunological activities of the target
cells. In some embodiments, nanoparticles encapsulate nucleic acids
that provide immunological tolerance to one or more target
antigens. These nanoparticles can be targeted to dendritic cells to
provide tolerogenic therapies. The nanoparticles induce immunogenic
tolerance through the delivery of inhibitory RNA that preclude or
otherwise moderate the maturation of dendritic cells to produce
semi-mature (i.e., tolerogenic) dendritic cell phenotype. The
nanoparticles simultaneously deliver one or more mRNAs encoding a
target antigen, to which immunological tolerance is desired. In
some embodiments, the target antigen is expressed within the
tolerogenic dendritic cells, which subsequently induce regulatory T
leukocytes (T-regs), and/or induce clonal deletion of T cells
within the host. An exemplary target antigen is a viral capsid
protein, for example, associated with viruses used as vectors for
gene therapy. Therefore, in certain embodiments, the nanoparticles
induce tolerogenic dendritic cells to enhance the efficacy of gene
therapy. Exemplary viral vector capsids include capsid proteins
from Lentiviruses, Retroviruses, adeno-associated virus (AAV)
serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, or AAV11. Exemplary capsid proteins include VP1, VP2, or VP3
capsid proteins.
[0017] In other embodiments, the nanoparticles deliver mRNAs and
inhibitory RNAs that simultaneously treat and prevent one or more
cancers. The nanoparticles induce expression of one or more tumor
suppressors, and simultaneously inhibit or reduce one or more
oncogenes and/or tumorigenic drivers. Nanoparticles effective for
treatment and prevention of cancer are loaded with mRNAs and siRNAs
specific to the cancer that is to be treated. For example, in some
embodiments, nanoparticles deliver siRNA to reduce or block
expression or function of the Androgen Receptor, and mRNA that
reconstitutes one or more lost tumor suppressor genes such as PTEN,
or RB and/or P53 for the treatment of prostate cancers. In other
embodiments, nanoparticles deliver siRNA to reduce or block
expression or function of the EGFR and/or KRAS, and mRNA that
reconstitutes one or more of p53, RB genes, p16 gene, or FHIT gene
for the treatment of lung cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic illustration of the polymeric
nanoparticles, micelles (FIG. 1A), lipid/polymer hybrid
nanoparticles, PEGylated lipid/polymer hybrid nanoparticles (FIG.
1B), and lipid micelles, liposomes and PEGylated liposomes (FIG.
1C).
[0019] FIG. 2 is a schematic illustration of the modification of
nucleic acids by a ligand.
[0020] FIG. 3 is a graph showing size distribution of siRNA loaded
PDSA8-1 NPs.
[0021] FIG. 4 is a graph showing size change profile (Diameter) of
siRNA loaded PDSA8-1 NPs incubated in PBS (.box-solid.), or 1 mM
(.tangle-solidup.) or 10 mM (.circle-solid.) GSH solution.
[0022] FIGS. 5A and 5B are graphs showing (FIG. 5A)
pharmacokinetics of naked DY647-siRNA (.circle-solid.), and
DY647-siRNA loaded PDSA8-1 NPs (.box-solid.); (FIG. 5B)
biodistribution of PDSA8-1 NPs in the tumors and main organs of the
PC3 xenograft tumor-bearing nude mice sacrificed 24 h
post-injection of DY677-siRNA loaded PDSA8-1 NPs.
[0023] FIGS. 6A and 6B are schematics showing (FIG. 6A) molecular
structures of the ultra pH-responsive polymer,
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14), and the tumor-penetrating
polymer, iRGD-PEG-b-PDPA; (FIG. 6B) ultra pH-responsive and
tumor-penetrating nanoplatform for nucleic acid loading and
release.
[0024] FIGS. 7A and 7B are graphs showing (FIG. 7A) normalized
fluorescence intensity as a function of pH for the Cy.5.5-labelled
NPs of PDPA80; (FIG. 7B) in vitro nucleic acid release at a pH of
7.4 (.box-solid.), and 6.0 (.circle-solid.) over time (hours) from
the NPs of PDPA80 at 37.degree. C.
[0025] FIGS. 8A and 8B are graphs showing expression of integrins
.alpha.v.beta.3 and .alpha.v.beta.5 on Luc-HeLa (FIG. 8A) and PC3
(FIG. 8B) cells determined by flow cytometry analysis.
[0026] FIGS. 9A and 9B are bar graphs showing mean fluorescence
intensity (MFI) of Luc-HeLa (FIG. 9A) and PC3 (FIG. 9B) cells
incubated with DY547-siRNA-loaded NPs80 and iRGD-NPs80 for 4 h at a
10 nM siRNA dose. *p<0.05
[0027] FIGS. 10A and 10B are graphs showing (FIG. 10A)
pharmacokinetics of naked siRNA (.tangle-solidup.), Nanoparticles
(NPs) (.box-solid.), and siRNA-loaded NPs (.circle-solid.); (FIG.
10B) Biodistribution of NPs in the PC3 xenograft tumor-bearing mice
sacrificed at 24 h post-injection of naked siRNA, and siRNA-loaded
NPs.
[0028] FIGS. 11A and 11B are schematic illustrations of (FIG. 11A)
molecular structures of the oligoarginine-functionalized sharp
pH-responsive polymer, Meo-PEG-b-P(DPA-co-GMA-Rn), and PCa-specific
polymer, ACUPA-PEG-b-PDPA; (FIG. 11B) the multifunctional
envelope-type NP platform for in vivo PCa-specific nucleic acid
delivery and therapy.
[0029] FIGS. 12A, 12B and 12C are graphs showing (FIG. 12A) size
and polydispersity (PDI) of GL3 siRNA loaded NPs of
Meo-PEG-b-P(DPA-co-GMA-Rn); (FIG. 12B) Zeta potential (0 and
encapsulation efficiency (EE %) of GL3 siRNA loaded NPs of
Meo-PEG-b-P(DPA-co-GMA-Rn); and (FIG. 12C) In vitro release of
DY745-siRNA from the NPsR10 at a pH of 6.0 (.box-solid.) and 7.4
(.circle-solid.).
[0030] FIG. 13 is a graph showing normalized fluorescence intensity
as a function of pH for the Cy.5.5-labelled NPs R10.
[0031] FIG. 14 is a bar graph showing the fluorescence intensity of
PSMA in Luc-HeLa, PC3, DU145, 22RV1, and LNCaP cells. Blank: cells
incubated with free medium; MFI-mean fluorescence intensity.
[0032] FIG. 15 is a flow cytometry profile of Luc-HeLa cells
incubated with the DY547-siRNA-loaded NPsR10 and ACUPA-NPsR10 for 4
h.
[0033] FIGS. 16A-16B are flow cytometry profiles of PC3 (FIG. 16A)
and DU145 cells (FIG. 16B) incubated with the DY547-siRNA-loaded
NPsR10 and ACUPA-NPsR10 for 4 h.
[0034] FIGS. 17A-17B are graphs showing (FIG. 17A) pharmacokinetics
of Nanoparticles (NPs) (.circle-solid.), naked DY647-siRNA
(.box-solid.), and DY647-siRNA loaded NPsR10 and ACUPA-NPsR10
(.tangle-solidup.); (FIG. 17B) biodistribution of the NPs
quantified from (FIG. 17C).
[0035] FIG. 18 is a schematic illustration of the redox-responsive
nanoparticle-mediated systemic siRNA and mRNA co-delivery for
concurrent upregulation and suppression of genetic causes of
cancer.
[0036] FIG. 19 is a graph showing Luciferase expression in Luc-HeLa
cells transfected with Luc siRNA loaded PDSA NPs at a 1 nM siRNA
dose.
[0037] FIGS. 20A-20C are plots showing the size (diameter in nm)
distribution (intensity) of the redox-responsive PDSA8-1
nanoparticles loaded with siRNA only (FIG. 20A), mRNA only (FIG.
20B), or co-loaded with siRNA/mRNA (FIG. 20C).
[0038] FIG. 21 is a bar graph showing the siRNA and mRNA release
from the siRNA/mRNA co-loaded NPs with the addition of different
concentrations of GSH. Including 2 .mu.M GSH mRNA (.box-solid.), 2
.mu.M GSH siRNA (.circle-solid.), 10 mM GSH mRNA (.tangle-solidup.)
and 10 mM GSH sRNA ().
[0039] FIG. 22 is a bar graph showing luciferase expression (%) in
luciferase-expressing HeLa cells treated with mRNA only (left bar
in each dosage cluster), siRNA/mRNA (center bar in each dosage
cluster), and siRNA only (right bar in each dosage cluster)
nanoparticle as a function of siRNA dose (nM).
[0040] FIG. 23 is a bar graph showing GFP expression in HeLa cells
treated with mRNA only (left bar in each dosage cluster) and
siRNA/mRNA (right bar in each dosage cluster) as a function of mRNA
dose (ng).
[0041] FIG. 24 is a graph showing proliferation rate of NCI-1650
cells over a period of 8 days post transfection with control NPs
(Control .box-solid.), PTEN mRNA loaded NPs (mRNA NPs
.circle-solid.), PHB1 siRNA loaded NPs (siRNA NPs
.tangle-solidup.), and PHB1 siRNA/PTEN mRNA loaded NPs (siRNA/mRNA
NPs ).
[0042] FIG. 25 is a bar graph showing the proliferation of LNCaP
cells treated with the AR siRNA loaded, PTEN mRNA loaded and AR
siRNA/PTEN mRNA co-loaded NPs.
[0043] FIG. 26 is a bar graph showing the blood circulation time of
the free siRNA, free mRNA, and siRNA/mRNA co-loaded NPs.
[0044] FIG. 27 is a bar graph showing biodistribution of
DY677-siRNA loaded NPs, Cy5-mRNA loaded NPs, and
DY677-siRNA/Cy5-mRNA co-loaded NPs in tissues and organs including
tumor, kidney, lung, spleen, liver, and heart of the NCI-1650
xenograft tumor-bearing mice.
[0045] FIG. 28 is a bar graph showing the biodistribution of the
naked DY677-siRNA, naked Cy5-mRNA, and DY677-siRNA/Cy5-mRNA
co-loaded NPs in the tissues and organs including tumor, kidney,
lung, spleen, liver, and heart of the LNCaP xenograft tumor-bearing
mice.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0046] Binding" refers to the interaction between a corresponding
pair of molecules or portions thereof that exhibit mutual affinity
or binding capacity, typically due to specific or non-specific
binding or interaction, including, but not limited to, biochemical,
physiological, and/or chemical interactions. "Binding partner"
refers to a molecule that can undergo binding with a particular
molecule. "Biological binding" defines a type of interaction that
occurs between pairs of molecules including proteins, peptides,
nucleic acids, glycoproteins, carbohydrates, or endogenous small
molecules. "Specific binding" refers to molecules, such as
polynucleotides, that are able to bind to or recognize a binding
partner (or a limited number of binding partners) to a
substantially higher degree than to other, similar biological
entities.
[0047] "Encapsulation efficiency" (EE) is the fraction of initial
drug that is encapsulated by the nanoparticles (NPs). "Loading" as
used herein refers to the mass fraction of encapsulated agent in
the NPs.
[0048] A "polymer," is given its ordinary meaning as used in the
art, i.e., a molecular structure including one or more repeat units
(monomers), connected by covalent bonds. A "biocompatible polymer"
is used here to refer to a polymer that does not typically induce
an adverse response when inserted or injected into a living
subject, for example, without significant inflammation and/or acute
rejection of the polymer by the immune system, for instance, via a
T-cell response. A "copolymer" refers to a polymer formed of two or
more different monomers. The different units may be arranged in a
random order, in an alternating order, or as a "block" copolymer,
i.e., including one or more regions each including a first repeat
unit (e.g., a first monomer or block of monomers), and one or more
regions each including a second repeat unit (e.g., a second block),
etc. Block copolymers may have two (a diblock copolymer), three (a
triblock copolymer), or more numbers of distinct blocks.
[0049] The term "amphiphilic" refers to a molecule has both a polar
portion and a non-polar portion. In some embodiments, the polar
portion is soluble in water, while the non-polar portion is
insoluble in water. The polar portion may have either a formal
positive charge, or a formal negative charge. Alternatively, the
polar portion may have both a formal positive and a negative
charge, and be a zwitterion or inner salt.
[0050] A "particle" refers to a particle, microcapsule, or
microsphere. As used herein, nanoparticles typically have a longest
dimension (e.g., diameter) of 1000 nm or less. In some embodiments,
nanoparticles have a diameter of between 40 and 500 nm, more
preferably 50 to 300 nm. In preferred embodiments, polymeric
particles can be formed using a solvent emulsion, spray drying, or
precipitation in bulk or microfluids, wherein the solvent is
removed to no more than an insignificant residue, leaving a solid
(which may, or may not, be hollow or have a liquid filled interior)
polymeric particle, unlike a micelle whose form is dependent upon
being present in an aqueous solution.
[0051] The term "carrier" or "excipient" refers to an organic or
inorganic ingredient, natural or synthetic inactive ingredient in a
formulation, with which one or more active ingredients are
combined.
[0052] The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredients.
[0053] The terms "sufficient" and "effective", are used
interchangeably, and refer to an amount (e.g. mass, volume, dosage,
concentration, and/or time period) needed to achieve one or more
desired result(s). The term "therapeutically effective amount"
means a dosage sufficient to alleviate one or more symptoms of a
disorder, disease, or condition being treated, or to otherwise
provide a desired pharmacologic and/or physiologic effect. The
precise dosage will vary according to a variety of factors such as
subject-dependent variables (e.g., age, immune system health,
etc.), the disease or disorder being treated, as well as the route
of administration and the pharmacokinetics of the agent being
administered.
[0054] The term "modulate" refers to the ability of a compound to
change an activity in some measurable way as compared to an
appropriate control. As a result of the presence of compounds in
the assays, activities can increase or decrease as compared to
controls in the absence of these compounds.
[0055] The terms "inhibit" and "reduce" mean to reduce or decrease
in activity or expression. This can be a complete inhibition or
reduction of activity or expression, or a partial inhibition or
reduction. Inhibition or reduction can be compared to a control or
to a standard level.
[0056] The term "prevention" or "preventing" means to administer a
composition to a subject or a system at risk for or having a
predisposition for one or more symptom caused by a disease or
disorder to cause cessation of a particular symptom of the disease
or disorder, a reduction or prevention of one or more symptoms of
the disease or disorder, a reduction in the severity of the disease
or disorder, the complete ablation of the disease or disorder,
stabilization or delay of the development or progression of the
disease or disorder.
[0057] The terms "bioactive agent" and "active agent", are used
interchangeablyand include physiologically or pharmacologically
active substances that act locally or systemically in the body. A
bioactive agent is a substance used for the treatment (e.g.,
therapeutic agent), prevention (e.g., prophylactic agent),
diagnosis (e.g., diagnostic agent), cure or mitigation of disease
or illness, a substance which affects the structure or function of
the body, or pro-drugs, which become biologically active or more
active after they have been placed in a predetermined physiological
environment.
[0058] The term "protein" "polypeptide" or "peptide" refers to a
natural or synthetic molecule comprising two or more amino acids
linked by the carboxyl group of one amino acid to the alpha amino
group of another.
[0059] The term "polynucleotide" or "nucleic acid sequence" refers
to a natural or synthetic molecule comprising two or more
nucleotides linked by a phosphate group at the 3' position of one
nucleotide to the 5' end of another nucleotide. The polynucleotide
is not limited by length, and thus the polynucleotide can include
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
II. Particle Delivery Vehicles
[0060] Synthetic nanoparticles for co-delivery of one or more
nucleic acids for suppressing one or more target genes, and one or
more nucleic acids for over-expression of target genes or a protein
in parallel are provided. Preferred vehicles include polymeric
nanoparticles, micelles, lipid micelles, liposomes, and hybrid
lipid-polymer nanoparticles. As demonstrated in the following
examples, co-delivery can be obtained using polymeric or
lipid-polymer hybrid nanoparticles wherein the polymer can be
either responsive (like the presented data) or non-responsive.
[0061] A. Polymeric Particles
[0062] Nanoparticles can be formed of biodegradable, biocompatible
polymers for co-delivery of the nucleic acids.
[0063] Typically the nanoparticles are formed of one or more
hydrophobic polymers, optionally including amphiphilic polymers in
the form of a blend where the hydrophilic polymers orient to the
exterior of the nanoparticle, and/or hydrophilic polymers on the
surface to avoid uptake by the reticuloendothelial system and
enhance phagocytosis. Cationic polymers may be utilized to increase
encapsulation of the nucleic acids.
[0064] Hydrophobic cationic material, hydrophobic polymer and/or
the hydrophobic portion of amphiphilic materials provide a
non-polar polymer matrix for loading non-polar drugs, protect and
promoting siRNA molecule retention inside the NP core, and control
drug release. The hydrophilic portion of the amphiphilic material
can form a corona around the particle which prolongs circulation of
the particles in the blood stream and decreases uptake by the RES.
In one embodiment, the amphiphilic material is a hydrophobic,
biodegradable polymer terminated with a hydrophilic block.
[0065] Biocompatible polymers include, but are not limited to,
polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,
polyalkylene oxides, polyalkylene terepthalates, polyvinyl
alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides,
polyvinylpyrrolidone, polylactides, polyglycolides, polysiloxanes,
polyurethanes and copolymers thereof, celluloses including alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, and
cellulose sulphate sodium salt; polyacrylic acid polymers such as
polymers of acrylic and methacrylic esters such as poly (methyl
methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate),
poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyalkylenes
such as polyethylene, polypropylene poly(ethylene glycol),
poly(ethylene oxide), and poly(ethylene terephthalate), poly(vinyl
alcohols), poly(vinyl acetate), poly vinyl chloride polystyrene and
polyvinylpryrrolidone, derivatives thereof, linear and branched
copolymers and block copolymers thereof, and blends thereof.
[0066] Exemplary biodegradable polymers include, but are not
limited to, polyesters, poly(ortho esters), poly(ethylene imines),
poly(caprolactones), poly(hydroxybutyrates),
poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids),
polyglycolides, poly(urethanes), polycarbonates, polyphosphate
esters, polyphosphazenes, derivatives thereof, linear and branched
copolymers and block copolymers thereof, and blends thereof. In
particularly preferred embodiments the polymeric core contains
biodegradable hydrophobic polyesters such as poly(lactic acid),
poly(glycolic acid), and poly(lactic-co-glycolic acid), and/or
these polymers conjugated to polyalkylene oxides such as
polyethylene glycol or block copolymers such as the polypropylene
oxide-polyethylene oxide PLURONICs.RTM..
[0067] The molecular weight of the biodegradable oligomeric or
polymeric segment or polymer can be varied to tailor the properties
of the polymer. Exemplary molecular weights include between about
150 Da and about 100 kDa, more preferably between about 1 kDa and
about 75 kDa, most preferably between about 5 kDa and about 50
kDa.
[0068] In some embodiments, the hydrophilic polymers or segment(s)
or block(s) include, but are not limited to, homo polymers or
copolymers of polyalkene glycols, such as poly(ethylene glycol),
poly(propylene glycol), poly(butylene glycol), and acrylates and
acrylamides, such as hydroxyethyl methacrylate and
hydroxypropyl-methacrylamide. The hydrophilic polymer segment
typically has a molecular weight of between about 150 Da and about
20 kDa, more preferably between about 500 Da and about 10 kDa, most
preferably between about 1 kDa and about 5 kDa.
[0069] The nanoparticles can be formed of a mixture or blend of
polymers. In preferred embodiments, these are a blend of
amphiphilic polymers, preferably copolymers of modified
polyethylene glycol (PEG) and polyesters, such as various forms of
PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as
"PEGylated polymers", some hydrophobic polymer such as PLGA, PLA or
PGA, and/or some may be hydrophilic polymer such as a PEG or PEG
derivative. Some may be modified by conjugation to a targeting
agent, a cell adhesion or a cell penetrating peptide.
[0070] In some embodiments, the cationic material is a material
that is cationic at the time the hydrophobic cationic material is
prepared or becomes cationic under physiological conditions. In
some embodiments, the cationic material contains one or more amine
containing moieties, such as amine containing small molecules,
amine-containing polymers, such as PEI, and amine-containing
macromolecules, such as dendrimers (see the structures below). The
cationic moieties are functionalized with one or more
hydrophobic/lipid moieties, such as lipophilic alkyl chains (e.g.,
C.sub.6-C.sub.30, preferably C.sub.6-C.sub.24, more preferably
C.sub.6-Cis), cholesterol, saturated or unsaturated fatty acids,
etc. The cationic moiety promotes retention of the siRNA in the
core through electrostatic interaction while the hydrophobic moiety
provides controlled release of the siRNA as well as any active
agents in the shell.
[0071] Stimuli responsive polymers are well known in the art.
Stimuli responsive amphiphilic polymers are responsive to a
stimulus such as a pH change, redox change, temperature change,
exposure to light or other stimuli, including binding to a target.
Stimuli responsive polymers are reviewed by James, et al., Acta
Pharma. Sinica B 4(2):120-127 (2014). The following is a list of
exemplary polymers categorized by responsive to various stimuli:
Temperature: POLOXAMERS, poly(N-alkylacrylamide)s,
poly(N-vinylcaprolactam)s, cellulose, xyoglucan, and chitosan; pH:
poly(methacrylic acid)s, poly(vinylpyridine)s, and
poly(vinylimmidazole)s; light: modified poly(acrylamide)s; electric
field: sulfonated polystyrenes, poly(thiophene)s, and
poly(ethyloxazoline)s; ultrasound: ethylenevinylacetate.
[0072] Exemplary pH dependent polymers include dendrimers formed of
poly(lysine), poly(hydroxyproline), PEG-PLA, Poly(propyl acrylic
acid), Poly(ethacrylic acid), CARBOPOLL.RTM., Polysilamine,
EUDRAGIT.RTM. S-100 EUDRAGIT.RTM. L-100, Chitosan, PMAA-PEG
copolymer, sodium alginate (Ca2+). The ionic pH sensitive polymers
are able to accept or release protons in response to pH changes.
These polymers contain acid groups (carboxylic or sulfonic) or
basic groups (ammonium salts) so that the pH sensitive polymers are
polyelectrolytes that have in their structure acid or basic groups
that can accept or release protons in response to pH changes in the
surrounding environment. pH values from several tissues and cell
compartments can be used to trigger release in these tissues. For
example, the pH of blood is 7.4-7.5; stomach is 1.0-3.0; duodenum
is 4.8-8.2; colon is 7.0-7.5; lysosome is 4.5-5.0; Golgi complex is
6.4; tumor--extracellular medium is 6.2-7.2. pH is typically lower
in areas of infection or inflammation. Examples of thermosensitive
polymers include the poly(N-substituted acrylamide) polymers such
as poly(N-isopoprylacrilamide) (PNIPAAm), poly (N,N'-diethyl
acrylamide), poly (dimethylamino ethyl methacrylate and poly
(N-(L)-(1-hydroxymethyl) propyl methacrylamide).
[0073] Biologically responsive polymer systems are increasingly
important in various biomedical applications. The major advantage
of bioresponsive polymers is that they can respond to the stimuli
that are inherently present in the natural system. Bioresponsive
polymeric systems mainly arise from common functional groups that
are known to interact with biologically relevant species, and in
other instances the synthetic polymer is conjugated to a biological
component. Bioresponsive polymers include antigen-responsive
polymers, glucose-sensitive polymers, and enzyme-responsive
polymers.
[0074] B. Lipid-Based Delivery Vehicles
[0075] Nanoparticles may include one or more lipids, may be in the
form of a liposome, may include a lipid monolayer or bilayer, or be
formed of micelles. In some embodiments, nanoparticles include a
polymeric core surrounded by a lipid layer (e.g., lipid bilayer,
lipid monolayer, etc.). In some embodiments, a nanoparticle
includes a non-polymeric core (e.g., metal particle, quantum dot,
ceramic particle, bone particle, etc.) surrounded by a lipid layer
(e.g., lipid bilayer, lipid monolayer, etc.).
[0076] The percent of lipid in the nanoparticles can be from
greater than 0% to 99% by weight, inclusive, from 10% to 99% by
weight, from 25% to 99% by weight, from 50% to 99% by weight, or
from 75% to 99% by weight. In some embodiments, the percent of
lipid in nanoparticles is approximately 1% by weight, approximately
2% by weight, approximately 3% by weight, approximately 4% by
weight, approximately 5% by weight, approximately 10% by weight,
approximately 15% by weight, approximately 20% by weight,
approximately 25% by weight, or approximately 30% by weight.
[0077] In some embodiments, lipids are biocompatible oils. Suitable
oils for use include plant oils and butyl stearate, caprylic
triglyceride, capric triglyceride, cyclomethicone, diethyl
sebacate, dimethicone 360, isopropyl myristate, mineral oil,
octyldodecanol, oleyl alcohol, silicone oil, and combinations
thereof.
[0078] Oils may include one or more fatty acid groups or salts
thereof. In some embodiments, a fatty acid group is digestible,
long chain (e.g., C8-050), substituted or unsubstituted
hydrocarbons. In some embodiments, a fatty acid group is a C10-C20
fatty acid, C15-C20 fatty acid, or C15-C25 fatty acid or salt
thereof. The fatty acid group can be unsaturated, monounsaturated,
or polyunsaturated. In some embodiments, a double bond of an
unsaturated fatty acid group is in the cis conformation. In some
embodiments, a double bond of an unsaturated fatty acid is in the
trans conformation.
[0079] In some embodiments, a fatty acid group is one or more of
butyric, caproic, caprylic, capric, lauric, myristic, palmitic,
stearic, arachidic, behenic, or lignoceric acid. In some
embodiments, a fatty acid group is one or more of palmitoleic,
oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic,
arachidonic, gadoleic, arachidonic, eicosapentaenoic,
docosahexaenoic, or erucic acid. In some embodiments, the oil is a
liquid triglyceride.
[0080] In some embodiments, a lipid is a steroid (e.g.,
cholesterol, bile acid), vitamin (e g vitamin E), phospholipid
(e.g. phosphatidyl choline), sphingolipid (e.g. ceramides), or
lipoprotein (e.g. apolipoprotein). In some embodiments, a lipid is
a lipid-like material (also called lipidoid). See Akinc, et al.,
Nat Biotechnol., 2008; 26(5):561-9; Love, et al., Proc Natl Acad
Sci U SA. 2010; 107(5):1864-9; and Whitehead, et al., Nat. Commun.,
2014; 5:4277.
[0081] In certain embodiments, the lipid is phosphatidylcholine,
lipid A, cholesterol, dolichol, sphingosine, sphingomyelin,
ceramide, glycosylceramide, cerebroside, sulfatide,
phytosphingosine, phosphatidyl-ethanolamine, phosphatidylglycerol,
phosphatidylinositol, phosphatidylserine, cardiolipin, phosphatidic
acid, and/or lyso-phophatides.
[0082] In some embodiments, nanoparticle-stabilized liposomes are
used to deliver the disclosed nucleic acid content. By allowing
small charged nanoparticles (1 nm-30 nm) to adsorb on liposome
surface, liposome-nanoparticle complexes have not only the merits
of bare liposomes, but also tunable membrane rigidity and
controllable liposome stability. When small charged nanoparticles
approach the surface of liposomes carrying either opposite charge
or no net charge, electrostatic or charge-dipole interaction
between nanoparticles and membrane attracts the nanoparticles to
stay on the membrane surface, being partially wrapped by lipid
membrane. This induces local membrane bending and globule surface
tension of liposomes, both of which enable tuning of membrane
rigidity. Adsorbed nanoparticles form a charged shell which
protects liposomes against fusion, thereby enhancing liposome
stability. In certain embodiments, small nanoparticles are mixed
with liposomes under gentle vortex, and the nanoparticles stick to
liposome surface spontaneously.
[0083] C. Lipid-Polymer Delivery Vehicles
[0084] In some embodiments, nanoparticles include one or more
polymers associated covalently, or non-covalently with one or more
lipids, preferably phospholipids.
[0085] In some embodiments, a polymeric matrix can be surrounded by
a lipid coating layer (e.g., liposome, lipid monolayer, micelle,
etc.). The lipid monolayer shell can include an amphiphilic
compound. In another embodiment, the amphiphilic compound is
lecithin. The lipid monolayer can be stabilized.
[0086] Phospholipids which may be used include, but are not limited
to, phosphatidic acids, phosphatidyl cholines with both saturated
and unsaturated lipids, phosphatidyl ethanolamines,
phosphatidylglycerols, phosphatidylserines, phosphatidylinositols,
lysophosphatidyl derivatives, cardiolipin, and .beta.-acyl-y-alkyl
phospholipids. In a particular embodiment, an amphiphilic component
that can be used to form an amphiphilic layer is lecithin, and, in
particular, phosphatidylcholine. Lecithin is an amphiphilic lipid
and, as such, forms a phospholipid bilayer having the hydrophilic
(polar) heads facing their surroundings, which are oftentimes
aqueous, and the hydrophobic tails facing each other. Lecithin has
an advantage of being a natural lipid that is available from, e.g.,
soybean, and already has FDA approval for use in other delivery
devices.
[0087] Examples of phospholipids include, but are not limited to,
phosphatidylcholines such as dioleoylphosphatidylcholine,
dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine
dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine
(DPPC), distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcho-line (DBPC),
ditricosanoylphosphatidylcholine (DTPC),
dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines
such as dioleoylphosphatidylethanolamine or
1-hexadecyl-2-palmitoylglycerophos-phoethanolamine, incorporated at
a ratio of between 0.01-60 (weight lipid/w polymer), most
preferably between 0.1-30 (weight lipid/w polymer). Synthetic
phospholipids with asymmetric acyl chains (e.g., with one acyl
chain of 6 carbons and another acyl chain of 12 carbons) may also
be used.
[0088] By covering the polymeric nanoparticles with a thin film of
small molecule amphiphilic compounds, the nanoparticles have merits
of both polymer- and lipid-based nanoparticles, while excluding
some of their limitations. The amphiphilic compounds form a tightly
assembled monolayer around the polymeric core. This monolayer
effectively prevents the carried agents from freely diffusing out
of the nanoparticle, thereby enhancing the encapsulation yield and
slowing drug release. Moreover, the amphiphilic monolayer also
reduces water penetration rate into the nanoparticle, which slows
the hydrolysis rate of the biodegradable polymers, thereby
increasing particle stability and lifetime.
[0089] In some embodiments, the nanoparticle include a polymeric
matrix, wherein the polymeric matrix includes a lipid-terminated
polymer such as polyalkylene glycol and/or a polyester. In some
embodiments, the nanoparticle includes an amphiphilic
lipid-terminated polymer, where a cationic and/or an aniotic lipid
is conjugated to a hydrophobic polymer. In one embodiment, the
polymeric matrix includes lipid-terminated PEG. In some
embodiments, the polymeric matrix includes lipid-terminated
copolymer. In another embodiment, the polymeric matrix includes
lipid-terminated PEG and PLGA. In one embodiment, the lipid is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof. In a preferred embodiment, the polymeric matrix includes
DSPE-terminated PEG. The lipid-terminated PEG can then, for
example, be mixed with PLGA to form a nanoparticle.
[0090] In some embodiments, long-circulating, optionally
cell-penetrating, and stimuli-responsive nanopaticles for effective
in vivo delivery of therapeutic, prophylactic and/or diagnostic
agents are used. In the preferred embodiment, the NPs are made of
an amphiphilic polymer, most preferably a PEGylated polymer, which
shows a response to a stimulus such as pH, temperature, or light,
such as an ultra pH-responsive characteristic with a pKa close to
the endosomal pH (6.0-6.5) (Wang Y et al, Nat Mater, 13, 204-212
(2014)). The polymer may include a targeting or cell penetrating or
adhesion molecule such as a tumor-penetrating peptide iRGD.
III. Therapeutic, Prophylactic and Diagnostic Agents
[0091] The nanoparticles contain both an inhibitory nucleic acid,
such as antisense, siRNA, miRNA, piRNA, etc., and a nucleic acid
stimulating or enhancing production of a gene product such as a
mRNA encoding an exogenous or endogenus antigen that is being
expressed for immune stimulation or immune tolerance. In another
example, the mRNA can be tumor suppressor-encoded mRNA which
suppresses cancer proliferation, metastasis, viability or genes
therein.
[0092] The experiments below show that when siRNA and mRNA are
delivered into a cell using a nanoparticle co-loaded with both the
siRNA and the mRNA, suppression of gene expression targeted by the
siRNA and expression of protein encoded by the mRNA are both
increased. Inhibition of expression of the target of siRNA was
reduced to a greater degree when co-delivered in combination with
mRNA, than when delivered alone using the same nanoparticle
composition. Similarly, protein expressed by the mRNA was higher
when delivered in combination with the siRNA, then when delivered
alone using the same nanoparticle composition.
[0093] In some embodiments, the NPs contain between about 1% and
about 70% weight/weight of cargo. Preferably, the NPs contain
between about 5% and about 50% weight/weight, most preferably
between about 10% and about 30% weight/weight of cargo.
[0094] Exemplary nucleic acid-based active agents are discussed in
greater detail below. However, it will be appreciated that in
addition to nucleic acid-based active agents, the particles can
further include other active agent cargos. Additionally or
alternatively nucleic acid-containing particles can be co-delivered
to a subject in combination with (i) particles containing other
active agents, or (ii) with other active agents not contained in
particles, or combination thereof. Active agent cargos to be
delivered include therapeutic, nutritional, diagnostic, and
prophylactic agents. The active agents can be small molecule active
agents or biomacromolecules, such as proteins, polypeptides, sugars
or carbohydrates, lipids, nucleic acids or small molecule compounds
(typically 1 kD or less, but may be larger). Suitable small
molecule active agents include organic and organometallic
compounds. The small molecule active agents can be a hydrophilic,
hydrophobic, or amphiphilic compound.
[0095] Active agents include synthetic and natural proteins
(including enzymes, peptide-hormones, receptors, growth factors,
antibodies, signaling molecules), and synthetic and natural nucleic
acids (including RNA, DNA, anti-sense RNA, triplex DNA, inhibitory
RNA (RNAi), and oligonucleotides), and biologically active portions
thereof. Suitable active agents have a size greater than about
1,000 Da for small peptides and polypeptides, more typically at
least about 5,000 Da and often 10,000 Da or more for proteins.
Nucleic acids are more typically listed in terms of base pairs or
bases (collectively "bp"). Nucleic acids with lengths above about
10 bp are typically used. More typically, useful lengths of nucleic
acids for probing or therapeutic use will be in the range from
about 20 bp (probes; inhibitory RNAs, etc.) to tens of thousands of
bp for genes and vectors. The active agents may also be hydrophilic
molecules, preferably having a low molecular weight.
[0096] In other embodiments, the nanoparticle contains only one
nucleic acid molecule which inhibits expression or function of a
co-stimulatory molecule to induce tolerance to an antigen within an
antigen presenting cell, or which expresses or up-regulates a
molecule which acts as an inhibitor of the expression or function
of a co-stimulatory molecule within an antigen presenting cell. The
down-regulation, silencing or blocking of the function of one or
more co-stimulatory molecules drives the antigen presenting cell
towards a tolerognic phenotype. For example, in some embodiments,
nanoparticles containing single nucleic acid species induce
potential tolerogenic dendritic cell. Simulataneous administration
of an antigen, either systemically or locally, in solution or in
particles administered as a suspension (e.g., in the form of
nanoparticles containing a single mRNA encoding the target
antigen), or by co-adminsitration of the target antigen to the same
host by other means (e.g., in the form of a conventional vaccine),
induces immunological tolerance to the target antigen in the
host.
[0097] In some embodiments, pharmaceutical formulations for
inducing tolerance to a target antigen include a combination of (i)
nanoparticles encapsulating a single inhibitory RNA species (e.g.,
siRNA) that down-regulates or silences a costimulatory molecule;
and (ii) a tolerogenic antigen in a non-nanparticle form for
administration to a subject in vivo. In other embodiments,
pharmaceutical formulations for inducing tolerance to a target
antigen include a combination of (i) nanoparticles encapsulating a
single mRNA species that encodes a protein that down-regulates or
silences a costimulatory molecule; and (ii) a tolerogenic antigen
in a non-nanparticle form for administration to a subject in
vivo.
[0098] In further embodiments, the nanoparticles are formulated in
a solution or administration to a subject in combination with on or
more additional non-nucleic-acid based active agents. The
non-nucleic-acid based active agents can be therapeutic,
prophylactic or diagnostic agents. The non-nucleic-acid based
active agents are encapsulated within the nanoparticles, or
otherwise encoproprated into the core, or shell, or onto the
surface of the particles, or combinations thereof. In some
embodiments, the non-nucleic acid active agents are not associated
with the nanoparticles, for example, they are administered as a
solution, gel or other mixture that also includes the particles, or
formulated for administration as a separate solution, solid or
powder, or for administration through a separate route to that of
the nanoparticles.
[0099] A. Nucleic Acid-Based Agents
[0100] An isolated nucleic acid can be, for example, a DNA, an RNA,
or a nucleic acid analog. Nucleic acid analogs can be modified at
the base moiety, sugar moiety, or phosphate backbone. Such
modification can improve, for example, stability, hybridization,
solubility, or targeting of the nucleic acid. Exemplary
modifications include, 2'O-methyl, 2' methoxyethyl,
phosphoramidate, methylphosphonate, and/or phosphorothioate
backbone chemistry. In some embodiments, nucleic acids are modified
to acquire one or more properties selected from the group
consisting of increase nuclease resistance, enhanced membrane
permeability, and reduced immunogenicity. In some embodiments, a
targeting moiety is conjugated with nucleic acid.
[0101] The chemical modifications include chemical modification of
nucleobases, sugar moieties, nucleotide linkages, or combinations
thereof. As used herein `modified nucleotide" or "chemically
modified nucleotide" defines a nucleotide that has a chemical
modification of one or more of the heterocyclic base, sugar moiety
or phosphate moiety constituents. In some embodiments, the charge
of the modified nucleotide is reduced compared to DNA or RNA
oligonucleotides of the same nucleobase sequence. For example, the
oligonucleotide can have low negative charge, no charge, or
positive charge.
[0102] Typically, nucleoside analogs support bases capable of
hydrogen bonding by Watson-Crick base pairing to standard
polynucleotide bases, where the analog backbone presents the bases
in a manner to permit such hydrogen bonding in a sequence-specific
fashion between the oligonucleotide analog molecule and bases in a
standard polynucleotide (e.g., single-stranded RNA or
single-stranded DNA). In some embodiments, the analogs have a
substantially uncharged, phosphorus containing backbone. Chemical
modifications of heterocyclic bases or heterocyclic base analogs
may be effective to increase the binding affinity or stability in
binding a target sequence. Chemically-modified heterocyclic bases
include, but are not limited to, inosine, 5-(1-propynyl) uracil
(pU), 5-(1-propynyl) cytosine (pC), 5-methylcytosine,
8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and
2-amino-5-(2'-deoxy-.beta.-D-ribofuranosyl)pyridine
(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine
derivatives.
