U.S. patent number 10,385,343 [Application Number 15/506,010] was granted by the patent office on 2019-08-20 for methods and compositions for the treatment of cancer.
This patent grant is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. The grantee listed for this patent is CHILDREN'S MEDICAL CENTER CORPORATION. Invention is credited to Adi Gilboa-Geffen, Judy Lieberman, Lee Adam Wheeler.
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United States Patent |
10,385,343 |
Lieberman , et al. |
August 20, 2019 |
Methods and compositions for the treatment of cancer
Abstract
Described herein are methods and compositions relating to the
treatment of cancer, e.g., breast cancer, using, e.g.,
aptamer-siRNA chimera molecules.
Inventors: |
Lieberman; Judy (Brookline,
MA), Gilboa-Geffen; Adi (Brookline, MA), Wheeler; Lee
Adam (Boston, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S MEDICAL CENTER CORPORATION |
Boston |
MA |
US |
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Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION (Boston, MA)
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Family
ID: |
55400670 |
Appl.
No.: |
15/506,010 |
Filed: |
August 28, 2015 |
PCT
Filed: |
August 28, 2015 |
PCT No.: |
PCT/US2015/047449 |
371(c)(1),(2),(4) Date: |
February 23, 2017 |
PCT
Pub. No.: |
WO2016/033472 |
PCT
Pub. Date: |
March 03, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170275629 A1 |
Sep 28, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62043803 |
Aug 29, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/1137 (20130101); C07H 21/00 (20130101); C07K
14/705 (20130101); A61P 35/00 (20180101); A61K
48/00 (20130101); C12N 15/113 (20130101); C12N
15/115 (20130101); C12N 2310/14 (20130101); A61K
2039/5152 (20130101); C12N 2310/16 (20130101); C12N
2310/3519 (20130101); C12N 2310/322 (20130101); C12N
2310/3533 (20130101) |
Current International
Class: |
C12N
15/11 (20060101); C07H 21/00 (20060101); C07K
14/705 (20060101); A61K 48/00 (20060101); C12N
15/115 (20100101); C12N 15/113 (20100101); A61K
39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101766817 |
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Jul 2010 |
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CN |
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103977433 |
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Aug 2014 |
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CN |
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2010/017319 |
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Feb 2010 |
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WO |
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2010/019446 |
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Feb 2010 |
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WO |
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2011/130458 |
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Oct 2011 |
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WO |
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2011/142970 |
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Nov 2011 |
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WO |
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WO-2012078637 |
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Jun 2012 |
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WO |
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2013/025930 |
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Feb 2013 |
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WO |
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2014/019025 |
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Feb 2014 |
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WO |
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WO-2014019025 |
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Feb 2014 |
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WO |
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2014/068408 |
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May 2014 |
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WO |
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2014/093698 |
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Jun 2014 |
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WO |
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2014/126160 |
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Aug 2014 |
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WO |
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2016/127216 |
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Aug 2016 |
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WO |
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Other References
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Primary Examiner: Poliakova-Georgantas; Ekaterina
Attorney, Agent or Firm: Nixon Peabody LLP Resnick; David S.
Kling; Nicole D.
Government Interests
GOVERNMENT SUPPORT
This invention was made with government support under grant number
W81XWH-09-1-0058 awarded by the U.S. Department of the Army. The
Government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. .sctn. 371 National Phase Entry
Application of International Application No. PCT/US15/047449 filed
Aug. 28, 2015, which designates the U.S. and claims benefit under
35 U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
62/043,803 filed Aug. 29, 2014, the contents of which are
incorporated herein by reference in their entirety.
Claims
What is claimed herein is:
1. A method of treating cancer, the method comprising administering
a chimeric molecule comprising an EPCAM binding aptamer domain and
an inhibitory nucleic acid domain, wherein the inhibitory nucleic
acid inhibits the expression of Plk1 and wherein the chimeric
molecule is an aptamer-siRNA chimera (AsiC) comprising the sequence
of SEQ ID NO: 1 or SEQ ID NO: 3.
2. The method of claim 1, wherein the cancer is an epithelial
cancer, breast cancer or triple-negative breast cancer.
3. The method of claim 1, wherein the administration is
subcutaneous.
4. The method of claim 1, wherein the subject is further
administered an additional cancer treatment.
5. The method of claim 4, wherein the cancer treatment is
paclitaxel.
6. The method of claim 1, wherein the 3' end of the chimeric
molecule comprises dTdT.
7. The method of claim 1, wherein the chimeric molecule comprises
at least one 2'-F pyrimidine.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Aug. 28,
2015, is named 701039-082401-PCT_SL.txt and is 8,984 bytes in
size.
TECHNICAL FIELD
The technology described herein relates to chimeric molecules
comprising an EpCAM binding-molecule and an inhibitory nucleic acid
and methods of using such compositions for the treatment of cancer,
e.g. epithelial cancer.
BACKGROUND
RNA interference (RNAi) has been explored for therapeutic use in
reducing gene expression in the liver. However, the liver is unique
in being easy to transfect with RNAi molecules. Delivery of small
RNAs and resulting gene knockdown in other tissues continues to be
inefficient and ultimately ineffective. In particular, the delivery
roadblock is a major obstacle to harnessing RNAi to treat
cancer.
SUMMARY
As described herein, the inventors have developed novel chimeric
aptamer-siRNA molecules (AsiCs). These AsiC's target cancer cell
markers to direct the siRNA specifically to the cancer cells,
increasing delivery efficacy and therapeutic effectiveness while
reducing the potential for side effects.
In one aspect, described herein is a chimeric molecule comprising a
cancer marker-binding aptamer domain and an inhibitory nucleic acid
domain. In some embodiments, the cancer marker is EpCAM or EphA2.
In some embodiments, the inhibitory nucleic acid specifically binds
to a gene product upregulated in a cancer cell. In some
embodiments, the inhibitory nucleic acid inhibits the expression of
a gene selected from the group consisting of: Plk1; MCL1; EphA2;
PsmA2; MSI1; BMI1; XBP1; PRPF8; PFPF38A; RBM22; USP39; RAN; NUP205;
and NDC80. In some embodiments, the cancer marker is EpCAM and the
inhibitory nucleic acid domain inhibits the expression of Plk1.
In some embodiments, the molecule is an aptamer-siRNA chimera
(AsiC). In some embodiments, the cancer marker-binding aptamer
domain comprises the sequence of SEQ ID NO: 33. In some
embodiments, the cancer marker-binding aptamer domain consists
essentially of the sequence of SEQ ID NO: 33. In some embodiments,
the inhibitory nucleic acid domain comprises the sequence of SEQ ID
NO: 2. In some embodiments, the inhibitory nucleic acid domain
consists essentially of the sequence of SEQ ID NO: 2. In some
embodiments, the molecule comprises the sequence of one of SEQ ID
NOs: 1-3. In some embodiments, the molecule consists essentially of
the sequence of one of SEQ ID NOs: 1-3.
In some embodiments, the 3' end of the molecule comprises dTdT. In
some embodiments, the molecule comprises at least one 2'-F
pyrimidine.