[0103] Sugar moiety modifications include, but are not limited to,
2'-O-aminoetoxy, 2'-O-amonioethyl (2'-OAE), 2'-O-methoxy,
2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-0,4'-C-methylene (LNA),
2'-O-(methoxyethyl) (2'-OME) and 2'-O--(N-(methyl)acetamido)
(2'-OMA).
[0104] In some embodiments, the functional nucleic acid is a
morpholino oligonucleotide. Morpholino oligonucleotides are
typically composed of two more morpholino monomers containing
purine or pyrimidine base-pairing moieties effective to bind, by
base-specific hydrogen bonding, to a base in a polynucleotide,
which are linked together by phosphorus-containing linkages, one to
three atoms long, joining the morpholino nitrogen of one monomer to
the 5' exocyclic carbon of an adjacent monomer. The purine or
pyrimidine base-pairing moiety is typically adenine, cytosine,
guanine, uracil or thymine. The synthesis, structures, and binding
characteristics of morpholino oligomers are detailed in U.S. Pat.
Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315,
5,521,063, and 5,506,337.
[0105] Important properties of the morpholino-based subunits
typically include: the ability to be linked in a oligomeric form by
stable, uncharged backbone linkages; the ability to support a
nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil
or inosine) such that the polymer formed can hybridize with a
complementary-base target nucleic acid, including target RNA, with
high T.sub.m, even with oligomers as short as 10-14 bases; the
ability of the oligomer to be actively transported into mammalian
cells; and the ability of an oligomer:RNA heteroduplex to resist
RNAse degradation.
[0106] Modifications to the phosphate backbone of DNA or RNA
oligonucleotides may increase the binding affinity or stability
oligonucleotides, reduce the susceptibility of oligonucleotides
nuclease digestion, or increase membrane permeability. Cationic
modifications, including, but not limited to,
diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP)
may be especially useful due to decrease electrostatic repulsion
between the oligonucleotide and a target. Modifications of the
phosphate backbone may also include the substitution of a sulfur
atom for one of the non-bridging oxygens in the phosphodiester
linkage. This substitution creates a phosphorothioate
internucleoside linkage in place of the phosphodiester linkage.
Oligonucleotides containing phosphorothioate internucleoside
linkages have been shown to be more stable in vivo.
[0107] Examples of modified nucleotides with reduced charge include
modified internucleotide linkages such as phosphate analogs having
achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P.
et al., Organic. Chem., 52:4202, (1987)), and uncharged
morpholino-based polymers having achiral intersubunit linkages
(see, e.g., U.S. Pat. No. 5,034,506), as discussed above. Some
internucleotide linkage analogs include morpholidate, acetal, and
polyamide-linked heterocycles.
[0108] The oligonucleotides can be locked nucleic acids. Locked
nucleic acids (LNA) are modified RNA nucleotides (see, for example,
Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNAs form hybrids
with DNA which are more stable than DNA/DNA hybrids, a property
similar to that of peptide nucleic acid (PNA)/DNA hybrids.
Therefore, LNA can be used just as PNA molecules would be. LNA
binding efficiency can be increased in some embodiments by adding
positive charges to it. Commercial nucleic acid synthesizers and
standard phosphoramidite chemistry are used to make LNAs.
[0109] In some embodiments, the oligonucleotides are composed of
peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic
DNA mimics in which the phosphate backbone of the oligonucleotide
is replaced in its entirety by repeating N-(2-aminoethyl)-glycine
units and phosphodiester bonds are typically replaced by peptide
bonds. The various heterocyclic bases are linked to the backbone by
methylene carbonyl bonds. PNAs maintain spacing of heterocyclic
bases that is similar to conventional DNA oligonucleotides, but are
achiral and neutrally charged molecules. Peptide nucleic acids are
formed of peptide nucleic acid monomers.
[0110] Other backbone modifications include peptide and amino acid
variations and modifications. Thus, the backbone constituents of
oligonucleotides such as PNA may be peptide linkages, or
alternatively, they may be non-peptide peptide linkages. Examples
include acetyl caps, amino spacers such as
8-amino-3,6-dioxaoctanoic acid (referred to herein as 0-linkers),
amino acids such as lysine are particularly useful if positive
charges are desired in the PNA, and the like. Methods for the
chemical assembly of PNAs are well known. See, for example, U.S.
Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336,
5,773,571 and 5,786,571.
[0111] Oligonucleotides optionally include one or more terminal
residues or modifications at either or both termini to increase
stability, and/or affinity of the oligonucleotide for its target.
Commonly used positively charged moieties include the amino acids
lysine and arginine, although other positively charged moieties may
also be useful. Oligonucleotides may further be modified to be end
capped to prevent degradation using a propylamine group. Procedures
for 3' or 5' capping oligonucleotides are well known in the
art.
[0112] The functional nucleic acid can be single stranded or double
stranded.
[0113] The nucleic acid molecule can exist as a separate molecule
independent of other sequences (e.g., a chemically synthesized
nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or
restriction endonuclease treatment), as well as recombinant DNA
that is incorporated into a vector, an autonomously replicating
plasmid, etc. The nucleic acid can be an engineered nucleic acid
such as a recombinant DNA molecule that is part of a hybrid or
fusion nucleic acid. In some embodiments, the nucleic acids contain
a fraction of AAV genome for enhanced packaging and delivery, for
example a 5' AAV inverted terminal repeat (ITR), a 3' AAV inverted
terminal repeat (ITR), a promoter and optional enhancer, a
polyadenylation signal. In some embodiments, the nucleic acid
includes AAV vector for example, a VP1, VP2, or VP3 capsid selected
from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAV10, AAV11, or mixtures, variants or derivatives
thereof.
[0114] 1. Functional Nucleic Acid Molecules
[0115] Functional nucleic acids are nucleic acid molecules that
have a specific function, such as binding a target molecule or
catalyzing a specific reaction. As discussed in more detail below,
functional nucleic acid molecules can be divided into the following
non-limiting categories: antisense molecules, RNAi including siRNA,
miRNA, shRNA, Dicer siRNA, and piRNA, aptamers, ribozymes, triplex
forming molecules, external guide sequences, and gene editing
compositions. The functional nucleic acid molecules can act as
effectors, inhibitors, modulators, and stimulators of a specific
activity possessed by a target molecule, or the functional nucleic
acid molecules can possess a de novo activity independent of any
other molecules.
[0116] Functional nucleic acid molecules can interact with any
macromolecule, such as DNA, RNA, polypeptides, or carbohydrate
chains. Thus, functional nucleic acids can interact with the mRNA
or the genomic DNA of a target polypeptide or they can interact
with the polypeptide itself. Often functional nucleic acids are
designed to interact with other nucleic acids based on sequence
homology between the target molecule and the functional nucleic
acid molecule. In other situations, the specific recognition
between the functional nucleic acid molecule and the target
molecule is not based on sequence homology between the functional
nucleic acid molecule and the target molecule, but rather is based
on the formation of tertiary structure that allows specific
recognition to take place.
[0117] The functional nucleic acids can be antisense molecules.
Antisense molecules are designed to interact with a target nucleic
acid molecule through either canonical or non-canonical base
pairing. The interaction of the antisense molecule and the target
molecule is designed to promote the destruction of the target
molecule through, for example, RNAse H mediated RNA-DNA hybrid
degradation. Alternatively the antisense molecule is designed to
interrupt a processing function that normally would take place on
the target molecule, such as transcription or replication.
Antisense molecules can be designed based on the sequence of the
target molecule. There are numerous methods for optimization of
antisense efficiency by finding the most accessible regions of the
target molecule. Exemplary methods include in vitro selection
experiments and DNA modification studies using DMS and DEPC. It is
preferred that antisense molecules bind the target molecule with a
dissociation constant (K.sub.d) less than or equal to 10.sup.-6,
10.sup.-8, 10.sup.-10, or 10.sup.-12 M.
[0118] The functional nucleic acids can be aptamers. Aptamers are
molecules that interact with a target molecule, preferably in a
specific way. Typically aptamers are small nucleic acids ranging
from 15-50 bases in length that fold into defined secondary and
tertiary structures, such as stem-loops or G-quartets. Aptamers can
bind small molecules, such as ATP and theophiline, as well as large
molecules, such as reverse transcriptase and thrombin. Aptamers can
bind very tightly with K.sub.d's from the target molecule of less
than 10.sup.-12 M. It is preferred that the aptamers bind the
target molecule with a K.sub.d less than 10.sup.-6, 10.sup.-8,
10.sup.-10, or 10.sup.-12 M. Aptamers can bind the target molecule
with a very high degree of specificity. For example, aptamers have
been isolated that have greater than a 10,000 fold difference in
binding affinities between the target molecule and another molecule
that differ at only a single position on the molecule. It is
preferred that the aptamer have a K.sub.d with the target molecule
at least 10, 100, 1000, 10,000, or 100,000 fold lower than the
K.sub.d with a background binding molecule. It is preferred when
doing the comparison for a molecule such as a polypeptide, that the
background molecule be a different polypeptide.
[0119] The functional nucleic acids can be ribozymes. Ribozymes are
nucleic acid molecules that are capable of catalyzing a chemical
reaction, either intramolecularly or intermolecularly. It is
preferred that the ribozymes catalyze intermolecular reactions.
There are a number of different types of ribozymes that catalyze
nuclease or nucleic acid polymerase type reactions which are based
on ribozymes found in natural systems, such as hammerhead
ribozymes. There are also a number of ribozymes that are not found
in natural systems, but which have been engineered to catalyze
specific reactions de novo. Preferred ribozymes cleave RNA or DNA
substrates, and more preferably cleave RNA substrates. Ribozymes
typically cleave nucleic acid substrates through recognition and
binding of the target substrate with subsequent cleavage. This
recognition is often based mostly on canonical or non-canonical
base pair interactions. This property makes ribozymes particularly
good candidates for target specific cleavage of nucleic acids
because recognition of the target substrate is based on the target
substrates sequence.
[0120] The functional nucleic acids can be triplex forming
molecules. Triplex forming functional nucleic acid molecules are
molecules that can interact with either double-stranded or
single-stranded nucleic acid. When triplex molecules interact with
a target region, a structure called a triplex is formed in which
there are three strands of DNA forming a complex dependent on both
Watson-Crick and Hoogsteen base-pairing. Triplex molecules are
preferred because they can bind target regions with high affinity
and specificity. It is preferred that the triplex forming molecules
bind the target molecule with a K.sub.d less than 10.sup.-6,
10.sup.-8, 10.sup.-10, or 10.sup.-12 M.
[0121] The functional nucleic acids can be external guide
sequences. External guide sequences (EGSs) are molecules that bind
a target nucleic acid molecule forming a complex, which is
recognized by RNase P, which then cleaves the target molecule. EGSs
can be designed to specifically target an RNA molecule of choice.
RNAse P aids in processing transfer RNA (tRNA) within a cell.
Bacterial RNAse P can be recruited to cleave virtually any RNA
sequence by using an EGS that causes the target RNA:EGS complex to
mimic the natural tRNA substrate. Similarly, eukaryotic EGS/RNAse
P-directed cleavage of RNA can be utilized to cleave desired
targets within eukarotic cells. Representative examples of how to
make and use EGS molecules to facilitate cleavage of a variety of
different target molecules are known in the art.
[0122] In some embodiments, the functional nucleic acids induce
gene silencing through RNA interference. Gene expression can also
be effectively silenced in a highly specific manner through RNA
interference (RNAi), which can generally be divided into three
major classes based on their processing mechanisms and partner
Argonaute proteins: micro RNAs (miRNAs), small interfering RNAs
(siRNAs), dicer siRNA, shRNA, and PIWI-interacting RNA (piRNAs)
(Czech and Hannon, Trends Biochem Sci., 2016 Jan. 19. pii:
50968-0004(15)00258-3. doi: 10.1016/j.tibs.2015.12.008.
[0123] RNAi silencing was originally observed with the addition of
double stranded RNA (dsRNA) (Fire, et al. (1998) Nature,
391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89; Hannon,
(2002) Nature, 418:244-51). Once dsRNA enters a cell, it is cleaved
by an RNase III-like enzyme, Dicer, into double stranded small
interfering RNAs (siRNA) 21-23 nucleotides in length that contains
2 nucleotide overhangs on the 3' ends (Elbashir, et al. (2001)
Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6;
Hammond, et al. (2000) Nature, 404:293-6). In an ATP dependent
step, the siRNAs become integrated into a multi-subunit protein
complex, commonly known as the RNAi induced silencing complex
(RISC), which guides the siRNAs to the target RNA sequence
(Nykanen, et al. (2001) Cell, 107:309-21). At some point the siRNA
duplex unwinds, and it appears that the antisense strand remains
bound to RISC and directs degradation of the complementary mRNA
sequence by a combination of endo and exonucleases (Martinez, et
al. (2002) Cell, 110:563-74). However, the effect of iRNA or siRNA
or their use is not limited to any type of mechanism.
[0124] Short Interfering RNA (siRNA) is a double-stranded RNA that
can induce sequence-specific post-transcriptional gene silencing,
thereby decreasing or even inhibiting gene expression. In one
example, a siRNA triggers the specific degradation of homologous
RNA molecules, such as mRNAs, within the region of sequence
identity between both the siRNA and the target RNA. For example, WO
02/44321 discloses siRNAs capable of sequence-specific degradation
of target mRNAs when base-paired with 3' overhanging ends.
[0125] Sequence specific gene silencing can be achieved in
mammalian cells using synthetic, short double-stranded RNAs that
mimic the siRNAs produced by the enzyme dicer. siRNA can be
chemically or in vitro-synthesized or can be the result of short
double-stranded hairpin-like RNAs (shRNAs) that are processed into
siRNAs inside the cell. Synthetic siRNAs are generally designed
using algorithms and a conventional DNA/RNA synthesizer. The
production of siRNA from a vector is more commonly done through the
transcription of a short hairpin RNAse (shRNAs).
[0126] Micro RNAs (abbreviated miRNA) are small non-coding RNA
molecules (containing about 22 nucleotides) that function in RNA
silencing and post-transcriptional regulation of gene expression.
miRNAs resemble siRNAs of the RNA interference (RNAi) pathway,
except miRNAs derive from regions of RNA transcripts that fold back
on themselves to form short hairpins, whereas siRNAs derive from
longer regions of double-stranded RNA (Bartel, et al., Cell,
116:281-297 (2004)). The biogenesis of miRNAs and siRNAs typically
depends on RNase III type enzymes that convert their
double-stranded RNA precursors into functional small RNAs. By
contrast, piRNAs derive from single-stranded RNAs and,
consequently, require alternative processing machinery.
[0127] Synthetic piRNAs can be used to block the synthesis of
target proteins by binding to mRNAs, as has been attempted with
miRNAs, might have the advantage of not requiring processing by
enzymes such as Dicer, which is required by miRNAs. Potential
advantages of piRNAs over miRNAs include the possibility of targets
with better specificity because each miRNA regulates several mRNAs
and there is the potential to access undesirable long non-coding
RNAs with possible implications in disease processes (Assumpcao, et
al., Epigenomics, 7(6):975-984 (2015)). miRNA and piRNA can be the
therapeutic agent or can be target sequences for
post-transcriptional silencing. For example, synthetic piRNAs
designed to couple to PIWI proteins and exert genomic silencing on
PIWI genes at a transcriptional level is a possible strategy.
[0128] In some embodiments, the functional nucleic acid is siRNA,
shRNA, miRNA, or piRNA. In some embodiments, the composition
includes a vector expressing the functional nucleic acid. Methods
of making and using vectors for in vivo expression of functional
nucleic acids such as antisense oligonucleotides, siRNA, shRNA,
miRNA, piRNA, EGSs, ribozymes, and aptamers are known in the
art.
[0129] 2. Gene Editing Compositions
[0130] In some embodiments the functional nucleic acids are gene
editing compositions. Gene editing compositions can include nucleic
acids that encode an element or elements that induce a single or a
double strand break in the target cell's genome, and optionally a
polynucleotide. The compositions can be used, for example, to
reduce or otherwise modify expression of a gene target.
[0131] In some embodiments, the element that induces a single or a
double strand break in the target cell's genome is a CRISPR/Cas
system. CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) is an acronym for DNA loci that contain multiple, short,
direct repetitions of base sequences. The prokaryotic CRISPR/Cas
system has been adapted for use as gene editing (silencing,
enhancing or changing specific genes) for use in eukaryotes (see,
for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek,
et al., Science, 337(6096):816-21 (2012)). By transfecting a cell
with the required elements including a cas gene and specifically
designed CRISPRs, the organism's genome can be cut and modified at
any desired location. Methods of preparing compositions for use in
genome editing using the CRISPR/Cas systems are described in detail
in WO 2013/176772 and WO 2014/018423, which are specifically
incorporated by reference herein in their entireties.
[0132] In general, "CRISPR system" refers collectively to
transcripts and other elements involved in the expression of or
directing the activity of CRISPR-associated ("Cas") genes,
including sequences encoding a Cas gene, a tracr (trans-activating
CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a
tracr-mate sequence (encompassing a "direct repeat" and a
tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR system), a guide sequence (also referred to as a
"spacer" in the context of an endogenous CRISPR system), or other
sequences and transcripts from a CRISPR locus. One or more tracr
mate sequences operably linked to a guide sequence (e.g., direct
repeat-spacer-direct repeat) can also be referred to as pre-crRNA
(pre-CRISPR RNA) before processing or crRNA after processing by a
nuclease.
[0133] In some embodiments, a tracrRNA and crRNA are linked and
form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused
to a partial tracrRNA via a synthetic stem loop to mimic the
natural crRNA:tracrRNA duplex as described in Cong, Science,
15:339(6121):819-823 (2013) and Jinek, et al., Science,
337(6096):816-21 (2012)). A single fused crRNA-tracrRNA construct
can also be referred to as a guide RNA or gRNA (or single-guide RNA
(sgRNA)). Within an sgRNA, the crRNA portion can be identified as
the `target sequence` and the tracrRNA is often referred to as the
`scaffold`.
[0134] There are many resources available for helping practitioners
determine suitable target sites once a desired DNA target sequence
is identified. For example, numerous public resources, including a
bioinformatically generated list of about 190,000 potential sgRNAs,
targeting more than 40% of human exons, are available to aid
practitioners in selecting target sites and designing the associate
sgRNA to affect a nick or double strand break at the site. See
also, http://crispr.i2bc.paris-saclay.fr, a tool designed to help
scientists find CRISPR targeting sites in a wide range of species
and generate the appropriate crRNA sequences.
[0135] In some embodiments, one or more vectors driving expression
of one or more elements of a CRISPR system are introduced into a
target cell such that expression of the elements of the CRISPR
system direct formation of a CRISPR complex at one or more target
sites. While the specifics can be varied in different engineered
CRISPR systems, the overall methodology is similar. CRISPR
technology can be used to target a DNA sequence by inserting a
short DNA fragment containing the target sequence into a guide RNA
expression plasmid. The sgRNA expression plasmid contains the
target sequence (about 20 nucleotides), a form of the tracrRNA
sequence (the scaffold) as well as a suitable promoter and
necessary elements for proper processing in eukaryotic cells. Such
vectors are commercially available (see, for example, Addgene).
Many of the systems rely on custom, complementary oligos that are
annealed to form a double stranded DNA and then cloned into the
sgRNA expression plasmid. Co-expression of the sgRNA and the
appropriate Cas enzyme from the same or separate plasmids in
transfected cells results in a single or double strand break
(depending of the activity of the Cas enzyme) at the desired target
site.
[0136] In some embodiments, the element that induces a single or a
double strand break in the target cell's genome is a nucleic acid
construct or constructs encoding a zinc finger nucleases (ZFNs).
ZFNs are typically fusion proteins that include a DNA-binding
domain derived from a zinc-finger protein linked to a cleavage
domain.
[0137] The most common cleavage domain is the Type IIS enzyme Fok1.
Fok1 catalyzes double-stranded cleavage of DNA, at 9 nucleotides
from its recognition site on one strand and 13 nucleotides from its
recognition site on the other. See, for example, U.S. Pat. Nos.
5,356,802; 5,436,150 and 5,487,994; as well as Li et al. Proc.,
Natl. Acad. Sci. USA 89 (1992):4275-4279; Li et al. Proc. Natl.
Acad. Sci. USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad.
Sci. USA. 91:883-887 (1994a); Kim et al. J. Biol. Chem.
269:31,978-31,982 (1994b). One or more of these enzymes (or
enzymatically functional fragments thereof) can be used as a source
of cleavage domains.
[0138] The DNA-binding domain, which can, in principle, be designed
to target any genomic location of interest, can be a tandem array
of Cys.sub.2His.sub.2 zinc fingers, each of which generally
recognizes three to four nucleotides in the target DNA sequence.
The Cys.sub.2His.sub.2 domain has a general structure: Phe
(sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)-Phe
(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino
acids)-His. By linking together multiple fingers (the number
varies: three to six fingers have been used per monomer in
published studies), ZFN pairs can be designed to bind to genomic
sequences 18-36 nucleotides long.
[0139] Engineering methods include, but are not limited to,
rational design and various types of empirical selection methods.
Rational design includes, for example, using databases including
triplet (or quadruplet) nucleotide sequences and individual zinc
finger amino acid sequences, in which each triplet or quadruplet
nucleotide sequence is associated with one or more amino acid
sequences of zinc fingers which bind the particular triplet or
quadruplet sequence. See, for example, U.S. Pat. Nos. 6,140,081;
6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617;
U.S. Published Application Nos. 2002/0165356; 2004/0197892;
2007/0154989; 2007/0213269; and International Patent Application
Publication Nos. WO 98/53059 and WO 2003/016496.
[0140] In some embodiments, the element that induces a single or a
double strand break in the target cell's genome is a nucleic acid
construct or constructs encoding a transcription activator-like
effector nuclease (TALEN). TALENs have an overall architecture
similar to that of ZFNs, with the main difference that the
DNA-binding domain comes from TAL effector proteins, transcription
factors from plant pathogenic bacteria. The DNA-binding domain of a
TALEN is a tandem array of amino acid repeats, each about 34
residues long. The repeats are very similar to each other;
typically they differ principally at two positions (amino acids 12
and 13, called the repeat variable diresidue, or RVD). Each RVD
specifies preferential binding to one of the four possible
nucleotides, meaning that each TALEN repeat binds to a single base
pair, though the NN RVD is known to bind adenines in addition to
guanine. TAL effector DNA binding is mechanistically less well
understood than that of zinc-finger proteins, but their seemingly
simpler code could prove very beneficial for engineered-nuclease
design. TALENs also cleave as dimers, have relatively long target
sequences (the shortest reported so far binds 13 nucleotides per
monomer) and appear to have less stringent requirements than ZFNs
for the length of the spacer between binding sites. Monomeric and
dimeric TALENs can include more than 10, more than 14, more than
20, or more than 24 repeats.
[0141] Methods of engineering TAL to bind to specific nucleic acids
are described in Cermak, et al, Nucl. Acids Res. 1-11 (2011). US
Published Application No. 2011/0145940, which discloses TAL
effectors and methods of using them to modify DNA. Miller et al.
Nature Biotechnol 29: 143 (2011) reported making TALENs for
site-specific nuclease architecture by linking TAL truncation
variants to the catalytic domain of Fold nuclease. The resulting
TALENs were shown to induce gene modification in immortalized human
cells. General design principles for TALE binding domains can be
found in, for example, WO 2011/072246.
[0142] The nuclease activity of the genome editing systems
described herein cleave target DNA to produce single or double
strand breaks in the target DNA. Double strand breaks can be
repaired by the cell in one of two ways: non-homologous end
joining, and homology-directed repair. In non-homologous end
joining (NHEJ), the double-strand breaks are repaired by direct
ligation of the break ends to one another. As such, no new nucleic
acid material is inserted into the site, although some nucleic acid
material may be lost, resulting in a deletion. In homology-directed
repair, a donor polynucleotide with homology to the cleaved target
DNA sequence is used as a template for repair of the cleaved target
DNA sequence, resulting in the transfer of genetic information from
a donor polynucleotide to the target DNA. As such, new nucleic acid
material can be inserted/copied into the site.
[0143] Therefore, in some embodiments, the genome editing
composition optionally includes a donor polynucleotide. The
modifications of the target DNA due to NHEJ and/or
homology-directed repair can be used to induce gene correction,
gene replacement, gene tagging, transgene insertion, nucleotide
deletion, gene disruption, gene mutation, etc.
[0144] Accordingly, cleavage of DNA by the genome editing
composition can be used to delete nucleic acid material from a
target DNA sequence by cleaving the target DNA sequence and
allowing the cell to repair the sequence in the absence of an
exogenously provided donor polynucleotide.
[0145] Alternatively, if the genome editing composition includes a
donor polynucleotide sequence that includes at least a segment with
homology to the target DNA sequence, the methods can be used to
add, i.e., insert or replace, nucleic acid material to a target DNA
sequence (e.g., to "knock in" a nucleic acid that encodes for a
protein, an siRNA, an miRNA, etc.), to add a tag (e.g.,
6.times.His, a fluorescent protein (e.g., a green fluorescent
protein; a yellow fluorescent protein, etc.), hemagglutinin (HA),
FLAG, etc.), to add a regulatory sequence to a gene (e.g.,
promoter, polyadenylation signal, internal ribosome entry sequence
(IRES), 2A peptide, start codon, stop codon, splice signal,
localization signal, etc.), to modify a nucleic acid sequence
(e.g., introduce a mutation), and the like. As such, the
compositions can be used to modify DNA in a site-specific, i.e.,
"targeted", way, for example gene knock-out, gene knock-in, gene
editing, gene tagging, etc. as used in, for example, gene
therapy.
[0146] In applications in which it is desirable to insert a
polynucleotide sequence into a target DNA sequence, a
polynucleotide including a donor sequence to be inserted is also
provided to the cell. By a "donor sequence" or "donor
polynucleotide" or "donor oligonucleotide" it is meant a nucleic
acid sequence to be inserted at the cleavage site. The donor
polynucleotide typically contains sufficient homology to a genomic
sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or
100% homology with the nucleotide sequences flanking the cleavage
site, e.g., within about 50 bases or less of the cleavage site,
e.g., within about 30 bases, within about 15 bases, within about 10
bases, within about 5 bases, or immediately flanking the cleavage
site, to support homology-directed repair between it and the
genomic sequence to which it bears homology. The donor sequence is
typically not identical to the genomic sequence that it replaces.
Rather, the donor sequence may contain at least one or more single
base changes, insertions, deletions, inversions or rearrangements
with respect to the genomic sequence, so long as sufficient
homology is present to support homology-directed repair. In some
embodiments, the donor sequence includes a non-homologous sequence
flanked by two regions of homology, such that homology-directed
repair between the target DNA region and the two flanking sequences
results in insertion of the non-homologous sequence at the target
region.
[0147] 3. Peptide and Protein Expression Constructs
[0148] In some embodiments, the active agent is a nucleic acid
encoding a protein or a polypeptide. Although discussed here in the
context of mRNA, it will be appreciated that the nucleic acid
active agent can itself be an mRNA, or can be a DNA or other
oligonucleotide encoding the mRNA (or a functional nucleic acid as
discussed above). As discussed in more detail below, the nucleic
acid active agents, including mRNA and functional nucleic acids,
can be encoded by a nucleic acid that encodes the RNA. The nucleic
acid can be operably linked to an expression control sequence. In
some embodiments, the nucleic acid is a vector, integration
construct, etc., that enables expression of the RNA in a cell.
[0149] The mRNA can be a mature mRNA or pre-mRNA. Thus in some
embodiments, the mRNA includes introns. The mRNA can be a naturally
occurring gene transcript, for example, a human gene transcript.
The mRNA can be an artificial sequence that is not normally
expressed in a naturally occurring organism. An exemplary
artificial sequence is one that contains portions of gene sequences
that are ligated together to form an open reading frame that
encodes a fusion protein. The portions of that are ligated together
can be from a single organism or from more than one organism.
[0150] The mRNA can encode a polypeptide that provides a
therapeutic or prophylactic effect to an organism or that can be
used to diagnose a disease or disorder in an organism. For example,
for treatment of cancer, autoimmune disorders, parasitic, viral,
bacterial, fungal or other infections, the polypeptide can be a
ligand or receptor for cells of the immune system, or can function
to stimulate or inhibit the immune system of an organism.
Typically, it is not desirable to have prolonged ongoing
stimulation of the immune system, nor necessary to produce changes
which last after successful treatment, since this may then elicit a
new problem. For treatment of an autoimmune disorder, it may be
desirable to inhibit or suppress the immune system during a
flare-up, but not long term, which could result in the patient
becoming overly sensitive to an infection. Thus in some
embodiments, delivery of mRNA for transient expression of the
protein (or functional nucleic acid) is preferred to sustained
expression by a vector or gene integration.
[0151] The mRNA can include a 5' cap. A 5' cap (also termed an RNA
cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified
guanine nucleotide that has been added to the "front" or 5' end of
a eukaryotic messenger RNA shortly after the start of
transcription. The 5' cap consists of a terminal group which is
linked to the first transcribed nucleotide. Its presence is
critical for recognition by the ribosome and protection from
RNases. Cap addition is coupled to transcription, and occurs
co-transcriptionally, such that each influences the other. Shortly
after the start of transcription, the 5' end of the mRNA being
synthesized is bound by a cap-synthesizing complex associated with
RNA polymerase. This enzymatic complex catalyzes the chemical
reactions that are required for mRNA capping. Synthesis proceeds as
a multi-step biochemical reaction. The capping moiety can be
modified to modulate functionality of mRNA such as its stability or
efficiency of translation. The 5' cap may, for example, be
m7G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')G or G(5')ppp(5')A
cap analogs, which are all commercially available. The 5' cap can
also be an anti-reverse-cap-analog (ARCA) (see, e.g., Stepinski, et
al., RNA, 7:1468-95 (2001)) or any other suitable analog. The 5'
cap is provided using techniques known in the art and described
herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001);
Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim
Biophys. Res. Commun., 330:958-966 (2005)). The mRNA can contain an
internal ribosome entry site (IRES) sequence. The IRES sequence may
be any viral, chromosomal or artificially designed sequence which
initiates cap-independent ribosome binding to mRNA and facilitates
the initiation of translation.
[0152] The mRNA can include a 5' untranslated region. The 5' UTR is
upstream from the coding sequence. Within the 5' UTR is a sequence
that is recognized by the ribosome which allows the ribosome to
bind and initiate translation. The mechanism of translation
initiation differs in Prokaryotes and Eukaryotes.
[0153] The mRNA includes an "open reading frame" or "ORF," which is
a series of nucleotides that contains a sequence of bases that
could potentially encode a polypeptide or protein. An open reading
frame is located between the start-code sequence (initiation codon
or start codon) and the stop-codon sequence (termination codon).
The ORF can be from a naturally occurring sequence from the genome
of an organism.
[0154] The mRNA can include a 3' untranslated region. The 3' UTR is
found immediately following the translation stop codon. The 3' UTR
plays an important role in translation termination as well as post
transcriptional gene expression.
[0155] In some embodiments, the mRNA is polyadenylated.
"Polyadenylation" refers to the covalent linkage of a polyadenylyl
moiety, or its modified variant, to a messenger RNA molecule. In
eukaryotic organisms, most messenger RNA (mRNA) molecules are
polyadenylated at the 3' end. The 3' poly(A) tail is a long
sequence of adenine nucleotides (often several hundred) added to
the pre-mRNA through the action of an enzyme, polyadenylate
polymerase. In higher eukaryotes, the poly(A) tail is added onto
transcripts that contain a specific sequence, the polyadenylation
signal. The poly(A) tail and the protein bound to it aid in
protecting mRNA from degradation by exonucleases. Polyadenylation
is also important for transcription termination, export of the mRNA
from the nucleus, and translation. Polyadenylation occurs in the
nucleus immediately after transcription of DNA into RNA, but
additionally can also occur later in the cytoplasm. After
transcription has been terminated, the mRNA chain is cleaved
through the action of an endonuclease complex associated with RNA
polymerase. The cleavage site is usually characterized by the
presence of the nucleotide base sequence AAUAAA near the cleavage
site. After the mRNA has been cleaved, adenosine residues are added
to the free 3' end at the cleavage site.
[0156] RNA, including mRNA and RNA-based functional nucleic acids,
can be prepared by in vitro transcription using, for example, a
purified linear DNA template containing a promoter, ribonucleotide
triphosphates, a buffer system that includes DTT and magnesium
ions, and an appropriate phage RNA polymerase. The template can be
a vector, PCR product, synthetic oligonucleotide, or cDNA.
[0157] 4. Genes Targeted for Expression or Suppression
[0158] The genetic material to be loaded into the particles is
chosen on the basis of the desired effect of that genetic material
on the cell into which it is intended to be delivered and the
mechanism by which that effect is to be carried out. For example,
the nucleic acid may be useful in gene therapy, for example in
order to express a desired gene in a cell or group of cells.
Nucleic acid can also be used in gene silencing. Such gene
silencing may be useful in therapy to switch off aberrant gene
expression. Nucleic acid can also be used for example to express
one or more antigens against which it is desired to produce an
immune response. Thus, the nucleic acid to be loaded into the
particle can encode one or more antigens against which is desired
to produce an immune response, including but not limited to tumour
antigens, antigens from pathogens such as viral, bacterial or
fungal pathogens, such as those discussed in more detail below.
Therapeutic strategies for treating cancer, inflammation, injury,
autoimmunity, and infections are discussed in more detail
below.
[0159] In some embodiments, the nanoparticles contain two or more
species of nucleic acids, where one species of nucleic acid has a
stimulatory effect upon one or more "target" genes, proteins or
other biological molecules, and the second (or further) nucleic
acid species suppresses the expression or function of one or more
"target" genes, proteins or other biological molecules. Exemplary
suppressor or inhibitory nucleic acids include functional nucleic
acids, such as silencing RNAs (siRNAs) specific to one or more
"target" genes or molecules. Exemplary stimulatory nucleic acids
include messenger RNAs (mRNAs) that directly express, or otherwise
induce expression of one or more "target" gene products or other
molecules.
[0160] Exemplary targets for suppressor/inhibitory nucleic acids
include co-stimulatory genes, proteins and molecules, for example,
to induce immunological tolerance, and tumorigenic driver genes,
proteins and activators, for example, to reduce the viability and
proliferation of cancer cells. Exemplary targets for stimulatory
nucleic acids include tolerogenic antigens and tumor suppressor
genes, proteins and molecules.
[0161] The desired effect of the inhibition or stimulation of a
target by the nucleic acid(s) can directly effect one or more
cellular molecules or processes, or can indirectly effect one or
more cellular molecules or processes. For example, in some
embodiments, the gene product(s) of a stimulatory nucleic acid act
to block, reduce or otherwise inhibit the function or expression of
a co-stimulatory molecule, for example, to induce immunogenic
tolerance against a target antigen. In other embodiments, the
suppressor/inhibitory nucleic acids block or reduce expression or
activation of genes or proteins that lead to the up-regulation of a
cellular molecuke or process, such as up-regulation of a tumor
suppressor.
[0162] a. Tumor Suppressors
[0163] In some embodiments, the mRNA can be tumor
suppressor-encoded mRNA. In some embodiments, the mRNA encodes one
or more exogenous or endogenus proteins that are expressed within
the recipient to prevent, reduce or otherwise minimize the
proliferation and/or viability of cancer cells within the
recipient. As used herein, a tumor suppressor is a protein that
acts to reduce the potential for cancer development and metastasis
by modulating tumor cell growth, by negative regulation of the cell
cycle or by promoting apoptosis. A number of tumor suppressors are
known in the art (see Table 1).
[0164] Therefore, in some embodiments the nanoparticles include
nucleic acids that encode tumor suppressor proteins, or activators
of tumor suppressor proteins. A non-limiting list of tumor
suppressor proteins that can be encoded by mRNAs within
nanoparticles is provided in Table 1. One skilled in the art will
understand that certain cancers are associated with or result from
deficiencies or other abnormal expression of one or more gebe
products. Thus, in some embodiments nanoparticles include mRNAs
encoding genes that are expressed in or around the cancer cells,
for example, to compensate or otherwise make up for the
deficiencies or otherwise abnormal expression of genes that results
in the cancer phenotype. In some embodiments, specific cancer cells
are targeted with nanoparticles including one or more mRNAs that
encode a gene product that is designed to suppress one or more of
the biological functions of the targeted cancer cell. In some
embodiments, the nanoparticles include two or more mRNAs encoding
two or more tumor suppressors. For example, the simultaneous
expression of the two or more tumor suppressor genes in the same
cancer cell results in reduced proliferation and/or viability of
the cell. In other embodiments, the nanoparticles include one or
more mRNAs encoding a tumor suppressor, and one or more nucleic
acids that suppresses one or more genes or gene products in the
cancer cell. For example, expression of the one or more tumor
suppressors and the simultaneous suppression of the one or more
targeted genes within the same cell results in reduced
proliferation and/or viability of the cell. In a preferred
embodiment, the nanoparticles include a combination of mRNA(s)
encoding tumor suppressors, and one or more siRNAs targeting an
oncogene and/or a tumorigenic driver element. Preferrably, the
simultaneous expression of one or more tumor suppressors, and the
silencing or down-regulating of one or more oncogenes and/or a
tumorigenic driver elements within a cancer cell results in
selective killing of the cancer cell.