In one aspect, described herein is a pharmaceutical composition
comprising a chimeric molecule as described herein and a
pharmaceutically acceptable carrier. In some embodiments, the
composition comprises at least two chimeric molecules as described
herein wherein the chimeric molecules have different aptamer
domains and/or inhibitory nucleic acid domains. In some
embodiments, the different apatmer or inhibitory nucleic acid
domains recognize different targets. In some embodiments, the
different apatmer or inhibitory nucleic acid domains have sequences
and recognize the same target.
In one aspect, described herein is a method of treating cancer, the
method comprising administering a chimeric molecule and/or
composition as described herein. In some embodiments, the cancer is
an epithelial cancer or breast cancer. In some embodiments, the
breast cancer is triple-negative breast cancer. In some
embodiments, the administration is subcutaneous. In some
embodiments, the subject is further administered an additional
cancer treatment. In some embodiments, the cancer treatment is
paclitaxel.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1H demonstrate that EpCAM aptamer specifically targets
Basal A breast cancer cells. Design of EpCAM-AsiC, containing an
EpCAM aptamer and a PLK1 siRNA (sense strand disclosed as SEQ ID
NO: 1 and antisense strand disclosed as SEQ ID NO: 2) (FIG. 1C).
Epithelial breast cancer cell line (BPLER) over express EpCAM
protein compared to normal breast epithelial cell line (BPE) (FIGS.
1A-1B). EpCAM-AsiC targeting GFP was Alexa647 or Cy3 labeled at the
3' end of the antisense siRNA strand and incubated with BPLER and
BPE cells. Uptake was assessed 24 hours later by flow cytometry
(FIG. 1D). Data are representative of 3 independent experiments.
Cy3 and Alexa647-labeled EpCAM-AsiC was taken up by MB468 and BPLER
(EpCAM+ cells) respectively and not by BPE (EpCAM-). MFI of each
peak is shown. To test for gene silencing, BPLER and BPE were
treated with EpCAM-AsiC targeting GFP (4 .mu.M) and compared to
Transfection controls using Dharmafect and GFP-siRNA (100 nM).
Knockdown was assessed by flow cytometry 72 hours after incubation.
Controls were mock and Dhrmafect only treatment (lipid). (n=4)
(FIG. 1D). EpCAM-AsiC targeting AKT1 selectively knocks-down AKT1
mRNA (FIG. 1E) and protein (FIG. 1F) expression in basal A and
luminal breast cancer cell lines and not in basal B or human
fibroblasts (hFb). Transfection with siRNA targeting AKT1 induces
gene knockdown in all cell lines, while treatment with EpCAM-AsiC
targeting GFP doesn't effect AKT1 mRNA and protein levels (*
p<0.05, p<0.01). Plots of AKT1 Protein and gene Knockdown
comparing the effect of EpCAM-AsiC to siRNA transfection.
EpCAM-AsiC induced knockdown correlates with EpCAM expression (FIG.
1E-H). (n=3; mean.+-.SEM normalized to mock; *P<0.05,
**P<0.01, 2-tailed t test).
FIGS. 2A-2E demonstrate that EpCAM AsiC targeting PLK1 specifically
inhibits cell proliferation in Basal A breast cancer cells. The
effect of EpCAM-AsiC targeting PLK1 on cell proliferation was
tested on 10 breast cancer cell lines representative of basal A, B
and luminal cell lines using cell-titer-glo assay (CTG). EpCAM-AsiC
targeting PLK1 decreased cell proliferation in both basal A and
luminal cell lines while having no effect on basal B cells (FIGS.
2A, 2C). A correlation was seen between EpCAM expression levels and
cell viability (FIG. 2B). Basal A (EpCAM+GFP-) cell were
co-cultured with BPE (EpCAM-GFP+) cells and treated with EpCAM-AsiC
targeting PLK1 or untreated. Untreated co-culture displayed a
similar ration of cells following EpCAM-AsiC targeting PLK1
treatment the ratio of EpCAM+ cells decreased and EpCAM- cells
increased. A representative flow cytometry plot (FIG. 2D), the
quantification of the experiment analyzed the ratio of GFP+/GFP-
cells in 4 different cell lines (FIG. 2E). (n=4, * p<0.05,
p<0.01).
FIGS. 3A-3D demonstrate that human TNBC tissue specifically takes
up Cy3-EpCAM aptamers. Experimental design; Cy3-EpCAM-AsiC
targeting GFP, Alexa647-siRNA-GFP or Alexa647-chol-siRNA-GFP (2
.mu.M of each) were added to breast cancer and control explants and
incubated for 24 h before tissue was digested with collagenase to a
single cell suspension and analyzed by flow cytometry (FIG. 3A).
Tumor biopsies over express EpCAM and cytokeratin, an epithelial
cell marker (FIG. 3B) Representative histograms from one of three
independent experiments show that siRNA and chol-siRNA penetrated
both tumor and healthy tissue with similar efficacy while
EpCAM-AsiC was selectively uptaken by the tumor tissue biopsy and
not by the healthy control tissue sample (FIG. 3C). The uptake
experiment was repeated in tumors from three different patients,
each biopsy receive was tested 3 times for each treatment. A
summary of all three patients (FIG. 3D). (n=3, mock, gray EpCAM,
red *P<0.05, **P<0.005, t-test CD4-AsiC versus mock
treatment).
FIGS. 4A-4C demonstrate that EpCAM AsiC targeting PLK1 specifically
inhibits tumor initiation in Basal A breast cancer cells. Colony
assays of breast cancer cell lines were treated with EpCAM-AsiC
targeting PLK1 or GFP (4 uM) or paclitaxel (100 nM) for 24 hr and
cultured for 8 days in drug-free medium. Treatment with paclitaxel
decreased colony formation in all cells lines while treatment with
EpCAM-AsiC targeting PLK1 only eliminated colony formation in
luminal (MCF7) and basal A (HCC1954) cells, treatment with
EpCAM-AsiC targeting GFP had no effect (FIG. 4A). The assay was
repeated in 3 more cells lines and results were reproducible (FIG.
4B). Sphere formation assay indicated similar results, EpCAM-AsiC
targeting PLK1 decreased the number of spheres only in basal A and
luminal cells and had no effect on basal B cells (FIG. 4C).
MB468-luc cells were treated for 24 h with EpCAM-AsiC targeting
either GFP or PLK1 and injected s.c. to the flank of nude mice.
Mice were imaged every 5 days for 20 days. Untreated mice and mice
treated with EpCAM-AsiC targeting GFP, displayed increase in tumor
initiation while mice injected with cell pretreated with EpCAM-AsiC
targeting PLK1 has no tumor initiation.
FIGS. 5A-5C demonstrate the selective uptake of
Alexa750-EpCAM-AsiCs into EpCAM+ tumors. FIG. 5A depicts the
experimental setup; nude mice were injected with MB468-luc (left
flank) and MB231-luc-mCherry (right flank) cells, 5 days post
injection Alexa750 labeled EpCAM-AsiC targeting GFP (0.5 mg/kg) was
injected s.c. in the neck area. The mice were imaged immediately
after injection and again after 24, 48 hr and 5 days. The Alexa750
labeled EpCAM-AsiC targeting GFP was co-localized with the
luciferase tumor in MB468-luc tumor (EpCAM+) and not the
MB231-luc-mCherry (EpCAM-) tumor. Analysis of 7 mice indicates a
significant increase of Alexa750 in MB468 (EpCAM+) tumors (FIG.