TABLE-US-00001 TABLE 1 Exemplary tumor suppressors that can be
enhanced or expressed Genetic Associated GenBank Acc No. GENE
Alteration(s) Cancer(s) mRNA Protein PTEN Point Prostate, breast,
AF067844.1 AAD13528.1 mutation, glioblastoma, deletion melanoma,
pancreatic cancer, colorectal cancer, leukemia APC Point
Adenomatous M74088.1 AAA03586.1 mutation, polyposis and deletion
sporadic colorectal tumors ARF Deletion Breast carcinomas,
AF208864.1 AAF64278.1 colorectal adenoma, glioblastoma BMPR Point
mutation Gastrointestinal NM_009758.4 NP_033888.2 cancer BRCA1
Point mutation Ductal breast U14680.1 AAA73985.1 cancers,
Epithelial ovarian cancers E-cadherin Point mutation Loss of
function Z13009.1 CAA78353.1 leads to metastasis EXT1,2 Point
Hereditary multiple S79639.1, AAB62283.1 mutation, exostoses, also
U62740.1 AAB07008.1 deletion, known as diaphyseal insertion aclasis
FBXW7 Point Breast cancer AF411971.1 AAL06290.1 mutation, deletion
FH Point mutation Hereditary BC003108.1 AAH03108.1 leiomyomatosis
and renal-cell cancer GPC3 Deletions, Lung carcinoma L47125.1
AAA98132.1 point mutation HIPK2 Point mutation Metastatic bladder
AF208291 AAG41236.1 cancer HRPT2 Point mutation Hereditary DQ366291
hyperparathyroidism- jaw tumor syndrome, Malignancy in sporadic
parathyroid tumors INPP4B Deletion, loss Epithelial U96922.1
AAB72153.1 of carcinomas and some heterozygosity, human basal-like
reduced breast carcinomas expression LKB1 Point Human Lung Cancer
U63333.1 AAB05809.1 mutation, (especially NSCLC), deletion cervical
carcinomas Inherited cancer disorder Peutz- Jeghers Syndrome MEN1
Point mutation Pituitary tumors U93236.1 AAC51228.1 MMR Point
Hereditary non- genes mutation, polyposis colon reduced cancer
expression MUTYH Point Lung and ovarian U63329.1 AAC50618.1
mutation, tumors, and deletion lymphomas NF1 Point Juvenile
NM_000267.3 NP_000258.1 mutation, myelomonocytic deletion leukemia,
Watson syndrome and breast cancer. NF2 Point Meningioma L11353.1
AAA36212.1 mutation, Thyroid cancer, deletion mesothelioma, and
melanoma p15, p16 Point mutation Colorectal cancer, AB060808.1
BAB91133.1 leukemia L27211.1 AAA92554.1 p53 Point Lung AF307851.1
AAG28785.1 mutation, Prostate deletion p57 Point mutation Beckwith-
D64137.1 BAA11014.1 Wiedemann syndrome Ptch Point mutation Cell
carcinomas of A1494442.1 Q13635 the skin, ovarian NM_000264.4
NP_000255.2 fibromas, and medulloblastomas Rb Point Prostate cancer
M15400.1 AAA69807.1 mutation, Pituitary deletion melanotroph tumors
RECQL4 Point mutation Osteosarcoma AB006532.1 BAA74453.1 SDH Point
Paraganglioma, renal U17248.1 AAA81167.1 mutation, cell carcinoma
deletion Smad2/3 Point Breast cancer U65019.1 AAB17054.1 mutation,
BC050743.1 AAH50743.1 deletions Smad4 Point mutation Pancreatic
U44378.1 AAA91041.1 Gastric Carcinoma Su(Fu) Point Brain tumor Not
available Not available mutation, deletion TGF.beta.R Point
mutation Head and neck Not available 5E92_A cancers, cervical and
ovarian carcinomas TSC1/TSC2 Point mutation Tuberous sclerosis
AF013168.1 AAC51674.1'' complex AB014460.1 BAA32694.1'' VHL Point
Renal carcinomas L15409.1 AAB64200 mutation, deletion, hyper-
methylation WT1 Point Haematological NM_000378.4 NP_000369.3
mutation, malignancies deletion Pediatric nephroblastoma Wilms
tumor XPA, C, D Point mutation Bladder cancer D14533.1 BAA03403.1
.alpha.-catenin Point mutation Basal-like breast HUMACA BAA02979
cancer RASSF1A Hyper- Lung, Cervical NM_007182 NP_009113
methylation, Cancer point mutation SDHB Point mutation Kidney
KR710096 Paragangliomas SIN3B Point mutation Prostate cancer
AAI10822 RGS12 Point mutation Prostate cancer AF035152 AAC39835
Kai1 Deletion, Prostate cancer HSU20770 CAG47051 metastasis
mutation and suppressor loss of expression ING1B Point mutation
Prostate cancer, AJ310392 NP_937861 Brain tumors Atg7 Deletion
Prostate cancer BC000091 ATG7_HUMAN JARID1D Point mutation Prostate
cancer Not available AAI46768 PALB2 Point mutation Breast cancer
NM_024675 AAH44254 53BP1 Point mutation Breast cancer NM_024675
AAH44254 RAD51 Point mutation Breast cancer HSU09477 1GZH_D XRCC4
Point mutation Breast cancer HUMRAD51 CAG38796 KEAP1 Point mutation
Liver cancer AB017445 BAB20668 ARIAD1A Point mutation Liver cancer
NM_012289 AAH15945 Ariad2 Point mutation Liver cancer Not available
Not available Rps6ka3 Point mutation Liver cancer Not available Not
available RAR.beta. Point mutation Lung cancer BC096303 BAC81131
FHIT Point mutation Lung cancer NM_001290276 BAH02279 PTCH1 Point
mutation Lung cancer HSU46922 AAH32336 DCC Point mutation
Colorectal cancer KY652975 AAH43542 Bax Point mutation Colorectal
cancer NM_005215 NP_005206 AML1 Point mutation Acute myeloid
HUMBAXA NP_620116 leukemia CDKN2A Point mutation Bladder X90981
BAA14022 Cdkn1b Point mutation Prostate cancer JQ694044 AFN61600
NKX3.1 Point mutation Prostate cancer NM_004064 CAG33680 P14 Point
mutation Melanoma NM_006167 AAB38747 CDK4 Point mutation Melanoma
NM_001098783 NP_008973 CDK6 Point mutation Melanoma NM_000075
CAG47043
[0165] In some embodiments, the nanoparticles include one or more
functional nucleic acids (such as siRNAs) that silence, or
otherwise alter the expression and/or function of tumor-associated
antigens. Exemplary tumor-associated antigens include, for example,
cellular oncogene-encoded products or aberrantly expressed
proto-oncogene-encoded products (e.g., products encoded by the neu,
ras, trk, and kit genes), or mutated forms of growth factor
receptor or receptor-like cell surface molecules (e.g., surface
receptor encoded by the c-erb B gene). Other tumor-associated
antigens that may be targeted fro genbe siliencing or modification
include molecules that may be directly involved in transformation
events, or molecules that may not be directly involved in oncogenic
transformation events but are expressed by tumor cells (e.g.,
carcinoembryonic antigen, CA-125, melonoma associated antigens,
etc.) (see, e.g., U.S. Pat. No. 6,699,475; Jager, et al., Int. J.
Cancer, 106:817-20 (2003); Kennedy, et al., Int. Rev. Immunol.,
22:141-72 (2003); Scanlan, et al. Cancer Immun., 4:1 (2004)).
[0166] Genes that encode cellular tumor associated antigens include
cellular oncogenes and proto-oncogenes that are aberrantly
expressed. In general, cellular oncogenes encode products that are
directly relevant to the transformation of the cell, and because of
this, these antigens are particularly preferred targets for
anticancer therapy. An example is the tumorigenic neu gene that
encodes a cell surface molecule involved in oncogenic
transformation. Other examples include the ras, kit, and trk genes.
The products of proto-oncogenes (the normal genes which are mutated
to form oncogenes) may be aberrantly expressed (e.g.,
overexpressed), and this aberrant expression can be related to
cellular transformation. Thus, the product encoded by
proto-oncogenes can be targeted. Some oncogenes encode growth
factor receptor molecules or growth factor receptor-like molecules
that are expressed on the tumor cell surface.
[0167] b. Vaccine Antigens
[0168] In some embodiments, the active agent is a nucleic acid
encoding an antigen designed to elicit a desired immune response.
In some embodiments the nucleic acid encodes a vaccine antigen thus
to elicit an immune response, and/or develop immune memory towards
the encoded antigen. An antigen can include any protein or peptide
that is foreign to the subject organism.
[0169] Preferred antigens can be presented at the surface of
antigen presenting cells (APC) of a subject for surveillance by
immune effector cells, such as leucocytes expressing the CD4
receptor (CD4 T cells) and Natural Killer (NK) cells. Typically,
the antigen is of viral, bacterial, protozoan, fungal, or animal
origin. In some embodiments the antigen encoded by the nucleic acid
is a cancer antigen. Cancer antigens can be antigens expressed only
on tumor cells and/or required for tumor cell survival.
[0170] Certain antigens are recognized by those skilled in the art
as immuno-stimulatory (i.e., stimulate effective immune
recognition) and provide effective immunity to the organism or
molecule from which they derive. The antigen can be derived from a
virus, bacterium, parasite, plant, protozoan, fungus, tissue or
transformed cell such as a cancer or leukemic cell and can be a
whole cell or immunogenic component thereof, e.g., cell wall
components or molecular components thereof. Suitable antigens are
known in the art and are available from commercial government and
scientific sources. Antigen encoded by nucleic acid can be all or
part of an antigenic protein. An antigen target may be provided as
single nucleic acid or may be provided in multiple nucleic
acids.
[0171] c. Antigens for Induction of Tolerance
[0172] In some embodiments, the active agent is a nucleic acid
encoding an antigen to which tolerance is desired. Suitable
antigens can be selected based on the desired therapeutic outcome
or the disease, disorder, or condition being treated. Exemplary
antigens are known in the art. See, for example, U.S. Published
Application No. 2014/0356384 which discusses:
[0173] Antigens to which tolerance is induced include
self-antigens, antigens associated with autoimmune disease such as
degenerative disease antigens, diseases such as gout where uricase
elicits an immune response, atopic disease antigens, self-antigens
such as those involved, and allergens such as insect toxins, drugs
such antibiotics like penicillin and erythromycin, and addictive
substances such as nicotine. Therefore, in some embodiments,
nanoparticles encapsulate one or more nucleic acids that silences
or expresses the urate oxidase enzyme. For example, an mRNA
encoding the uricase enzyme, or fragments thereof expresses
endogenous urate oxidase in a recipient, and a simultaneously
delivered siRNA tollerizes the host to this antigen, through
appropriate modulation of the dendritic cell phenotype in the
recipient.
[0174] Specific examples include antigen derived from naturally
occurring allergens such as pollen allergens (tree-, herb, weed-,
and grass pollen allergens), insect allergens (inhalant, saliva and
venom allergens), animal hair and dandruff allergens, and food
allergens. Important pollen allergens from trees, grasses and herbs
originate from the taxonomic orders of Fagales, Oleales, Pinales
and platanaceae including i.a. birch (Betula), alder (Alnus), hazel
(Corylus), hornbeam (Carpinus) and olive (Olea), cedar
(Cryptomeriaand Juniperus), Plane tree (Platanus), the order of
Poales including i.e. grasses of the genera Lolium, Phleum, Poa,
Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the
orders of Asterales and Urticales including i.a. herbs of the
genera Ambrosia, Artemisia, and Parietaria. Other allergen antigens
that may be used include allergens from house dust mites of the
genus Dermatophagoides and Euroglyphus, storage mite e.g.
Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches,
midges and fleas e.g. Blatella, Periplaneta, Chironomus and
Ctenocepphalides, those from mammals such as cat, dog and horse,
birds, venom allergens including such originating from stinging or
biting insects such as those from the taxonomic order of
Hymenoptera including bees (superfamily Apidae), wasps (superfamily
Vespidea), and ants (superfamily Formicoidae). Still other allergen
antigens that may be used include inhalation allergens from fungi
such as from the genera Alternaria and Cladosporium.
[0175] In some embodiments, the active agent is a nucleic acid
encoding a viral antigen such as those used in gene therapy or to
treat certain cancers. Nucleic acids encoding viral antigens can be
naturally isolated from a virus, amplified, synthesized, or
combinations thereof. Viral antigens can be derived from any virus
used for gene therapy, such as Adenovirus and Adeno Associated
Virus. Many gene therapy clinical trials rely on retroviruses or
adenoviruses to deliver the desired gene. Other viruses used as
vectors include adeno-associated viruses, lentiviruses, pox
viruses, alphaviruses, and herpes viruses. Gene therapy can also
include genes that induce an immune response to cancerous cells
which are delivered via a viral vaccine.
[0176] The tolerogenic antigen can be derived from a therapeutic
agent protein to which tolerance is desired. Examples are protein
drugs in their wild type, e.g., human factor VIII or factor IX, to
which patients did not establish central tolerance because they
were deficient in those proteins; or nonhuman protein drugs, used
in a human. Other examples are protein drugs that are glycosylated
in nonhuman forms due to production, or engineered protein drugs,
e.g., having non-native sequences that can provoke an unwanted
immune response. Examples of tolerogenic antigens that are
engineered therapeutic proteins not naturally found in humans
include human proteins with engineered mutations, e.g., mutations
to improve pharmacological characteristics. Examples of tolerogenic
antigens that contain nonhuman glycosylation include proteins
produced in yeast or insect cells.
[0177] The tolerogenic antigen can be derived from proteins that
are administered to humans that are deficient in the protein.
Deficient means that the patient receiving the protein does not
naturally produce enough of the protein. Moreover, the proteins may
be proteins for which a patient is genetically deficient. Such
proteins include, for example, antithrombin-III, protein C, factor
VIII, factor IX, growth hormone, somatotropin, insulin, pramlintide
acetate, mecasermin (IGF-1), .beta.-gluco cerebrosidase,
alglucosidase-.alpha., laronidase (.alpha.-L-iduronidase),
idursuphase (iduronate-2-sulphatase), galsulphase,
agalsidase-.beta. (.alpha.-galactosidase), .alpha.-1 proteinase
inhibitor, and albumin.
[0178] The tolerogenic antigen can be derived from therapeutic
antibodies and antibody-like molecules, including antibody
fragments and fusion proteins with antibodies and antibody
fragments. These include nonhuman (such as mouse) antibodies,
chimeric antibodies, and humanized antibodies. Immune responses to
even humanized antibodies have been observed in humans (Getts D R,
Getts M T, McCarthy D P, Chastain E M L, & Miller S D (2010),
mAbs, 2(6):682-694.). The tolerogenic antigen can be derived from
proteins that are nonhuman. Examples of such proteins include
adenosine deaminase, pancreatic lipase, pancreatic amylase,
lactase, botulinum toxin type A, botulinum toxin type B,
collagenase, hyaluronidase, papain, L-Asparaginase, rasburicase,
lepirudin, streptokinase, anistreplase (anisoylated plasminogen
streptokinase activator complex), antithymocyte globulin,
crotalidae polyvalent immune Fab, digoxin immune serum Fab,
L-arginase, and L-methionase.
[0179] The tolerogenic antigen can be derived from human allograft
transplantation antigens. Examples of these antigens are the
subunits of the various MHC class I and MHC class II haplotype
proteins, and single-amino-acid polymorphisms on minor blood group
antigens including RhCE, Kell, Kidd, Duffy and Ss.
[0180] The tolerogenic antigen can be a self-antigen against which
a patient has developed an autoimmune response or may develop an
autoimmune response. Examples are proinsulin (diabetes), collagens
(rheumatoid arthritis), myelin basic protein (multiple
sclerosis).
[0181] For example, Type 1 diabetes mellitus (T1D) is an autoimmune
disease whereby T cells that recognize islet proteins have broken
free of immune regulation and signal the immune system to destroy
pancreatic tissue. Numerous protein antigens that are targets of
such diabetogenic T cells have been discovered, including insulin,
GAD65, chromogranin-A, among others. In the treatment or prevention
of T1D, it would be useful to induce antigen-specific immune
tolerance towards defined diabetogenic antigens to functionally
inactivate or delete the diabetogenic T cell clones.
[0182] Tolerance and/or delay of onset or progression of autoimmune
diseases may be achieved for many proteins that are human
autoimmune proteins, a term referring to various autoimmune
diseases wherein the protein or proteins causing the disease are
known or can be established by routine testing.
[0183] The tolerogenic antigen can be one or more of the following
proteins, or a fragment or peptide derived therefrom. In type 1
diabetes mellitus, several main antigens have been identified:
insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65
(GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), and
insulinoma-associated protein 213 (IA-213); other antigens include
ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38,
chromogranin-A, FISP-60, caboxypeptidase E, peripherin, glucose
transporter 2, hepatocarcinoma-intestine-pancreas/pancreatic
associated protein, S100.beta., glial fibrillary acidic protein,
regenerating gene II, pancreatic duodenal homeobox 1, dystrophia
myotonica kinase, islet-specific glucose-6-phosphatase catalytic
subunit-related protein, and SST G-protein coupled receptors 1-5.
In autoimmune diseases of the thyroid, including Hashimoto's
thyroiditis and Graves' disease, main antigens include
thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin
receptor (TSHR); other antigens include sodium iodine symporter
(NIS) and megalin. In thyroid-associated ophthalmopathy and
dermopathy, in addition to thyroid autoantigens including TSHR, an
antigen is insulin-like growth factor 1 receptor. In
hypoparathyroidism, a main antigen is calcium sensitive receptor.
In Addison's disease, main antigens include 21-hydroxylase,
17.alpha.-hydroxylase, and P450 side chain cleavage enzyme
(P450scc); other antigens include ACTH receptor, P450c21 and
P450c17. In premature ovarian failure, main antigens include FSH
receptor and .alpha.-enolase. In autoimmune hypophysitis, or
pituitary autoimmune disease, main antigens include pituitary
gland-specific protein factor (PGSF) 1a and 2; another antigen is
type 2 iodothyronine deiodinase. In multiple sclerosis, main
antigens include myelin basic protein, myelin oligodendrocyte
glycoprotein and proteolipid protein. In rheumatoid arthritis, a
main antigen is collagen II. In immunogastritis, a main antigen is
H+, K+-ATPase. In pernicious angemis, a main antigen is intrinsic
factor. In celiac disease, main antigens are tissue
transglutaminase and gliadin. In vitiligo, a main antigen is
tyrosinase, and tyrosinase related protein 1 and 2. In myasthenia
gravis, a main antigen is acetylcholine receptor. In pemphigus
vulgaris and variants, main antigens are desmoglein 3, 1 and 4;
other antigens include pemphaxin, desmocollins, plakoglobin,
perplakin, desmoplakins, and acetylcholine receptor. In bullous
pemphigoid, main antigens include BP180 and BP230; other antigens
include plectin and laminin 5 In dermatitis herpetiformis Duhring,
main antigens include endomysium and tissue transglutaminase. In
epidermolysis bullosa acquisita, a main antigen is collagen VII. In
systemic sclerosis, main antigens include matrix metalloproteinase
1 and 3, the collagen-specific molecular chaperone heat-shock
protein 47, fibrillin-1, and PDGF receptor; other antigens include
Scl-70, U1 RNP, Th/To, Ku, Jol, NAG-2, centromere proteins,
topoisomerase I, nucleolar proteins, RNA polymerase I, II and III,
PM-Slc, fibrillarin, and B23. In mixed connective tissue disease, a
main antigen is U1snRNP. In Sjogren's syndrome, the main antigens
are nuclear antigens SS-A and SS-B; other antigens include fodrin,
poly(ADP-ribose) polymerase and topoisomerase. In systemic lupus
erythematosus, main antigens include nuclear proteins including
SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone
proteins and double-stranded DNA. In Goodpasture's syndrome, main
antigens include glomerular basement membrane proteins including
collagen IV. In rheumatic heart disease, a main antigen is cardiac
myosin. Other autoantigens revealed in autoimmune polyglandular
syndrome type 1 include aromatic L-amino acid decarboxylase,
histidine decarboxylase, cysteine sulfinic acid decarboxylase,
tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine
hydroxylase, hepatic P450 cytochromes P4501A2 and 2A6, SOX-9,
SOX-10, calcium-sensing receptor protein, and the type 1
interferons interferon alpha, beta and omega.
[0184] The tolerogenic antigen can be a foreign antigen against
which a patient has developed an unwanted immune response. Examples
are food antigens. Embodiments include testing a patient to
identify foreign antigen and creating a molecular fusion that
incorporates the antigen and treating the patient to develop
immunotolerance to the antigen or food. Examples of such foods
and/or antigens are provided. Examples are from peanut: conarachin
(Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin
(Ara h 6); from apple: 31 kda major allergen/disease resistance
protein homolog (Mal d 2), lipid transfer protein precursor (Mal d
3), major allergen Mal d 1.03D (Mal d 1); from milk:
.alpha.-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin
(Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d
2), kiwellin (Act d 5); from mustard: 2S albumin (Sin a 1), 11 S
globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin
a 4); from celery: profilin (Api g 4), high molecular weight
glycoprotein (Api g 5); from shrimp: Pen a 1 allergen (Pen a 1),
allergen Pen m 2 (Pen in 2), tropomyosin fast isoform; from wheat
and/or other cereals: high molecular weight glutenin, low molecular
weight glutenin, alpha- and gamma-gliadin, hordein, secalin,
avenin; from strawberry: major strawberry allergy Fra a 1-E (Fra a
1), from banana: profilin (Mus xp 1).
[0185] Many protein drugs that are used in human and veterinary
medicine induce immune responses, which create risks for the
patient and limit the efficacy of the drug. This can occur with
human proteins that have been engineered, with human proteins used
in patients with congenital deficiencies in production of that
protein, and with nonhuman proteins. It would be advantageous to
tolerize a recipient to these protein drugs prior to initial
administration, and it would be advantageous to tolerize a
recipient to these protein drugs after initial administration and
development of immune response. In patients with autoimmunity, the
self-antigen(s) to which autoimmunity is developed are known. In
these cases, it would be advantageous to tolerize subjects at risk
prior to development of autoimmunity, and it would be advantageous
to tolerize subjects at the time of or after development of
biomolecular indicators of incipient autoimmunity. For example, in
Type 1 diabetes mellitus, immunological indicators of autoimmunity
are present before broad destruction of beta cells in the pancreas
and onset of clinical disease involved in glucose homeostasis. It
would be advantageous to tolerize a subject after detection of
these immunological indicators prior to onset of clinical
disease.
[0186] In some embodiments, the nucleic acid encodes a tolerogenic
antigen that corresponding to one or more of the protein components
of a viral capsid. For example, in some embodiments, the
tolerogenic antigen is a viral capsid protein, or fragment of a
protein of a virus that is used to deliver one or more active
agents to the body. Therefore, in some embodiments, nanoparticles
deliver nucleic acids to induce tolerance to a viral vector used
for gene therapy. In some embodiments, the nanoparticles provide
tolerogenic therapy to enhance the efficacy of gene therapy, to
reduce the host immune response to vectors used in gene therapy, or
to facilitate selective cellular uptake/targeting of vectors used
for gene therapy. Viruses used for gene therapy are known in the
art, and include viral subtypes from adeno-associated viruses
(AAV), such as viruses of the Dendovirus genus, lentiviruses and
retroviruses, such as Human Immunodeficiency Virus (HIV), and
herpesviruses, such as HSV-1, HSV2 and Epstein-Barr virus (EBV)).
An exemplary viral capsid antigen is one or more of the VP1, VP2,
or VP3 capsid proteins from a virion of the AAV virus. For example,
in some embodiments, one or more nucleic acids loaded into a
nanoparticle, or administered in combination with a nanoparticle
encodes a tolerogenic antigen corresponding to one or more capsid
proteins from one or more AAV viruses, such as serotype AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or
mixtures, variants or derivatives thereof.
[0187] In an exemplary embodiment, the nanoparticle co-delivery of
siRNA and mRNA provides tolerogenic vaccines, wherein the mRNA
encodes an antigen designed to elicit a desired immune response,
and the siRNA silences the expression of co-stimulatory proteins.
In an exemplary embodiment, co-delivery of B7-1/7-2 siRNA and mRNA
encoding AAV capsid proteins (VP1, VP2 or VP3) induces tolerance
for AAV vectors for gene therapy. In another embodiment,
co-delivery of B7-1/7-2 siRNA and mRNA encoding GAD65, insulin,
proinsulin, HSP60, IA-2, ZnT8 or IGRP provides therapy for Type 1
diabetes. In an exemplary embodiment, nanoparticles include an
siRNA against 7-ketocholesterol (7KC). In another embodiment,
nanoparticles include an siRNA against collagenase enzymes. A
non-limiting list of tolerogenic antigens that can be up-regulated
or expressed by RNAs encapsulated in nanoparticles, or otherwise
presented for immune surveillance by tolerogenic antigen presenting
cells is provided in Table 2, below.
TABLE-US-00002 TABLE 2 Exemplary Tolerogenic Antigens that may be
induced or expressed by RNAs within nanoparticles Proteins for
which a patient is genetically deficient ANTITHROMBIN-III PROTEIN C
FACTOR VIII FACTOR IX GROWTH HORMONE SOMATOTROPIN INSULIN
PRAMLINTIDE ACETATE MECASERMIN (IGF-1) B-GLUCO CEREBROSIDASE
ALGLUCOSIDASE-A LARONIDASE (A-L-IDURONIDASE) GALSULPHASE
IDURSUPHASE (IDURONATE-2-SULPHATASE) AGALSIDASE-B (A-GALACTOSIDASE)
A-1 PROTEINASE INHIBITOR ALBUMIN Food antigens (Peanut) CONARACHIN
(ARA H 1) ALLERGEN II (ARA H 2) ARACHIS AGGLUTININ, CONGLUTIN (ARA
H 6) Other Food allergens HIGH MOLECULAR WEIGHT GLUTENIN LOW
MOLECULAR WEIGHT GLUTENIN ALPHA- AND GAMMA-GLIADIN HORDEIN SECALIN
AVENIN MAJOR STRAWBERRY ALLERGY FRA A 1-E (FRA A 1) PROFILIN (MUS
XP 1) A-LACTALBUMIN (ALA) 2S ALBUMIN (SIN A 1) LACTOTRANSFERRIN
ACTINIDIN (ACT C 1, ACT D 1) PHYTOCYSTATIN THAUMATIN-LIKE PROTEIN
(ACT D 2) KIWELLIN (ACT D 5) 11 S GLOBULIN (SIN A 2) LIPID TRANSFER
PROTEIN (SIN A 3) PROFILIN (SIN A 4); PROFILIN (API G 4),; HIGH
MOLECULAR WEIGHT GLYCOPROTEIN (API G 5) Exemplary Nonhuman protein
antigens ADENOSINE DEAMINASE PANCREATIC LIPASE, BOTULINUM TOXIN
TYPE A PANCREATIC AMYLASE LACTASE BOTULINUM TOXIN TYPE B
COLLAGENASE, L-ASPARAGINASE HYALURONIDASE CROTALIDAE POLYVALENT
IMMUNE FAB DIGOXIN IMMUNE SERUM FAB L-ARGINASE PAPAIN ANISTREPLASE
(ANISOYLATED PLASMINOGEN STREPTOKINASE ACTIVATOR COMPLEX)
ANTITHYMOCYTE GLOBULIN RASBURICASE LEPIRUDIN STREPTOKINASE
L-METHIONASE Human allograft transplantation antigens MHC class I
haplotype proteins MHC class II haplotype proteins Polymorphisms on
RhCE Polymorphisms on Kell Polymorphisms on Kidd Polymorphisms on
Duffy Food allergens (apple) 31 KDA MAJOR ALLERGEN/DISEASE
RESISTANCE PROTEIN HOMOLOG (MAL D 2), LIPID TRANSFER PROTEIN
PRECURSOR (MAL D 3) MAJOR ALLERGEN MAL D 1.03D (MAL D 1); Food
allergens (shrimp) PEN A 1 ALLERGEN (PEN A 1) ALLERGEN PEN M 2 (PEN
IN 2) TROPOMYOSIN FAST ISOFORM Viral Capsid Proteins AAV1 Capsid
(VP1, VP2, or VP3) AAV2 (VP1, VP2, or VP3) AAV3 (VP1, VP2, or VP3)
AAV4 (VP1, VP2, or VP3) AAV5 (VP1, VP2, or VP3) AAV6 (VP1, VP2, or
VP3) AAV7 (VP1, VP2, or VP3) AAV8 (VP1, VP2, or VP3) AAV9 (VP1,
VP2, or VP3) AAV10 (VP1, VP2, or VP3) AAV11 (VP1, VP2, or VP3)
Human Autoimmune Markers PROINSULIN COLLAGENS MYELIN BASIC PROTEIN
GLUTAMIC ACID DECARBOXYLASE-65 CHROMOGRANIN-A GLUTAMIC ACID
DECARBOXYLASE-67 SODIUM IODINE SYMPORTER (NIS) GLIAL FIBRILLARY
ACIDIC PROTEIN MEGALIN INSULINOMA-ASSOCIATED PROTEIN 2 (IA-2)
INSULINOMA-ASSOCIATED PROTEIN 2B (IA-213) ICA69 ICA12 (SOX-13)
CARBOXYPEPTIDASE H IMOGEN 38 GLIMA 38 CHROMOGRANIN-A FISP-60
CABOXYPEPTIDASE E, PERIPHERIN GLUCOSE TRANSPORTER 2
HEPATOCARCINOMA-INTESTINE-PANCREAS/PANCREATIC ASSOCIATED PROTEIN
S100.beta. REGENERATING GENE II, THYROGLOBULIN (TG) THYROID
PEROXIDASE (TPO) THYROTROPIN RECEPTOR (TSHR) PANCREATIC DUODENAL
HOMEOBOX 1 DYSTROPHIA MYOTONICA KINASE ISLET-SPECIFIC
GLUCOSE-6-PHOSPHATASE CATALYTIC SUBUNIT-RELATED PROTEIN SST
G-PROTEIN COUPLED RECEPTORS 1-5 21-HYDROXYLASE 17A-HYDROXYLASE P450
SIDE CHAIN CLEAVAGE ENZYME (P450SCC) ACTH RECEPTOR P450C21 P450C17
FSH RECEPTOR .alpha.-ENOLASE H+, K+-ATPase TISSUE TRANSGLUTAMINASE
GLIADIN TYROSINASE (also TYROSINASE RELATED PROTEIN 1 and 2)
ACETYLCHOLINE RECEPTOR
[0188] d. Costimulatory Molecules
[0189] In some embodiments, the nucleic acid suppresses, enhances
or otherwise alters the function of one or more co-stimulatory
molecules, such as those associated with the activation and
immunological functions of Leukocytes. For example, in some
embodiments, nanoparticles include one or more siRNAs that silence
or down-regulate one or more genes responsible for the activation
of antigen presenting cells, such as dendritic cells.
[0190] Activation of naive T cells requires at least two signals.
The first signal is antigen displayed by antigen presenting cells
(APCs) in the form of peptides bound to histocompatibility
molecules. The second signal, the co-stimulatory signal, is antigen
nonspecific and is provided by molecules on APCs that engage
particular costimulatory receptors on T cells, leading to T cell
stimulation in conjunction with antigen. Costimulation is important
for an effective immune response of adaptive immunity, and drives T
cell proliferation, differentiation and survival. Activation of T
cells without costimulation may lead to T cell anergy, T cell
deletion or the development of immune tolerance.
[0191] One of the best characterized costimulatory receptors
expressed by T cells is CD28, which interacts with CD80 (B7-1) and
CD86 (B7-2) on the membrane of APCs. CD28 is constitutively
expressed on almost all T cells, and is the major costimulatory
receptor for naive T cells. The costimulatory molecules B7-1 and
B7-2 are expressed mainly on APCs, including dendritic cells,
macrophages, and B cells. The expression of B7-1 and B7-2 on APCs
can be increased by the presence of microbes and by cytokines that
are produced in response to microbes. B7 costimulator expression
ensures that T cells respond best only when necessary, that is,
when faced with pathogens.
[0192] Another costimulatory receptor expressed by T cells is ICOS
(Inducible Costimulator), which interacts with ICOS-L. ICOS, which
is a member of the CD28 family of costimulatory molecules. Induced
upon activation, ICOS is a desirable target for modifying
T-cell-mediated immune responses. The activation and effector
function of ICOS for both Th1 and Th2 cell indicates it contributes
to generation and maintenance of humoral immunity. Numerous
costimulatory molecules have been identified playing a role in the
initiation of immune responses by T and B lymphocytes. For example,
activation of B cells requires CD40-CD40L interactions for proper
antibody response: promoting survival, cytokine receptor
expression, and inducing antibody class switch. Without this
costimulation B cells do not further proliferate. Recent
discoveries have illustrated the contrasting roles of costimulatory
molecules: stimulatory (costimulation) verse inhibitory
(coinhibition), and various aspects of immune dysfunction in cancer
are related to the presence of coinhibitory (ex: PD-1, PD-L1,
CTLA-4, BTLA) and costimulatory (ex: CD28, ICOS, 4-1BB, CD40, OX40,
CD27) signaling.
[0193] Costimulatory molecules can be organized by family.
[0194] B7/CD28 family member include, for example, B7-1/CD80,
B7-2/CD86, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28,
CTLA-4, ICOS, PD-1, PD-L1/B7-H1, PD-L2/B7-DC, PDCD6, TMIGD2/CD28H,
and VISTA/B7-H5/PD-1H.
[0195] Butyrophilins include, for example, BTN1A1/Butyrophilin,
BTN2A1, BTN2A2/Butyrophilin 2A2, BTN3A1/2, BTN3A2, BTN3A3,
BTNL2/Butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9,
BTNL10, and CD277/BTN3A1.
[0196] LAIR Family members include, for example, LAIR1 and
LAIR2.
[0197] Nectin and Nectin-like Ligand/Receptor co-signaling
molecules include, for example, CD96, CD155/PVR, CRTAM,
DNAM-1/CD226, Nectin-2/CD112, Nectin-3, PVRIG, and TIGIT.
[0198] Regulation of T cell co-stimulation by ILT/CD85 family
proteins include, for example, LILRA3/CD85e, LILRA4/CD85g/ILT7,
LILRB1/CD85j/ILT2, LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5, and
LILRB4/CD85k/ILT3.
[0199] Regulation of T cell co-stimulation by TNF superfamily
members include, for example, 4-1BB/TNFRSF9/CD137, 4-1BB
Ligand/TNFSF9, BAH-IBLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7,
CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5,
CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR
Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14,
Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4,
RELT/TNI-RSF19L, TACl/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, and TNF
RII/TNFRSF1B.
[0200] SLAM family members include, for example, 2B4/CD244/SLAMF4,
BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3,
CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, and
SLAM/CD150.
[0201] TIM family co-Signaling molecules include, for example,
TIM-1/KIM-1/HAVCR, TIM-3, and TIM-4.
[0202] Other co-stimulatory molecules include, for example, CD7,
CD160, CD200, CD300a/LMIR1, CD300d/LMIR4, CLECL1/DCAL-1, DAP12,
Dectin-1/CLEC7A, DPPIV/CD26, EphB6, Integrin alpha 4 beta 1,
Integrin alpha 4 beta 7/LPAM-1, LAG-3, and TSLP R. A non-limiting
list of co-stimulatory molecules that can be down-regulated,
silenced, up-reglated, or otherwise modified by nucleic acids
(e.g., siRNAs) encapsulated within the described nanoparticles is
provided in Table 3, below.
TABLE-US-00003 TABLE 3 Exemplary Co-stimulatory molecules that can
be silenced, down- regulated, or otherwise moderated by nucleic
acids delivered within nanoparticles. Costimulatory molecules are
arranged by super family. B7/CD28 Family B7-1/CD80 B7-2/CD86 B7-H2
B7-H3 B7-H4 B7-H6 B7-H7/HHLA2 BTLA CD28 CTLA-4 ICOS PD-1
PD-L1/B7-H1 PD-L2/B7-DC PDCD6 TMIGD2/CD28H VISTA/B7-H5/PD-1H
Butyrophilins BTN1A1/Butyrophilin BTN2A1 BTN2A2/Butyrophilin 2A2
BTN3A1/2 BTN3A2 BTN3A3 BTNL2/Butyrophilin-like 2 BTNL3 BTNL4 BTNL6
BTNL9 BTNL10 CD277/BTN3A1 LAIR Family members LAIR1 LAIR2 Nectin
and Nectin-like Ligand/Receptor CD155/PVR CRTAM DNAM-1/CD226
Nectin-2/CD112 Nectin-3 PVRIG TIGIT CD96 TNF superfamily members
4-1BB/TNFRSF9/CD137 4-1BB Ligand/TNFSF9 BAFF/BLyS/TNFSF13B BAFF
R/TNFRSF13C CD27 Ligand/TNFSF7 CD27/TNFRSF7 CD30/TNFRSF8 CD30
Ligand/TNFSF8 CD40/TNFRSF5 CD40 Ligand/TNFSF5 DR3/TNFRSF25
GITR/TNFRSF18 GITR Ligand/TNFSF18 HVEM/TNFRSF14 LIGHT/TNFSF14
Lymphotoxin-alpha/TNF-beta OX40/TNFRSF4 RII/TNFRSF1B OX40
Ligand/TNFSF4 RELT/TNFRSF19L TACI/TNFRSF13B TL1A/TNFSF15 TNF-alpha
TNF RII/TNFRSF1B SLAM family members BLAME/SLAMF8 2B4/CD244/SLAMF4
CD2 CD2F-10/SLAMF9 CD48/SLAMF2 CD58/LFA-3 CD84/SLAMF5 CD229/SLAMF3
CRACC/SLAMF7 NTB-A/SLAMF6 SLAM/CD150 TIM family co-Signaling
molecules TIM-1/KIM-1/HAVCR TIM-3 TIM-4. Other co-stimulatory
molecules CD7 CD160 CD200 CD300a/LMIR1 CD300d/LMIR4 CLECL1/DCAL-1
DAP12 Dectin-1/CLEC7A DPPIV/CD26 EphB6 Integrin alpha 4 beta 1
Integrin alpha 4 beta 7/LPAM-1 LAG-3 TSLP R ILT/CD85 family
proteins LILRA3/CD85e LILRA4/CD85g/ILT7 LILRB1/CD85j/ILT2
LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5 LILRB4/CD85k/ILT3
[0203] In some embodiments, the active agent is a nucleic acid
encoding or targeting one or more costimulatory molecules.
Exemplary preferred costimulatory molecules include costimulatory
molecules of the B7 family e.g, CD80 (B7-1), CD86 (B7-2),
ICOS-ligand (B7-H2), B7-H3, B7-H4, PD-L1 (B7-H1), PD-L2 (B7-DC);
costimulatory molecules of the TNF-receptor family e.g., CD40, OX40
ligand (OX40L), LIGHT, 4-1BBL, CD30L, CD70; SLAM family members,
and combinations thereof.
[0204] 2. Nucleic Acid Composition
[0205] The nucleic acid cargos can be deoxyribonucleic acid (DNA)
or ribonucleic acid (RNA) nucleotides which typically include a
heterocyclic base (nucleic acid base), a sugar moiety attached to
the heterocyclic base, and a phosphate moiety which esterifies a
hydroxyl function of the sugar moiety. The principal
naturally-occurring nucleotides include uracil, thymine, cytosine,
adenine and guanine as the heterocyclic bases, and ribose or
deoxyribose sugar linked by phosphodiester bonds.
[0206] In some embodiments, the oligonucleotides are composed of
nucleotide analogs that have been chemically modified to improve
stability, half-life, or specificity or affinity for a target
receptor, relative to a DNA or RNA counterpart. The chemical
modifications include chemical modification of nucleobases, sugar
moieties, nucleotide linkages, or combinations thereof. As used
herein `modified nucleotide" or "chemically modified nucleotide"
defines a nucleotide that has a chemical modification of one or
more of the heterocyclic base, sugar moiety or phosphate moiety
constituents. In some embodiments, the charge of the modified
nucleotide is reduced compared to DNA or RNA oligonucleotides of
the same nucleobase sequence. For example, the oligonucleotide can
have low negative charge, no charge, or positive charge.
[0207] Typically, nucleoside analogs support bases capable of
hydrogen bonding by Watson-Crick base pairing to standard
polynucleotide bases, where the analog backbone presents the bases
in a manner to permit such hydrogen bonding in a sequence-specific
fashion between the oligonucleotide analog molecule and bases in a
standard polynucleotide (e.g., single-stranded RNA or
single-stranded DNA). In some embodiments, the analogs have a
substantially uncharged, phosphorus containing backbone.