5B). FIG. 5C depicts a graph of Alexa750 uptake rates.
FIGS. 6A-6B demonstrate the EpCAM AsiC targeting PLK1 specifically
inhibits tumor growth in Basal A breast cancer cells. FIG. 6A
depicts the experimental design. Nude mice injected with either
MB231-luc-mCherry cells (5.times.10.sup.5) or MB468-luc cells
(5.times.10.sup.6) were treated with 5 mg/Kg of either EpCAM AsiC
targeting PLK1 or GFP every 72 h or left untreated. FIG. 6B:
MB468-luc tumors treated with EpCAM-AsiC targeting PLK1 shrunk in
size as early as 6 days post treatment and in many mice completely
disappeared after 14 days, Untreated tumors both EpCAM+ and
EpCAM-increased in size over the 14 days.
FIG. 7 demonstrates that EpCAM AsiCs are stable in human and mouse
serum. eGFP EpCAM-AsiCs synthesized using 2'-fluoro-pyrimidines,
chemically-stabilized cholesterol-conjugated eGFP siRNAs
(chol-siRNA), or unmodified eGFP siRNAs were incubated with an
equal volume of human or mouse serum. Aliquots were removed at
regular intervals and resuspended in gel loading buffer and stored
at -80.degree. C. before electrophoresis on denaturing PAGE gels.
The average intensity (+S.E.M.) of bands from 2 independent
experiments quantified by densitometry after staining is shown.
FIGS. 8A-8B demonstrate that injection of EpCAM AsiCs does not
stimulate innate immunity in mice. Mice were injected sc with eGFP
EpCAM-AsiCs (5 mg/kg, n=3) or ip with Poly(I:C) (5 or 50 mg/kg
(n=2/dose). FIG. 8A: Serum samples, collected at baseline and 6 and
16 hr after treatment were assessed for IFN.beta., IL-6 and IP-10
by multiplex immunoassay. * p<0.05. ** p<0.01, ***
p<0.001, compared to baseline. FIG. 8B: mRNA expression of
cytokine and IFN-induced genes, relative to gapdh was assayed by
qRT-PCR in total splenocytes harvested 16 hr post treatment. **
p<0.01, compared to untreated (NT, n=3).
FIG. 9 depicts a table of sequences. (SEQ ID NOS 1-2 and 23-32,
respectively, in order of appearance).
FIGS. 10A-10B depict aptamers-siRNA chimera (AsiC). FIG. 10A
depicts a diagram of the AsiC (aptamer covalently linked to one
strand of an siRNA) specifically recognizing a cancer cell surface
receptor, being endocytosed and then released to the cytosol, where
it is processed like endogenous pre-miRNAs to knockdown a target
gene. Bars indicate the 2 delivery hurdles--cell uptake and release
from endosomes to the cytosol where Dicer and the RNA induced
silencing complex (RISC) are located. FIG. 10B depicts the design
of the EpCAM AsiC targeting PLK1. (sense strand disclosed as SEQ ID
NO: 1 and antisense strand disclosed as SEQ ID NO: 2).
FIGS. 11A-11D demonstrate that EpCAM-AsiC knockdown and antitumor
effect correlates with EpCAM levels and inhibits epithelial breast
tumor T-ICs. FIGS. 11A-11B: Representative experiment (FIG. 11A)
and AKT1 knockdown comparing EpCAM-AsiC with lipid siRNA
transfection (FIG. 11B). FIG. 11C: Anti-proliferative effect of
EpCAM-AsiCs knocking down PLK1 only in EpCAM+ cell lines. D PLK1
EpCAM-AsiCs inhibit colony formation in luminal MCF and basal-A
TNBC HCC1143, but not in mesenchymal basal-B MB231 cells.
FIGS. 12A-12B demonstrate the identification of a functional EphA2
aptamer FIG. 12A: Incubation of EphA2+ basal-B MB231 cells with
EphA2 aptamer (EphA2apt) leads to EphA2 degradation and a transient
decrease in active Akt (pAkt). FIG. 12B: EphA2+ breast cancer cells
incubated for 2 h with EphA2apt (0 to 100 nM), but not control
nonbinding aptamer (ctl), show reduced EphA2. Addition of Ephrin A
was used as a positive control for EphA2 degradation.
FIGS. 13A-13C EpCAM-AsiCs knockdown GFP protein (FIG. 13A) and AKT1
mRNA (FIGS. 13B-13C) only in EpCAM+ cell lines, but not in
immortalized breast epithelial cell line (BPE) or mesenchymal basal
B TNBC or human fibroblasts. A transfected siRNA is nonspecific in
its knockdown. *, P<0.05
FIG. 14. Normal breast tissue and basal-A TNBC tumor biopsies from
the same subject were incubated with Cy3-labeled EpCAM-AsiC and
single cell suspensions were analyzed 3 d later for uptake by flow
cytometry. Naked siRNAs were not taken up by either,
cholesterol-conjugated siRNAs were equally taken up, but
EpCAM-AsiCs were specifically taken up by the tumor. Representative
tissues are shown at left.
FIGS. 15A-15C. Treatment of EpCAM+, but with not EpCAM-, breast
cancer lines with PLK1 EpCAM-AsiCs inhibits colony (FIGS. 15A, 15B)
and mammosphere (FIG. 15C) function, in vitro assays of T-IC
function.
FIG. 16 demonstrates that ex vivo treatment of MB468 cells with
PLK1 EpCAM-AsiCs eliminated their ability to form tumors in nude
mice. An equal number of viable cells were implanted the day after
treatment.
FIGS. 17A-17B demonstrate that EpCAM-AsiCs are selectively taken up
into EpCAM+, but not EpCAM-, TNBC tumors. FIG. 17A depicts the
experimental scheme. FIG. 17B depicts the concentration of
EpCAM-AsiCs in excised tumors at sacrifice.
FIG. 18A-18B demonstrate that PLK1 EpCAM-AsiCs caused complete
tumor regression of EpCAM+ TNBC xenografts, but had no effect on
EpCAM- basal-B xenografts. FIG. 18A depicts the experimental
design. Imaging of luciferase activity of left and right flank
tumors was performed sequentially over 2 wks. FIG. 18B depicts a
graph of tumor size by luciferase activity. All the EpCAM+ tumors
in mice treated with PLK1 AsiCs rapidly regressed, while the other
tumors continued to grow.
FIGS. 19A-19C demonstrate that basal dependency genes include 4
tri-snRNP spliceosome complex genes (PFPF8, PRPF38A, RBM22, USP39),
2 nuclear export genes (NUP205, RAN), and a kinetochore gene
(NDC80). FIG. 19A depicts cell viability, 3 d after knockdown,
normalized to control siRNA. FIG. 19B depicts colony formation
assessed by plating viable cells 2 d after knockdown. FIG. 19C
depicts caspase activation 2 d after knockdown is specific for
MB468 and does not occur in BPE cells.
FIG. 20 depicts some possible designs for multimerized EpCAM-AsiCs
to improve endocytosis. In these designs the sense and antisense
strands could be exchanged and the linkers could be varied.
FIGS. 21A-21D demonstrate that EpCAM aptamer specifically targets
Basal A breast cancer cells. FIG. 21A depicts the design of
EpCAM-AsiC, containing an EpCAM aptamer and a PLK1 siRNA (sense
strand disclosed as SEQ ID NO: 1 and antisense strand disclosed as
SEQ ID NO: 2). FIG. 21B depicts graphs demonstrateing that
epithelial breast cancer cell line (BPLER) over express EpCAM
protein compared to normal breast epithelial cell line (BPE).
EpCAM-AsiC targeting GFP was Alexa647 or Cy3 labeled at the 3' end
of the antisense siRNA strand and incubated with BPLER and BPE
cells. Uptake was assessed 24 hours later by flow cytometry (FIG.
21C). Data are representative of 3 independent experiments. Cy3 and
Alexa647-labeled EpCAM-AsiC was taken up by MB468 and BPLER (EpCAM+
cells) respectively and not by BPE (EpCAM-). MFI of each peak is
shown (mock, gray). FIG. 21D depicts graphs of experiments in
which, to test for gene silencing, BPLER and BPE were treated with
EpCAM-AsiC targeting GFP (4 .mu.M) and compared to Transfection
controls using Dharmafect and GFP-siRNA (100 nM). Knockdown was
assessed by flow cytometry 72 hours after incubation. Controls were
mock and Dhrmafect only treatment (lipid). (n=4).
FIG. 22 depicts graphs demonstrating that EpCAM aptamers do not
bind mouse EpCAM. Mouse ESA (EpCAM) levels were determined using
flow cytometry with a mCD326 antibody. 4T1 cell an epithelial mouse
breast cancer cell line displayed high expression levels of EpCAM.
Both RAW (mouse monocyte cell line) and MB468 (human basal A cell
line) displayed an increase in EpCAM expression but much smaller
than 4T1 cells. A mouse mesanchymal cancer cell line (67NR)
displayed a minimal increase in EpCAM expression. Uptake
experiments demonstrated that EpCAM-Aptamer was not taken up by
neither 4T1 nor 67NR cells.
FIG. 23 depicts graphs demonstrating that EpCAM is over expressed
in basal A and luminal but not basal B breast cancer cell lines.
Representative FACS plots of 8 different breast cancer cell lines,
testing EpCAM expression levels by flow cytometery using a hEpCAM
Antibody. EpCAM is over expressed in all basal A and luminal cells
lines and not in basal B. (mock, shaded gray EpCAM, black)
FIGS. 24A-24F demonstrate that EpCAM AsiC specifically silences
gene expression in Basal A breast cancer cells. EpCAM-AsiC
targeting AKT1 selectively knocks-down AKT1 mRNA (FIG. 24A) and
protein (FIGS. 24B, 24C) expression in basal A and luminal breast
cancer cell lines and not in basal B or human fibroblasts (hFb).
Transfection with siRNA targeting AKT1 induces gene knockdown in
all cell lines, while treatment with EpCAM-AsiC targeting GFP
doesn't effect AKT1 mRNA and protein levels (* p<0.05,
p<0.01). Plots of AKT1 Protein and gene Knockdown comparing the
effect of EpCAM-AsiC to siRNA transfection. EpCAM-AsiC induced
knockdown correlates with EpCAM expression (FIG. 24D, 24E). (n=3;
mean.+-.SEM normalized to mock; *P<0.05, **P<0.01, 2-tailed t
test). FIG. 24F depicts the results of flow cytometry analysis.
FIGS. 25A-25E demonstrate that human TNBC tissue specifically takes
up Cy3-EpCAM aptamers. FIG. 25A depicts the experimental design;
Cy3-EpCAM-AsiC targeting GFP, Alexa647-siRNA-GFP or
Alexa647-chol-siRNA-GFP (2 .mu.M of each) were added to breast
cancer and control explants and incubated for 24 h before tissue
was digested with collagenase to a single cell suspension and
analyzed by flow cytometry. FIG. 25B depicts graphs demonstrating
that tumor biopsies over express EpCAM and cytokeratin, an
epithelial cell marker. FIG. 25C depicts representative histograms
from one of three independent experiments show that siRNA and
chol-siRNA penetrated both tumor and healthy tissue with similar
efficacy while EpCAM-AsiC was selectively uptaken by the tumor
tissue biopsy and not by the healthy control tissue sample. The
uptake experiment was repeated in tumors from three different
patients, each biopsy received was tested 3 times for each
treatment. FIG. 25D depicts representative tumors. A summary of all
three patients is depicted in FIG. 25E. (n=3, *P<0.05,
**P<0.005, t-test CD4-AsiC versus mock treatment).
FIG. 26 depicts graphs demonstrating that EpCAM-AsiC is taken up by
both healthy and colon cancer biopsies. Cy3-EpCAM-AsiC targeting
GFP, Alexa647-siRNA-GFP or Alexa647-chol-siRNA-GFP (2 .mu.M of
each) were added to colon cancer and control explants and incubated
for 24 h before tissues were digested with collagenase to a single
cell suspension and analyzed by flow cytometry. Representative
histograms show that EpCAM-AsiC, siRNA and chol-siRNA penetrated
both tumor and healthy tissue with similar efficacy.
FIGS. 27A-27D demonstrate that EpCAM AsiC targeting PLK1
specifically inhibits cell proliferation in Basal A breast cancer
cells. The effect of EpCAM-AsiC targeting PLK1 on cell
proliferation was tested on 10 breast cancer cell lines
representative of basal A, B and luminal cell lines using
cell-titer-glo assay (CTG). EpCAM-AsiC targeting PLK1 decreased
cell proliferation in both basal A and luminal cell lines while
having no effect on basal B cells (FIG. 27A). A correlation was
seen between EpCAM expression levels and cell viability (FIG. 27B).
Basal A (EpCAM+GFP-) cell were co-cultured with BPE (EpCAM-GFP+)
cells and treated with EpCAM-AsiC targeting PLK1 or untreated.
Untreated co-culture displayed a similar ration of cells following
EpCAM-AsiC targeting PLK1 treatment the ratio of EpCAM+ cells
decreased and EpCAM- cells increased. FIG. 27C depicts
representative flow cytometry plots, and FIG. 27D depicts a graph
of the quantification of the experiment analyzed the ratio of
GFP+/GFP- cells in 4 different cell lines. (n=4, * p<0.05,
p<0.01).
FIG. 28 depicts a graph demonstrating specific decrease in cell
viability in Basal A breast cancer cell lines is PLK1 dependent.
Ten different breast cancer cell lines representing basal A, B and
luminal cells were treated with either EpCAM-AsiC targeting PLK1 or
just the EpCAM-aptamer and compared to untreated controls. None of
the cell lines treated with EpCAM-aptamer displayed decrease in
cell viability, while basal A and luminal cell lines displayed a
decrease in cell viability following treatment with EpCAM-AsiC
targeting PLK1.