[0208] a. Heterocyclic Bases
[0209] The principal naturally-occurring nucleotides include
uracil, thymine, cytosine, adenine and guanine as the heterocyclic
bases. The oligonucleotides can include chemical modifications to
their nucleobase constituents. Chemical modifications of
heterocyclic bases or heterocyclic base analogs may be effective to
increase the binding affinity or stability in binding a target
sequence. Chemically-modified heterocyclic bases include, but are
not limited to, inosine, 5-(1-propynyl) uracil (pU), 5-(1-propynyl)
cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine,
pseudoisocytosine, 5 and
2-amino-5-(2'-deoxy-.beta.-D-ribofuranosyl)pyridine
(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine
derivatives.
[0210] b. Sugar Modifications
[0211] Oligonucleotides can also contain nucleotides with modified
sugar moieties or sugar moiety analogs. Sugar moiety modifications
include, but are not limited to, 2'-O-aminoetoxy, 2'-O-amonioethyl
(2'-0AE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE),
2'-0,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME) and
2'-O--(N-(methyl)acetamido) (2'-OMA).
[0212] In some embodiments, the functional nucleic acid is a
morpholino oligonucleotide. Morpholino oligonucleotides are
typically composed of two more morpholino monomers containing
purine or pyrimidine base-pairing moieties effective to bind, by
base-specific hydrogen bonding, to a base in a polynucleotide,
which are linked together by phosphorus-containing linkages, one to
three atoms long, joining the morpholino nitrogen of one monomer to
the 5' exocyclic carbon of an adjacent monomer. The purine or
pyrimidine base-pairing moiety is typically adenine, cytosine,
guanine, uracil or thymine. The synthesis, structures, and binding
characteristics of morpholino oligomers are detailed in U.S. Pat.
Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315,
5,521,063, and 5,506,337.
[0213] Important properties of the morpholino-based subunits
typically include: the ability to be linked in a oligomeric form by
stable, uncharged backbone linkages; the ability to support a
nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil
or inosine) such that the polymer formed can hybridize with a
complementary-base target nucleic acid, including target RNA, with
high T.sub.m, even with oligomers as short as 10-14 bases; the
ability of the oligomer to be actively transported into mammalian
cells; and the ability of an oligomer:RNA heteroduplex to resist
RNAse degradation.
[0214] In some embodiments, oligonucleotides employ
morpholino-based subunits bearing base-pairing moieties, joined by
uncharged linkages, as described above.
[0215] c. Internucleotide Linkages
[0216] Oligonucleotides connected by an internucleotide bond that
refers to a chemical linkage between two nucleoside moieties.
Modifications to the phosphate backbone of DNA or RNA
oligonucleotides may increase the binding affinity or stability
oligonucleotides, reduce the susceptibility of oligonucleotides
nuclease digestion, or increase membrane permeability. Cationic
modifications, including, but not limited to,
diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP)
may be especially useful due to decrease electrostatic repulsion
between the oligonucleotide and a target. Modifications of the
phosphate backbone may also include the substitution of a sulfur
atom for one of the non-bridging oxygens in the phosphodiester
linkage. This substitution creates a phosphorothioate
internucleoside linkage in place of the phosphodiester linkage.
Oligonucleotides containing phosphorothioate internucleoside
linkages have been shown to be more stable in vivo.
[0217] Examples of modified nucleotides with reduced charge include
modified internucleotide linkages such as phosphate analogs having
achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P.
et al., Organic. Chem., 52:4202, (1987)), and uncharged
morpholino-based polymers having achiral intersubunit linkages
(see, e.g., U.S. Pat. No. 5,034,506), as discussed above. Some
internucleotide linkage analogs include morpholidate, acetal, and
polyamide-linked heterocycles.
[0218] The oligonucleotides can be locked nucleic acids. Locked
nucleic acids (LNA) are modified RNA nucleotides (see, for example,
Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNAs form hybrids
with DNA which are more stable than DNA/DNA hybrids, a property
similar to that of peptide nucleic acid (PNA)/DNA hybrids.
Therefore, LNA can be used just as PNA molecules would be. LNA
binding efficiency can be increased in some embodiments by adding
positive charges to it. Commercial nucleic acid synthesizers and
standard phosphoramidite chemistry are used to make LNAs.
[0219] In some embodiments, the oligonucleotides are composed of
peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic
DNA mimics in which the phosphate backbone of the oligonucleotide
is replaced in its entirety by repeating N-(2-aminoethyl)-glycine
units and phosphodiester bonds are typically replaced by peptide
bonds. The various heterocyclic bases are linked to the backbone by
methylene carbonyl bonds. PNAs maintain spacing of heterocyclic
bases that is similar to conventional DNA oligonucleotides, but are
achiral and neutrally charged molecules. Peptide nucleic acids are
formed of peptide nucleic acid monomers.
[0220] Other backbone modifications include peptide and amino acid
variations and modifications. Thus, the backbone constituents of
oligonucleotides such as PNA may be peptide linkages, or
alternatively, they may be non-peptide peptide linkages. Examples
include acetyl caps, amino spacers such as
8-amino-3,6-dioxaoctanoic acid (referred to herein as 0-linkers),
amino acids such as lysine are particularly useful if positive
charges are desired in the PNA, and the like. Methods for the
chemical assembly of PNAs are well known. See, for example, U.S.
Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336,
5,773,571 and 5,786,571.
[0221] Oligonucleotides optionally include one or more terminal
residues or modifications at either or both termini to increase
stability, and/or affinity of the oligonucleotide for its target.
Commonly used positively charged moieties include the amino acids
lysine and arginine, although other positively charged moieties may
also be useful. Oligonucleotides may further be modified to be end
capped to prevent degradation using a propylamine group. Procedures
for 3' or 5' capping oligonucleotides are well known in the art.
The functional nucleic acid can be single stranded or double
stranded.
[0222] B. Non-Nucleic Acid-Based Agents
[0223] In some embodiments, non-nucleic acid based active agents
are delivered within, or in combination with the nanoparticles
including nucleic acids.
[0224] It will be appreciated that in addition to nucleic
acid-based active agents, the particles can further include other
active agent cargos. Additionally or alternatively nucleic
acid-containing particles can be co-delivered to a subject in
combination with (i) particles containing other active agents, or
(ii) with active agents not contained in particles, or combination
thereof. Active agent cargos to be delivered include therapeutic,
nutritional, diagnostic, and prophylactic agents. The active agents
can be small molecule active agents or biomacromolecules, such as
proteins, polypeptides, sugars or carbohydrates, lipids, nucleic
acids or small molecule compounds (typically 1 kD or less, but may
be larger). Suitable small molecule active agents include organic
and organometallic compounds. The small molecule active agents can
be a hydrophilic, hydrophobic, or amphiphilic compound.
[0225] Active agents include synthetic and natural proteins
(including enzymes, peptide-hormones, receptors, growth factors,
antibodies, signaling molecules), and synthetic and natural nucleic
acids (including RNA, DNA, anti-sense RNA, triplex DNA, inhibitory
RNA (RNAi), and oligonucleotides), and biologically active portions
thereof. Suitable active agents have a size greater than about
1,000 Da for small peptides and polypeptides, more typically at
least about 5,000 Da and often 10,000 Da or more for proteins.
Nucleic acids are more typically listed in terms of base pairs or
bases (collectively "bp"). Nucleic acids with lengths above about
10 bp are typically used. More typically, useful lengths of nucleic
acids for probing or therapeutic use will be in the range from
about 20 bp (probes; inhibitory RNAs, etc.) to tens of thousands of
bp for genes and vectors. The active agents may also be hydrophilic
molecules, preferably having a low molecular weight.
[0226] Exemplary therapeutic agents that can be co-delivered with
the particles include cytokines, chemotherapeutic agents,
radionuclides, monoclonal antibodies or other immunotherapeutics,
enzymes, antibiotics, antivirals, anti-parasites (helminths,
protozoans), growth factors, growth inhibitors, hormones, hormone
antagonists, antibodies and bioactive fragments thereof (including
humanized, single chain, and chimeric antibodies), antigen and
vaccine formulations (including adjuvants), peptide drugs,
anti-inflammatories, immunomodulators (including ligands that bind
to Toll-Like Receptors (including, but not limited to, CpG
oligonucleotides) to activate the innate immune system, molecules
that mobilize and optimize the adaptive immune system, molecules
that activate or up-regulate the action of cytotoxic T lymphocytes,
natural killer cells and helper T-cells, and molecules that
deactivate or down-regulate suppressor or regulatory T-cells),
agents that promote uptake of particles into cells, and
nutraceuticals such as vitamins.
[0227] Exemplary diagnostic agents include paramagnetic molecules,
fluorescent compounds, magnetic molecules, and radionuclides, x-ray
imaging agents, and contrast agents. An imaging, detectable or
sensing moiety, i.e., a moiety that can be determined in some
fashion, either directly or indirectly, may be bound to the NPs or
to the polymers forming the NPs, or encapsulated therein.
Representative imaging entities include, but are not limited to,
fluorescent, radioactive, electron-dense, magnetic, or labeled
members of a binding pair or a substrate for an enzymatic reaction,
which can be detected. In some cases, the imaging entity itself is
not directly determined, but instead interacts with a second entity
in order to effect determination; for example, coupling of the
second entity to the imaging entity may result in a determinable
signal. Non-limiting examples of imaging moieties include, but are
not limited to, fluorescent compounds such as FITC or a FITC
derivative, fluorescein, green fluorescent protein ("GFP"),
radioactive atoms such as .sup.3H, .sup.14C, .sup.33P, .sup.32P,
.sup.125I, .sup.131I, .sup.35S, or a heavy metal species, for
example, gold or osmium. An imaging moiety may be a gold
nanoparticle. A diagnostic or imaging tag such as a fluorescent tag
can be chemically conjugated to a polymer to yield a fluorescently
labeled polymer. For imaging, radioactive materials such as
Technetium99 (.sup.99mTc) or magnetic materials such as
Fe.sub.2O.sub.3 could be used. Examples of other materials include
gases or gas emitting compounds, which are radioopaque.
IV. Tissue Targeting Ligands, Cell Adhesion Ligands, and Endosomal
Escape Ligands
[0228] 1. Targeting Moieties
[0229] The nanoparticles, cargo they contain, or a combination
thereof, can include a targeting moiety, i.e., a moiety able to
bind to or otherwise associate with a target, for example, a
membrane component, a cell surface receptor, or a molecule at a
site where delivery is to occur. In one embodiment, the targeting
moiety has a specificity (as measured via a disassociation
constant) of less than about 1 micromolar, at least about 10
micromolar, or at least about 100 micromolar. Numerous examples of
targeting moieties are known, some of which are more selective than
others. The ligand can be selected based on the disease to be
treated, the target cells, tissue or organ, and the desired
delivery strategy (e.g., into a cells or into the extracellular
space). The particles or cargo can include two, three, or more
targeting moieties. In some embodiments, some polymers of the
particle have a targeting moiety attached thereto and others do
not. In this way, the density of the targeting moiety on the
surface of the particle can be manipulated.
[0230] The targeting signal can include a sequence of monomers that
facilitates in vivo localization of the molecule. The monomers can
be amino acids, nucleotide or nucleoside bases, or sugar groups
such as glucose, galactose, and the like which form carbohydrate
targeting signals. Exemplary targeting molecules include small
molecules, peptides, aptamers, polynucleotides, and antibodies and
antigen binding fragments thereof. In certain embodiments, the
antibody is polyclonal, monoclonal, linear, humanized, chimeric or
a fragment thereof. Representative antibody fragments are those
fragments that bind the antibody binding portion such as Fab, Fab',
F(ab'), Fv diabodies, linear antibodies, single chain antibodies
and bispecific antibodies.
[0231] Targeting signals or sequences can be specific for a host,
tissue, organ, cell, organelle, non-nuclear organelle, or cellular
compartment. For example, in some embodiments, the particle or
cargo includes a cell-specific targeting domain, an organelle
specific targeting domain to enhance delivery to a subcellular
organelle, or a combination thereof. For example, the particle can
include targeting moiety that directs the particle to a
microenvironment where the cargo is released. A second targeting
moiety on the cargo can then enhance delivery to cargo into a
target cell or cell(s) in the microenvironment. In some embodiment,
the particle includes a moiety that targets it to a tissue, cell or
organ, and the cargo includes a moiety that enhances delivery of
the cargo to a subcellular location such as an organelle.
[0232] General classes and methods of targeting are discussed here,
and specific exemplary cell, tissue, organ, and microenvironment
specific targets are discussed in more detail and the sections
below devoted to therapeutic strategies and in the working
Examples.
[0233] Another embodiment provides an antibody or antigen binding
fragment thereof bound to the proteins of interest acting as the
targeting signal. The antibodies or antigen binding fragment
thereof are useful for directing the vector to a cell type or cell
state. In one embodiment, the polypeptide of interest possesses an
antibody binding domain, for example, from proteins known to bind
antibodies such as Protein A and Protein G from Staphylococcus
aureus.
[0234] In some embodiments, the targeting domain includes all or
part of an antibody that directs the vector to the desired target
cell type or cell state. Antibodies can be monoclonal or
polyclonal, but are preferably monoclonal. For human gene therapy
purposes, antibodies are derived from human genes and are specific
for cell surface markers, and are produced to reduce potential
immunogenicity to a human host as is known in the art. For example,
transgenic mice which contain the entire human immunoglobulin gene
cluster are capable of producing "human" antibodies can be
utilized. In one embodiment, fragments of such human antibodies are
employed as targeting signals. In a preferred embodiment, single
chain antibodies modeled on human antibodies are prepared in
prokaryotic culture.
[0235] In one embodiment, the targeting ligand is a fusion protein.
The fusion protein can include, for example, a
polynucleotide-binding polypeptide, a protein transduction domain,
and optionally one or more targeting signals. Other exemplary
fusion proteins containing a mitochondrial transcription factor
polypeptide are disclosed in U.S. Pat. Nos. 8,039,587, 8,062,891,
8,133,733.
[0236] The particles, the cargo, or a combination thereof can be
modified to target a specific cell type or population of cells. For
example, the particles and cargo can be modified with
galactosyl-terminating macromolecules to target the polypeptide of
interest to the liver or to liver cells. The modified particles and
cargo selectively enters hepatocytes after interaction of the
carrier galactose residues with the asialoglycoprotein receptor
present in large amounts and high affinity only on these cells. The
eukaryotic cell includes a number of distinct cell surface
molecules. The structure and function of each molecule can be
specific to the origin, expression, character and structure of the
cell. Determining the unique cell surface complement of molecules
of a specific cell type can be determined using techniques well
known in the art.
[0237] In some embodiments, the targeting signal binds to its
ligand or receptor which is located on the surface of a target cell
such as to bring the composition and cell membranes sufficiently
close to each other to allow penetration of the composition into
the cell. One skilled in the art will appreciate that the tropism
of the particles and cargo can be altered by changing the targeting
signal. For example, the compositions can be modified to include
cell surface antigen specific antibodies. Exemplary cell surface
antigens are disclosed in Wagner et al., Adv Gen, 53:333-354
(2005). Tumor antigens discussed in more detail below.
[0238] It is known in the art that nearly every cell type in a
tissue in a mammalian organism possesses some unique cell surface
receptor or antigen. Thus, it is possible to incorporate nearly any
ligand for the cell surface receptor or antigen as a targeting
signal. For example, peptidyl hormones can be used a targeting
moieties to target delivery to those cells which possess receptors
for such hormones. Chemokines and cytokines can similarly be
employed as targeting signals to target delivery of the complex to
their target cells. A variety of technologies have been developed
to identify genes that are preferentially expressed in certain
cells or cell states and one of skill in the art can employ such
technology to identify targeting signals which are preferentially
or uniquely expressed on the target tissue of interest
[0239] The targeting signal can be directed to cells of the nervous
system, including the brain and peripheral nervous system. Cells in
the brain include several types and states and possess unique cell
surface molecules specific for the type. Furthermore, cell types
and states can be further characterized and grouped by the
presentation of common cell surface molecules. The targeting signal
can be directed to specific neurotransmitter receptors expressed on
the surface of cells of the nervous system. The distribution of
neurotransmitter receptors is well known in the art and one so
skilled can direct the compositions described by using
neurotransmitter receptor specific antibodies as targeting signals.
Furthermore, given the tropism of neurotransmitters for their
receptors, in one embodiment the targeting signal consists of a
neurotransmitter or ligand capable of specifically binding to a
neurotransmitter receptor.
[0240] The targeting signal can be specific to cells of the nervous
system which may include astrocytes, microglia, neurons,
oligodendrites and Schwann cells. These cells can be further
divided by their function, location, shape, neurotransmitter class
and pathological state. Cells of the nervous system can also be
identified by their state of differentiation, for example stem
cells. Exemplary markers specific for these cell types and states
are well known in the art and include, but are not limited to CD133
and Neurosphere.
[0241] The targeting signal can be directed to cells of the
musculoskeletal system. Muscle cells include several types and
possess unique cell surface molecules specific for the type and
state. Furthermore, cell types and states can be further
characterized and grouped by the presentation of common cell
surface molecules. For example, the targeting signal can be
directed to specific neurotransmitter receptors expressed on the
surface of muscle cells. The distribution of neurotransmitter
receptors is well known in the art and one so skilled can direct
the compositions described by using neurotransmitter receptor
specific antibodies as targeting signals. Furthermore, given the
tropism of neurotransmitters for their receptors, in some
embodiments the targeting signal consists of a neurotransmitter.
Exemplary neurotransmitters expressed on muscle cells that can be
targeted include but are not limited to acetycholine and
norepinephrine.
[0242] The targeting signal can be specific to muscle cells which
consist of two major groupings, Type I and Type II. These cells can
be further divided by their function, location, shape, myoglobin
content and pathological state. Muscle cells can also be identified
by their state of differentiation, for example muscle stem cells.
Exemplary markers specific for these cell types and states are well
known in the art include, but are not limited to MyoD, Pax7 and
MR4.
[0243] In some embodiments, the particle, cargo, or a combination
thereof is modified to target a subcellular organelle. Targeting of
the disclosed composition to organelles can be accomplished by
modifying the composition to contain specific organelle targeting
signals. These sequences can target organelles, either specifically
or non-specifically. In some embodiments the interaction of the
targeting signal with the organelle does not occur through a
traditional receptor-ligand interaction.
[0244] The eukaryotic cell includes a number of discrete membrane
bound compartments, or organelles. The structure and function of
each organelle is largely determined by its unique complement of
constituent polypeptides. However, the vast majority of these
polypeptides begin their synthesis in the cytoplasm. Thus organelle
biogenesis and upkeep require that newly synthesized proteins can
be accurately targeted to their appropriate compartment. This is
often accomplished by amino-terminal signaling sequences, as well
as post-translational modifications and secondary structure.
[0245] Organelles can have single or multiple membranes and exist
in both plant and animal cells. Depending on the function of the
organelle, the organelle can consist of specific components such as
proteins and cofactors. The composition delivered to the organelle
can enhance or inhibit to the functioning of the organelle.
Exemplary organelles include the nucleus, mitochondrion,
chloroplast, lysosome, peroxisome, Golgi, endoplasmic reticulum,
and nucleolus. Some organelles, such as mitochondria and
chloroplasts, contain their own genome. Nucleic acids are
replicated, transcribed, and translated within these organelles.
Proteins are imported and metabolites are exported.
[0246] There can be an exchange of material across the membranes of
organelles. Synthetic organelles can be formed from lipids and can
contain specific proteins within the lipid membranes. Additionally,
the content of synthetic organelles can be manipulated to contain
components for the translation of nucleic acids.
[0247] In certain embodiments the particle, the cargo, or a
combination thereof specifically target mitochondria. Mitochondria
contain the molecular machinery for the conversion of energy from
the breakdown of glucose into adenosine triphosphate (ATP). The
energy stored in the high energy phosphate bonds of ATP is then
available to power cellular functions. Cells with high metabolic
activity, such as heart muscle, have many well developed
mitochondria.
[0248] Mitochondrial targeting agents can include a sequence of
highly positively charged amino acids. This allows the protein to
be targeted to the highly negatively charged mitochondria. Unlike
receptor-ligand approaches that rely upon stochastic Brownian
motion for the ligand to approach the receptor, such targeting
signals are drawn to mitochondria because of charge. Therefore, in
some embodiments, the mitochondrial targeting agent is a protein
transduction domain including but not limited to the protein
transduction domains discussed in more detail below.
[0249] Mitochondrial targeting agents also include short peptide
sequences (Yousif, et al., Chembiochem., 10(13):2131 (2009)), for
example, mitochondrial transporters-synthetic cell-permeable
peptides, also known as mitochondria-penetrating peptides (MPPs),
that are able to enter mitochondria. MPPs are typically cationic,
but also lipophilic; this combination of characteristics
facilitates permeation of the hydrophobic mitochondrial membrane.
For example, MPPs can include alternating cationic and hydrophobic
residues (Horton, et al., Chem Biol., 15(4):375-82 (2008)). Some
MPPs include delocalized lipophilic cations (DLCs) in the peptide
sequence instead of, or in addition to natural cationic amino acids
(Kelley, et al., Pharm. Res., 2011 Aug. 11 [Epub ahead of print]).
Other variants can be based on an oligomeric carbohydrate scaffold,
for example attaching guanidinium moieties due to their delocalized
cationic form (Yousif, et al., Chembiochem., 10(13):2131
(2009).
[0250] Mitochondrial targeting agents also include mitochondrial
localization signals or mitochondrial targeting signals. Many
mitochondrial proteins are synthesized as cytosolic precursor
proteins containing a leader sequence, also known as a presequence,
or peptide signal sequence. Many sequences are known in the art,
see for example, U.S. Pat. No. 8,039,587. The identification of the
specific sequences necessary for translocation of a linked compound
into a mitochondrion can be determined using predictive software
known to those skilled in the art.
[0251] In some embodiments the target moiety directs the
composition to the nucleus. Nuclear localization signals (NLS) or
domains are known in the art and include for example, SV 40 T
antigen or a fragment thereof. The NLS can be simple cationic
sequences of about 4 to about 8 amino acids, or can be bipartite
having two interdependent positively charged clusters separated by
a mutation resistant linker region of about 10-12 amino acids.
[0252] In some embodiments, the particles, cargo, or a combination
thereof additionally or alternatively include a moiety that
enhances escape from endosomes or macropinosomes. In some
embodiments, particles enter cells through endocytosis and are
entrapped in endosomes. These early endosomes subsequently fuse
with sorting endosomes, which in turn transfer their contents to
the late endosomes. Late endosomal vesicles are acidified (pH 5-6)
by membrane-bound proton-pump ATPases. If the particles are not
released from the endosome, for example, by pH-induced degradation
and the associated "sponge" effect as discussed in more detail
below, the endosomal content can be relocated to the lysosomes,
which are further acidified (pH .about.4.5) and contain various
nucleases that promote the degradation of nucleic acids. To avoid
lysosomal degradation of cargo, particularly nucleic acid cargo,
the particle including the cargo, or the cargo itself (following
release from the particle) escapes from the endosome into the
cytosol. This is particularly preferred for mRNA and functional
nucleic acid cargos which may rely on cytosolic cellular machinery
for their activity.
[0253] Strategies to promote endosomal release are known in the
art, and include, for example, the use of fusogenic lipids,
polymers with high buffering capacity and membrane-interacting
peptides (exemplary strategies are reviewed in Dominska and
Dykxhoom, J Cell Sci, 123: 1183-1189 (2010)). In particularly
preferred embodiments, the endosomal escape sequence is a membrane
interacting peptide. In some embodiments, the endosomal escape
sequence is a protein transduction domain. Thus in some embodiments
the endosomal escape sequence is part of, or consecutive with, the
protein transduction domain. In some embodiments, the endosomal
escape sequence is non-consecutive with the protein transduction
domain or provided in the absence of a protein transduction domain.
In some embodiments the endosomal escape sequence includes a
portion of the hemagglutinin peptide from influenza (HA).
[0254] Examples of endosomal escape sequences are known in the art.
See, for example, WO 2013/103972. Hatakeyama, et al., have
described a fusogenic PEG-peptide-DOPE (PPD) construct and a
pH-sensitive fusogenic GALA peptide (Hatakeyama, et al., J.
Control. Release 139, 127-132 (2009)) and that PPD constructs can
be cleaved by matrix metalloproteinases that are specifically
secreted by cancer cells, enhancing the delivery of siRNA complexed
with this carrier to tumor cells (Hatakeyama, et al., Gene Ther.,
14, 68-77 (2007)).
[0255] Another membrane-destabilization mechanism takes advantage
of the pore-forming ability of viroporins, highly hydrophobic
proteins that create channels and facilitate ion flow across
biological membranes (Gonzalez and Carrasco, FEBS Lett. 552, 28-34
(2003)). For example, peptides derived from the endodomain of the
HIV gp41 envelope glycoprotein (sequence corresponding to residues
783-806 of gp160) form pores in the cell membrane by adopting an
amphipathic .alpha.-helical structure (Costin et al., Virol. J.,
4:123 (2007)) and (Kwon et al., Bioconjugate Chem., 19, 920-927
(2008)).
[0256] The influenza-derived fusogenic peptide diINF-7 has also
been shown to enhance endosomal release (Oliveira et al., Int. J.
Pharm. 331, 211-214 (2007)).
[0257] The particles, any of the active agents, but particularly
protein and nucleic acid agents, or a combination thereof can
include a protein transduction domain to improve delivery of the
active agent across extracellular membranes, intracellular
membranes, or the combination thereof. As used herein, a "protein
transduction domain" or PTD refers to a polypeptide,
polynucleotide, carbohydrate, organic or inorganic compound that
facilitates traversing a lipid bilayer, micelle, cell membrane,
organelle membrane, or vesicle membrane. A PTD attached to another
molecule facilitates the molecule traversing membranes, for example
going from extracellular space to intracellular space, or cytosol
to within an organelle.
[0258] The protein transduction domain can be a polypeptide
including positively charged amino acids and can be cationic or
amphipathic. Protein transduction domains (PTD), also known as a
cell penetrating peptides (CPP), are typically polypeptides
including positively charged amino acids. PTDs are known in the
art
IV. Nanoparticle Formation
[0259] The nanoparticles are typically formed using an emulsion
process, single or double, using an aqueous and a non-aqueous
solvent or self-assembly of amphiphilic polymers or a mixture of
amphiphlic polymers and hydrophobic polymers. Typically, the
nanoparticles contain a minimal amount of the non-aqueous solvent
after solvent removal. Preferred methods of preparing these
nanoparticles are described in the examples.
[0260] In one embodiment, nanoparticles are prepared using emulsion
solvent evaporation method. A polymeric material is dissolved in a
water immiscible organic solvent and mixed with a drug solution or
a combination of drug solutions. The water immiscible organic
solvent is preferably a GRAS ingredient such as chloroform,
dichloromethane, and acyl acetate. The drug can be dissolved in,
but is not limited to, one or a plurality of the following:
acetone, ethanol, methanol, isopropyl alcohol, acetonitrile and
Dimethyl sulfoxide (DMSO). An aqueous solution is then added into
the resulting mixture solution to yield emulsion solution by
emulsification. The emulsification technique can be, but not
limited to, probe sonication or homogenization through a
homogenizer.
[0261] In another embodiment, nanoparticles are prepared using
nanoprecipitation methods or microfluidic devices. A polymeric
material is mixed with a drug or drug combinations in a water
miscible organic solvent. The water miscible organic solvent can be
one or more of the following: acetone, ethanol, methanol, isopropyl
alcohol, acetonitrile and Dimethyl sulfoxide (DMSO). The resulting
mixture solution is then added to an aqueous solution to yield
nanoparticle solution. The agents may be associated with the
surface of, encapsulated within, surrounded by, and/or distributed
throughout the polymeric matrix of the particles.
[0262] In another embodiment, nanoparticles are prepared by the
self-assembly of the amphiphilic polymers, optionally including
hydrophilic and/or hydrophobic polymers, using emulsion solvent
evaporation, a single-step nanoprecipitation method, or
microfluidic devices.
[0263] Two methods to incorporate targeting moieties into the
nanoparticles include: i) conjugation of targeting ligands to the
hydrophilic region (e.g. PEG) of polymers prior to nanoparticle
preparation; and ii) incorporation of targeting molecules onto
nanoparticles where the PEG layer on the nanoparticle surface can
be cleaved in the presence of a chemical or enzyme at tissues of
interest to expose the targeting molecules.
[0264] In some embodiments, the polymer or lipid forming the
particle can be couple to targeting agents or other molecules as
discussed above using a linker. The linker may bea cleavable linker
hydrolyzed by a chemical or enzymatic process. Interactive release
can be engineered using a linker cleaved by hydrogen peroxide,
which is produced at sites of inflammation or areas of high
neutrophil concentration.
[0265] The average diameters of the nanoparticles are typically
between about 40 nm and about 150 nm, preferably between about 50
nm and about 100 nm. In some embodiments, the average diameters of
the nanoparticles are about 100 nm. The zeta potential of the
nanoparticles is typically between about -50 mV and about +50 mV,
preferably between about -25 mV and +25 mV, most preferably between
about -10 mV and about +10 my.
V. Formulations and Methods of Administration
[0266] A. Formulations
[0267] Formulations are prepared using a pharmaceutically
acceptable "carrier" composed of materials that are considered safe
and effective and may be administered to an individual without
causing undesirable biological side effects or unwanted
interactions. The "carrier" is all components present in the
pharmaceutical formulation other than the active ingredient or
ingredients. The term "carrier" includes but is not limited to
diluents, binders, lubricants, desintegrators, fillers, and coating
compositions.
[0268] Pharmaceutical compositions can be for administration by
parenteral (intramuscular, intraperitoneal, intravenous (IV) or
subcutaneous injection), routes of administration and can be
formulated in dosage forms appropriate for each route of
administration. The compositions are most typically administered
systemically.
[0269] Compounds and pharmaceutical compositions thereof can be
administered in an aqueous solution, by parenteral injection. The
formulation may also be in the form of a suspension or emulsion,
optionally including pharmaceutically acceptable diluents,
preservatives, solubilizers, emulsifiers, adjuvants and/or
carriers. Diluents include sterile water, buffered saline of
various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and
ionic strength; and optionally, additives such as detergents and
solubilizing agents (e.g., TWEEN.RTM. 20, TWEEN.RTM. 80 also
referred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite), and preservatives. Examples of
non-aqueous solvents or vehicles are propylene glycol, polyethylene
glycol, vegetable oils, such as olive oil and corn oil, gelatin,
and injectable organic esters such as ethyl oleate. The
formulations may be lyophilized and redissolved/resuspended
immediately before use. The formulation may be sterilized by, for
example, filtration through a bacteria retaining filter, by
incorporating sterilizing agents into the compositions, by
irradiating the compositions, or by heating the compositions.
[0270] Active agent(s) and compositions thereof can be formulated
for pulmonary or mucosal administration. The administration can
include delivery of the composition to the lungs, nasal, oral
(sublingual, buccal), vaginal, or rectal mucosa.
[0271] Suitable parenteral administration routes include
intravascular administration; subcutaneous injection or deposition
including subcutaneous infusion (such as by osmotic pumps); or
direct application by a catheter or other placement device (e.g.,
an implant comprising a porous, non-porous, or gelatinous
material).
[0272] The formulation can be administered in a single dose or in
multiple doses. Dosage levels on the order of about 1 mg/kg to 100
mg/kg of body weight per administration are useful in the treatment
of a disease. One skilled in the art can also readily determine an
appropriate dosage regimen for administering the disclosed
polynucleotides to a given subject. For example, the formulation
can be administered to the subject once, e.g., as a single
injection, infusion or bolus. Alternatively, the formulation can be
administered once or twice daily to a subject for a period of from
about three to about twenty-eight days, or from about seven to
about ten days.
[0273] B. Exemplary Applications of the Technology
[0274] The compositions and methods are particularly useful for
modulating gene expression in cells. Inhibitory nucleic acids can
be introduced into cells to reduce gene expression. The expressed
gene can encode a protein, or can encode a non-coding nucleic acid,
for example, a functional non-coding RNA molecule such as transfer
RNA, ribosomal RNA, or regulatory RNA. The inhibitory nucleic acid
can specifically target the expressed gene (or an RNA expressed
therefrom) and reduce expression of the gene product.
Alternatively, mRNA and functional non-coding RNA molecules such as
transfer RNA, ribosomal RNA, and regulatory RNA can be introduced
into a cell to increase expression of the protein or polypeptide
encoded by the mRNA, or increase the presence of the functional RNA
in the cell. Table 4 shows four examples of co-delivery of siRNA
and mRNA or DNA for various disease treatments.
TABLE-US-00004 TABLE 4 Examples of co-delivery of siRNA and mRNA or
DNA for treatments. Species 1 Species 2 Application Example 1 siRNA
against AR mRNA encoding Prostate cancer PTEN therapy Example 2
siRNA against DNA encoding Non-small cell PHB1 p53 lung cancer
(NSCLC) therapy Example 3 siRNA against B7- mRNA encoding Tolerance
for 1 and/or B7-2 AAV capsid AAV vectors for proteins (VP1, gene
therapy VP2 and VP3) Example 4 siRNA against B7- mRNA encoding Type
1 diabetes 1 and/or B7-2 antigens (e.g., therapy insulin, glutamic
acid decarboxylase, insulinoma antigen-2, and ZnT8) Example 5 siRNA
an mRNA encoding a Cancer therapy oncogene tumor suppressor Example
6 siRNA against one mRNA encoding a Induction of or more co-
antigen (e.g., a tolerance against stimulatory tolerogenic the
antigen molecules (e.g., antigen). immune activating molecules
including, but not limited to, B71, B72, etc).
[0275] The disclosed compositions and methods can be used to
deliver multiple different nucleic acid species into a single cell
at the same time. Preferably the particles are loaded with two or
more species of nucleic acids. Although the compositions and
methods include co-loading two or more mRNA or two or more
functional nucleic acids into a single particle, in the most
preferred embodiments, the particle is loaded with at least one
functional nucleic acid, preferable an inhibitory RNA such as a
siRNA, and at least on one mRNA.
[0276] As illustrated in the experiments below, co-delivery of
siRNA and mRNA into cells can enhance the function of the siRNA and
the mRNA. In some embodiments, the effect of a nucleic acid
delivered by particles loaded with two or more species of nucleic
acid is compared to the effect of the nucleic acid when delivered
in particles absent the other nucleic acid species. In some
embodiments, the effect of the nucleic acid is increased 5, 10, 15,
20, 25, 30, 35, 40, 50, 75, 100, or more percent. In some
embodiments, individual particles are loaded with two of more
different species of nucleic acids. In some embodiments, individual
particles are loaded with one species of nucleic acid, and two or
more species of particles (and thus two or more species of nucleic
acids) are co-delivered to a subject in need thereof.
[0277] The co-delivery of a functional nucleic acid and an mRNA can
be used to reduce expression of one target and increase expression
of another target simultaneously. The targets can be in the same or
different molecular pathways. In some embodiments, the two targets
are in the same or related cellular processes, for example, cell
communication, cellular senescence, DNA repair, gene expression,
metabolism, necrosis, and programmed cell death (apoptosis), etc.
Thus strategies can be designed such that one of the nucleic acid
species increases expression or is an inducer or activator of a
pathway or process, and the second nucleic acid species decreases
expression of an inhibitor or a checkpoint in the pathway or
process. For example, in an exemplary embodiment apoptosis can be
increased by increasing expression of a pro-apoptotic factor (e.g.,
introducing mRNA encoding Bax, BAD, Bak, Bok, etc.) while also
decreasing expression of an anti-apoptotic factor (e.g., an
inhibitory nucleic acid that targets Bcl-2 proper, Bcl-xL, Bcl-w,
etc.).
[0278] The two species of nucleic acids can also target different
pathways or processes. In such embodiments, the targets are
selected based disease to be treated or another desired outcome.
For example, in the treatment of cancer, strategies may be designed
to reduce expression of anti-apoptotic or other survival signaling,
reduce proliferation, increase sensitivity to anticancer agents,
modulate DNA damage and repair pathways, alter cellular metabolism,
etc., in combinations of two or more. Targeting two or more of
these pathways separately, but simultaneously, can be more
effective at treating the disease or disorder than targeting only
pathway. In one pathway, the nucleic acids can decrease the
expression of disease-associated protein expression. These proteins
can include kinesin spindle protein, RRM2, keratin 6a, HER1, ErbB2,
VEGFR1, VEGFR3), PDGFR-.alpha., PDGFR-.beta., EGFR, FGFR1, FGFR2,
FGFR3, FGFR4, EphA2, EphA3, EphA4, HER2, HER3, HER4, INS-R, IGF-1R,
IR-R, CSF1R, KIT, FLK-II, KDR/FLK-1, FLK-4, flt-1, c-Met, Ron, Sea,
TRKA, TRKB, TRKC, FLT3, VEGFR/Flt2, Flt4, EphA1, EphB2, EphB4,
Pim1, Pim2, Pim3, Tie2PKN3, PLK1, PLK2, PLK3, Src, Fyn, Lck, Fgr,
Btk, Fak, SYK, FRK, JAK, Abl, Kit, KDR, CaM-kinase, phosphorylase
kinase, MEKK, ERK, MAP kinase, PI3K, Akt1, Akt2, Akt3,
TGF-.alpha.R, KRAS, BRAF, CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK9,
GSK3, CLK1, CLK4, Aurora A, Aurora B, Aurora C, MEK1, MEK2, mTOR,
protein kinase A, protein kinase C, protein kinase G, PHB1,
survivin, KIF11, and BRD4. In a preferred embodiment, the nucleic
acids encapsulated are siRNA targeting PHB1.
[0279] In another pathway, the nucleic acids can increase the
expression of disease-suppressed protein expression (e.g., see
Table 1). These proteins can be but not only adenomatous polyposis
coli (APC), tumor protein p53 (TP53), cyclin-dependent kinase
inhibitor 2A (CDKN2A), phosphatase and tensin homolog (PTEN),
retinoblastoma 1 (RB 1), mothers against DPP homolog 4 (SMAD4), von
Hippel-Lindau protein (pVHL), CD95, suppression of tumorigenicity 5
(ST5), suppression of tumorigenicity 7 (ST7), suppression of
tumorigenicity (ST14), and Yippee-like 3 (YPEL3). In a preferred
embodiment, the nucleic acids encapsulated are mRNA encoding
PTEN.
[0280] In some embodiments, the nanoparticles are
stimuli-responsive nanoparticles. Stimuli-responsive nanoparticles,
which can undergo shape, structure and property change upon
encountering endogenous or exogenous stimuli, can be used in
diverse range of biomedical applications, such as drug controlled
release, nucleic acid delivery, imaging, and diagnostics. The
stimuli-responsive characteristic provides spatiotemporal control
over the macroscopic properties of the nanoparticles, and thus the
release of the encapsulated cargo can occur directly at the desired
site, minimizing toxic and side effects in surrounding, healthy
tissue. Dissociation of the particle and release of its cargo, can
be driven by, for example, pH-, redox-, light-, temperature-,
enzyme-, or ultrasound-responsive polymers composing the
particles.