FIGS. 29A-29C demonstrate that EpCAM AsiC targeting PLK1
specifically inhibits tumor initiation in Basal A breast cancer
cells. Colony assays of breast cancer cell lines were treated with
EpCAM-AsiC targeting PLK1 or GFP (4 uM) or paclitaxel (100 nM) for
24 hr and cultured for 8 days in drug-free medium. Treatment with
paclitaxel decreased colony formation in all cells lines while
treatment with EpCAM-AsiC targeting PLK1 only eliminated colony
formation in luminal (MCF7) and basal A (HCC1954) cells, treatment
with EpCAM-AsiC targeting GFP had no effect. FIG. 29A depicts
images of the assay results. The assay was repeated in 3 more cells
lines and results were reproducible, as demonstrated in the graph
depicted in FIG. 29B. FIG. 29C depicts a graph demonstrating that
sphere formation assay indicated similar results, EpCAM-AsiC
targeting PLK1 decreased the number of spheres only in basal A and
luminal cells and had no effect on basal B cells. MB468-luc cells
were treated for 24 h with EpCAM-AsiC targeting either GFP or PLK1
and injected s.c. to the flank of nude mice. Mice were imaged every
5 days for 20 days. Untreated mice and mice treated with EpCAM-AsiC
targeting GFP, displayed increase in tumor initiation while mice
injected with cell pretreated with EpCAM-AsiC targeting PLK1 has no
tumor initiation.
FIGS. 30A-30B demonstrate that EpCAM AsiC is stable in human and
mouse serum for 36 hours. EpCAM-AsiC targeting GFP synthesized
using 2'-fluoro-pyrimidines, chemically-stabilized 21-mer
cholesterol-conjugated GFP-siRNAs (chol-siRNA), and unmodified
21-mer GFP-siRNA, each in 100 ul PBS, which were added to 100 .mu.l
of of human or mouse serum. At regular intervals, 20 .mu.L was
removed, and resuspended in gel loading buffer and frozen at
-80.degree. C. before being electrophoresed on a denaturing PAGE
gel. FIG. 30A depicts representative PAGE gels and FIG. 30B depicts
graphs of the average intensity (+S.E.M.) of bands from two
independent experiments analyzed by densitometry. Both the
stabilized cholesterol-conjugated siRNA and the EpCAM-AsiC are
stable over the 36 h of the experiment.
FIGS. 31A-31B demonstrate selective uptake of Alexa750-EpCAM-AsiCs
into EpCAM+ tumors. FIG. 31A depicts the experimental setup; nude
mice were injected with MB468-luc (left flank) and
MB231-luc-mCherry (right flank) cells, 5 days post injection
Alexa750 labeled EpCAM-AsiC targeting GFP (0.5 mg/kg) was injected
s.c. in the neck area. The mice were imaged immediately after
injection and again after 24, 48 hr and 5 days. The Alexa750
labeled EpCAM-AsiC targeting GFP was co-localized with the
luciferase tumor in MB468-luc tumor (EpCAM+) and not the
MB231-luc-mCherry (EpCAM-) tumor. FIG. 31B depicts a graph of
analysis of 7 mice indicating a significant increase of Alexa750 in
MB468 (EpCAM+) tumors. At day 5 the tumors were removed and
visualized to validate that the Alexa750 labeled EpCAM-AsiC
targeting GFP indeed entered the tumors. Increased level of
Alexa750 is negatively correlated with mCherry levels. (n=8,
*P<0.05, t-test EpCAM+ versus EpCAM- cells).
FIGS. 32A-32B demonstrate that EpCAM AsiC targeting PLK1
specifically inhibits tumor growth in Basal A breast cancer cells.
FIG. 32A depicts the experimental setup; nude mice injected with
either MB231-luc-mCherry cells (5.times.10.sup.5) or MB468-luc
cells (5.times.10.sup.6) were treated with 5 mg/Kg of either EpCAM
AsiC targeting PLK1 or GFP every 72 h or left untreated. Mice were
imaged using the IVIS Spectra imaging system every 72 h for 14
days. FIG. 32B depicts a graph demonstrating that MB468-luc tumors
treated with EpCAM-AsiC targeting PLK1 shrunk in size as early as 6
days post treatment and in many mice completely disappeared after
14 days, Untreated tumors both EpCAM+ and EpCAM- increased in size
over the 14 days.
FIG. 33 depicts graphs of tumor growth demonstrating that MB468
tumors regress only after treatment with PLK1 EpCAM-AsiC. Mice with
sc MB468 tumors were treated with 5 mg/kg RNA 2.times./wk beginning
when tumors became palpable. PLK1 EpCAM-AsiC, GFP SpCAM-AsiC, EpCAM
aptamer, PLK1 siRNA, and mock treated samples were analyzed as
indicated.
FIG. 34 demonstrates that PLK1 siRNA associates with Argonaute
(AGO) in cells treated with PLK1 EpCAM-AsiCs. MB-468 cells, treated
with PLK1 EPCAM-AsiC or siRNA for 2 days, were lysed, and cell
lysates were immunoprecipitated with pan-AGO antibody or IgG
isotype control. The amount of PLK1 siRNA in the immunoprecipitates
was quantified by Taqman qRT-PCR, presented as log.sub.2 mean with
SEM, relative to miR-16. **, P<0.01 by Student's t-test relative
to siRNA-treated cells. ND, not detectable. PLK1 siRNA was found in
the RISC after treatment with PLK1 EpCAM-AsiCs. However, the Ago
immunoprecipitation did not significantly deplete PLK1 siRNAs from
the supernatant. This is likely because most RNAs that are taken up
by cells are not released from endosomes to the cytosol (A. Wittrup
et al., Visualizing lipid-formulated siRNA release from endosomes
and target gene knockdown. Nature Biotechnology 2015, in
press).
FIG. 35 demonstrates that PLK EpCAM AsiC suppresses MCF10CA1a
(CA1a) tumor growth. The top panel depicts the experimental scheme.
In this experiment the AsiCs were injected sc in the flank near the
tumor, but not into the tumor. The bottom panel depicts a graph of
Log.sub.e total luminescent photon flux of the tumors (N=4); *,
P<0.05 by Student's t-test.
DETAILED DESCRIPTION
The inventors have demonstrated the suprising efficacy of AsiCs
(aptamer-siRNA chimeric molecules) in treating cancer. The AsiC's
described herein utilize an aptamer that targets the chimeric
molecule specifically to cancer cells, providing effective and
on-target suppression of the gene targeted by the siRNA.
In particular, the aptamers described herein, e.g. those targeting
EpCAM and EphA2, permit the therapy to target tumor-initiating
cells (also referred to as cancer stem cells). These cells are
responsible not only for tumor initiation, replapse, and
metastasis, but are also relatively resistant to conventional
cytotoxic therapy. Thus, the compositions and methods described
herein permit effective treatment of the underlying pathology in a
way that existing therapies fail to do. The success of the AsiC's
described herein is particularly suprising in that direct targeting
of EpCAM with antibodies has been previously investigated and found
to lack effectiveness.
Moreover, the AsiC's described herein are demonstrated to be
surprisingly efficacious in the treatment of epithelial cancers,
e.g. breast cancer (e.g. triple negative breast cancer (TNBC)).
There are no current targeted therapies for TNBC and what
treatments are available typically result in metastasis within 3
years, leading to death. The AsiC's described herein demonstrated
effective gene knockdown specifically in luminal and basal-A TNBC
cells as compared to healthy cells, suppressed colony and
mammosphere formation in vitro and abrogated tumor initiation ex
vivo. In vitro treatment with the AsiC's resulted in targeted
delivery of the therapeutic and rapid tumor regression.