[0281] The stimuli that drive a response by the particle can be
present within a cell (e.g., intracellularly) or outside cells in
the extracellular microenvironment, or can be an external stimuli
for example, light, heat, ultrasound, etc., which can be applied by
the user to the target site. The particles can optionally include a
targeting moiety or ligand. For embodiments in which intracellular
release is desired, the targeting moiety or ligand is typically one
that preferentially binds to the surface of a target cell and
induces or allows the particle to be absorbed or internalized by,
for example, endocytosis or micropinocytosis (Vranic et al.,
Particle and Fibre Toxicology, 10(2):(12 page) (2013)). For
embodiments in which extracellular release is desired, the
targeting moiety or ligand can be one that preferentially binds to
an extracellular target in the desired microenvironment.
[0282] C. Exemplary Environments for Selective Delivery
[0283] 1. Acidic Environment
[0284] pH responsive nanoparticles can be used to target tissues
with acidic extracellular pH. Although the nanoparticles can
optionally include a cell, tissue, organ, or extracellular
matrix-specific targeting moiety or ligand, a targeting moiety or
ligand is not requirement. The pH responsive nanoparticles can be
designed to have spherical morphology at a pH above pKa to protect
cargo during systemic circulation and infiltration into tissues
with extracellular pH at or around neutral or physiological pH. The
particles can dissociate at a pH below pKa, releasing its cargo
into the microenvironment. Low pH is associated with infection,
cancer and some other conditions. In this way, the particles
selectively release their cargo at the target site.
[0285] pH responsive nanoparticles can also be used to deliver
cargo into cells. Particles, preferable with a targeting moiety or
ligand, can bind to a target cell and be absorbed or internalized.
Upon encountering an acidic intracellular environment such as that
of endosomes, the pH responsive particles can dissociate and
release their cargo. The particles can also optionally include a
moiety that enhances endosomal escape, such as oligoarinine. As
illustrated in the working Examples below, particle dissociation
within the endosome is believe to induce swelling of the endosome
via "sponge" effect, thus achieving fast and high efficacy delivery
of their cargo into the cytosol. Using an intracellular
endosomal-release strategy, virtually any cell with endosomes (or
another equivalently acidic intracellular environment, compartment,
or organelle) can be the target cell. The addition of a targeting
moiety can be used to accomplish selective delivery of the particle
into target cells over non-target cells. pH responsive
intracellular release can be most effective when the extracellular
pH does not induce nanoparticle dissociation thus allowing the
particles to absorbed or internalized by cells.
[0286] In some embodiments, cargo is released below physiological
pH (e.g., 7.4, or 7.2), or below neutral pH (e.g., 7.0), or in a pH
range of about 5.8 to about 7.3, or about 5.8 to about 6.9, or
about 6.0 to about 6.5, or about 6.5 to about 6.9.
[0287] 2. Temperature
[0288] In embodiments, cargo release is driven by a change in
temperature. In the biomedical setting, a change in temperature
will can be an increase or decrease from the physiological
temperature of the subject being treated. Normal human body
temperature, also referred to as normothermia or euthermia, depends
upon the place in the body at which the measurement is made, the
time of day, as well as the activity level of the person. Typically
values for oral measurement (under the tongue) are
36.8.+-.0.4.degree. C. (98.2.+-.0.72.degree. F.) and internal
(rectal, vaginal) measurement are 37.0.degree. C. (98.6.degree. F.)
(Harrison's Principles of Internal Medicine, 18e, Longo, Editor,
Fauci, et al., Editor, Kasper). Human temperature classifications
can be, for example, Hypothermia <35.0.degree. C. (95.0.degree.
F.); Normal 36.5-37.5.degree. C. (97.7-99.5.degree. F.), Fever
>37.5 or 38.3.degree. C. (99.5 or 100.9.degree. F.),
Hyperthermia >37.5 or 38.3.degree. C. (99.5 or 100.9.degree.
F.), Hyperpyrexia >40.0 or 41.5.degree. C. (104.0 or
106.7.degree. F.). The particles can be designed for release within
one or more of these temperature classifications, or a sub-range
thereof. It will be appreciated that a subject's normal body
temperature can fluctuate, for example, with the time of day, sleep
vs. wake, eating vs. fasting, exercise, the amount of clothing
being worn, the ambient temperature, the anxiety or excitement
level of the subject, etc., as is known in the art. The particles
can be tuned for release when body temperature drops below or
exceeds a predetermined threshold, and therefore selectively
release cargo during certain times of the day or night, caloric
intake (or lack thereof), during exercise, anxiety, etc. The
release can be local so systemic.
[0289] In addition of more global changes in overall body
temperature, such as those introduced above, the particles can be
tuned for release at sites of local temperature changes. For
example, local, tissue-specific increase in tissue temperature can
occur at sites of inflammation, injury, infection, and cancer
(e.g., tumor) (Chapter Nine, Inflammation, Tissue Repair, and
Fever, pages 150-167). The change in temperature can be relative to
unaffected tissue and may occur in the presence or absence of a
global change in body temperature.
[0290] 3. Reduction-Oxidation (Redox)
[0291] The release of nanoparticle cargo can be induced by a
reduction-oxidation ("redox") reaction. In some embodiments, the
polymers composing the particles include one or more disulfide
bonds. The particles can release their cargo when disulfide bond is
reduced upon exposure to a reducing agent. In some embodiments, the
reducing agent is a glutathione. L-Glutathione (GSH) is a
tripeptide molecule that can also act as an antioxidant. In cells,
GSH reduces the disulfide bonds formed within cytoplasmic proteins
to cysteines and reacts to other oxidized GSH to an oxidized form
of glutathione disulfide (GSSG), also called L(-)-glutathione
(Traverso, et al., Oxidative Medicine and Cellular Longevity,
Volume 2013 (2013), Article ID 972913, 10 pages). As discussed in
more detail below, intracellular levels of glutathione (GSH) are
100-1000 fold higher in cancer cells than in normal tissue, and
thus redox-sensitive particles can be used to selective release
cargo in cells with higher-than-normal GSH, such as cancer cells.
For example, one study showed that intracellular GHS levels in
normal lung cells were about 11.20.+-.0.58 (SEM) nmol GSH/mg
protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg
protein, while GHS level in adenocarcinomas was 8.83.+-.0.96
nmol/mg protein (8 patients); large cell carcinomas was
8.25.+-.2.51 nmol/mg protein (3 patients); and squamous cell
carcinomas 23.25.+-.5.99 nmol/mg protein (8 patients) (Cook, et
al., Cancer Research, 51:4287-4294 (1991).
[0292] The Examples below show that cargo can be released
redox-sensitive particles in matter of minutes in the presences of
10 nM GSH.
[0293] In some embodiments, the reducing agent is not endogenous to
the cell, tissue, organ, or other microenvironment. For example, in
some embodiments, the reducing agent is administered locally or
systemically to trigger release of the cargo from the particles in
a local or systemic fashion. In addition to GSH, other reducing
agents can also induce release of the cargo, however, it will be
appreciated that in some embodiments, the use, or the amount that
can be used, of certain reducing agents is limited in biological
and therapeutic applications by their toxicity.
[0294] 4. External Stimuli
[0295] As introduced above, release of nanoparticle cargo can be
induced by external stimuli, such as light, temperature, or
ultrasound. The stimuli can be applied globally, for example to the
entire subject, or preferably to a more limited or local aspect
thereof. For example, light, heat (or cold), or ultrasound can be
administered to a specific tissue(s), location(s), or combination
thereof to modulate selective release of cargo from particles
accumulating or passing through the targeted tissue or location.
For example, heat (or cold) can be applied to the target tissue or
location to cause a local temperature shift that induces
dissociation of the particle and release of its cargo. Radiation at
different frequencies along the electromagnetic spectrum can also
be used to release cargo. For example, particles can be formed that
are sensitive to ionizing radiation, visible light, microwaves, or
radiowaves. In particular embodiments, the particles are sensitive
to visible light (e.g., near ultraviolet, near infrared, mid
infrared, far infrared). Particles can also be formed that are
sensitive to sound waves. For example, in particular embodiments,
the particles release cargo in response to ultrasound.
[0296] In particular embodiments, the particles are sensitive to
ultraviolet light. Ultraviolet (UV) light is an electromagnetic
radiation with a wavelength shorter than that of visible light but
longer than X-rays. The wavelength of UV light is typically from
about 400 nm (750 THz) to about 10 nm (30 PHz). UV radiation can be
divided into five categories: UV-A is about 320-400 nm, UV-B is
290-320 nm, UV-C is 220-209 nm, Far UV is 190-220 nm, and vacuum UV
40-190 nm. In some embodiments, the particles are sensitive to
UV-A, UV-B, UV-C, or a combination thereof. The Examples below
illustrate that particles can be formed that the release their
cargo after exposure to UV light, for example 365 nm UV light (16
W), for different time periods. In some embodiments, the source
provides a specific desired wavelength. In some embodiments, the
source provides a range of wavelength.
[0297] The external stimuli can be provided by the practitioner
using, for example, a piece of equipment that provides the stimuli.
The stimuli can also be provided by the environment and may or may
not be under the control of practitioner or user. For example, the
sun generates visible light, heat, and UV radiation. Thus, in some
embodiments, the particles are designed to release their cargo in
response to the sun.
[0298] Exposure to external stimuli can be carried out over
minutes, hours, days or weeks. In some embodiments, the exposure is
between about 1 and about 120 minutes, for example, 10, 15, 30, 45,
60, 90, or 120 minutes. In some embodiments, the exposure is
between about 1 and 48 hours, for example, 1, 2, 3, 4, 5, 10, 12.5,
15, 20, 24, 36, or 48 hours. In some embodiments, the exposure is
over two or more days.
[0299] D. Exemplary Tissues to Target and Therapeutic
Strategies
[0300] Particles co-loaded with two or more nucleic acid species
can be used to in a variety of therapeutic applications. Suitable
methods can include administering a subject an effective amount of
nanoparticles containing a therapeutic cargo to reduce or alleviate
one or more symptoms of the disease or disorder to be treated. As
discussed above, the particles can be used to selectively target
cells, tissues, organs, or microenvironments thereof. The selective
release of cargo at a target site can be used in strategies to
treat a variety of diseases and disorders. In the most preferred
embodiments, nanoparticles containing nucleic acids cross the
cellular membrane and their contents are released into the cytosol.
Strategies can include targeting certain intracellular and/or
extracellular environments for selective release based on
response-inducing stimuli alone, or in combination with one or more
targeting moieties that enhance delivery to a desired cell type,
tissue, organ, microenvironment, subcellular organelle, or a
combination thereof.
[0301] 1. Tumor Targeting
[0302] Methods of treating cancer are provided. The nanoparticles
can be designed, for example, for release in the tumor
microenvironment or within a tumor cells, or in an immune response
microenvironment or within an immune cell. Suitable methods can
include administering a subject an effective amount of
nanoparticles containing a therapeutic cargo to reduce or alleviate
one or more symptoms of the cancer. The effect of the particles on
the cancer can be direct or indirect. The compositions and methods
described herein are useful for treating subjects having benign or
malignant tumors by delaying or inhibiting the growth of a tumor in
a subject, reducing the growth or size of the tumor, inhibiting or
reducing metastasis of the tumor, and/or inhibiting or reducing
symptoms associated with tumor development or growth.
[0303] The tumor microenvironment is the cellular environment in
which the tumor exists, and can include surrounding blood vessels,
immune cells, fibroblasts, bone marrow-derived inflammatory cells,
lymphocytes, signaling molecules and the extracellular matrix
(ECM). The tumor and the surrounding microenvironment are closely
related and interact constantly. Tumors can modulate the
microenvironment by releasing extracellular signals, promoting
tumor angiogenesis and inducing peripheral immune tolerance, while
the immune cells in the microenvironment can affect the growth and
evolution of cancerous cells. The microenvironment in tumor tissue
is different from the normal tissues. Compared to normal tissues,
the pH in tumor tissue is more acidic, the tissue temperature is
relatively higher, and some specific enzymes or chemicals are
over-expressed. The interstitial fluid of tumors and abscesses also
has shown pH values of less than 6.0, averaging 0.2-0.6 units lower
than mean extracellular pH of normal tissues (Kraus and Wolf,
Tumour Biol, 17,133-154 (1996)). Tumors commonly have an
extracellular environment with a pH in the range of, for example,
6.5-6.9. See, for example, Balkwill, et al., Journal of Cell
Science, 125(23):5591-6 (2012) and Kato, et al., Cancer Cell
International, 13(89) (8 pages) (2013). Thus, in some embodiments
pH-sensitive nanoparticles are used to selectively delivery cargo
to an acidic tumor microenvironment.
[0304] Tumors can also have elevated temperatures relative to the
surround or otherwise normal or non-malignant tissue (see, e.g.,
Stefanadis, JCO, 19(3):676-681 (2001)). Therefore,
temperature-responsive particles can also be utilized to
selectively target tumors.
[0305] The intracellular levels of glutathione (GSH) are 100-1000
fold higher in cancer cells than in normal tissue. Redox-sensitive
approach is particularly promising to enhance the exposure of
cancer cells to therapeutic molecules.
[0306] a. Tumor Targeting Moieties
[0307] In addition or alternative to selectively targeting cancer
cells by targeting an acidic microenvironment, or one with an
elevated temperature, cancer cells or their microenvironment can be
specifically targeted relative to healthy or normal cells by
including a targeting moiety. Tumor or tumor-associated
neovasculature targeting domains can be ligands that bind to cell
surface antigens or receptors that are specifically expressed on
tumor cells or tumor-associated neovasculature or microenvironment,
or are overexpressed on tumor cells or tumor-associated
neovasculature or microenvironment as compared to normal tissue.
Tumors also secrete a large number of ligands into the tumor
microenvironment that affect tumor growth and development.
Receptors that bind to ligands secreted by tumors, including, but
not limited to growth factors, cytokines and chemokines, including
the chemokines provided below, can also be used. Ligands secreted
by tumors can be targeted using soluble fragments of receptors that
bind to the secreted ligands. Soluble receptor fragments are
fragments polypeptides that may be shed, secreted or otherwise
extracted from the producing cells and include the entire
extracellular domain, or fragments thereof. In some embodiments,
the targeting moiety is an antibody, for example a single chain
antibody, the binds to the target.
[0308] i. Cancer Antigens
[0309] Cancer antigens that can be targeted are well known in the
art. The antigen expressed by the tumor may be specific to the
tumor, or may be expressed at a higher level on the tumor cells as
compared to non-tumor cells. Antigenic markers such as
serologically defined markers known as tumor associated antigens,
which are either uniquely expressed by cancer cells or are present
at markedly higher levels (e.g., elevated in a statistically
significant manner) in subjects having a malignant condition
relative to appropriate controls, are contemplated for use in
certain embodiments.
[0310] Tumor-associated antigens may include, for example, cellular
oncogene-encoded products or aberrantly expressed
proto-oncogene-encoded products (e.g., products encoded by the neu,
ras, trk, and kit genes), or mutated forms of growth factor
receptor or receptor-like cell surface molecules (e.g., surface
receptor encoded by the c-erb B gene). Other tumor-associated
antigens include molecules that may be directly involved in
transformation events, or molecules that may not be directly
involved in oncogenic transformation events but are expressed by
tumor cells (e.g., carcinoembryonic antigen, CA-125, melonoma
associated antigens, etc.) (see, e.g., U.S. Pat. No. 6,699,475;
Jager, et al., Int. J. Cancer, 106:817-20 (2003); Kennedy, et al.,
Int. Rev. Immunol., 22:141-72 (2003); Scanlan, et al. Cancer
Immun., 4:1 (2004)).
[0311] Genes that encode cellular tumor associated antigens include
cellular oncogenes and proto-oncogenes that are aberrantly
expressed. In general, cellular oncogenes encode products that are
directly relevant to the transformation of the cell, and because of
this, these antigens are particularly preferred targets for
anticancer therapy. An example is the tumorigenic neu gene that
encodes a cell surface molecule involved in oncogenic
transformation. Other examples include the ras, kit, and trk genes.
The products of proto-oncogenes (the normal genes which are mutated
to form oncogenes) may be aberrantly expressed (e.g.,
overexpressed), and this aberrant expression can be related to
cellular transformation. Thus, the product encoded by
proto-oncogenes can be targeted. Some oncogenes encode growth
factor receptor molecules or growth factor receptor-like molecules
that are expressed on the tumor cell surface. An example is the
cell surface receptor encoded by the c-erbB gene. Other
tumor-associated antigens may or may not be directly involved in
malignant transformation. These antigens, however, are expressed by
certain tumor cells and may therefore provide effective targets.
Some examples are carcinoembryonic antigen (CEA), CA 125
(associated with ovarian carcinoma), and melanoma specific
antigens.
[0312] In ovarian and other carcinomas, for example, tumor
associated antigens are detectable in samples of readily obtained
biological fluids such as serum or mucosal secretions. One such
marker is CA125, a carcinoma associated antigen that is also shed
into the bloodstream, where it is detectable in serum (e.g., Bast,
et al., N. Eng. J. Med., 309:883 (1983); Lloyd, et al., Int. J.
Canc., 71:842 (1997). CA125 levels in serum and other biological
fluids have been measured along with levels of other markers, for
example, carcinoembryonic antigen (CEA), squamous cell carcinoma
antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN
mucin (STN), and placental alkaline phosphatase (PLAP), in efforts
to provide diagnostic and/or prognostic profiles of ovarian and
other carcinomas (e.g., Sarandakou, et al., Acta Oncol., 36:755
(1997); Sarandakou, et al., Eur. J. Gynaecol. Oncol., 19:73 (1998);
Meier, et al., Anticancer Res., 17(4B):2945 (1997); Kudoh, et al.,
Gynecol. Obstet. Invest., 47:52 (1999)). Elevated serum CA125 may
also accompany neuroblastoma (e.g., Hirokawa, et al., Surg. Today,
28:349 (1998), while elevated CEA and SCC, among others, may
accompany colorectal cancer (Gebauer, et al., Anticancer Res.,
17(4B):2939 (1997)).
[0313] The tumor associated antigen, mesothelin, defined by
reactivity with monoclonal antibody K-1, is present on a majority
of squamous cell carcinomas including epithelial ovarian, cervical,
and esophageal tumors, and on mesotheliomas (Chang, et al., Cancer
Res., 52:181 (1992); Chang, et al., Int. J. Cancer, 50:373 (1992);
Chang, et al., Int. J. Cancer, 51:548 (1992); Chang, et al., Proc.
Natl. Acad. Sci. USA, 93:136 (1996); Chowdhury, et al., Proc. Natl.
Acad. Sci. USA, 95:669 (1998)). Using MAb K-1, mesothelin is
detectable only as a cell-associated tumor marker and has not been
found in soluble form in serum from ovarian cancer patients, or in
medium conditioned by OVCAR-3 cells (Chang, et al., Int. J. Cancer,
50:373 (1992)). Structurally related human mesothelin polypeptides,
however, also include tumor-associated antigen polypeptides such as
the distinct mesothelin related antigen (MRA) polypeptide, which is
detectable as a naturally occurring soluble antigen in biological
fluids from patients having malignancies (see WO 00/50900).
[0314] A tumor antigen may include a cell surface molecule. Tumor
antigens of known structure and having a known or described
function, include the following cell surface receptors: HER1
(GenBank Accession No. U48722), HER2 (Yoshino, et al., J. Immunol.,
152:2393 (1994); Disis, et al., Canc. Res., 54:16 (1994); GenBank
Acc. Nos. X03363 and M17730), HER3 (GenBank Acc. Nos. U29339 and
M34309), HER4 (Plowman, et al., Nature, 366:473 (1993); GenBank
Acc. Nos. L07868 and T64105), epidermal growth factor receptor
(EGFR) (GenBank Acc. Nos. U48722, and K03193), vascular endothelial
cell growth factor (GenBank No. M32977), vascular endothelial cell
growth factor receptor (GenBank Acc. Nos. AF022375, 1680143, U48801
and X62568), insulin-like growth factor-I (GenBank Acc. Nos.
X00173, X56774, X56773, X06043, European Patent No. GB 2241703),
insulin-like growth factor-II (GenBank Acc. Nos. X03562, X00910,
M17863 and M17862), transferrin receptor (Trowbridge and Omary,
Proc. Nat. Acad. USA, 78:3039 (1981); GenBank Acc. Nos. X01060 and
M11507), estrogen receptor (GenBank Acc. Nos. M38651, X03635,
X99101, U47678 and M12674), progesterone receptor (GenBank Acc.
Nos. X51730, X69068 and M15716), follicle stimulating hormone
receptor (FSH-R) (GenBank Acc. Nos. Z34260 and M65085), retinoic
acid receptor (GenBank Acc. Nos. L12060, M60909, X77664, X57280,
X07282 and X06538), MUC-1 (Barnes, et al., Proc. Nat. Acad. Sci.
USA, 86:7159 (1989); GenBank Acc. Nos. M65132 and M64928) NY-ESO-1
(GenBank Acc. Nos. AJ003149 and U87459), NA 17-A (PCT Publication
No. WO 96/40039), Melan-A/MART-1 (Kawakami, et al., Proc. Nat.
Acad. Sci. USA, 91:3515 (1994); GenBank Acc. Nos. U06654 and
U06452), tyrosinase (Topalian, et al., Proc. Nat. Acad. Sci. USA,
91:9461 (1994); GenBank Acc. No. M26729; Weber, et al., J. Clin.
Invest, 102:1258 (1998)), Gp-100 (Kawakami, et al., Proc. Nat.
Acad. Sci. USA, 91:3515 (1994); GenBank Acc. No. 573003, Adema, et
al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen, et
al., Science, 254:1643 (1991)); GenBank Acc. Nos. U93163, AF064589,
U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690,
U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339,
L18920, U03735 and M77481), BAGE (GenBank Acc. No. U19180; U.S.
Pat. Nos. 5,683,886 and 5,571,711), GAGE (GenBank Acc. Nos.
AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144,
U19143 and U19142), any of the CTA class of receptors including in
particular HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank
Acc. Nos. X86175, U90842, U90841 and X86174), carcinoembryonic
antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1985);
GenBank Acc. Nos. M59710, M59255 and M29540), and PyLT (GenBank
Acc. Nos. J02289 and J02038); p97 (melanotransferrin) (Brown, et
al., J. Immunol., 127:539-46 (1981); Rose, et al., Proc. Natl.
Acad. Sci. USA, 83:1261-61 (1986)).
[0315] Additional tumor associated antigens include prostate
surface antigen (PSA) (U.S. Pat. Nos. 6,677,157; 6,673,545);
.beta.-human chorionic gonadotropin .beta.-HCG) (McManus, et al.,
Cancer Res., 36:3476-81 (1976); Yoshimura, et al., Cancer,
73:2745-52 (1994); Yamaguchi, et al., Br. J. Cancer, 60:382-84
(1989): Alfthan, et al., Cancer Res., 52:4628-33 (1992));
glycosyltransferase .beta.-1,4-N-acetylgalactosaminyltransferases
(GalNAc) (Hoon, et al., Int. J. Cancer, 43:857-62 (1989); Ando, et
al., Int. J. Cancer, 40:12-17 (1987); Tsuchida, et al., J. Natl.
Cancer, 78:45-54 (1987); Tsuchida, et al., J. Natl. Cancer,
78:55-60 (1987)); NUC18 (Lehmann, et al., Proc. Natl. Acad. Sci.
USA, 86:9891-95 (1989); Lehmann, et al., Cancer Res., 47:841-45
(1987)); melanoma antigen gp75 (Vijayasardahi, et al., J. Exp.
Med., 171:1375-80 (1990); GenBank Accession No. X51455); human
cytokeratin 8; high molecular weight melanoma antigen (Natali, et
al., Cancer, 59:55-63 (1987); keratin 19 (Datta, et al., J. Clin.
Oncol., 12:475-82 (1994)).
[0316] Tumor antigens of interest include antigens regarded in the
art as "cancer/testis" (CT) antigens that are immunogenic in
subjects having a malignant condition (Scanlan, et al., Cancer
Immun., 4:1 (2004)). CT antigens include at least 19 different
families of antigens that contain one or more members and that are
capable of inducing an immune response, including but not limited
to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5);
NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88
(CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26);
HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43);
and TPTE (CT44).
[0317] Additional tumor antigens that can be targeted, including a
tumor-associated or tumor-specific antigen, include, but not
limited to, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8,
beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein,
EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion
protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and
3, neo-PAP, myosin class I, OS-9, pml-RARc fusion protein, PTPRK,
K-ras, N-ras, Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7,
GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88,
NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100
(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1,
GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40,
PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK,
MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus
(HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6,
p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA
72-4, CAM 17.1, NuMa, K-ras, .beta.-Catenin, CDK4, Mum-1, p16,
TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72,
.alpha.-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,
G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K,
NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin
C-associated protein), TAAL6, TAG72, TLP, and TPS. Other
tumor-associated and tumor-specific antigens are known to those of
skill in the art and are suitable for targeting the disclosed
nanoparticles.
[0318] ii. Antigens Associated with Tumor Neovasculature
[0319] The antigen may be specific to tumor neovasculature or may
be expressed at a higher level in tumor neovasculature when
compared to normal vasculature. Exemplary antigens that are
over-expressed by tumor-associated neovasculature as compared to
normal vasculature include, but are not limited to, VEGF/KDR, Tie2,
vascular cell adhesion molecule (VCAM), endoglin and
.alpha..sub.5.beta..sub.3 integrin/vitronectin. Other antigens that
are over-expressed by tumor-associated neovasculature as compared
to normal vasculature are known to those of skill in the art and
are suitable for targeting by the nanoparticles.
[0320] iii. Chemokines/Chemokine Receptors
[0321] In another embodiment, the particles contain a domain that
specifically binds to a chemokine or a chemokine receptor.
Chemokines are soluble, small molecular weight (8-14 kDa) proteins
that bind to their cognate G-protein coupled receptors (GPCRs) to
elicit a cellular response, usually directional migration or
chemotaxis. Tumor cells secrete and respond to chemokines, which
facilitate growth that is achieved by increased endothelial cell
recruitment and angiogenesis, subversion of immunological
surveillance and maneuvering of the tumoral leukocyte profile to
skew it such that the chemokine release enables the tumor growth
and metastasis to distant sites. Thus, chemokines are vital for
tumor progression.
[0322] Based on the positioning of the conserved two N-terminal
cysteine residues of the chemokines, they are classified into four
groups namely CXC, CC, CX3C and C chemokines. The CXC chemokines
can be further classified into ELR+ and ELR- chemokines based on
the presence or absence of the motif `glu-leu-arg (ELR motif)`
preceding the CXC sequence. The CXC chemokines bind to and activate
their cognate chemokine receptors on neutrophils, lymphocytes,
endothelial and epithelial cells. The CC chemokines act on several
subsets of dendritic cells, lymphocytes, macrophages, eosinophils,
natural killer cells but do not stimulate neutrophils as they lack
CC chemokine receptors except murine neutrophils. There are
approximately 50 chemokines and only 20 chemokine receptors, thus
there is considerable redundancy in this system of ligand/receptor
interaction.
[0323] Chemokines elaborated from the tumor and the stromal cells
bind to the chemokine receptors present on the tumor and the
stromal cells. The autocrine loop of the tumor cells and the
paracrine stimulatory loop between the tumor and the stromal cells
facilitate the progression of the tumor. Notably, CXCR2, CXCR4,
CCR2 and CCR7 play major roles in tumorigenesis and metastasis.
CXCR2 plays a vital role in angiogenesis and CCR2 plays a role in
the recruitment of macrophages into the tumor microenvironment.
CCR7 is involved in metastasis of the tumor cells into the sentinel
lymph nodes as the lymph nodes have the ligand for CCR7, CCL21.
CXCR4 is mainly involved in the metastatic spread of a wide variety
of tumors.
[0324] b. Cancers to be Treated
[0325] The types of cancer that can be treated with the provided
compositions and methods include, but are not limited to, the
following: bladder, brain, breast, cervical, colo-rectal,
esophageal, kidney, liver, lung, nasopharangeal, pancreatic,
prostate, skin, stomach, uterine, ovarian, testicular and the like.
Administration is not limited to the treatment of an existing
tumors but can also be used to prevent or lower the risk of
developing such diseases in an individual, i.e., for prophylactic
use. Potential candidates for prophylactic vaccination include
individuals with a high risk of developing cancer, i.e., with a
personal or familial history of certain types of cancer.
[0326] Malignant tumors which may be treated are classified herein
according to the embryonic origin of the tissue from which the
tumor is derived. Carcinomas are tumors arising from endodermal or
ectodermal tissues such as skin or the epithelial lining of
internal organs and glands. Sarcomas, which arise less frequently,
are derived from mesodermal connective tissues such as bone, fat,
and cartilage. The leukemias and lymphomas are malignant tumors of
hematopoietic cells of the bone marrow. Leukemias proliferate as
single cells, whereas lymphomas tend to grow as tumor masses.
Malignant tumors may show up at numerous organs or tissues of the
body to establish a cancer.
[0327] In some embodiments, the nucleci acid cargo can be
functional nucleic acids having an anticancer activity for example
siRNAs that target one or more oncogenese, and one or more mRNAs
that encode proteins having anti-proliferative activity, a
pro-apoptotic activity, or other cytotoxic activity. In some
embodiments the nanoparticles are delivered in addition to one or
more chemotherapeutic drugs or therapeutic regimens, surgeries or
methods.
[0328] In a particularly preferred example, a cancer therapy
includes co-delivery of RNA encoding one or more tumor suppressors
and functional nucleic acid (e.g., siRNA) that targets and reduces
expression of one or more oncogenes. Suitable tumor supressors and
oncogenes are well known in the art. Exemplary oncogenes and tumor
suppressors are illustrated in Table 5, adapted from Supplemental
FIG. 3 (compiled from the CancerGenes website) of Walker, et al.,
Cancer Res., 72(3):636-644 (2012) and supplemental information,
DOI: 10.1158/0008-5472.CAN-11-2266.
TABLE-US-00005 TABLE 5 Oncogenes and Tumor Suppressors Gene Symbol
- Oncogenes Gene Symbol - Tumor Suppressors ABL1 EVI1 MYC APC IL2
TNFAIP3 ABL2 EWSR1 MYCL1 ARHGEF12 JAK2 TP53 AKT1 FEV MYCN ATM
MAP2K4 TSC1 AKT2 FGFR1 NCOA4 BCL11B MDM4 TSC2 ATF1 FGFR1OP NFKB2
BLM MEN1 VHL BCL11A FGFR2 NRAS BMPR1A MLH1 WRN BCL2 FUS NTRK1 BRCA1
MSH2 WT1 BCL3 GOLGA5 NUP214 BRCA2 NF1 BCL6 GOPC PAX8 CARS NF2 BCR
HMGA1 PDGFB CBFA2T3 NOTCH1 BRAF HMGA2 PIK3CA CDH1 NPM1 CARD11 HRAS
PIM1 CDH11 NR4A3 CBLB IRF4 PLAG1 CDK6 NUP98 CBLC JUN PPARG CDKN2C
PALB2 CCND1 KIT PTPN11 CEBPA PML CCND2 KRAS RAF1 CHEK2 PTEN CCND3
LCK REL CREB1 RB1 CDX2 LMO2 RET CREBBP RUNX1 CTNNB1 MAF ROS1 CYLD
SDHB DDB2 MAFB SMO DDX5 SDHD DDIT3 MAML2 SS18 EXT1 SMARCA4 DDX6
MDM2 TCL1A EXT2 SMARCB1 DEK MET TET2 FBXW7 SOCS1 EGFR MITF TFG FH
STK11 ELK4 MLL TLX1 FLT3 SUFU ERBB2 MPL TPR FOXP1 SUZ12 ETV4 MYB
USP6 GPC3 SYK ETV6 IDH1 TCF3
[0329] 2. Inflammation and Infection
[0330] Methods of treating inflammation and infection are provided.
The nanoparticles can be designed, for example, for release in the
microenvironment of inflammation, injury, and infection, or immune
or pro-inflammatory cells, or within immune or inflammatory cells
themselves. Suitable methods can include administering a subject an
effective amount of nanoparticles containing a therapeutic cargo to
reduce or alleviate one or more symptoms of the inflammation,
injury, or infection. The effect on the inflammation, injury, or
infection can be direct or indirect. Administration is not limited
to the treatment of an existing inflammation, injury, and
infection, but can also be used to prevent or lower the risk of
developing such diseases in an individual, i.e., for prophylactic
use. A characteristic feature of the inflammation is local
acidosis, which is attributed to the local increase of lactic-acid
production by the anaerobic, glycolytic activity of infiltrated
neutrophils and to the presence of short-chain, fatty acid
by-products of bacterial metabolism (Grinstein, et al., Clin.
Biochem. 24,241-247 (1991) and Ehrich, W. E. (1961) Inflammation
Allgower, M. eds. Progress in Surgery vol. 1,1-70 S. Karger Basel,
Switzerland). An acidic extracellular pH is also found in the
epidermis and plays an important protective role against bacterial
infection (Lardner, et al., Journal of Leukocyte Biology,
69(4):522-530 (2001)). As discussed above, local, tissue-specific
increase in tissue temperature can occur at site of inflammation,
injury, and infection. Similar to selectively targeting the tumor
microenvironment, the pH and temperature sensitive particles can be
utilized to delivery and selectively release cargo at sites of
inflammation, injury, and infection.
[0331] As with cancer, in addition or alternative to selectively
targeting cancer cells by targeting an acidic microenvironment, or
one with an elevated temperature, cancer cells or their
microenvironment can be specifically targeted relative to healthy
or normal cells by including a targeting moiety. Preferred
targeting domains target the molecule to areas of inflammation,
injury, or infection. Exemplary targeting domains are antibodies,
or antigen binding fragments thereof that are specific for inflamed
tissue or to a proinflammatory cytokine including but not limited
to IL17, IL-4, IL-6, IL-12, IL-21, IL-22, and IL-23. In the case of
neurological disorders such as Multiple Sclerosis, the targeting
domain may target the molecule to the CNS or may bind to VCAM-1 on
the vascular epithelium. Additional targeting domains can be
peptide aptamers specific for a proinflammatory molecule. In other
embodiments, the particles can include a binding partner specific
for a polypeptide displayed on the surface of an immune cell, for
example a T cell. In still other embodiments, the targeting domain
specifically targets activated immune cells. Preferred immune cells
that are targeted include Th0, Th1, Th17 and Th22 T cells, other
cells that secrete, or cause other cells to secrete inflammatory
molecules including, but not limited to, IL-1.beta., TNF-.alpha.,
TGF-beta, IFN-.gamma., IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs,
and Tregs. For example, a targeting domain for Tregs may bind
specifically to CD25.
[0332] In some embodiments, the target site is neutrophils, which
may phagocytize the particles to release a therapeutic and/or
diagnostic agent at the site of inflammation.
[0333] Proteins constitutively expressed on the surface of
neutrophils that are important for recognition of the endothelial
inflammatory signals include the glycoprotein P-selectin
glycoprotein ligand-1 (PSGL-1) and L-selectin. Other agents to be
targeted include those associated with the disease. For example, a
plaque targeted peptide can be one or more of the following:
Collagen IV, CREKA (SEQ ID NO: 1), LyP-I, CRKRLDRNC (SEQ ID NO:2),
or their combinations at various molar ratios.
[0334] In another embodiment, particles can contain a targeting
domain to target the molecule to an organ or tissue that is being
transplanted. For example, the targeting domain can be an antibody,
antigen binding fragment thereof, or another binding partner
specific for a polypeptide displayed on the surface of cells
specific to the type of organ or tissue being transplanted.
[0335] a. Inflammation
[0336] Inflammation is typically a localized physical condition in
which part of the body becomes reddened, swollen, hot, and often
painful, especially as a reaction to injury or infection.
Inflammation is a protective response that involves immune cells,
blood vessels, and molecular mediators, the purpose of which is to
eliminate the cause of cell injury, remove necrotic cells and
tissues damaged from the injury and the inflammatory process, and
to initiate tissue repair. The compositions can be used to treat
acute and chronic inflammation.
[0337] The inflammation can be caused by an infection such as those
described below or can be caused by a non-infectious mechanism. For
example, inflammation is associated with atherosclerosis, type III
hypersensitivity, trauma, and ischaemia. Inflammation can be
associated with autoimmune diseases, transplantation, graft verse
host disease, and conditions driven by immune responses. In some
embodiments, the particles are used to deliver a cargo for
treatment of an inflammatory or autoimmune disease or disorder such
as rheumatoid arthritis, systemic lupus erythematosus, alopecia
areata, anklosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome (alps), autoimmune thrombocytopenic
purpura (ATP), Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome
immune deficiency, syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy, cicatricial pemphigoid, cold
agglutinin disease, Crest syndrome, Crohn's disease, Dego's
disease, dermatomyositis, dermatomyositis--juvenile, discoid lupus,
essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,
grave's disease, guillain-barre, hashimoto's thyroiditis,
idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura
(ITP), Iga nephropathy, insulin dependent diabetes (Type I),
juvenile arthritis, Meniere's disease, mixed connective tissue
disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris,
pernicious anemia, polyarteritis nodosa, polychondritis,
polyglancular syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant
cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo,
and Wegener's granulomatosis. In some embodiments, the cargo is a
functional nucleic acid that targets a factor that contributes to
inflammation, the activation or persistence of pro-inflammatory
cells, a pro-inflammatory response, active immune response, an
autoimmune response, etc. Specific targets include, for example,
pro-inflammatory molecules such as IL-1.beta., TNF-.alpha.,
TGF-beta, IFN-.gamma., IL-17, IL-6, IL-23, IL-22, IL-21, and
MMPs.
[0338] Additionally or alternatively, an RNA can be introduced to
reduce the inflammation or autoimmune response. These can be
introduced into cells that inhibit the development of naive T cells
into Th1, Th17, Th22 or other cells that secrete, or cause other
cells to secrete, inflammatory molecules. The cargo can increase
the number or activity of Tregs.
[0339] The cargo can promote or enhance production of IL-10 or
another anti-inflammatory cytokine. For example, Kamaly, et al.,
ACS Nano 2016, 10, 5280-5292, DOI: 10.1021/acsnano.6b01114,
describes controlled-release polymeric nanoparticles incorporating
IL-10 for targeted delivery to atherosclerotic plaques.
Nanoparticles were nanoengineered via self-assembly of
biodegradable polyester polymers by nanoprecipitation using a rapid
micromixer chip capable of producing nanoparticles with retained
IL-10 bioactivity post-exposure to organic solvent, reducing acute
inflammation and preventing plaque formation in disease models.
Thus, in some embodiments, the cargo that can promote or enhance
production of IL-10 is RNA encoding IL-10.
[0340] In some embodiments, the cargo enhances the differentiation,
recruitment and/or expansion of Treg cells in the region of
inflammation, autoimmune activity, or tissue engraftment. Exemplary
functional nucleic acid targets for treating autoimmune disease are
reviewed in Pauley and Cha, Pharmaceuticals 2013, 6(3), 287-294;
and discussed in, for example, Kim, et al., Molecular Therapy,
(2010) 18 5, 993-1001, Laroui, et al., Molecular Therapy (2014); 22
1, 69-80, Ponnappa, et al., Curr Opin Investig Drugs. 2009 May;
10(5):418-24; Abrams, et al., Molecular Therapy, (2010) 18 1,
171-180, Leuschner, et al., Nature biotechnology 29.11 (2011):
1005-1010. PMC. Web. 29 Mar. 2016. In some embodiments, the cargo
is a nucleic acid that encodes an anti-inflammatory cytokine, for
example, (IL)-1 receptor antagonist, IL-4, IL-6, IL-10, IL-11, or
IL-13 (Opal and DePalo, et al., Chest. (2000)117(4):1162-72).
[0341] In some embodiments, the methods includes delivery an
inflammation resolution mediator. Resolution mediators are known in
the art and discussed in, for example, Fredman, et al., Sci Transl
Med. 2015 Feb. 18; 7(275): 275ra20.
doi:10.1126/scitranslmed.aaa1065). One class of resolution
mediators includes fatty acid-derived lipids called lipoxins,
resolvins, protectins, and maresins, which are collectively
referred to as specialized proresolving lipid mediators (Buckley,
et al., Immunity. 2014; 40:315-327). Several of these mediators,
notably RvE1, have shown benefit in preclinical models of certain
chronic inflammatory diseases, including asthma, rheumatoid
arthritis, and periodontal disease (Hasturk, et al., FASEB J. 2006;
20:401-403; Haworth, et al., Nat. Immunol. 2008; 9:873-879.). In
humans, proresolving lipid mediators have shown benefit in dry eye
syndrome, a chronic inflammatory disease affecting the ocular
surface. Another class of proresolving mediators includes proteins
such as transforming growth factor-.beta. (TGF-.beta.) and annexin
A1 (Perretti, et al., Nat. Rev. Immunol. 2009; 9:62-70). Endogenous
annexin A1, a 37-kD protein, mediates inflammation resolution in
several disease models, and administration of a 25-amino acid
peptide encompassing its pharmacophore N-terminal region, called
Ac2-26, can mimic the effects of annexin A1 (Perretti, et al., Br.
J. Pharmacol. 2009; 158:936-946.). Annexin A1 and Ac2-26 bind and
activate a specific G protein-coupled receptor (GPCR) called
N-formyl peptide receptor 2 (FPR2/ALX) to evoke their protective
actions, and intriguingly, this is the same receptor used by two
lipid mediators: resolvin D1 (RvD1) and lipoxin A4 (LXA4) (Cooray,
et al., Proc. Natl. Acad. Sci. U.S.A. 2013; 110:18232-18237;
Perretti, et al., Nat. Med. 2002; 8:1296-1302; Fiore, et al., J.
Exp. Med. 1994; 180:253-260; Krishnamoorthy, et al., Proc. Natl.
Acad. Sci. U.S.A. 2010; 107:1660-1665).
[0342] Thus, in some embodiments, the composition and method of
resolution of inflammation include particle delivery of a
inflammation resolution mediator, for example, RNA encoding a
transforming growth factor-.beta. (TGF-.beta.) or annexin A1
protein such as Ac2-26, alone or in combination with RNA encoding
IL-10.
[0343] A number of chronic diseases, including atherosclerosis,
type 2 diabetes, and Alzheimer's disease, have aninflammatory
component. In some cases, the inflammatory stimulus is unknown and,
if known, is difficult to remove. Thus, there is interest in
therapeutically targeting the inflammatory response. The basic
priniciples of the inflammatory response and inflammation
resolution, and the principles of anti-inflammatory therapy in
chronic autoimmune inflammatory diseases are reviewed in Tabas and
Glass, Science. 2013 Jan. 11; 339(6116): 166-172.
doi:10.1126/science.1230720, which also provides specific targets
and strategies for targeting inflammation in chronic disease with
an inflammatory component not triggered by autoimmunity. The
targets discussed in Tabas and Glass can be modulated using the
disclosed compositions and methods for treating inflammation.
[0344] In some embodiments, the particles are targeted to the site
of inflammation, for example, atherosclerosis, using a targeting
moiety. An exemplary targeting moiety is one that binds to Col IV,
e.g., the Col IV-binding heptapeptide discussed in Chan, et al.,
Proc. Natl. Acad. Sci. U.S.A. 2010; 107:2213-2218.
[0345] b. Infections
[0346] Similarly, in some embodiments, the particles are used to
deliver a cargo for treatment of an infectious disease. Infectious
diseases that can be treated, prevented, and/or managed using the
disclosed nanoparticles can be caused by infectious agents
including but not limited to bacteria, fungi, protozae, and
viruses. Viral diseases include, for example, those caused by
hepatitis type A, hepatitis type B, hepatitis type C, influenza
(e.g., influenza A or influenza B), varicella, adenovirus, herpes
simplex type I (HSV-I), herpes simplex type II (HSV-II),
rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial
virus, papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,
rubella virus, polio virus, small pox, Epstein Barr virus, human
immunodeficiency virus type I (HIV-I), human immunodeficiency virus
type II (HIV-II), and agents of viral diseases such as viral
meningitis, encephalitis, dengue or small pox.
[0347] Bacterial diseases can be caused by bacteria (e.g.,
Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus,
Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans,
and Pseudomonas aeruginosa) include, for example, mycobacteria
rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia
burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,
streptococcus, staphylococcus, mycobacterium, pertissus, cholera,
plague, diptheria, chlamydia, S. aureus and legionella.
[0348] Protozoal diseases caused by protozoa include, for example,
leishmania, kokzidioa, trypanosome schistosoma or malaria.
Parasitic diseases caused by parasites include chlamydia and
rickettsia.
[0349] Fungal infections include, but are not limited to, Candida
infections, zygomycosis, Candida mastitis, progressive disseminated
trichosporonosis with latent trichosporonemia, disseminated
candidiasis, pulmonary paracoccidioidomycosis, pulmonary
aspergillosis, Pneumocystis carinii pneumonia, cryptococcal
meningitis, coccidioidal meningoencephalitis and cerebrospinal
vasculitis, Aspergillus niger infection, Fusarium keratitis,
paranasal sinus mycoses, Aspergillus fumigatus endocarditis, tibial
dyschondroplasia, Candida glabrata vaginitis, oropharyngeal
candidiasis, X-linked chronic granulomatous disease, tinea pedis,
cutaneous candidiasis, mycotic placentitis, disseminated
trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic
keratitis, Cryptococcus neoformans infection, fungal peritonitis,
Curvularia geniculata infection, staphylococcal endophthalmitis,
sporotrichosis, and dermatophytosis.
[0350] In some embodiments, the cargo is a functional nucleic acid
that targets a factor that contributes to anti-infective drug
resistance, for example, drug efflux pumps, anti-apoptotic defense
mechanisms, etc., or infected cells or the pathogens themselves. In
some embodiments, the functional nucleic acid specifically targets
a gene expressed by the pathogen. See, for example, Fischer, et
al., Cell Research, (2004) 14, 460-466, which describes RNAi
strategies for targeting viral infection. Additionally or
alternatively, an RNA can be introduced to enhance the fight
against the infection. As in described above in the context of
cancer, these can be introduced into cells that induce, program, or
activate cells to resolve an infection. For example, in some
embodiments, the cargo is a nucleic acid that primes T cells or
other immune cells for immunotherapy against the infection
Immunotherapeutic methods, including CAR T cell therapy and other
strategies for activation of immune cells against target antigens,
and inhibition of immune check points leading to T cell exhaustion,
anergy, or deactivation were well known in the art. The particles
can be used in vitro or in vivo to introduce nucleic acids into
targets including immune cells, to, for example, increase
antigen-specific proliferation of T cells, enhance cytokine
production by T cells, stimulate differentiation, stimulate
effector functions of T cells, promote T cell survival, overcome T
cell exhaustion, overcome T cell anergy or a combination thereof.
Immune cells, including but not limited to, neutrophils,
lymphocytes, dendritic cells, macrophages, eosinophils, natural
killer cells, can be the target of therapy.
[0351] 3. Modifying Immune Responses to Antigen
[0352] Methods for modifying, or enhancing antigen presentation by
antigen presenting cells, and modifying the immunological responses
to a specific antigen are provided. Methods for inducing or
stimulating an immune response to an exogenous antigen a subject
are also provided. Typically, the methods include administering to
the subject pharmaceutical compositions including the nanoparticles
carrying one or more nucleic acids and a pharmaceutically
acceptable excipient in an amount sufficient to induce an immune
response in the subject, preferably via the antigen presenting
cells in the subject. Exemplary antigen presenting cells include
dendritic cells, neutrophils, macrophages, Langerhans cells, and
lymphocytes. Exemplary antigens include viral antigens, bacterial
antigens, protozoan antigens, fungal antigens, nematode antigens
and cancer antigens. In some embodiments, the nanoparticles include
more than one antigen.
[0353] Preferably, in some embodiments, the nanoparticles carrying
one or more nucleic acids expressing desired antigens further
include one or more nucleic acids that condition cells, or cellular
environment to modulate an immunological response. Depending on the
applications, in some embodiments, the desired immunological
response is to enhance immunity against an antigen, in others, to
suppress immunity or to induce tolerance.
[0354] A growing body of evidence suggests that mTOR regulates
functional outcome in a wide range of immune cells, including T
cells, B cells, dendritic cells, macrophages, neutrophils, mast
cells and natural killer cells. Thus, in some embodiments, the
nanoparticles can include one or more nucleic acids that
overexpress protein targets that upregulated by rapamycin, and/or
suppress protein targets that are downregulated or inhibited by
rapamycin.
[0355] The mammalian target of rapamycin (mTOR) is a
serine/threonine kinase that controls cell proliferation and
metabolism in response to a diverse range of extracellular stimuli
such as the availability of nutrients, growth factors and stress.
mTOR is physiologically active in complex with accessory proteins
that determine the functional outcomes of mTOR signaling. The two
currently recognized multi-molecular signaling forms of mTOR, mTOR
complex 1 (mTORC1) and mTOR complex 2 (mTORC2), are differentially
activated by distinct extracellular and intracellular signals (Xu X
et al., Semin Immunol. 24(6):429-35(2012)).
[0356] mTORC1 includes mTOR and four subunits, including the
scaffolding protein, regulatory-associated protein of mTOR
(RAPTOR), DEP-containing mTOR-interacting protein (DEPTOR),
mammalian lethal with Sec13 protein 8 (mLST8) and the Proline-Rich
Akt Substrate 40 kDa (PRAS40). mTORC2 includes mTOR and five
subunits, including the scaffold protein Raptor-Independent
Companion of TOR (RICTOR), mammalian stress-activated protein
kinase interacting protein 1 (mSIN1), DEPTOR, mLST8 and the Protein
Observed with RICTOR (PROTOR). Classically, growth factors,
cytokines or other co-stimulatory signals activate PI3 kinase
(PI3K), leading to activation of the protein kinase Akt. Akt in
turn activates mTOR through inhibition of the mTORC1 repression
factor Tuberous Sclerosis Complex (TSC). TSC is a hetero-dimeric
complex, comprising the TSC1 and TSC2 subunits, which functions as
a GTPase-activating protein by inhibition of the GTP-binding
protein Rheb. Rheb is an essential component of mTORC1 activation.
Upon de-activation of TSC, the active, GTP-bound Rheb interacts
with mTORC1 to promote signaling. Akt can further phosphorylate
PRAS40, relieving it from inhibiting mTORC1. Reciprocal to the
activating signals from Akt, a decrease in the ATP/ADP ratio can
activate AMP-activated Protein Kinase (AMPK) which in turn inhibits
mTOR activity by phosphorylation of TSC2 and/or RAPTOR.
[0357] The mTORC1 signaling pathway controls the expression of a
diverse range of genes that promote cellular growth and
proliferation. One of these pathways is the phosphorylation of
p70-S6 kinase (S6K) and eukaryotic initiation factor 4E binding
protein (4E-BP), which promotes protein translation. Active mTORC1
also down-regulates the autophagy pathway and promotes lipid
biosynthesis as well as mitochondrial biogenesis. Enhanced
mitochondrial biogenesis was observed upon increased mTORC1
activity by genetically deleting the mTORC1 repression factor, TSC1
in hematopoietic stem cells. Signaling events involving mTORC2 are
less well-characterized. mTORC2 signaling has been associated with
cell survival and cytoskeleton organization.
[0358] In some embodiments, the nanoparticles can include one or
more nucleic acids that target one or more upstream targets of
mTOC1 to mimic the effect of rapamycin. For example, the one or
more nucleic acid can suppress PI3 kinase, Akt, and/or Rheb.
Alternatively, the one or more nucleic acid can overexpress TSC to
inhibit mTORC1 activation. In some embodiments, the nanoparticles
can include one or more nucleic acids that promote autophagy,
reduce glycolytic metabolism, and/or promote fatty acid oxidation
of target cells.
[0359] In some embodiments, the molecules to be overexpressed or
suppressed are molecules of professional APC such as dendritic
cells. Exemplary costimulatory molecules include B7-1, B7-2, B7-H3,
B7-H4, CD40, OX40L, ICOS-L, PD-L1, PD-L2, LIGHT, CD70, 4-1BBL,
CD30L, SLAM, and combinations thereof. The nucleic acid can also
target one or more of DC markers. Exemplary DC markers include, but
are not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD1c (M241,
R7); CD1d (R3); CDle (R2); CD11b (.alpha.M Integrin chain, CR3,
Mo1, C3niR, Mac-1); CD11c (aX Integrin, p150, 95, AXb2); CDw117
(Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35 (CR1,
C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39
(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA,
T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d
(VLA-4.alpha., .alpha.4 Integrin); CD49e (VLA-5a, a5 Integrin);
CD58 (LFA-3); CD64 (Fc.gamma.RI); CD72 (Ly-19.2, Ly-32.2, Lyb-2);
CD73 (Ecto-5'nucloticlase); CD74 (Ii, invariant chain); CD80 (B7,
B7-1, BB1); CD81 (TAPA-1); CD83 (HB15); CD85a (ILT5, LIR3, HL9);
CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3,
LIRS, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97 (BL-KDD/F12); CD101
(IGSF2, P126, V7); CD116 (GM-CSFRa); CD120a (TMFRI, p55); CD120b
(TNFRII, p75, TNFR p80); CD123 (IL-3Ra); CD139; CD148 (HPTP-.eta.,
p260, DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP);
CD157 (MoS, BST-1); CD167a (DDR1, trkE, cak); CD168 (RHAMM, IHABP,
HMMR); CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171
(L1CAM, NILE); CD172 (SIRP-1a, MyD-1); CD172b (SIRP(3); CD180
(RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196
(CCR6); CD197 (CCR7 (ws CDw197)); CDw197 (CCR7, EBIL BLR2); CD200
(OX2); CD205 (DEC-205); CD206 (MMR); CD207 (Langerin); CD208
(DC-LAMP); CD209 (DC-SIGN); CDw218a (IL18Ra); CDw218b (IL8R.beta.);
CD227 (MUC1, PUM, PEM, EMA); CD230 (Prion Protein (PrP)); CD252
(OX40L, TNF (ligand) superfamily, member 4); CD258 (LIGHT, TNF
(ligand) superfamily, member 14); CD265 (TRANCE-R, TNF-R
superfamily, member 11a); CD271 (NGFR, p75, TNFR superfamily,
member 16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2,
ICOSL); CD276 (B7H3); CD277 (BT3.1, B7 family: Butyrophilin 3);
CD283 (TLR3, TOLL-like receptor 3); CD289 (TLR9, TOLL-like receptor
9); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase (33 submit); CD300a
(CMRF-35H); CD300c (CMRF-35A); CD301 (MGL1, CLECSF14); CD302
(DCL1); CD303 (BDCA2); CD304 (BDCA4); CD312 (EMR2); CD317 (BST2);
CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68 (gp110, Macrosialin);
class II MHC; BDCA-1; Siglec-H; wherein the names listed in
parentheses represent alternative names.
[0360] a. Enhancing Vaccination Efficacy
[0361] Methods of using the nanoparticle compositions for
vaccination are provided. Nanoparticles can carry one or more
nucleic acids encoding the desired antigen(s) are suitable for use
in vaccination. Methods for enhancing vaccine efficacy are also
provided. Nanoparticles carrying one or more nucleic acids encoding
the desired antigen(s) can further include one or more nucleic
acids target molecules that boost the immunological response
towards the antigen(s). The methods typically include administering
a subject in a need thereof an effective amount of the composition
for an enhanced vaccination response.
[0362] Small molecules such as rapamycin and metformin have shown
to improve CD8 T-cell differentiation and memory formation (Araki K
et al., Nature. 460(7251):108-12 (2009); Pearce E L et al., Nature.
460, 103-107 (2009)). Thus, in some embodiments, the nanoparticles
can include one or more nucleic acids that overexpress protein
targets that upregulated by rapamycin, and/or suppress protein
targets that are downregulated or inhibited by rapamycin. For
example, the nanoparticle can carry one nucleic acid encoding
yellow fever vaccine, and an additional nucleic acid encoding siRNA
targeting raptor (which mimics rapamycin function in inhibiting
mTORC1 function). In further embodiments, the nanoparticles can
include one or more nucleic acids that overexpress protein targets
that upregulated by metformin, and/or suppress protein targets that
are downregulated or inhibited by metformin.
[0363] In some embodiments, the one or more nucleic acids
incorporated in the nanoparticles can enhance antigen presentation
by MHC molecules. Exemplary molecules to overexpress to promote
activation include surface molecules of antigen presenting cells
CD80, CD86, CD40, OX40L, CLII, and ICOSL; soluble factors IL-12,
IL-6, IL-1, IFN-.gamma., TNF-.alpha., IL-18, and IL-2. Exemplary
targets to suppress in DC to induce maturation include surface
molecules ILT3, ILT4, PD-L1, PD-L2, and CD275; soluble factors
TGF13, IL-10. In some embodiments, the nucleic acids incorporated
in the nanoparticles can promote cellular metabolism to be more
glycolytic such as increase mTORC1 activities.
[0364] In some embodiments, the one or more nucleic acids
incorporated in the nanoparticles can enhance antigen presentation
by promoting autophagy. Autophagy has been shown to mediate both
MHC-I and MHC-II presentation.
[0365] In some embodiments, co-delivery of siRNA and mRNA,
preferably in the form of nanoparticles can be used as tolerogenic
vaccines, wherein mRNA encodes an antigen designed to elicit a
desired immune response and siRNA silences the expression of
co-stimulatory proteins. For example, co-delivery of B7-1/7-2 siRNA
and mRNA encoding adeno-associated virus (AAV) capsid proteins
(VP1, VP2 or VP3) can be developed for the application of tolerance
for AAV vectors for gene therapy (Table 2). In another embodiment,
co-delivery of B7-1/7-2 siRNA and mRNA encoding GAD65, insulin,
proinsulin, HSP60, IA-2, ZnT8 or IGRP are used for Type 1 diabetes
therapy (Table 2).
[0366] b. Inducing Immune Tolerance
[0367] In some embodiments, the nanoparticles can be used to
suppress the immune system and/or promote tolerance in a subject.
Methods for inducing immune tolerance are provided. Methods
typically include administering a subject in a need thereof an
effective amount of the composition for inducing tolerance. In
preferred embodiments, methods include administering a subject in a
need thereof an effective amount of the composition to induce
regulatory T cells. In some embodiments, the subject in need
thereof has an allergic disease or condition. In some embodiments,
the subject in need thereof is about to, is undergoing, or has
undergone stem cell or tissue transplantation.
[0368] Nanoparticles carrying one or more nucleic acids encoding
the desired antigen(s) can further include one or more nucleic
acids target molecules to induce tolerance. In some embodiments,
the nucleic acids target molecules to induce tolerance can induce
regulatory T cells and/or acquisition of tolerance to the antigen.
In some embodiments, the therapy includes introducing a nucleic
acid (e.g., mRNA) that increases expression of cohihibitory
molecule (e.g., PD-1, PD-L1, CTLA-4, BTLA), or functional nucleic
acid (e.g., siRNA) that decreases expression of a costimulatory
molecule (e.g., CD80 (B7-1), CD86 (B7-2), CD28, ICOS, 4-1BB, CD40,
OX40, CD27) signaling. For example, a siRNA that downregulates one
or more costimulatory molecules such as CD80 (B7-1), CD86 (B7-2)
can be incorporated to help induce immunological tolerance towards
the antigen on a dendritic cell.
[0369] The ability of dendritic cells (DC) to regulate Ag-specific
immune responses via their influence on T regulatory cells (Treg)
may be key to their potential as therapeutic tools or targets for
the promotion/restoration of tolerance. It has been shown that
rapamycin-conditioned DCs have impaired ability to stimulate
effector CD4 T cells but promoted antigen-specific Foxp3+T
Regulatory Cells (Turnquist H R et al., J Immunol.
178(11):7018-31(2007)). In some embodiments, the nanoparticles
include one or more nucleic acids to induce tolerogenic DC function
for example by increasing the expression of immunosuppressive
cytokine IL-10, or suppressing the expression of costimulatory
molecules. Exemplary targets to overexpress in DC to induce
tolerance include surface molecules ILT3, ILT4, PD-L1, PD-L2, and
CD275; soluble factors TGF.beta., IL-10. Exemplary molecules to
suppress include surface molecules of antigen presenting cells
CD80, CD86, CD40, OX40L, CLII, ICOSL; soluble factors IL-12, IL-6,
IL-1, IFN-.gamma., TNF-.alpha., IL-18, and IL-2. In some
embodiments, the nucleic acids incorporated in the nanoparticles to
induce tolerance are to promote less glycolytic metabolism, and/or
more fatty acid oxidation. In some embodiments, the nucleic acids
incorporated in the nanoparticles to induce tolerance are effective
in suppressing activities of mTOC1. In some embodiments, the
nucleic acids incorporated in the nanoparticles to induce tolerance
are effective in increasing activities of AMPK, and/or
autophagy.
[0370] Thus, in some embodiments, to induce immune tolerance, the
disclosed nanoparticles can include one or more nucleic acids that
overexpress protein targets that upregulated by rapamycin, and/or
suppress protein targets that are downregulated or inhibited by
rapamycin. For example, the nanoparticles carrying nucleic acids
that express one or more desired antigens further include nucleic
acids that suppress the expression of mTORC1, for example via siRNA
targeting raptor, which is an essential component of mTORC1
complex. In some embodiments, the downstream effectors of mTORC1
are the inhibitory targets such as S6K1, S6, and eIF4E. In some
embodiments, the nanoparticles include one or more nucleic acid
that promotes one or more of the downstream pathways that are
inhibited by mTORC1 such as 4E-BP, ULK1.
[0371] A particularly preferred therapeutic strategy includes
co-administration of a nucleic acid (e.g., mRNA) encoding an
antigenic protein to which tolerance is desired in combination with
a functional nucleic acid (e.g., an siRNA) that reduces
costimulatory signaling. Exemplary antigens are discussed above and
include, for example, autoimmune antigens, food antigens,
allergens, etc. Exemplary costimulatory signaling molecules are
also discussed above and include, but are not limited to CD80
(B7-1), CD86 (B7-2).
[0372] C. Diagnositic and Prognostic Uses
[0373] As introduced above, upon exposure to plamsa, cells, tissue,
or other biological material, particles rapid absorb proteins and
other biomolecules such as nucleic acids and lipids, forming a
corona around the particle. In some embodiments, empty or loaded
particles are utilized in diagonistic or prognostic
applications.
[0374] In some embodiments, analysis of the corona is used to
characterize the effecicay of a treatment. For example, in some
embodiments, a biomarker associated with a disease or condition to
be treated forms part of the corona of injected particles.
Particles can be injected and later collected before, during, or
after treatment with a therapeutic composition. The therapeutic
composition can be one disclosed herein or a different therapy, for
example, a conventional drug treatment regimen. A change in the
level of the biomarker in the corona can indicate an efficacious
treatment, while no change in the level of the biomarker in the
protein corona can indicate a non-efficacious treatment.
[0375] The present invention will be further understood by
reference to the following non-limiting examples.
Example 1: Fast Redox-Responsive Hybrid Nanoparticles
[0376] Methods
[0377] Synthesis of the L-Cystine-Based Poly(Disulfide) (PDSA)
Polymers
[0378] PDSA polymers were prepared by one-step polycondensation of
L-cystine dimethyl ester dihydrochloride ((H-Cys-OMe)2.2HCl) and
dichlorides or Bis-nitrophenol esters of different fatty diacids. A
standard synthesis procedure was carried out as follows:
(H-Cys-OMe)2.2HCl (10.0 mmol) and triethylamine (15 mmol) were
dissolved in 20.0 mL DMSO, then the dichloride of fatty acid (10.0
mmol) DMSO solution (10.0 mL) was added into the cystine mixture
solution dropwise. The solution was stirred for 15 mins to obtain a
uniform mixture, precipitated twice in 250 mL of cold ethyl ether,
and dried under reduced atmosphere. The final product was a yellow
or brown yellow powder. The synthesis scheme is shown below.
##STR00001##
[0379] (PDSA)Redox-Responsive Behavior of the PDSA Polymers
[0380] GPC analysis was used to study the redox-responsive behavior
of the PDSA polymers. The polymer (1 mg) was dissolved in 2 mL of
DMF/H2O (9:1, V/V) and then GSH (6.2 mg, 0.02 mmol) was added to
obtain a solution with GSH concentration of 10 mM. At predetermined
intervals, 100 .mu.L of the solution was taken for GPC
analysis.
[0381] Preparation and Characterization of Nanoparticles (NPs)
[0382] The PDSA polymers were dissolved in DMF or DMSO to form a
homogenous solution with a concentration of 20 mg/mL. Subsequently,
200 .mu.L of this solution was taken and mixed with 140 .mu.L of
DSPE-PEG3000 (20 mg/mL in DMF), 50 .mu.L of G0-C14 (5 mg/mL in DMF)
and 1 nmol siRNA (0.1 nmol/.mu.L aqueous solution). Under
vigorously stirring (1000 rpm), the mixture was added dropwise to 5
mL of deionized water. The NP dispersion formed was transferred to
an ultrafiltration device (EMD Millipore, MWCO 100 K) and
centrifuged to remove the organic solvent and free compounds. After
washing with PBS (pH 7.4) solution (3.times.5 mL), the siRNA loaded
NPs were dispersed in 1 mL of phosphate buffered saline (PBS, pH
7.4) solution. Size and zeta potential were determined by DLS. The
morphology of NPs was visualized on TEM. To determine the siRNA
encapsulation efficiency, DY547-labelled GL3 siRNA (DY547-siRNA)
loaded NPs were prepared according to the method described above. A
small volume (50 .mu.L) of the NP solution was withdrawn and mixed
with 20-fold DMSO. The fluorescence intensity of DY547-siRNA was
measured using a Synergy HT multi-mode microplate reader (BioTek
Instruments) and compared to the free DY-547 labelled GL3 siRNA
solution (1 nmol/mL PBS solution).
[0383] Redox-Responsive Behavior of the NPs
[0384] The siRNA loaded NPs were prepared as described above and
dispersed in PBS containing 10 mM GSH. At pre-determined time
point, the particle size was examined by DLS and the particle
morphology was observed on TEM. To evaluate the intracellular
redox-responsive behavior, the NPs with Nile red and coumarin 6
encapsulated in their hydrophobic cores were prepared and then
incubated with HeLa cells for different time. The fluorescence of
Nile red and coumarin 6 was observed a FV1000 confocal laser
scanning microscope (CLSM, Olympus). If the NPs respond to redox
stimulus, the Nile red and coumarin 6 will release and only green
fluorescence of coumarin 6 can be observed under CLSM. If the NPs
are intact, the fluorescence of coumarin 6 will be quenched by Nile
red and only red fluorescence can be observed under CLSM.
[0385] Evaluation of Endosomal Escape
[0386] Luc-HeLa cells (20,000 cells) were seeded in discs and
incubated in 1 mL of RPMI 1640 medium containing 10% FBS for 24 h.
Subsequently, the DY547-siRNA-loaded NPs were added, and the cells
were allowed to incubate for 1 or 2 h. After removing the medium
and subsequently washing with PBS (pH 7.4) solution thrice, the
endosomes and nuclei were stained with lysotracker green and
Hoechst 33342, respectively. The cells were then viewed under
CLSM.
[0387] In Vitro siRNA Release
[0388] DY547-siRNA-loaded NPs were prepared as described above.
Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and
then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100
kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree. C.
At a predetermined interval, 5 .mu.L of the NP solution was
withdrawn and mixed with 20-fold DMSO. The fluorescence intensity
of DY547-siRNA was determined by Synergy HT multi-mode microplate
reader.
[0389] PC3 Xenograft Tumor Model
[0390] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of LNCaP cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density of
2.times.10.sup.6 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the PC3 tumor xenograft
reached .about.50 mm.sup.3, the mice were used for the following in
vivo experiments.
[0391] Pharmacokinetics Study
[0392] Healthy male BALB/c mice were randomly divided into two
groups (n=3) and given an intravenous injection of either (i) free
DY647-labelled GL3 siRNA (DY647-siRNA) and (ii) DY647-siRNA-loaded
NPs at a 650 .mu.g/kg siRNA dose. At predetermined time intervals,
orbital vein blood (20 .mu.L) was withdrawn using a tube containing
heparin, and the wound was pressed for several seconds to stop the
bleeding. The fluorescence intensity of DY-647 labelled siRNA in
the blood was determined using a microplate reader. The blood
circulation half-life (t1/2) was calculated by first-order decay
fit.
[0393] Biodistribution
[0394] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and given an intravenous injection of
either (i) free DY677-labelled GL3 siRNA (DY677-siRNA) or (ii)
DY677-siRNA-loaded NPs at a 650 .mu.g/kg siRNA dose. Twenty-four
hours after the injection, the mice were imaged using the Maestro 2
In-Vivo Imaging System (Cri Inc). Main organs and tumors were then
harvested and imaged. To quantify the accumulation of NPs in tumors
and organs, the fluorescence intensity of each tissue was
quantified by Image-J.
[0395] Results
[0396] Redox-responsive hydrophobic polymer was synthesized which
could co-assemble with lipid-PEG to form spherical NPs for gene
delivery and cancer therapy. The intracellular levels of
glutathione (GSH) are 100-1000 fold higher in cancer cells than in
normal tissue. Redox-sensitive approach is particularly promising
to enhance the exposure of cancer cells to therapeutic molecules.
In this example, L-cystine dimethyl ester and fatty diacid were
used to synthesize a library of L-cystine-based poly(disulfide
amide) polymers (PDSA). The success in the polymer synthesis was
confirmed by .sup.1HNMR spectrum.
[0397] Feed compositions and molecular weight of the PDSA polymers
are summarized in Table 6. Taking PDSA8-1, for example, with the
presence of many disulfide bonds, there is a significant decrease
in the molecule weight of PDSA8-1 after incubation in 10 mM
glutathione (GSH) solution for 4 hours, shifting from 5700
(PDI=1.43) to 870 (PDI=1.65) based on the retention time on GPC.
When mixing this redox-responsive polymer with DSPE-PEG3000, siRNA
and cationic lipid (Xu X et al. Proc Natl Acad Sci USA, 110,
18638-18643(2013)) in water miscible solvent such as DMF, DMSO,
THF, etc., spherical NPs with an average size of .about.100 nm
(FIG. 3) can be formed via nanoprecipitation method, in which
hydrophilic PEG chains are on the outer shell and siRNA is
encapsulated in the hydrophobic core. The physiochemical properties
of other PDSA polymers are summarized in Table 7. With the
redox-responsive characteristic to induce the breakage of the NPs
of PDSA8-1, the size of NPs increases when incubated in 10 mM
glutathione (GSH) solution (FIG. 4). In vitro experiment results
show that the siRNA loaded NPs of PDSA8-1 have efficient endosomal
escape ability as seen in fluorescent images of HeLa cells
incubated with the siRNA loaded NPs of PDSA8-1 at 4 hour time
point. In vivo experiment results demonstrated that these NPs have
a long blood circulation (FIG. 5A) and show high accumulation in
PC3 xenograft tumor of mice as seen in overlaid fluorescent image
of the PC3 xenograft tumor-bearing nude mice 24 h post systemic
injection of naked DY677-siRNA, and DY677-siRNA loaded NPs of
PDSA8-1 (FIG. 5B).
TABLE-US-00006 TABLE 6 Feed compositions and molecular weight of
the PDSA polymers. Poly(disulfide amide) M.sub.n.sup.a
M.sub.w.sup.a Polydispersity.sup.a m = 4 PDSA4 2900 4300 1.48 m = 6
PDSA6 3900 5700 1.46 m = 8 PDSA8-1 5700 7300 1.43 m = 10 PDSA10
9100 13200 1.45 m = 8 PDSA8-2 4700 7800 1.66 m = 8 PDSA8-3 9300
15200 1.63 m = 8 PDSA8-4 11700 16600 1.42 .sup.aDetermined by GPC
using DMF as the eluent.
TABLE-US-00007 TABLE 7 Size, siRNA encapsulation efficiency (EE %)
and zeta potential of the NPs of PDSA polymers. PDSA4 PDSA6 PDSA8-1
PDSA10 PDSA8-2 PDSA8-3 PDSA8-4 Size.sub.a(nm) 155.7 134.5 102.9
87.6 118.9 99.4 93.4 EE %.sup.b 29.7 35.1 55.9 82.9 46.3 79.4 88.2
.xi. (mV) -6.79 -8.08 -11.21 -15.05 -9.79 -12.05 -20.01 .sub.aN:P
ratio is 20:1; .sup.bsiRNA encapsuiation efficiency.
Example 2: Ultra pH-Responsive and Tumor-Penetrating Polymeric
Nanoparticles
[0398] Methods and Materials
[0399] Materials
[0400] Methoxyl-polyethylene glycol (Meo-PEG113-OH) and hydroxyl
polyethylene glycol carboxylic acid (HO-PEG113-COOH) were purchased
from JenKem Technology and used as received. Internalizing RGD
(iRGD) with the sequence CRGDRGPDC (SEQ ID NO: 3) was obtained from
GL Biochem Ltd. 2-(Diisopropyl amino) ethyl methacrylate (DPA-MA),
glycidyl methacrylate (GMA), and methyl methacrylate (MMA) were
provided by Sigma-Aldrich and passed over an alumina column before
use in order to remove the hydroquinone inhibitors.
.quadrature.-Bromoisobutyryl bromide, triethylamine (TEA),
N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA), copper (I)
bromide (CuBr), N,N'-dimethylformamide (DMF),
tetraethylenepentamine (TEPA), 1,2-epoxyhexadecane, isopropyl
alcohol, and dichloromethane (DCM) were acquired from Sigma-Aldrich
and used directly. Lipofectamine 2000 (Lipo2K) was purchased from
Invitrogen. Steady-Glo luciferase assay system was provided by
Promega. GL3, fluorescent dye (DY547, DY647 and DY677) labeled GL3
siRNAs were acquired from Dharmacon. The siRNA sequences are as
follows: GL3 siRNA, 5'-CUU ACG CUG AGU ACU UCG AdTdT-3' (SEQ ID NO:
4) (sense) and 5'-UCG AAG UAC UCA GCG UAA GdTdT-3' (SEQ ID NO: 5)
(antisense); The fluorescent dyes DY547 and DY647 were labeled at
the 5'-end of the sense strand of GL3 siRNA. DY677 was labeled at
the 5'-end of both the sense and antisense strands of GL3 siRNA.
HeLa cells stably expressing firefly and Renilla luciferase
(Luc-HeLa) were obtained from Alnylam Pharmaceuticals, Inc. The
cells were incubated in RPMI-1640 medium (Invitrogen) with 10%
fetal bovine serum (FBS, Sigma-Aldrich) and 1%
penicillin/streptomycin (Sigma-Aldrich). All other reagents and
solvents are of analytical grade and used without further
purification.
[0401] Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0402] Meo-PEG113-OH (8 g, 1.6 mmol) and TEA (1.3 mL, 9.6 mmol)
were dissolved in 250 mL of DCM. In an ice-salt bath,
.alpha.-bromoisobutyryl bromide (1 mL, 8 mmol) dissolved in 10 mL
of DCM was added dropwise. After stirring for 24 h, the mixture was
washed with 1 M NaOH (3.times.50 mL), 1 M HCl (3.times.50 mL), and
deionized water (3.times.50 mL), respectively. After drying over
anhydrous MgSO.sub.4, the solution was concentrated, and cold ether
was added to precipitate the product. After re-precipitation
thrice, the product was collected as white powder after drying
under vacuum. The synthesis of Br-PEG-COOH was carried out
according to a method similar to that described above, by changing
Meo-PEG113-OH with HO-PEG113-COOH. The synthesis scheme of
Br-PEG-COOH is shown below.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA))
[0403] Meo-PEG-b-P(DPA-co-GMA) copolymers with different
compositions were synthesized by atom transfer radical
polymerization (ATRP). Meo-PEG113-b-P(DPA80-co-GMA5) is used as an
example to illustrate the procedure. DPA-MA (2.6 g, 12 mmol), GMA
(0.11 g, 0.75 mmol), Meo-PEG-Br (0.75 g, 0.15 mmol), and PMDETA
(31.5 .mu.L, 0.15 mmol) were added to a polymerization tube. DMF (3
mL) and 2-propanol (3 mL) were then added to dissolve the monomer
and initiator. After three cycles of freeze-pump-thaw to remove
oxygen, CuBr (21.6 mg, 0.15 mmol) was added under nitrogen
atmosphere and the polymerization tube was sealed under vacuum.
After polymerization at 40.degree. C. for 24 h, tetrahydrofuran
(THF) was added to dilute the product, which was then passed
through a neutral Al.sub.2O.sub.3 column to remove the catalyst.
The resulting THF solution was concentrated and the residue was
dialyzed against THF, followed by deionized water. The expected
copolymer was collected as a white powder after freeze-drying under
vacuum. The synthesis scheme is shown below. The feed compositions
of the copolymers are summarized in Table 8.
TABLE-US-00008 TABLE 8 Feed compositions and characterizations of
Meo-PEG-b- P(DPA-co-GMA) Repeat Repeat unit unit Mn, GPC Mn, NMR
No. (DPA) a (GMA) a (.times. 10.sup.-4 Da) b PDI b (.times.