In one aspect, described herein is a chimeric molecule comprising a
cancer marker-binding domain and an inhibitory nucleic acid domain.
As used herein, "cancer marker-binding domain" refers to a domain
and/or molecule that can bind specifically to a molecule more
highly expressed on the surface of a cancer cell as compared to a
healthy cell of the same type (a cancer marker). In some
embodiments, the cancer marker can be a protein and/or polypeptide.
In some embodiments, the cancer marker can be selected from EpCAM
or EphA2. In some embodiments, the cancer marker-binding domain can
be an aptamer.
As used herein, "EpCAM" or "epithelial cell adhesion molecule"
refers to a transmembrane glycoprotein mediating Ca2+-independent
homotypic cell-cell adhesion in epithelial cells. Sequences for
EpCAM are known for a variety of species, e.g., human EpCAM (see,
e.g., NCBI Gene ID:4072; protein sequence: NCBI Ref Seq:
NP_002345.2).
As used herein, "EphA2" or "EPH receptor A2" refers to a ephirin
type protein-tyrosine kinase receptor. EphA2 binding ephrin-A
ligands and permits entry of Kaposi sarcoma-associated herpesvirus
into host cells. Sequences for EphA2 are known for a variety of
species, e.g., human EphA2 (see, e.g., NCBI Gene ID:1969; protein
sequence: NCBI Ref Seq: NP_004422.2).
As used herein, "inhibitory nucleic acid domain" refers to a domain
comprising an inhibitory nucleic acid. In some embodiments, the
inhibitory nucleic acid can be a siRNA.
The inhibitory nucleic acid domain can inhibit, e.g., can target,
the expression of a gene product that is upregulated in a cancer
cell and/or the expression of a gene that is required for cell
growth and/or survival. In some embodiments, the inhibitory nucleic
acid domain can inhibit the expression of a gene selected from Plk1
(e.g. "polo-like kinase 1"; NCBI Gene ID: 5347); MCL1 (e.g. myeloid
cell leukemia 1; NCBI Gene ID: 4170); EphA2 (NCBI Gene ID: 1969);
PsmA2 (e.g. proteasome subunit alpha 2; NCBI Gene ID: 5683); MSI1
(e.g., musashi RNA-binding protein 1; NCBI Gene ID: 4440); BMI1
(e.g., B lymphoma Mo-MLV insertion 1, NCBI Gene ID: 648); XBP1
(X-boxn binding protein 1; NCBI Gene ID: 7494); PRPF8 (e.g.,
pre-mRNA processing factor 8; NCBI Gene ID:10594), PFPF38A (e.g.,
pre-mRNA processing factor 38A; NCBI Gene ID: 84950), RBM22 (e.g.,
RNA binding motif protein 22; NCBI Gene ID: 55696), USP39 (e.g.,
ubiquitin specific peptidase 39; NCBI Gene ID: 10713); RAN (e.g.,
ras-related nuclear protein; NCBI Gene ID: 5901); NUP205 (e.g.,
nucleoporin 205 kDa; NCBI Gene ID: 23165), and NDC80 (e.g., NDC80
kinetochore complex component; NCBI Gene ID: 10403). Sequences of
these genes, e.g., the human mRNAs, are readily obtained from the
NCBI database and can be used by one of skill in the art to design
inhibitory nucleic acids. Furthermore, provided herein are
exemplary inhibitory nucleic acid domains, e.g. a nuleic acid
having the sequence of SEQ ID NO: 2.
In some embodiments, a composition as described herein can comprise
a cancer marker-binding domain comprising an aptamer and an
inhibitory nucleic acid domain comprising an siRNA, e.g. the
composition can comprise an aptamer-siRNA chimera (AsiC).
In some embodiments, the methods described herein relate to
treating a subject having or diagnosed as having cancer with a
composition as described herein. Subjects having cancer can be
identified by a physician using current methods of diagnosing
cancer. Symptoms and/or complications of cancer which characterize
these conditions and aid in diagnosis are well known in the art and
include but are not limited to, for example, in the case of breast
cancer a lump or mass in the breast tissue, swelling of all or part
of a breast, skin irritation, dimpling of the breast, pain in the
breast or nipple, nipple retraction, redness, scaliness, or
irritation of the breast or nipple, and nipple discharge. Tests
that may aid in a diagnosis of, e.g. breast cancer include, but are
not limited to, mammograms, x-rays, MRI, ultrasound, ductogram, a
biopsy, and ductal lavage. A family history of cancer or exposure
to risk factors for cancer (e.g. smoke, radiation, pollutants,
BRCA1 mutation, etc.) can also aid in determining if a subject is
likely to have cancer or in making a diagnosis of cancer.
The terms "malignancy," "malignant condition," "cancer," or
"tumor," as used herein, refer to an uncontrolled growth of cells
which interferes with the normal functioning of the bodily organs
and systems.
As used herein, the term "cancer" relates generally to a class of
diseases or conditions in which abnormal cells divide without
control and can invade nearby tissues. Cancer cells can also spread
to other parts of the body through the blood and lymph systems.
A "cancer cell" or "tumor cell" refers to an individual cell of a
cancerous growth or tissue. A tumor refers generally to a swelling
or lesion formed by an abnormal growth of cells, which may be
benign, pre-malignant, or malignant. Most cancer cells form tumors,
but some, e.g., leukemia, do not necessarily form tumors. For those
cancer cells that form tumors, the terms cancer (cell) and tumor
(cell) are used interchangeably.
A subject that has a cancer or a tumor is a subject having
objectively measurable cancer cells present in the subject's body.
Included in this definition are malignant, actively proliferative
cancers, as well as potentially dormant tumors or micrometastatses.
Cancers which migrate from their original location and seed other
vital organs can eventually lead to the death of the subject
through the functional deterioration of the affected organs.
Hemopoietic cancers, such as leukemia, are able to out-compete the
normal hemopoietic compartments in a subject, thereby leading to
hemopoietic failure (in the form of anemia, thrombocytopenia and
neutropenia) ultimately causing death.
Examples of cancer include but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma,
biliary tract cancer; bladder cancer; bone cancer; brain and CNS
cancer; breast cancer; cancer of the peritoneum; cervical cancer;
choriocarcinoma; colon and rectum cancer; connective tissue cancer;
cancer of the digestive system; endometrial cancer; esophageal
cancer; eye cancer; cancer of the head and neck; gastric cancer
(including gastrointestinal cancer); glioblastoma (GBM); hepatic
carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal
cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g.,
small-cell lung cancer, non-small cell lung cancer, adenocarcinoma
of the lung, and squamous carcinoma of the lung); lymphoma
including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma;
neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and
pharynx); ovarian cancer; pancreatic cancer; prostate cancer;
retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the
respiratory system; salivary gland carcinoma; sarcoma; skin cancer;
squamous cell cancer; stomach cancer; testicular cancer; thyroid
cancer; uterine or endometrial cancer; cancer of the urinary
system; vulval cancer; as well as other carcinomas and sarcomas; as
well as B-cell lymphoma (including low grade/follicular
non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;
intermediate grade/follicular NHL; intermediate grade diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high
grade small non-cleaved cell NHL; bulky disease NHL; mantle cell
lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome. In some embodiments, the cancer
can be epithelial cancer. In some embodiments, the cancer can be
breast cancer. In some embodiments, the cancer can be triple
negative breast cancer.
A "cancer cell" is a cancerous, pre-cancerous, or transformed cell,
either in vivo, ex vivo, or in tissue culture, that has spontaneous
or induced phenotypic changes that do not necessarily involve the
uptake of new genetic material. Although transformation can arise
from infection with a transforming virus and incorporation of new
genomic nucleic acid, or uptake of exogenous nucleic acid, it can
also arise spontaneously or following exposure to a carcinogen,
thereby mutating an endogenous gene. Transformation/cancer is
associated with, e.g., morphological changes, immortalization of
cells, aberrant growth control, foci formation, anchorage
independence, malignancy, loss of contact inhibition and density
limitation of growth, growth factor or serum independence, tumor
specific markers, invasiveness or metastasis, and tumor growth in
suitable animal hosts such as nude mice. See, e.g., Freshney,
CULTURE ANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994).
The compositions and methods described herein can be administered
to a subject having or diagnosed as having cancer. In some
embodiments, the methods described herein comprise administering an
effective amount of compositions described herein, to a subject in
order to alleviate a symptom of a cancer. As used herein,
"alleviating a symptom of a cancer" is ameliorating any condition
or symptom associated with the cancer. As compared with an
equivalent untreated control, such reduction is by at least 5%,
10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by
any standard technique. A variety of means for administering the
compositions described herein to subjects are known to those of
skill in the art. Such methods can include, but are not limited to
oral, parenteral, intravenous, intramuscular, subcutaneous,
transdermal, airway (aerosol), pulmonary, cutaneous, topical,
injection, or intratumoral administration. Administration can be
local or systemic. In some embodiments, the administration is
subcutaneous. In some embodiments, the administration of an AsiC as
described herein is subcutaneous.
The term "effective amount" as used herein refers to the amount of
of a composition needed to alleviate at least one or more symptom
of the disease or disorder, and relates to a sufficient amount of
pharmacological composition to provide the desired effect. The term
"therapeutically effective amount" therefore refers to an amount
that is sufficient to provide a particular anti-cancer effect when
administered to a typical subject. An effective amount as used
herein, in various contexts, would also include an amount
sufficient to delay the development of a symptom of the disease,
alter the course of a symptom disease (for example but not limited
to, slowing the progression of a symptom of the disease), or
reverse a symptom of the disease. Thus, it is not generally
practicable to specify an exact "effective amount". However, for
any given case, an appropriate "effective amount" can be determined
by one of ordinary skill in the art using only routine
experimentation.
Effective amounts, toxicity, and therapeutic efficacy can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dosage can
vary depending upon the dosage form employed and the route of
administration utilized. The dose ratio between toxic and
therapeutic effects is the therapeutic index and can be expressed
as the ratio LD50/ED50. Compositions and methods that exhibit large
therapeutic indices are preferred. A therapeutically effective dose
can be estimated initially from cell culture assays. Also, a dose
can be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of a composition) which achieves a half-maximal inhibition of
symptoms) as determined in cell culture, or in an appropriate
animal model. Levels in plasma can be measured, for example, by
high performance liquid chromatography. The effects of any
particular dosage can be monitored by a suitable bioassay, e.g.,
assay for tumor size, among others. The dosage can be determined by
a physician and adjusted, as necessary, to suit observed effects of
the treatment.
In some embodiments, the technology described herein relates to a
pharmaceutical composition as described herein, and optionally a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers and diluents include saline, aqueous buffer solutions,
solvents and/or dispersion media. The use of such carriers and
diluents is well known in the art. Some non-limiting examples of
materials which can serve as pharmaceutically-acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose,
methylcellulose, ethyl cellulose, microcrystalline cellulose and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;
(7) lubricating agents, such as magnesium stearate, sodium lauryl
sulfate and talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)
esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)
buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)
isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)
pH buffered solutions; (21) polyesters, polycarbonates and/or
polyanhydrides; (22) bulking agents, such as polypeptides and amino
acids (23) serum component, such as serum albumin, HDL and LDL;
(22) C.sub.2-C.sub.12 alcohols, such as ethanol; and (23) other
non-toxic compatible substances employed in pharmaceutical
formulations. Wetting agents, coloring agents, release agents,
coating agents, sweetening agents, flavoring agents, perfuming
agents, preservative and antioxidants can also be present in the
formulation. The terms such as "excipient", "carrier",
"pharmaceutically acceptable carrier" or the like are used
interchangeably herein. In some embodiments, the carrier inhibits
the degradation of the active agent, e.g. as described herein.
In some embodiments, the pharmaceutical composition as described
herein can be a parenteral dose form. Since administration of
parenteral dosage forms typically bypasses the patient's natural
defenses against contaminants, parenteral dosage forms are
preferably sterile or capable of being sterilized prior to
administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions. In addition, controlled-release parenteral dosage
forms can be prepared for administration of a patient, including,
but not limited to, DUROS.RTM.-type dosage forms and
dose-dumping.
Suitable vehicles that can be used to provide parenteral dosage
forms as disclosed within are well known to those skilled in the
art. Examples include, without limitation: sterile water; water for
injection USP; saline solution; glucose solution; aqueous vehicles
such as but not limited to, sodium chloride injection, Ringer's
injection, dextrose Injection, dextrose and sodium chloride
injection, and lactated Ringer's injection; water-miscible vehicles
such as, but not limited to, ethyl alcohol, polyethylene glycol,
and propylene glycol; and non-aqueous vehicles such as, but not
limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl
oleate, isopropyl myristate, and benzyl benzoate. Compounds that
alter or modify the solubility of a pharmaceutically acceptable
salt can also be incorporated into the parenteral dosage forms of
the disclosure, including conventional and controlled-release
parenteral dosage forms.
Pharmaceutical compositions can also be formulated to be suitable
for oral administration, for example as discrete dosage forms, such
as, but not limited to, tablets (including without limitation
scored or coated tablets), pills, caplets, capsules, chewable
tablets, powder packets, cachets, troches, wafers, aerosol sprays,
or liquids, such as but not limited to, syrups, elixirs, solutions
or suspensions in an aqueous liquid, a non-aqueous liquid, an
oil-in-water emulsion, or a water-in-oil emulsion. Such
compositions contain a predetermined amount of the pharmaceutically
acceptable salt of the disclosed compounds, and may be prepared by
methods of pharmacy well known to those skilled in the art. See
generally, Remington: The Science and Practice of Pharmacy, 21st
Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa.
(2005).
Conventional dosage forms generally provide rapid or immediate drug
release from the formulation. Depending on the pharmacology and
pharmacokinetics of the drug, use of conventional dosage forms can
lead to wide fluctuations in the concentrations of the drug in a
patient's blood and other tissues. These fluctuations can impact a
number of parameters, such as dose frequency, onset of action,
duration of efficacy, maintenance of therapeutic blood levels,
toxicity, side effects, and the like. Advantageously,
controlled-release formulations can be used to control a drug's
onset of action, duration of action, plasma levels within the
therapeutic window, and peak blood levels. In particular,
controlled- or extended-release dosage forms or formulations can be
used to ensure that the maximum effectiveness of a drug is achieved
while minimizing potential adverse effects and safety concerns,
which can occur both from under-dosing a drug (i.e., going below
the minimum therapeutic levels) as well as exceeding the toxicity
level for the drug. In some embodiments, the composition can be
administered in a sustained release formulation.