10.sup.-4 Da) a pKa c PDPA40-GMA5 39 5 1.44 1.19 1.42 6.34
PDPA50-GMA5 50 5 1.68 1.12 1.66 6.31 PDPA60-GMA5 58 5 1.69 1.18
1.83 6.29 PDPA70-GMA5 69 5 1.94 1.24 2.06 6.26 PDPA80-GMA5 80 5
2.19 1.29 2.29 6.24 PDPA100- 99 5 2.87 1.14 2.71 6.21 a Determined
by 1HNMR using CDCl3 as solvent. b Number-averaged (Mn) and
polydispersity index (PDI) were determined by GPC using THF as the
eluent.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA)
[0404] Meo-PEG-b-P(DPA-co-GMA-TEPA) was synthesized via the ring
opening reaction between TEPA and the epoxy group of GMA repeating
unit. In brief, Meo-PEG-b-P(DPA-co-GMA) (1.5 g) dissolved in DMF
(20 mL) was added dropwise to the DMF solution (5 mL) of TEPA
(30-fold molar excess relative to the GMA repeating unit). After
reaction at 60.degree. C. for 7 h, the mixture was transferred to a
dialysis tube and then dialyzed against deionized water. The
Meo-PEG-b-P(DPA-co-GMA-TEPA) was finally collected as a white
powder after freeze-drying under vacuum. The synthesis route of
Meo-PEG-b-P(DPA-co-GMA-TEPA) is shown below.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)
[0405] Meo-PEG-b-P(DPA-co-GMA-TEPA) (1 g) and 1,2-epoxyhexadecane
(equal molar amount relative to TEPA repeating unit) were dissolved
in DMF (20 mL) and the solution was stirred at 70.degree. C. for 5
h. Subsequently, the solution was transferred to a dialysis tube
and then dialyzed against DMF, followed by deionized water. The
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was obtained as a white powder
after freeze-drying under vacuum. The detailed synthesis of
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) is shown below.
[0406] Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5)
[0407] Meo-PEG-b-P(DPA-co-GMA-TEPA) (0.2 g) and Cy5.5 NHS ester
(1.5-fold molar excess relative to the TEPA repeating unit) were
well dissolved in 5 mL of THF. After constantly stirring in dark
for 48 h, the solution was dialyzed against deionized water and the
product was collected after freeze-drying. The synthesis of
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) is shown below.
##STR00002##
Synthesis of HOOC-PEG-b-PDPA
[0408] HOOC-PEG-b-PDPA copolymers were also synthesized by the ATRP
method. DPA-MA (1.73 g, 8 mmol), Br-PEG-COOH (0.5 g, 0.1 mmol), and
PMDETA (21 .mu.L, 0.1 mmol) were added to a polymerization tube.
Subsequently, DMF (2 mL) and 2-propanol (2 mL) were added to
dissolve the monomer and initiator. After three cycles of
freeze-pump-thaw to remove oxygen, CuBr (14.4 mg, 0.1 mmol) was
added under nitrogen atmosphere and the polymerization tube was
sealed under vacuum. After polymerization at 40.degree. C. for 24
h, tetrahydrofuran (THF) was added to dilute the product, which was
then passed through a neutral Al.sub.2O.sub.3 column to remove the
catalyst. The obtained THF solution was concentrated and the
residue was dialyzed against deionized water. The HOOC-PEG-b-PDPA
was obtained as a white powder after freeze-drying under vacuum.
The synthesis scheme is shown below. The feed compositions are
summarized in Table 9.
TABLE-US-00009 TABLE 9 Feed compositions and characterizations of
HOOC-PEG-b- PDPA Repeat Mn, NMR unit Mn, GPC (.times.10-4 Da) No.
(DPA) a (.times.10-4 Da) b PDI b a HOOC-PEG-b-PDPA40 36 1.31 1.34
1.27 HOOC-PEG-b-PDPA50 45 1.49 1.28 1.48 HOOC-PEG-b-PDPA60 55 1.76
1.29 1.69 HOOC-PEG-b-PDPA70 64 1.92 1.27 1.89 HOOC-PEG-b-PDPA80 76
2.04 1.24 2.14 HOOC-PEG-b-PDPA100 92 2.57 1.19 2.48 a Determined by
1HNMR using CDCl3 as solvent. b Number-averaged (Mn) and
polydispersity index (PDI) were determined by GPC using THF as the
eluent.
Synthesis of iRGD-PEG-b-PDPA
[0409] HOOC-PEG-b-PDPA copolymer (0.2 g), iRGD peptide (1.5-fold
molar excess relative to the terminal carboxylic acid group),
EDC.HCl (3-fold molar excess relative to the terminal carboxylic
acid group), and NHS (3-fold molar excess relative to the terminal
carboxylic acid group) were well dissolved in pH 5.0 water. The
mixture was stirred at room temperature for 48 h. The solution was
subsequently dialyzed against deionized water and the expected
iRGD-PEG-PDPA was collected after freeze-drying.
##STR00003##
Synthesis of Control Copolymers
[0410] The control copolymers, methoxyl-polyethylene glycol-b-poly
(methyl methacrylate-co-glycidyl methacrylate)
(Meo-PEG113-b-P(MMA80-co-GMA5))
Meo-PEG113-b-P(MMA80-co-GMA5-TEPA5), HOOC-PEG113-b-PMMA80,
iRGD-PEG113-b-PMMA80, and Meo-PEG113-b-P(MMA80-co-GMA5-TEPA5-C145)
were synthesized according to the method described above, by
changing the monomer DPA-MA with MMA. The chemical structure of
iRGD-PEG113-b-PMMA80 and Meo-PEG113-b-P(MMA80-co-GMA5-TEPA5-C145)
is shown below.
##STR00004##
[0411] Gel Permeation Chromatography (GPC)
[0412] Number- and weight-average molecular weights (Mn and Mw,
respectively) of the polymers were determined by a gel permeation
chromatographic system equipped with a Waters 2690D separations
module and a Waters 2410 refractive index detector. THF was used as
the eluent at a flow rate of 0.3 mL/min Waters millennium module
software was used to calculate molecular weight on the basis of a
universal calibration curve generated by polystyrene standard of
narrow molecular weight distribution.
[0413] .sup.1H Nuclear Magnetic Resonance (.sup.1HNMR)
[0414] The 1HNMR spectra of the polymers were recorded on a Mercury
VX-300 spectrometer at 400 MHz (Varian, USA), using CDCl3 as a
solvent and TMS as an internal standard.
[0415] Acid-Base Titration
[0416] Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was dispersed in deionized
water, and a concentrated HCl aqueous solution was added until
complete dissolution of the copolymer (1 mg/mL). Subsequently, 1 M
NaOH aqueous solution was added in 1-5 .mu.L increments. After each
addition, the solution was constantly stirred for 3 min, and the
solution pH was measured using a pH meter. The pKa of the copolymer
was determined as the pH at which 50% copolymer turns ionized.
Preparation and Characterization of Nanoparticles (NPs)
[0417] Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was dissolved in THF to
form a homogenous solution with a concentration of 4 mg/mL.
Subsequently, a certain volume of this THF solution was taken and
mixed with 1 nmol siRNA (0.1 nmol/.mu.L aqueous solution) in a N/P
molar ratio of 40:1. Under vigorous stirring (1000 rpm), the
mixture was added dropwise to 2.5 mL of deionized water. The NP
dispersion formed was transferred to an ultrafiltration device (EMD
Millipore, MWCO 100 K) and centrifuged to remove the organic
solvent and free compounds. After washing with PBS (pH 7.4)
solution (3.times.5 mL), the siRNA loaded NPs were dispersed in 1
mL of phosphate buffered saline (PBS, pH 7.4) solution. Size and
zeta potential were determined by dynamic light scattering (DLS,
Brookhaven Instruments Corporation). The morphology of NPs was
visualized on a Tecnai G2 Spirit BioTWIN transmission electron
microscope (TEM). Before observation, the sample was stained with
1% uranyl acetate and dried under air. To determine siRNA
encapsulation efficiency, DY547-labelled GL3 siRNA loaded NPs were
prepared according to the method described above. A small volume
(50 .mu.L) of the NP solution was withdrawn and mixed with 20-fold
DMSO. The fluorescence intensity of DY547-labelled GL3 siRNA was
measured using a Synergy HT multi-mode microplate reader (BioTek
Instruments) and compared to the free DY547-labelled GL3 siRNA
solution (1 nmol/mL PBS solution).
[0418] To prepare the iRGD-NPs, Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (4
mg/mL in THF) was mixed with 1 nmol siRNA (0.1 nmol/.mu.L aqueous
solution) in a N/P molar ratio of 40:1. Then iRGD-PEG-b-PDPA (4
mg/mL in THF, 10 mol % compared to
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)) was added, and the mixture was
added dropwise to 2.5 mL of deionized water. The iRGD-NPs were
purified by an ultrafiltration device (EMD Millipore, MWCO 100 K)
and finally dispersed in 1 mL of PBS. The siRNA encapsulation
efficiency was examined by replacing the siRNA with DY547-labelled
GL3 siRNA.
[0419] Evaluation of pH Responsiveness
[0420] The THF solution of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (4
mg/mL) and Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) (4 mg/mL) was mixed
in a volume ratio of 8:2. Under vigorously stirring (1000 rpm), 0.5
mL of the mixture was added dropwise to 5 mL of deionized water.
After collection and purification by an ultrafiltration device (EMD
Millipore, MWCO 100 kDa), the NPs formed were dispersed in 1 mL of
deionized water. Subsequently, 1 M NaOH or HCl was added in 1-5
.mu.L increments, and the fluorescence intensity of the NPs was
measured on a Synergy HT multi-mode microplate reader. The
normalized fluorescence intensity (NH) vs. pH profile was used to
quantitatively assess the pH responsiveness. NH is calculated as
follows:
NFI=(F-Fmin)/(Fmax-Fmin)
[0421] where F is the fluorescence intensity of the NPs at any
given pH value and Fmax and Fmin are the maximal and minimal
fluorescence intensity of the NPs, respectively.
[0422] In Vitro siRNA Release
[0423] DY547-labelled GL3 siRNA-loaded NPs were prepared as
described above. Subsequently, the NPs were dispersed in 1 mL of
PBS (pH 7.4) and then transferred to a Float-a-lyzer G2 dialysis
device (MWCO 100 kDa, Spectrum) that was immersed in PBS (pH 7.4)
at 37.degree. C. At a predetermined interval, 5 .mu.L of the NP
solution was withdrawn and mixed with 20-fold DMSO. The
fluorescence intensity of DY547-labelled siRNA was determined by
Synergy HT multi-mode microplate reader.
[0424] Cell Culture
[0425] Human cervical cancer cell line with the expression of
luciferase (Luc-HeLa) and prostate cancer cell line (PC3) were
incubated in RPMI1640 medium with 10% FBS at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2.
[0426] Determination of the Expression of Integrins .alpha.v.beta.3
and .alpha.v.beta.5
[0427] Luc-HeLa and PC3 cells were seeded in 6-well plates (50,000
cells per well) and incubated in 1 mL of RPMI1640 medium containing
10% FBS for 24 h. Thereafter, 10 .mu.L of FITC-conjugated
anti-human CD51/61 antibody (BioLegend) or FITC-conjugated
anti-human integrin .alpha.v.beta.5 antibody (EMD Millipore) were
added, and the cells were allowed to incubate for another 4 h.
After removing the medium and washing with PBS (pH 7.4) solution
thrice, the cells were collected for flow cytometry quantitative
analysis (BD FACSAria.TM. III, USA).
[0428] Confocal Laser Scanning Microscope (CLSM)
[0429] Luc-HeLa and PC3 cells (20,000 cells) were seeded in discs
and incubated in 1 mL of RPMI1640 medium containing 10% FBS for 24
h. Subsequently, the DY547-labelled GL3 siRNA-loaded NPs or
iRGD-NPs were added, and the cells were allowed to incubate for 1
or 4 h. After removing the medium and subsequently washing with PBS
(pH 7.4) solution thrice, the endosomes and nuclei were stained by
lysotracker green and Hoechst 33342, respectively. The cells were
then viewed under a FV1000 CLSM (Olympus).
[0430] Flow Cytometry
[0431] Luc-HeLa and PC3 cells were seeded in 6-well plates (50,000
cells per well) and incubated in 1 mL of RPMI1640 medium containing
10% FBS for 24 h. Subsequently, the DY547-labelled GL3 siRNA-loaded
NPs or iRGD-NPs were added, and the cells were allowed to incubate
for another 4 h. After removing the medium and subsequently washing
with PBS (pH 7.4) solution thrice, the cells were collected for
flow cytometry quantitative analysis.
[0432] Animals
[0433] Healthy male BALB/c mice (4-5 weeks old) were purchased from
Charles River Laboratories. All in vivo studies were performed in
accordance with National Institutes of Health animal care
guidelines and in strict pathogen-free conditions in the animal
facility of Brigham and Women's Hospital. Animal protocol was
approved by the Institutional Animal Care and Use Committees on
animal care (Harvard Medical School).
[0434] PC3 Xenograft Tumor Model
[0435] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of PC3 cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density
1.times.10.sup.7 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the PC3 tumor xenograft
reached .about.100 mm.sup.3, the mice were used for the following
in vivo experiments.
[0436] Pharmacokinetics Study
[0437] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and given an intravenous injection of either (i) free
DY647-labelled GL3 siRNA, (ii) DY647-labelled GL3 siRNA-loaded NPs,
or (iii) DY647-labelled GL3 siRNA-loaded iRGD-NP at 650 .mu.g siRNA
dose per kg mouse weight. At predetermined time intervals, orbital
vein blood (20 .mu.L) was withdrawn using a tube containing
heparin, and the wound was pressed for several seconds to stop the
bleeding. The fluorescence intensity of DY647-labelled siRNA in the
blood was determined by microplate reader. The blood circulation
half-life (t1/2) was calculated by first-order decay fit.
[0438] Biodistribution
[0439] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into three groups (n=3) and given an intravenous injection
of either (i) free DY677-labelled GL3 siRNA, (ii) DY677-labelled
GL3 siRNA-loaded NPs or (iii) DY677-labelled GL3 siRNA-loaded
iRGD-NPs at 650 .mu.g siRNA dose per kg mouse weight. Twenty-four
hours after the injection, the mice were imaged using the Maestro 2
In-Vivo Imaging System (Cri Inc). Organs and tumors were then
harvested and imaged. To quantify the accumulation of NPs in tumors
and organs, the fluorescence intensity of each tissue was
quantified by Image-J.
[0440] Immunofluorescence Staining
[0441] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into three groups (n=3) and intravenously injected with
either (i) free DY677-labelled GL3 siRNA, (ii) DY677-labelled GL3
siRNA-loaded NPs or (iii) DY677-labelled GL3 siRNA-loaded iRGD-NPs
at 650 .mu.g siRNA dose per kg mouse weight. Four hours after
injection, the mice were sacrificed and the tumors were harvested,
followed by fixing with 4% paraformaldehyde, embedding in paraffin,
and cutting into sections. To image the tumor vasculature, the
slices were heated at 60.degree. C. for 1 h and washed with xylene,
ethanol, and PBS thrice. After blocking with 10% FBS for 1.5 h, the
slices were incubated with rat anti-mouse CD31 antibody (Abcam) at
4.degree. C. for 1 h. After washing with PBS/0.2% triton X-100
thrice, Alexa Flour 488-conjugated secondary antibody (Goat
anti-rat IgG, Abcam) was added for 1 h to stain the slices.
Thereafter, the slices were washed with PBS thrice and then stained
with Hoechst 33342. The images of the tumor vasculature were viewed
on a FLV1000 CLSM.
[0442] Results
[0443] A long-circulating, optionally cell-penetrating, and
stimuli-responsive NP platform for effective in vivo delivery of
therapeutic, prophylactic and/or diagnostic agents is made of an
amphiphilic polymer, most preferably a PEGylated polymer, which
shows a response to a stimulus such as pH, temperature, or light,
such as an ultra pH-responsive characteristic with a pKa close to
the endosomal pH (6.0-6.5) (Wang Y et al, Nat Mater, 13, 204-212
(2014)). The polymer may include a targeting or cell penetrating or
adhesion molecule such as a tumor-penetrating peptide iRGD.
[0444] As demonstrated by example 2, after encapsulating the
agent(s) to be delivered, the resulting delivery system shows four
unique features (FIGS. 6A-6B):
[0445] i) the surface-encoded iRGD peptide endows the NPs with
tumor-targeting and tumor-penetrating abilities;
[0446] ii) the hydrophilic PEG shells prolong the blood
circulation;
[0447] iii) a small population of cationic lipid-like grafts
randomly dispersed in the hydrophobic poly(2-(diisopropylamino)
ethylmethacrylate) (PDPA) segment can entrap siRNA in the
hydrophobic cores of the NPs; and
[0448] iv) the rapid protonation of the ultra pH-responsive PDPA
segment induces the endosomal swelling via the "proton sponge"
effect, which synergizes with the insertion of the cationic
lipid-like grafts into endosomal membrane to induce membrane
destabilization (Zhu X et al., Proceedings of the National Academy
of Sciences, 112, 7779-7784 (2015)) and efficient endosomal
escape.
[0449] The amphiphilic polymer, methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA)) was first synthesized (Table 8), which
was further grafted by tetraethylenepentamine (TEPA) and
1,2-epoxyhexadecane to obtain Meo-PEG-b-P(DPA-co-GMA-TEPA-C14).
##STR00005##
[0450] The length of PDPA segment was varied to adjust siRNA
encapsulation efficiency (EE %). As the PDPA length increases, the
EE % and size of the resulting NPs increase (Table 10), possibly
because the increased PDPA length leads to an increase in the size
of the hydrophobic core. Specifically, the EE % reaches almost 100%
for the polymer with 80 (PDPA80) or 100 (PDPA100) DPA repeat units.
Notably, using a mixture of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (90
mol %) and tumor-penetrating polymer (iRGD-PEG-b-PDPA, 10 mol %) to
prepare NPs does not cause obvious change in the EE % or particle
size (Table 11).
TABLE-US-00010 TABLE 10 Size, zeta potential, siRNA encapsulation
efficiency (EE %), and pH responsiveness of the NPs prepared from
Meo-PEG-b-P(DPA-co-GMA- TEPA-C14) DPA Size Zeta Polymer repeating
pKa of (nm) potential No. abbreviation units .sup.a polymer .sup.b
c (mv) EE% .sup.d .DELTA.pH.sub.10%-90% NPs40 PDPA40 39 6.34 62.5
4.79 54.6 0.45 NPs50 PDPA50 50 6.31 69.6 5.26 59.6 0.40 NPs60
PDPA60 58 6.29 75.9 3.13 65.6 0.37 NPs70 PDPA70 69 6.26 66.0 6.44
69.7 0.35 NPs80 PDPA80 80 6.24 69.7 3.81 99.7 0.34 NPs100 PDPA100
99 6.21 82.3 9.26 100 0.33 .sup.a Determined by 1HNMR shown in
Table 8. .sup.b Determined by acid-base titration c Determined by
dynamic light scattering (DLS). .sup.d DY547-labelled GL3 siRNA was
used to examine the EE %.
[0451] The polymer, PDPA80 (pKa 6.24, Table 10), was chosen for pH
response evaluation by incorporating a near-infrared dye, Cy5.5,
into its PDPA segment. Due to the quenching of the aggregated
fluorophores inside the hydrophobic cores of the NPs (Wang Y et al,
Nat Mater, 13, 204-212 (2014)), there is no fluorescence signal at
a pH above pKa of PDPA80 (FIG. 7A). In contrast, at a pH below pKa,
the protonated PDPA segment induces the disassembly of the NPs and
a dramatic increase in the fluorescence signal. Measurement of the
fluorescence intensity upon pH change reveals that the pH
difference from 10 to 90% fluorescence activation
(.DELTA.pH.sub.10-90%) is 0.34 (Wang Y et al, Nat Mater, 13,
204-212 (2014)) (FIG. 7A), which is much smaller than that of small
molecule dyes (about 2 pH units) (Urano Y et al., Nat Med, 15,
104-109 (2009)), indicating the ultra-fast pH response of PDPA80.
This characteristic is confirmed by transmission electron
microscope (TEM). The spherical siRNA-loaded NPs could be
visualized at a pH of 6.5, with an average size of 69.7 nm
determined by dynamic light scattering (DLS, Table 10). If altering
pH to 6.0, there are no observable NPs after 20 min incubation.
With this morphological change, the NPs offer super-fast release of
DY547-labelled GL3 siRNA (DY547-siRNA). Around 90% loaded siRNA has
been released within 4 h at a pH of 6.0. Within the same time
frame, less than 30% of the loaded siRNA is released at a pH of 7.4
(FIG. 7B).
TABLE-US-00011 TABLE 11 Size, zeta potential and siRNA
encapsulation efficiency (EE %) of the iRGD-NPs of prepared from
the mixture of Meo-PEG-b-P(DPA-co- GMA-TEPA-C14) and
iRGD-PEG-b-PDPA.sup.a No. Size (nm).sup.b Zeta potential (mv) EE
%.sup.c iRGD-NPs40 64.2 3.26 55.1 iRGD-NPs50 68.3 3.98 59.7
iRGD-NPs60 82.1 5.69 66.4 iRGD-NPs70 76.5 7.18 69.6 iRGD-NPs80 70.7
5.26 99.8 iRGD-NPs100 86.3 8.93 100 .sup.aThe molar ratio of
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) and iRGD-PEG-b-PDPA is 9:1.
.sup.bDetermined by dynamic light scattering (DLS).
.sup.cDY547-labelled GL3 siRNA was used to examine the EE %.
[0452] Flow cytometry was employed to evaluate its in vitro
tumor-targeting ability. With the specific recognition between
integrins (.alpha.v.beta.3 and .alpha.v.beta.5, FIGS. 8A-8B) on
Luc-HeLa cells and iRGD, the uptake of DY547-siRNA-loaded
iRGD-NPs80 is 3-fold higher than that of iRGD-absent NPs80 (FIGS.
9A-9B), demonstrating the excellent tumor-targeting ability of
iRGD-NPs80. Endosomal escape ability was assessed by staining the
endosomes with lysotracker green. Fluorescent images of Luc-HeLa
cells incubated with the siRNA-loaded iRGD-NPs80 showed that a
majority of the internalized siRNA-loaded NPs entered the cytoplasm
after 4 h incubation, indicating the effective endosomal escape of
the iRGD-NPs80.
[0453] After validating the in vitro tumor-targeting ability of
these ultra pH-responsive NPs, their in vivo tumor-targeting
ability was assessed. Pharmacokinetics was first examined by
intravenous injection of DY647-siRNA-loaded NPs. The blood
half-life (t.sub.1/2) of iRGD-NPs80 is around 3.56 h (FIG. 10A),
which is far longer than that of naked siRNA (t.sub.1/2<30 min).
This prolonged blood circulation is mainly due to the protection of
PEG outer layer and small particle size (Knop K et al., Angewandte
Chemie International Edition, 49, 6288-6308(2010)). The in vivo
tumor-targeting ability was evaluated by intravenously injecting
DY677-siRNA-loaded NPs into PC3 xenograft tumor-bearing mice.
Overlaid fluorescent image of PC3 xenograft tumor-bearing mice at
24 h post-injection of naked siRNA and siRNA-loaded NPs showed
that, with the iRGD-mediated tumor-targeting, the iRGD-NPs80 show a
much higher tumor accumulation than that of NPs80 at 24 h
post-injection. The tumors and main organs were harvested. Naked
siRNA has a characteristic biodistribution, i.e., high accumulation
in kidney but extremely low accumulation in tumor. With the
specific recognition between iRGD and integrins .alpha.v.beta.3 and
.alpha.v.beta.5 over-expressed on tumor cells and angiogenic tumor
vasculature (Wang Y et al., Nat Mater, 13, 204-212 (2014); Sugahara
K N et al., Cancer Cell, 16, 510-520 (2009)), the tumor
accumulation of the iRGD-NPs80 is around 3-fold higher that of
NPs80 (FIG. 10B).
[0454] To evaluate the tumor-penetrating ability of the iRGD-NPs80,
the tumors were collected at 4 h post-injection of the
DY677-siRNA-loaded NPs and then sectioned for immunofluorescence
staining There is nearly no naked siRNA in the tumor section. For
the NPs80, the number of NPs in tumor section is very low.
Additionally, most of these NPs are positioned in the tumor
vessels, and only a small number reach the extravascular tumor
parenchyma. In contrast, highly concentrated iRGD-NPs80 with bright
red fluorescence could be visualized in the tumor section.
Remarkably, a majority of these NPs can cross tumor vessels and
reach the extravascular tumor parenchyma, strongly demonstrating
the deep tumor-penetrating characteristic of iRGD-NPs80.
[0455] In summary, an ultra pH-responsive and tumor-penetrating
nanoplatform for targeted systemic gene delivery has been
developed. The in vitro and in vivo results demonstrate that this
polymeric NP has a long blood circulation, and can efficiently
target tumor and penetrate tumor parenchyma.
Example 3: Ultra pH-Responsive, Membrane-Penetrating, and Prostate
Cancer Specific Polymeric Nanoparticles
[0456] Methods and Materials
[0457] Materials
[0458] Methoxyl-polyethylene glycol (Meo-PEG113-OH) and hydroxyl
polyethylene glycol carboxylic acid (HO-PEG113-COOH) were purchased
from JenKem Technology and used as received. Oligoarginine
(NH2-Rn-CONH2, n=6, 8, 10, 20, 30) was provided by MIT Biopolymer
facility. Allyl protected
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA) was kindly provided by BIND Therapeutics as a gift.
2-(Diisopropyl amino) ethyl methacrylate (DPA-MA) and glycidyl
methacrylate (GMA) were provided by Sigma-Aldrich and passed over
an alumina column before use in order to remove the hydroquinone
inhibitors. .quadrature.-Bromoisobutyryl bromide,
N,N'-dimethylformamide (DMF), triethylamine (TEA),
N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA), copper (I)
bromide (CuBr), tetraethylenepentamine (TEPA), isopropyl alcohol,
p-toluenesulfinate tetrahydrate (PTSF),
tetrakis(triphenylphosphine) palladium (Pd(PPh3)4) and
dichloromethane (DCM) were acquired from Sigma-Aldrich and used
directly. Lipofectamine 2000 (Lipo2K) was purchased from
Invitrogen. Steady-Glo luciferase assay system was provided by
Promega. GL3, fluorescent dye (DY547, DY647 and Cy5.5) labeled GL3
and PHB1 siRNAs were acquired from Dharmacon. The siRNA sequences
are as follows: GL3 siRNA, 5'-CUU ACG CUG AGU ACU UCG AdTdT-3' (SEQ
ID NO: 4) (sense) and 5'-UCG AAG UAC UCA GCG UAA GdTdT-3' (SEQ ID
NO: 5) (antisense). The fluorescent dyes DY-547 and DY-647 were
labeled at the 5'-end of the sense strand of GL3 siRNA. Cy5.5 was
labeled at the 5'-end of both the sense and antisense strands of
GL3 siRNA. HeLa cells stably expressing firefly and Renilla
luciferase (Luc-HeLa) were obtained from Alnylam Pharmaceuticals,
Inc. The cells were incubated in RPMI 1640 medium (Invitrogen) with
10% fetal bovine serum (FBS, Sigma-Aldrich). All other reagents and
solvents are of analytical grade and used without further
purification.
Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0459] Meo-PEG113-OH (8 g, 1.6 mmol) and TEA (1.3 mL, 9.6 mmol)
were dissolved in 250 mL of DCM. In an ice-salt bath,
.quadrature..quadrature.bromoisobutyryl bromide (1 mL, 8 mmol)
dissolved in 10 mL of DCM was added dropwise. After stirring for 24
h, the mixture was washed with 1 M NaOH (3.times.50 mL), 1 M HCl
(3.times.50 mL), and deionized water (3.times.50 mL). After drying
over anhydrous MgSO.sub.4, the solution was concentrated, and cold
ether was added to precipitate the product. After re-precipitating
thrice, the product was collected as white powder after drying
under vacuum. The synthesis of Br-PEG-COOH was carried out
according to a method similar to that described above, by changing
Meo-PEG113-OH with HO-PEG113-COOH. The synthesis scheme of
Meo-PEG-Br is shown below.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA))
[0460] Meo-PEG-b-P(DPA-co-GMA) copolymer was synthesized by atom
transfer radical polymerization (ATRP). DPA-MA (2.6 g, 12 mmol),
GMA (0.07 g, 0.45 mmol), Meo-PEG-Br (0.75 g, 0.15 mmol), and PMDETA
(31.5 .mu.L, 0.15 mmol) were added to a polymerization tube. DMF (3
mL) and 2-propanol (3 mL) were then added to dissolve the monomer
and initiator. After three cycles of freeze-pump-thaw to remove
oxygen, CuBr (21.6 mg, 0.15 mmol) was added under nitrogen
atmosphere and the polymerization tube was sealed under vacuum.
After polymerization at 40.degree. C. for 24 h, tetrahydrofuran
(THF) was added to dilute the product, which was then passed
through a neutral Al.sub.2O.sub.3 column to remove the catalyst.
The resulting THF solution was concentrated and the residue was
dialyzed against THF, followed by deionized water. The expected
copolymer was collected as a white powder after freeze-drying under
vacuum. The synthesis scheme is shown below.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-Rn)
[0461] Meo-PEG-b-P(DPA-co-GMA-Rn) was synthesized via the ring
opening reaction between the amino group of NH2-Rn-CONH2 and the
epoxy group of the GMA repeating unit. In brief,
Meo-PEG-b-P(DPA-co-GMA) (1 g) dissolved in DMF (15 mL) was added
dropwise to the DMF solution (10 mL) of NH2-Rn-CONH2 (10-fold molar
excess relative to the GMA repeating unit). After reaction at
60.degree. C. for 7 h, the mixture was transferred to a dialysis
tube and then dialyzed against deionized water. The
Meo-PEG-b-P(DPA-co-GMA-Rn) was finally collected as a white powder
after freeze-drying under vacuum.
[0462] The synthesis route of Meo-PEG-b-P(DPA-co-GMA-Rn) is shown
below.
##STR00006##
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA)
[0463] Meo-PEG-b-P(DPA-co-GMA-TEPA) was synthesized via the ring
opening reaction between TEPA and the epoxy group of the GMA
repeating unit. In brief, Meo-PEG-b-P(DPA-co-GMA) (1 g) dissolved
in DMF (15 mL) was added dropwise to the DMF solution (5 mL) of
TEPA (30-fold molar excess relative to the GMA repeating unit).
After reacting at 60.degree. C. for 7 h, the mixture was
transferred to a dialysis tube and then dialyzed against deionized
water. The Meo-PEG-b-P(DPA-co-GMA-TEPA) was finally collected as a
white powder after freeze-drying under vacuum. The synthesis route
of Meo-PEG-b-P(DPA-co-GMA-TEPA) is shown below.
##STR00007##
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5)
[0464] Meo-PEG-b-P(DPA-co-GMA-TEPA) (0.2 g) and Cy5.5 NHS ester
(1.5-fold molar excess relative to the TEPA repeating unit) were
well dissolved in 5 mL of THF. After constantly stirring in dark
for 48 h, the solution was dialyzed against deionized water and the
product was collected after freeze-drying.
[0465] The synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) is shown
below.
##STR00008##
Synthesis of HOOC-PEG-b-PDPA
[0466] HOOC-PEG-b-PDPA copolymers were also synthesized by the ATRP
method. For example, DPA-MA (1.73 g, 8 mmol), Br-PEG-COOH (0.5 g,
0.1 mmol), and PMDETA (21 .mu.L, 0.1 mmol) were added to a
polymerization tube. Subsequently, DMF (2 mL) and 2-propanol (2 mL)
were added to dissolve the monomer and initiator. After three
cycles of freeze-pump-thaw to remove oxygen, CuBr (14.4 mg, 0.1
mmol) was added under nitrogen atmosphere and the polymerization
tube was sealed under vacuum. After polymerization at 40.degree. C.
for 24 h, tetrahydrofuran (THF) was added to dilute the product,
which was then passed through a neutral Al.sub.2O.sub.3 column to
remove the catalyst. The obtained THF solution was concentrated and
the residue was dialyzed against deionized water. The
HOOC-PEG-b-PDPA was obtained as a white powder after freeze-drying
under vacuum. The synthesis route of HOOC-PEG-b-PDPA is shown
below.
Synthesis of Allyl-Protected ACUPA-PEG-b-PDPA
[0467] HOOC-PEG-b-PDPA copolymer (1 g), allyl protected ACUPA
(5-fold molar excess relative to the terminal carboxylic acid
group), EDC.HCl (3-fold molar excess relative to the terminal
carboxylic acid group), and NHS (3-fold molar excess relative to
the terminal carboxylic acid group) were well dissolved in 15 mL of
THF. The mixture was stirred at room temperature for 48 h. The
solution was subsequently dialyzed against DMF for 48 h followed by
deionized water. The expected allyl-protected ACUPA-PEG-PDPA was
collected after freeze-drying. The synthesis route of
Allyl-protected ACUPA-PEG-b-PDPA is shown below.
Synthesis of ACUPA-PEG-b-PDPA
[0468] Allyl-protected ACUPA-PEG-PDPA (1 g) was well dissolved in
15 mL of THF and Pd(PPh3).sub.4 (42 mg) was added. Under stirring,
PTSF (155 mg) dissolved in 2.5 mL of methanol was added to the
suspension of Allyl protected ACUPA-PEG-PDPA and Pd(PPh3).sub.4.
After reacting in dark for 2 h, the suspension was transferred to a
dialysis tube (MWCO 3500) and dialyzed against toluene for 48 h.
Thereafter, the solution was removed by rotary evaporation and the
residue was dissolved in 15 mL of THF. After dialyzing against
deionized water for 48 h, the ACUPA-PEG-PDPA was collected through
freeze-drying.
[0469] The synthesis route of ACUPA-PEG-b-PDPA is shown below.
##STR00009##
[0470] Gel Permeation Chromatography (GPC)
[0471] Number- and weight-average molecular weights (Mn and Mw,
respectively) of the polymers were determined by a gel permeation
chromatographic system equipped with a Waters 2690D separations
module and a Waters 2410 refractive index detector. THF was used as
the eluent at a flow rate of 0.3 mL/min Waters millennium module
software was used to calculate molecular weight on the basis of a
universal calibration curve generated by polystyrene standard of
narrow molecular weight distribution.
[0472] .sup.1H Nuclear Magnetic Resonance (.sup.1HNMR)
[0473] The 1HNMR spectra of the polymers were recorded on a Mercury
VX-300 spectrometer at 400 MHz (Varian, USA), using CDCl3 as a
solvent and TMS as an internal standard.
[0474] Acid-Base Titration
[0475] Meo-PEG-b-P(DPA-co-GMA-Rn) was dispersed in deionized water,
and a concentrated HCl aqueous solution was added until the
copolymer was completely dissolved (1 mg/mL). Subsequently, 1 M
NaOH aqueous solution was added in 1-5 .mu.L increments. After each
addition, the solution was constantly stirred for 3 min, and the
solution pH was measured using a pH meter. The pKa of the copolymer
was determined as the pH at which 50% of the copolymer turns
ionizes.
[0476] Preparation and Characterization of Nanoparticles (NPs)
[0477] Meo-PEG-b-P(DPA-co-GMA-Rn) was dissolved in THF to form a
homogenous solution with a concentration of 4 mg/mL. Subsequently,
a certain volume of this THF solution was taken and mixed with 1
nmol siRNA (0.1 nmol/.mu.L aqueous solution) in an N/P molar ratio
of 80:1. Under vigorously stirring (1000 rpm), the mixture was
added dropwise to 4 mL of deionized water. The NP dispersion formed
was transferred to an ultrafiltration device (EMD Millipore, MWCO
100 K) and centrifuged to remove the organic solvent and free
compounds. After washing with PBS (pH 7.4) solution (3.times.5 mL),
the siRNA loaded NPs were dispersed in 1 mL of phosphate buffered
saline (PBS, pH 7.4) solution. Size and zeta potential were
determined by dynamic light scattering (DLS, Brookhaven Instruments
Corporation). The morphology of NPs was visualized on a Tecnai G2
Spirit BioTWIN transmission electron microscope (TEM). Before
observation, the sample was stained with 1% uranyl acetate and
dried under air. To determine the siRNA encapsulation efficiency,
DY547-labelled GL3 siRNA (DY547-siRNA) loaded NPs were prepared
according to the method described above. A small volume (50 .mu.L)
of the NP solution was withdrawn and mixed with 20-fold DMSO. The
fluorescence intensity of DY547-siRNA was measured using a Synergy
HT multi-mode microplate reader (BioTek Instruments) and compared
to the free DY-547 labelled GL3 siRNA solution (1 nmol/mL PBS
solution).
[0478] To prepare the ACUPA-NPs, Meo-PEG-b-P(DPA-co-GMA-Rn) (4
mg/mL in THF) was mixed with 1 nmol siRNA (0.1 nmol/.mu.L aqueous
solution) in a N/P molar ratio of 80:1. Then ACUPA-PEG-b-PDPA (4
mg/mL in THF, 10 mol % compared to Meo-PEG-b-P(DPA-co-GMA-Rn)) was
added, and the mixture was added dropwise to 4 mL of deionized
water. The ACUPA-NPs were purified by an ultrafiltration device
(EMD Millipore, MWCO 100 K) and finally dispersed in 1 mL of PBS.
The siRNA encapsulation efficiency was examined by replacing the
siRNA with DY-547 labelled GL3 siRNA.
[0479] Evaluation of pH Sensitivity
[0480] The THF solution of Meo-PEG-b-P(DPA-co-GMA-Rn) (4 mg/mL) and
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) (4 mg/mL) was mixed in a volume
ratio of 8:2. Under vigorously stirring (1000 rpm), 0.2 mL of the
mixture was added dropwise to 2 mL of deionized water. After
collection and purification by an ultrafiltration device (EMD
Millipore, MWCO 100 kDa), the NPs formed were dispersed in 1 mL of
deionized water. Subsequently, 1 M NaOH or HCl was added in 1-5
.mu.L increments, and the fluorescence intensity of the NPs was
measured on a Synergy HT multi-mode microplate reader. The
normalized fluorescence intensity (NFI) vs. pH profile was used to
quantitatively assess the pH responsiveness. NFI is calculated as
follows:
NFI=(F-Fmin)/(Fmax-Fmin)
[0481] where F is the fluorescence intensity of the NPs at any
given pH value and Fmax and Fmin are the maximal and minimal
fluorescence intensity of the NPs, respectively.
[0482] In Vitro siRNA Release
[0483] DY547-siRNA-loaded NPs were prepared as described above.
Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and
then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100
kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree. C.
At a predetermined interval, 5 .mu.L of the NP solution was
withdrawn and mixed with 20-fold DMSO. The fluorescence intensity
of DY547-siRNA was determined by Synergy HT multi-mode microplate
reader.
[0484] Cell Culture
[0485] Human cervical cancer cell line with the expression of
luciferase (Luc-HeLa) and prostate cancer (PCa) cell lines (LNCaP,
PC3, DU145, 22RV1) were incubated in RPMI 1640 medium with 10% FBS
at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2.
[0486] Determination of the Expression of Prostate Specific
Membrane Antigen (PSMA)
[0487] The PCa cell lines were seeded in 6-well plates (50,000
cells per well) and incubated in 1 mL of RPMI 1640 medium
containing 10% FBS for 24 h. Thereafter, 10 .mu.L of PE-conjugated
anti-human PSMA antibody (BioLegend) was added, and the cells were
allowed to incubate for another 4 h. After removing the medium and
washing with PBS (pH 7.4) solution thrice, the cells were collected
for flow cytometry quantitative analysis (DXP11 Analyzer).
[0488] Evaluation of Endosomal Escape
[0489] Luc-HeLa cells (20,000 cells) were seeded in discs and
incubated in 1 mL of RPMI 1640 medium containing 10% FBS for 24 h.
Subsequently, the DY547-siRNA-loaded NPs were added, and the cells
were allowed to incubate for 1 or 2 h. After removing the medium
and subsequently washing with PBS (pH 7.4) solution thrice, the
endosomes and nuclei were stained with lysotracker green and
Hoechst 33342, respectively. The cells were then viewed under a
FV1000 confocal laser scanning microscope (CLSM, Olympus).
[0490] Flow Cytometry
[0491] Luc-HeLa and PCa cell lines (LNCaP, PC3, DU145) were seeded
in 6-well plates (50,000 cells per well) and incubated in 1 mL of
RPMI 1640 medium containing 10% FBS for 24 h. Subsequently, the
DY547-siRNA-loaded NPs or ACUPA-NPs were added, and the cells were
allowed to incubate for another 4 h. After removing the medium and
subsequently washing with PBS (pH 7.4) solution thrice, the cells
were collected for flow cytometry quantitative analysis.
[0492] Animals
[0493] Healthy male BALB/c mice (4-5 weeks old) were purchased from
Charles River Laboratories. All in vivo studies were performed in
accordance with National Institutes of Health animal care
guidelines and in strict pathogen-free conditions in the animal
facility of Brigham and Women's Hospital. Animal protocol was
approved by the Institutional Animal Care and Use Committees on
animal care (Harvard Medical School).
[0494] LNCaP Xenograft Tumor Model
[0495] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of LNCaP cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density of
3.times.10.sup.7 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the PC3 tumor xenograft
reached .about.50 mm.sup.3, the mice were used for the following in
vivo experiments.
[0496] Pharmacokinetics Study
[0497] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and given an intravenous injection of either (i) free
DY647-labelled GL3 siRNA (DY647-siRNA), (ii) DY647-siRNA-loaded
NPs, or (iii) DY647-siRNA-loaded ACUPA-NPs at a 650 .mu.g/kg siRNA
dose. At predetermined time intervals, orbital vein blood (20
.mu.L) was withdrawn using a tube containing heparin, and the wound
was pressed for several seconds to stop the bleeding. The
fluorescence intensity of DY-647 labelled siRNA in the blood was
determined using a microplate reader. The blood circulation
half-life (t1/2) was calculated by first-order decay fit.
[0498] Biodistribution
[0499] LNCaP tumor-bearing male BALB/c nude mice were randomly
divided into four groups (n=3) and given an intravenous injection
of either (i) free Cy5.5-labelled GL3 siRNA (Cy5.5-siRNA), (ii)
Cy5.5-siRNA-loaded NPs, (iii) Cy5.5-siRNA-loaded ACUPA-NPs or (iv)
PSMA antibody (5 mg/kg dose) 15 min followed by Cy5.5-siRNA loaded
ACUPA-NPs at a 650 siRNA dose. Twenty-four hours after the
injection, the mice were imaged using the Maestro 2 In-Vivo Imaging
System (Cri Inc). Main organs and tumors were then harvested and
imaged. To quantify the accumulation of NPs in tumors and organs,
the fluorescence intensity of each tissue was quantified by
Image-J.
[0500] Results
[0501] A high loading, biosafe and long-circulating siRNA delivery
nanoplatform that shows high prostate specificity and excellent
endosomal escape capability for PCa therapy is developed. To
construct this robust nanoplatform, a library of ultra
pH-responsive PEGylated polymers were developed, containing
membrane-penetrating oligoarginine grafts and an
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA) terminus. ACUPA is a small molecule target ligand that can
specifically bind to prostate specific membrane antigen (PSMA),
which is abundantly expressed in PCa, in both its metastatic form
and the hormone-refractory form (Israeli, R et al., Cancer
Research, 53, (2), 227-230 (1993); Murphy, G P et al., Cancer, 83,
(11), 2259-2269 (1998); Dhar, S et al., Proceedings of the National
Academy of Sciences, 105, (45), 17356-17361 (2008)). The resulting
polymeric nanoplatform is expected to have the following unique
features (FIGS. 11A-11B): i) the surface-encoded ACUPA moieties
endow the NPs with high PCa specificity and selectivity; ii) the
hydrophilic PEG shells allow the NPs to escape immunological
recognition, thus improving blood circulation (Knop, K et al.,
Angewandte Chemie International Edition, 49, (36), 6288-6308
(2010); Guo, X et al., Accounts of Chemical Research, 45, 971-979
(2012); Bertrand, N et al., Advanced Drug Delivery Reviews, 66,
2-25(2014); iii) a small population of cationic
membrane-penetrating oligoarginine grafts randomly dispersed in the
hydrophobic poly(2-(diisopropylamino) ethylmethacrylate) (PDPA)
segment can strongly entrap a high amount of siRNA into the
hydrophobic cores of the NPs; iv) the rapid protonation of the
ultra pH-responsive PDPA segment with a pKa close to endosomal pH
(6.0-6.5) causes the swelling of endosomes via the "proton sponge"
effect (Yu, H et al., ACS Nano, 5, 9246-9255 (2011); Zhou, K et
al., Angewandte Chemie International Edition, 50, 6109-6114
(2011)), which works alongside the membrane-penetrating
oligoarginine grafts to induce efficient and fast release of siRNA
in cytoplasm to inhibit tumor growth (Chen, J X et al., ACS Applied
Materials &Interfaces, 6, (1), 593-598 (2014); Chen, J X et
al., Biomaterials, 32, (6), 1678-1684 (2011); Lim, Y B et al., M.
Angewandte Chemie International Edition, 46, 9011-9014(2007).
[0502] Atom-transfer radical polymerization (ATRP) was employed to
synthesize the PEGlyated polymer, methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate) (Meo-PEG-b-P(DPA-co-GMA)). The epoxy group was
subsequently subjected to attack by oligoarginine (Rn, n=6, 8, 10,
20, 30) to endow the resulting polymer (Meo-PEG-b-P(DPA-co-GMA-Rn)
with siRNA loading and endosomal membrane-penetrating abilities.
The PCa-specific PEGylated polymer, ACUPA-PEG-b-PDPA was also
prepared by ATRP, followed by conjugation with ACUPA.
[0503] The length of the oligoarginine grafts was varied to adjust
the siRNA loading ability and physiochemical properties of the NPs.
The siRNA-loaded NPs were prepared by mixing siRNA aqueous solution
with the tetrahydrofuran (THF) solution of
Meo-PEG-b-P(DPA-co-GMA-Rn) at a N/P molar ratio of 80:1. The
amphiphilic nature of the polymers induces self-assembly into NPs
with siRNA entrapped in the hydrophobic cores. As the number of
arginine residues increases from 6 to 30, the size of the resulting
NPs increases from 56.6 to 179.9 nm (Table 12, FIG. 12A), but siRNA
encapsulation efficiency (EE %) decreases from 90.6% to 49.7%
(Table 12, FIG. 12B). One possible reason is that enhancing the
whole hydrophilicity of the amphiphilic polymers by increasing the
length of the oligoarginine grafts leads to the formation of looser
NPs with weaker siRNA loading ability. This also results in an
increased zeta potential. Notably, there is no obvious change in
the EE % or size of the NPs made with the mixture of
Meo-PEG-b-P(DPA-co-GMA-Rn) (90 mol %) and ACUPA-PEG-b-PDPA (10 mol
%) (Table 13).
TABLE-US-00012 TABLE 12 Size, zeta potential, siRNA encapsulation
efficiency (EE %), and pH responsiveness of the NPs prepared from
Meo-PEG-b-P(DPA-co- GMA-Rn) No. Size (nm).sup.a Zeta potential (mv)
EE %.sup.b pKa.sup.c .DELTA.pH.sub.10-90% NPsR6 56.6 7.09 90.6 6.24
0.32 NPsR8 83.4 8.26 84.4 6.27 0.36 NPsR10 90.8 9.13 72.7 6.31 0.39
NPsR20 117.8 13.74 54 6.42 0.46 NPsR30 179.9 14.01 49.7 6.49 0.51
.sup.aDetermined by dynamic light scattering (DLS).
.sup.bDY-547-labelled GL3 siRNA was used to examine the EE %.
.sup.cCorresponding to the pKa of the polymer determined by
acid-base titration.
TABLE-US-00013 TABLE 13 Size, zeta potential and siRNA
encapsulation efficiency (EE %) of the iRGD-NPs of prepared from
the mixture of Meo-PEG-b-P(DPA-co- GMA-Rn) and
ACUPA-PEG-b-PDPA.sup.a No. Size (nm).sup.b Zeta potential (mv) EE
%.sup.c ACUPA-NPsR6 58.7 6.97 92.1 ACUPA-NPsR8 85.9 7.92 86.9
ACUPA-NPsR10 93.6 8.87 76.1 ACUPA-NPsR20 119.4 13.46 58.2
ACUPA-NPsR30 184.1 13.78 51.8 .sup.aThe molar ratio of
Meo-PEG-b-P(DPA-co-GMA-Rn) and ACUPA-PEG-b-PDPA is 9:1.
.sup.bDetermined by dynamic light scattering (DLS).
.sup.cDY-547-labelled GL3 siRNA was used to examine the EE %.
[0504] The amphiphilic polymer, Meo-PEG-b-P(DPA-co-GMA-R10) (pKa
6.31) was used to investigate its pH sensitivity. The transmission
electron microscope (TEM) image of the GL3 siRNA-loaded NPs of
Meo-PEG-b-P(DPA-co-GMA-R10) incubated in PBS buffer at a pH of 6.5
indicated that this amphiphilic copolymer was able to assemble with
siRNA to form spherical NPs at a pH of 6.5, with an average size of
90.8 nm determined by dynamic light scattering (DLS). When the
solution pH decreases to 6.0, there are no observable NPs after 20
min incubation using TEM imaging, indicating a very fast pH
sensitivity. To further evaluate the pH sensitivity, a
near-infrared dye, Cy5.5-conjugated PEGylated polymer, was mixed
with Meo-PEG-b-P(DPA-co-GMA-R10) to prepare the NPs with the
aggregation of fluorophores inside the hydrophobic cores.
Fluorescent images of the Cy.5.5 labelled NPs of
Meo-PEG-b-P(DPA-co-GMA-R10) at different pH indicated that, with
the quenching of the fluorophores, fluorescence signal is absent at
a pH above pKa. However, protonation of the PDPA segment at pH
below pKa causes the NPs to disassemble, leading to a dramatic
increase in the fluorescence signal. Measuring the fluorescence
intensity upon the pH change reveals that the pH difference from 10
to 90% fluorescence activation (.DELTA.pH.sub.10-90%) is 0.39 (FIG.
13) (Wang, Y et al., Nat Mater, 13, (2), 204-212 (2014)). This
value is much smaller than that of small molecule dyes (about 2 pH
units) with the same degree of fluorescence intensity change
(Urano, Y et al., Nat Med, 15, (1), 104-109 (2009)), demonstrating
the ultra-fast pH response rate of Meo-PEG-b-P(DPA-co-GMA-R10).
This characteristic allows the NPs of this polymer to show a
super-fast release of DY547-labelled GL3 siRNA (DY547-siRNA) at a
pH below pKa. Around 80% of the loaded siRNA has been released
within 3 h at a pH of 6.0. Within the same time frame, less than
30% of the loaded siRNA is released at a pH of 7.4 (FIG. 12C).
[0505] To validate the ultra pH-sensitivity of the NPs, their
endosomal escape ability was examined by using lysotracker green
the endosomes. The confocal laser scanning microscope (CLSM) images
of Luc-HeLa cells incubated with the DY547-siRN-loaded NPsR10 for 2
h showed that a majority of the internalized siRNA-loaded NPs enter
the cytoplasm after 2 h incubation, clearly demonstrating the
excellent endosomal escape ability of the NPs.
[0506] After demonstrating the excellent endosomal escape ability
of the NPs, their PCa specificity was evaluated. LNCaP cells, a PCa
cell line with over-expressed PSMA (FIG. 14) (Farokhzad, O C et
al., Proceedings of the National Academy of Sciences, 103, (16),
6315-6320 (2006)), were chosen for incubation with the NPs. LNCaP
cells show around 5-fold stronger uptake of the DY547-siRNA-loaded
ACUPA-NPsR10 than that of NPsR10 (FIG. 15). If the cells are
pre-treated with the PSMA antibody, there is no obvious difference
in cellular uptake between ACUPA-NPsR10 and NPsR10 (FIG. 15),
indicating that the high cellular uptake of ACUPA-NPsR10 is built
on the specific recognition between the ACUPA ligand and the
over-expressed PSMA on LNCaP cells. To further validate this
ACUPA-mediated PCa specificity, two other PCa cell lines with
extremely low PSMA expression, PC3 and DU145 cells, were also
incubated with the DY547-siRNA-loaded NPs. With the absence of
specific interaction between the ACUPA ligand and PSMA, these two
cell lines show similar ability to internalize the ACUPA-NPsR10 and
NPsR10 (FIGS. 16A-16B).
[0507] After proving the in vitro PCa-specificity of the
ACUPA-NPsR10, their pharmacokinetics and in vivo PCa-specificity
was evaluated. The pharmacokinetics of the ACUPA-NPsR10 was
examined by intravenous injection of DY647 labelled GL3 siRNA
(DY647-siRNA) loaded NPs to healthy mice (650 .mu.g/kg siRNA dose,
n=3). The blood half-life (t.sub.1/2) of ACUPA-NPsR10 is around
4.56 h (FIG. 17A), far longer than naked siRNA (t.sub.1/2<30
min). This better stability is mainly attributed to protection by
the PEG outer layer and small particle size (Knop, K et al.,
Angewandte Chemie International Edition, 49, 6288-6308 (2010); Guo,
X et al., Accounts of Chemical Research, 45, 971-979 (2012);
Bertrand, N et al., Advanced Drug Delivery Reviews, 66, 2-25(2014).
Moreover, due to the negative nature of the surface-encoded ACUPA
ligand with three carboxylic acid groups, the ACUPA-NPsR10 show
longer blood circulation than NPsR10 (t.sub.1/2=4.18 h). The in
vivo PCa-specificity of ACUPA-NPsR10 was assessed by intravenously
injecting Cy5.5 labelled GL3 siRNA (Cy5.5-siRNA) loaded NPs to
LNCaP xenograft tumor-bearing mice (650 .mu.g/kg siRNA dose, n=3).
Overlaid fluorescent images of the LNCaP xenograft tumor-bearing
nude mice 24 h post-injection of naked Cy5.5-siRNA,
Cy5.5-siRNA-loaded NPsR10 and ACUPA-NPsR10, and PSMA antibody
followed by Cy5.5-siRNA-loaded ACUPA-NPsR10 show that there is
almost no accumulation of naked siRNA in the tumor. However, the
ACUPA-NPsR10 shows high accumulation in the tumor corresponding to
the bright fluorescence. In the absence of the PSMA-specific ACUPA
ligand, the accumulation of NPsR10 in the tumor is much lower
compared to ACUPA-NPsR10. If first injecting the PSMA antibody (5
mg/kg dose) followed by ACUPA-NPsR10, the blocked PSMA leads to a
decrease in the accumulation of ACUPA-NPsR10 in tumor, highlighting
the important effect of specific interaction between PSMA and the
ACUPA ligand on the PCa-specificity of ACUPA-NPsR10. To analyze the
accumulation of NPs in tumor and other organs, the tumor and main
organs of mice were harvested 24 h post-injection of DY677-siRNA
loaded ACUPA-NPsR10 and NPsR10, PSMA antibody followed by
DY677-siRNA loaded ACUPA-NPsR10, and naked DY677-siRNA and the
biodistribution of the NPs determined (FIG. 17B). The naked siRNA
presents a characteristic biodistribution, i.e., high accumulation
in kidney but extremely low accumulation in tumor (Zhu X et al.,
Proceedings of the National Academy of Sciences, 112, (25),
7779-7784 (2015)). With the specific recognition between the ACUPA
ligand and PSMA over-expressed on LNCaP xenograft tumor, the
accumulation of ACUPA-NPsR10 in tumor is around 3-fold higher than
that of NPsR10 or that found in mice pre-treated with PSMA antibody
(FIG. 17B).
[0508] In conclusion, a PCa-specific and ultra pH-responsive
nanoplatform has been developed. This nanoplatform can specifically
deliver cargos such as siRNA to PCa through the recognition between
the ACUPA ligand and over-expressed PSMA on PCa cells. With the
endosome swelling induced by ultra pH-responsive characteristic
along with the oligoarginine-mediated endosomal membrane
penetration, this nanoplatform can efficiently escape from
endosomes and rapidly release therapeutic molecules in the
cytoplasm. The targeted membrane-penetrating and ultra
pH-responsive nanoplatform is effective as a robust delivery
vehicle for PCa-specific therapy.
Example 4: Redox-Responsive Nanoparticle-Mediated Systemic siRNA
and mRNA Co-Delivery for Concurrent Upregulation and Suppression of
Genetic Causes of Cancer
[0509] Methods
Synthesis of the L-Cystine-Based Poly(Disulfide) (PDSA)
Polymers
[0510] PDSA polymers were prepared by using the same method
described former example 1.
[0511] Preparation and Characterization of Nanoparticles (NPs)
[0512] The PDSA polymers were dissolved in DMF or DMSO to form a
homogenous solution with a concentration of 20 mg/mL. Subsequently,
200 .mu.L of this solution was taken and mixed with 140 .mu.L of
DSPE-PEG3000 (20 mg/mL in DMF), 50 .mu.L of G0-C14 (5 mg/mL in DMF)
and 1 nmol siRNA (0.1 nmol/.mu.L aqueous solution). Under
vigorously stirring (1000 rpm), the mixture was added dropwise to 5
mL of deionized water. The NP dispersion formed was transferred to
an ultrafiltration device (EMD Millipore, MWCO 100 K) and
centrifuged to remove the organic solvent and free compounds. After
washing with PBS (pH 7.4) solution (3.times.5 mL), the siRNA loaded
NPs were dispersed in 1 mL of phosphate buffered saline (PBS, pH
7.4) solution. Size and zeta potential were determined by DLS. The
morphology of NPs was visualized on TEM. To determine the siRNA
encapsulation efficiency, DY547-labelled GL3 siRNA (DY547-siRNA)
loaded NPs were prepared according to the method described above. A
small volume (50 .mu.L) of the NP solution was withdrawn and mixed
with 20-fold DMSO. The fluorescence intensity of DY547-siRNA was
measured using a Synergy HT multi-mode microplate reader (BioTek
Instruments) and compared to the free DY-547 labelled GL3 siRNA
solution (1 nmol/mL PBS solution).
[0513] Redox-Responsive Behavior of the NPs
[0514] The siRNA loaded NPs were prepared as described above and
dispersed in PBS containing 10 mM GSH. At pre-determined time
point, the particle size was examined by DLS and the particle
morphology was observed on TEM. To evaluate the intracellular
redox-responsive behavior, the NPs with Nile red and coumarin 6
encapsulated in their hydrophobic cores were prepared and then
incubated with HeLa cells for different time. The fluorescence of
Nile red and coumarin 6 was observed a FV1000 confocal laser
scanning microscope (CLSM, Olympus). If the NPs respond to redox
stimulus, the Nile red and coumarin 6 will release and only green
fluorescence of coumarin 6 can be observed under CLSM. If the NPs
are intact, the fluorescence of coumarin 6 will be quenched by Nile
red and only red fluorescence can be observed under CLSM.
[0515] Luciferase Silencing
[0516] Luc-HeLa cells were seeded in 96-well plates (5,000 cells
per well) and incubated in 0.1 mL of RPMI 1640 medium with 10% FBS
for 24 h. Thereafter, the GL3 siRNA-loaded NPs were added. After
incubating for 24 h, the cells were washed with fresh medium and
allowed to incubate for another 48 h. The expression of firefly
luciferase in HeLa cells was determined using Steady-Glo luciferase
assay kits. Cytotoxicity was measured using the Alamarblue assay
according to the manufacturer's protocol. The luminescence or
fluorescence intensity was measured using a microplate reader, and
the average value of five independent experiments was collected. As
a control, the silencing effect of Lipo2K/GL3 siRNA complexes was
also evaluated according to the procedure described above and
compared to that of GL3 siRNA-loaded NPs.
[0517] Preparation and Characterization of siRNA/mRNA Co-Loaded
NPs
[0518] The PDSA polymers were dissolved in DMF or DMSO to form a
homogenous solution with a concentration of 20 mg/mL. Subsequently,
200 .mu.L of this solution was taken and mixed with 140 .mu.L of
DSPE-PEG3000 (20 mg/mL in DMF), 50 .mu.L of G0-C14 (5 mg/mL in
DMF), 1 nmol siRNA (0.1 nmol/.mu.L aqueous solution) and 10 .mu.g
mRNA (1 mg/mL aqueous solution). Under vigorously stirring (1000
rpm), the mixture was added dropwise to 5 mL of deionized water.
The NP dispersion formed was transferred to an ultrafiltration
device (EMD Millipore, MWCO 100 K) and centrifuged to remove the
organic solvent and free compounds. After washing with PBS (pH 7.4)
solution (3.times.5 mL), the siRNA loaded NPs were dispersed in 1
mL of phosphate buffered saline (PBS, pH 7.4) solution. Size and
zeta potential were determined by DLS. The morphology of NPs was
visualized on TEM. To determine the encapsulation efficiency of
siRNA and mRNA, DY547-labelled GL3 siRNA (DY547-siRNA) and
Cy5-labelled GFP mRNA (Cy5-mRNA) were used and siRNA/mRNA co-loaded
NPs were prepared according to the method described above. A small
volume (50 .mu.L) of the NP solution was withdrawn and mixed with
20-fold DMSO. The fluorescence intensity of DY547-siRNA and
Cy5-mRNA was measured using a Synergy HT multi-mode microplate
reader (BioTek Instruments) and compared to the free DY-547 siRNA
(1 nmol/mL PBS solution) and Cy5.5-mRNA solution (10
.quadrature.g/mL PBS solution).
[0519] In Vitro siRNA and mRNA Release
[0520] DY547-siRNA/Cy5-mRNA co-loaded NPs were prepared as
described above. Subsequently, the NPs were dispersed in 1 mL of
PBS (pH 7.4) and then transferred to a Float-a-lyzer G2 dialysis
device (MWCO 100 kDa, Spectrum) that was immersed in PBS (pH 7.4)
at 37.degree. C. At a predetermined interval, 5 .mu.L of the NP
solution was withdrawn and mixed with 20-fold DMSO. The
fluorescence intensity of DY547-siRNA and Cy5-mRNA was determined
by Synergy HT multi-mode microplate reader.
[0521] Luciferase Silencing and GFP Expression
[0522] Luc-HeLa cells were seeded in 96-well plates (5,000 cells
per well) and incubated in 0.1 mL of RPMI 1640 medium with 10% FBS
for 24 h. Thereafter, the GL3 siRNA loaded NPs, GFP mRNA loaded NPs
and GL3 siRNA/GFP mRNA co-loaded NPs were added. After incubating
for 24 h, the cells were washed with fresh medium and allowed to
incubate for another 48 h. The expression of firefly luciferase in
HeLa cells was determined using Steady-Glo luciferase assay kits.
Cytotoxicity was measured using the alamarBlue assay according to
the manufacturer's protocol. The luminescence or fluorescence
intensity was measured using a microplate reader, and the average
value of five independent experiments was collected. To examine the
GFP expression, the cells were digested by trypsin and flow
cytometry was employed to examine the GFP expression in the
collected cells.
[0523] In Vitro PHB1 Silencing and PTEN Expression
[0524] PHB1-positive and PTEN negative Lung cancer cells (NCI-1650
cells) were seeded in 6-well plates (50,000 cells per well) and
incubated in 2 mL of RPMI 1640 medium containing 10% FBS for 24 h.
Subsequently, the cells were transfected with the PHB1 siRNA loaded
NPs, PTEN mRNA loaded NPs, and PHB1 siRNA/PTEN mRNA co-loaded NPs
for 24 h. After washing the cells with PBS thrice, the cells were
further incubated in fresh medium for another 48 h. Thereafter, the
cells were digested by trypsin and the proteins were extracted
using modified radioimmunoprecipitation assay lysis buffer (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.25% sodium
deoxycholate, 1 mM sodium fluoride, 1 mM Na3VO4, 1 mM EDTA),
supplemented with protease inhibitor cocktail and 1 mM
phenylmethanesulfonyl fluoride (PMSF). The expression of PHB1 and
PTEN was examined using the western blot analysis.
[0525] In Vitro AR Silencing and PTEN Expression
[0526] AR-positive and PTEN-negative Pprostate cancer cells (LNCaP)
were seeded in 6-well plates (50,000 cells per well) and incubated
in 2 mL of RPMI 1640 medium containing 10% FBS for 24 h.
Subsequently, the cells were transfected with the AR siRNA loaded
NPs, PTEN mRNA loaded NPs and AR siRNA/PTEN mRNA co-loaded NPs for
24 h. After washing the cells with PBS thrice, the cells were
further incubated in fresh medium for another 48 h. Thereafter, the
cells were digested by trypsin and the proteins were extracted
using modified radioimmunoprecipitation assay lysis buffer (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.25% sodium
deoxycholate, 1 mM sodium fluoride, 1 mM Na3VO4, 1 mM EDTA),
supplemented with protease inhibitor cocktail and 1 mM
phenylmethanesulfonyl fluoride (PMSF). The expression of AR and
PTEN was examined using the western blot analysis.
[0527] In Vitro NCI-1650 Cell Proliferation
[0528] NCI-1650 cells were seeded in 6-well plates (20,000 cells
per well) and incubated in 1 mL of RPMI 1640 medium containing 10%
FBS for 24 h. Thereafter, the cells were transfected with the NPs
loading PHB1 siRNA, PTEN mRNA, or PHB1 siRNA/PTEN mRNA for 24 h and
then washed with fresh medium for further incubation. At
predetermined intervals, the cytotoxicity was measured using the
alamarBlue assay according to the manufacturer's protocol. After
each measurement, the alamarBlue agent was removed and the cells
were incubated in fresh medium for further proliferation.
[0529] In Vitro LNCaP Cell Proliferation
[0530] LNCaP cells were seeded in 6-well plates (20,000 cells per
well) and incubated in 2 mL of RPMI 1640 medium containing 10% FBS
for 24 h. Thereafter, the cells were transfected with the AR siRNA
loaded NPs, PTEN mRNA loaded NPs and AR siRNA/PTEN mRNA co-loaded
NPs for 24 h, and then washed with fresh medium for further
incubation. At predetermined intervals, the cytotoxicity was
measured using the AlamarBlue assay according to the manufacturer's
protocol. After each measurement, the alamarBlue agent was removed
and the cells were incubated in fresh medium for further
proliferation.
[0531] Pharmacokinetics Study
[0532] Healthy male BALB/c mice were randomly divided into two
groups (n=3) and given an intravenous injection of either (i) naked
DY677-labelled GL3 siRNA (DY677-siRNA), (ii) naked Cy5-labelled GFP
mRNA (Cy5-mRNA), and (iii) DY677-siRNA/Cy5-mRNA co-loaded NPs at a
650 .mu.g/kg siRNA dose and 500 .mu.g/kg mRNA dose. At
predetermined time intervals, orbital vein blood (20 .mu.L) was
withdrawn using a tube containing heparin, and the wound was
pressed for several seconds to stop the bleeding. The fluorescence
intensity of DY677-siRNA and Cy5-mRNA in the blood was determined
using a microplate reader. The blood circulation half-life
(t.sub.1/2) was calculated by first-order decay fit.
[0533] NCI-1650 Xenograft Tumor Model
[0534] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of NCI-1650 cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density of
2.times.10.sup.6 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the NCI-1650 tumor xenograft
reached .about.50 mm.sup.3, the mice were used for the following in
vivo experiments.
[0535] LNCaP Xenograft Tumor Model
[0536] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of LNCaP cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density of
2.times.10.sup.6 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the LNCaP tumor xenograft
reached .about.50 mm.sup.3, the mice were used for the following in
vivo experiments.
[0537] Biodistribution in NCI-1650 Tumor-Bearing Mice
[0538] NCI-1650 tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and given an intravenous injection of
either (i) DY677-siRNA-loaded NPs, (ii) Cy5-mRNA-loaded NPs, or
(iii) Dy677-siRNA/Cy5-mRNA co-loaded NPs at a siRNA dose of 650
.mu.g/kg and mRNA dose of 500 .mu.g/kg. Twenty-four hours after the
injection, the mice were imaged using the Maestro 2 In-Vivo Imaging
System (Cri Inc). Main organs and tumors were then harvested and
imaged. To quantify the accumulation of NPs in tumors and organs,
the fluorescence intensity of each tissue was quantified by
Image-J.
[0539] Biodistribution in LNCaP Tumor-Bearing Mice
[0540] LNCaP tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and given an intravenous injection of
either (i) naked DY677-siRNA, (ii) naked Cy5-mRNA or (iii)
DY677-siRNA/Cy5-mRNA co-loaded NPs at a 650 .mu.g/kg siRNA dose and
500 .mu.g/kg mRNA dose. Twenty-four hours after the injection, the
mice were imaged using the Maestro 2 In-Vivo Imaging System (Cri
Inc). Main organs and tumors were then harvested and imaged. To
quantify the accumulation of NPs in tumors and organs, the
fluorescence intensity of each tissue was quantified by
Image-J.
[0541] In Vivo PBB1 Silencing and PTEN Expression
[0542] NCI-1650 tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and intravenously injected with (i)
PHB1 siRNA-loaded NPs, (ii) PTEN mRNA-loaded NPs or (iii) PHB1
siRNA/PTEM mRNA co-loaded NPs at a siRNA dose of 650 .mu.g/kg and
mRNA dose of 500 .mu.g/kg for three consecutive days. Twenty-four
hours post the final injection, mice were sacrificed and tumors
were harvested. The proteins in the tumor were extracted using
modified radioimmunoprecipitation assay lysis buffer (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.25% sodium
deoxycholate, 1 mM sodium fluoride, 1 mM Na.sub.3VO.sub.4, 1 mM
EDTA), supplemented with protease inhibitor cocktail and 1 mM
phenylmethanesulfonyl fluoride (PMSF). The expression of PHB1 and
PTEN was examined using the aforementioned western blot
analysis.
[0543] In Vivo AR Silencing and PTEN Expression
[0544] LNCaP tumor-bearing male BALB/c nude mice were randomly
divided into three groups (n=3) and intravenously injected with (i)
AR siRNA-loaded NPs, (ii) PTEN mRNA-loaded NPs or (iii) AR
siRNA/PTEN mRNA co-loaded NPs at a 650 .mu.g/kg siRNA dose and 500
.mu.g/kg mRNA dose for three consecutive days. Twenty-four hours
post the final injection, mice were sacrificed and tumors were
harvested. The proteins in the tumor were extracted using modified
radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM
sodium fluoride, 1 mM Na.sub.3VO.sub.4, 1 mM EDTA), supplemented
with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl
fluoride (PMSF). The expression of AR and PTEN was examined using
the aforementioned western blot analysis.
[0545] In Vivo Toxicity
[0546] Healthy male BALB/c mice were randomly divided into two
groups (n=3) and administered by intravenous injections of either
(i) PBS or (ii) PDSA NPs. Twenty-four hours after the injection,
the main organs were collected, fixed with 4% paraformaldehyde, and
embedded in paraffin.
[0547] Tissue sections were stained with hematoxylin-eosin
(H&E) and viewed under an optical microscope.
[0548] Results
[0549] FIG. 18 shows the scheme of redox-responsive
nanoparticle-mediated systemic siRNA and mRNA co-delivery for
concurrent upregulation and suppression of genetic causes of
cancer. With the redox-responsive characteristic to induce the
breakage of the NPs of PDSA8-1, the siRNA loaded NPs of PDSA8-1
show the highest efficacy in down-regulation of luciferase
expression in HeLa cells (FIG. 19, >90% knockdown at 1 nM siRNA
dose).
[0550] After obtaining the best gene silencing platform (PDSA8-1),
it was used to co-deliver siRNA and mRNA. Luciferase-expressing
HeLa cells were incubated with redox-responsive nanoparticles
loaded with a combination of siRNA targeting luciferase mRNA and
mRNA encoding GFP ("siRNA/mRNA NPs"). FIGS. 20A-20C shows the
change in size distribution of these three types of particles. The
size of siRNA/mRNA NPs (FIG. 20C) are slightly larger than that of
siRNA (FIG. 20A) or mRNA (FIG. 20B) loaded NPs. FIG. 21 shows the
siRNA and mRNA release profile of the co-delivery NPs. With the
addition of 10 mM GSH (intracellular GSH concentration) to provide
redox stimulus, the co-delivery NPs show fast siRNA and mRNA
release.
[0551] siRNA/mRNA means that there are both siRNA and mRNA in each
NP. The final NPs are homogeneous. Three experiments were performed
using single siRNA or mRNA loaded NPs. There is a significant
difference in the gene silencing (FIG. 22) and GFP expression (FIG.
23). The siRNA/mRNA co-loaded NPs improved the gene silencing
efficacy and GFP expression compared to single siRNA or mRNA loaded
NPs. FIG. 22 shows the luciferase expression in HeLa cells treated
with the three types of NPs. This is a bar graph illustrating the
relative reduction in luciferase expression in cells induced by
transfection with each particle population. From left to right each
cluster of bars grouped by siRNA dose (nM) represents siRNA only
(left bar), siRNA/mRNA (center bar), and mRNA only (right bar)
nanoparticle populations. The results indicate that the
encapsulation of mRNA in the NPs can improve the silencing efficacy
of siRNA. The result is seen most dramatically at a siRNA dose of
0.5 nM (right-most cluster of bars), where siRNA efficacy when
co-delivered with mRNA is about twice that of siRNA delivered
alone.
[0552] FIG. 23 is a bar graph illustrating the relative expression
of GFP in Luc-HeLa cells treated with the NPs loading mRNA (mRNA
NPs and siRNA/mRNA NPs). The bar at "0" mRNA dose (ng) shows the
mean fluorescence intensity (MFI) of control cells that not
transfected with mRNA. From left to right beginning at 14 ng dose
of mRNA, each pair of bars shows mRNA only (left bar) and
siRNA/mRNA (right bar) nanoparticle populations. The results
indicate that siRNA improved the transfection or expression of
mRNA, when the two RNAs are co-delivered to cells. The result was
evident at each dosage illustrated.
[0553] Protein expression of PHB1 in NCI-1650 cells treated by PHB1
siRNA loaded NPs, and PHB1 siRNA/PTEN mRNA co-loaded NPs
demonstrated that both of these NPs suppressed PHB1 expression
using western blotting analysis. The siRNA/mRNA NPs showed
relatively higher suppression efficacy in PHB1 expression at each
PHB1 siRNA dosage (10 nM, 15 nM, 20 nM, and 25 nM). Protein
expression of PTEN in NCI-1650 cells treated by PTEN mRNA loaded
NPs, and PHB1 siRNA/PTEN mRNA co-loaded NPs demonstrated that both
of these NPs improved PTEN expression using western blotting
analysis. The siRNA/mRNA NPs showed relatively higher gene
transfection efficacy and/or gene translation in NCI-1650 cells at
each PTEN mRNA dosage (14 ng, 21 ng, 29 ng, and 36 ng).
[0554] Expression of AR in LNCaP cells treated by AR siRNA loaded
NPs and AR siRNA/PTEN mRNA co-loaded NPs demonstrates that both of
these NPs can suppress AR expression. The siRNA/mRNA NPs especially
show relatively higher suppression efficacy in AR expression.
[0555] Expression of PTEN in the LNCaP cells treated by PTEN mRNA
loaded NPs and AR siRNA/PTEN mRNA co-loaded NPs demonstrates that
both of these NPs can improve PTEN expression. The siRNA/mRNA NPs
show especially relatively higher gene transfection in the LNCaP
cells.
[0556] Proliferation of NCI-1650 cells over a period of 8 days post
transfection indicated that although single RNA NPs including PHB1
siRNA loaded NPs, and PTEN mRNA loaded NPs significantly reduced
the rate of proliferation of NCI-1650 cells, siRNA/mRNA NPs were
most effective in reducing the rate of proliferation of these cells
compared to these single RNA loaded NPs (FIG. 24). The similar
result can be observed for LNCaP cells treated with the co-delivery
NPs. FIG. 25 shows the proliferation of the LNCaP cells incubated
with the AR siRNA/PTEN mRNA co-loaded NPs. Compared with single RNA
loaded NPs, the co-delivery NPs show better inhibition of the LNCaP
cell growth.
[0557] FIG. 26 shows the blood circulation ability of the
co-delivery NPs. Compared with the free siRNA or mRNA, the
siRNA/mRNA co-loaded NPs show much longer blood circulation
time.
[0558] Fluorescent imaging was carried out to determine the
biodistribution of NPs in various tissues and organs including
tumor, kidney, lung, spleen, liver, and heart of the NCI-1650 (FIG.
27) and LNCaP (FIG. 28) xenograft tumor-bearing mice. The
co-delivery NPs have a higher tumor accumulation in the tumor
tissues than naked siRNA and mRNA (FIG. 28). In addition, Western
blotting analysis of in vivo protein levels of PHB1 and PTEN shows
that co-delivery of PHB1 siRNA and PTEN mRNA in the same
nanoparticles enhanced the efficiency of expression of PTEN as well
as the suppression of PHB1 simultaneously compared to single RNA
loaded NPs. Western blotting analysis of in vivo protein levels of
AR and PTEN also shows that AR siRNA/PTEN mRNA co-loaded NPs have
stonger AR silencing and higher PTEN expression in the tumor
tissues compared to single RNA loaded NPs.
[0559] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0560] 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. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
515PRTArtificial SequenceTargeted peptide 1Cys Arg Glu Lys Ala1
529PRTArtificial SequenceTargeted peptide 2Cys Arg Lys Arg Leu Asp
Arg Asn Cys1 539PRTArtificial SequenceInternalizing peptide 3Cys
Arg Gly Asp Arg Gly Pro Asp Cys1 5419DNAArtificial SequenceGL3
siRNAmisc_feature(19)..(19)dTdT - deoxythymidine dinucleotide
overhang TT 4cuuacgcuga guacuucga 19519DNAArtificial SequenceGL3
siRNA (antisense)misc_feature(19)..(19)dTdT - deoxythymidine
dinucleotide overhang TT 5ucgaaguacu cagcguaag 19
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References