Controlled-release pharmaceutical products have a common goal of
improving drug therapy over that achieved by their non-controlled
release counterparts. Ideally, the use of an optimally designed
controlled-release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition in a minimum amount of time. Advantages of
controlled-release formulations include: 1) extended activity of
the drug; 2) reduced dosage frequency; 3) increased patient
compliance; 4) usage of less total drug; 5) reduction in local or
systemic side effects; 6) minimization of drug accumulation; 7)
reduction in blood level fluctuations; 8) improvement in efficacy
of treatment; 9) reduction of potentiation or loss of drug
activity; and 10) improvement in speed of control of diseases or
conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design,
2 (Technomic Publishing, Lancaster, Pa.: 2000).
Most controlled-release formulations are designed to initially
release an amount of drug (active ingredient) that promptly
produces the desired therapeutic effect, and gradually and
continually release other amounts of drug to maintain this level of
therapeutic or prophylactic effect over an extended period of time.
In order to maintain this constant level of drug in the body, the
drug must be released from the dosage form at a rate that will
replace the amount of drug being metabolized and excreted from the
body. Controlled-release of an active ingredient can be stimulated
by various conditions including, but not limited to, pH, ionic
strength, osmotic pressure, temperature, enzymes, water, and other
physiological conditions or compounds.
A variety of known controlled- or extended-release dosage forms,
formulations, and devices can be adapted for use with the salts and
compositions of the disclosure. Examples include, but are not
limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;
3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;
5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and
6,365,185 B1; each of which is incorporated herein by reference.
These dosage forms can be used to provide slow or
controlled-release of one or more active ingredients using, for
example, hydroxypropylmethyl cellulose, other polymer matrices,
gels, permeable membranes, osmotic systems (such as OROS.RTM. (Alza
Corporation, Mountain View, Calif. USA)), or a combination thereof
to provide the desired release profile in varying proportions.
The methods described herein can further comprise administering a
second agent and/or treatment to the subject, e.g. as part of a
combinatorial therapy. Non-limiting examples of a second agent
and/or treatment can include radiation therapy, surgery,
gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib,
AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737,
PI-103; alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegall (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.TM. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (Tykerb.TM.); inhibitors of PKC-alpha,
Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva.RTM.)) and VEGF-A that
reduce cell proliferation and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
In addition, the methods of treatment can further include the use
of radiation or radiation therapy. Further, the methods of
treatment can further include the use of surgical treatments.
In some embodiments of any of the aspects described herein, a
chimeric molecule as described herein can be administered in
combination with a taxane (e.g. docetaxel or paclitaxel). In some
embodiments of any of the aspects described herein, a chimeric
molecule as described herein can be administered in combination
with paclitaxel. In some embodiments of any of the aspects
described herein, an AsiC as described herein can be administered
in combination with a taxane. In some embodiments of any of the
aspects described herein, an AsiC as described herein can be
administered in combination with paclitaxel.
In certain embodiments, an effective dose of a composition as
described herein can be administered to a patient once. In certain
embodiments, an effective dose of a composition can be administered
to a patient repeatedly. For systemic administration, subjects can
be administered a therapeutic amount of a composition comprising
such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5
mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg,
40 mg/kg, 50 mg/kg, or more.
In some embodiments, after an initial treatment regimen, the
treatments can be administered on a less frequent basis. For
example, after treatment biweekly for three months, treatment can
be repeated once per month, for six months or a year or longer.
Treatment according to the methods described herein can reduce
levels of a marker or symptom of a condition, e.g. by at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80% or at
least 90% or more.
The dosage of a composition as described herein can be determined
by a physician and adjusted, as necessary, to suit observed effects
of the treatment. With respect to duration and frequency of
treatment, it is typical for skilled clinicians to monitor subjects
in order to determine when the treatment is providing therapeutic
benefit, and to determine whether to increase or decrease dosage,
increase or decrease administration frequency, discontinue
treatment, resume treatment, or make other alterations to the
treatment regimen. The dosing schedule can vary from once a week to
daily depending on a number of clinical factors, such as the
subject's sensitivity to the composition. The desired dose or
amount of activation can be administered at one time or divided
into subdoses, e.g., 2-4 subdoses and administered over a period of
time, e.g., at appropriate intervals through the day or other
appropriate schedule. In some embodiments, administration can be
chronic, e.g., one or more doses and/or treatments daily over a
period of weeks or months. Examples of dosing and/or treatment
schedules are administration daily, twice daily, three times daily
or four or more times daily over a period of 1 week, 2 weeks, 3
weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or
6 months, or more. A composition can be administered over a period
of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute,
or 25 minute period.
For convenience, the meaning of some terms and phrases used in the
specification, examples, and appended claims, are provided below.
Unless stated otherwise, or implicit from context, the following
terms and phrases include the meanings provided below. The
definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the claims.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. If there
is an apparent discrepancy between the usage of a term in the art
and its definition provided herein, the definition provided within
the specification shall prevail.
For convenience, certain terms employed herein, in the
specification, examples and appended claims are collected here.
The terms "decrease", "reduced", "reduction", or "inhibit" are all
used herein to mean a decrease by a statistically significant
amount. In some embodiments, "reduce," "reduction" or "decrease" or
"inhibit" typically means a decrease by at least 10% as compared to
a reference level (e.g. the absence of a given treatment) and can
include, for example, a decrease by at least about 10%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 98%, at
least about 99%, or more. As used herein, "reduction" or
"inhibition" does not encompass a complete inhibition or reduction
as compared to a reference level. "Complete inhibition" is a 100%
inhibition as compared to a reference level. A decrease can be
preferably down to a level accepted as within the range of normal
for an individual without a given disorder.
The terms "increased", "increase", "enhance", or "activate" are all
used herein to mean an increase by a statically significant amount.
In some embodiments, the terms "increased", "increase", "enhance",
or "activate" can mean an increase of at least 10% as compared to a
reference level, for example an increase of at least about 20%, or
at least about 30%, or at least about 40%, or at least about 50%,
or at least about 60%, or at least about 70%, or at least about
80%, or at least about 90% or up to and including a 100% increase
or any increase between 10-100% as compared to a reference level,
or at least about a 2-fold, or at least about a 3-fold, or at least
about a 4-fold, or at least about a 5-fold or at least about a
10-fold increase, or any increase between 2-fold and 10-fold or
greater as compared to a reference level. In the context of a
marker or symptom, a "increase" is a statistically significant
increase in such level.
As used herein, a "subject" means a human or animal. Usually the
animal is a vertebrate such as a primate, rodent, domestic animal
or game animal. Primates include chimpanzees, cynomologous monkeys,
spider monkeys, and macaques, e.g., Rhesus. Rodents include mice,
rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game
animals include cows, horses, pigs, deer, bison, buffalo, feline
species, e.g., domestic cat, canine species, e.g., dog, fox, wolf,
avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout,
catfish and salmon. In some embodiments, the subject is a mammal,
e.g., a primate, e.g., a human. The terms, "individual," "patient"
and "subject" are used interchangeably herein.
Preferably, the subject is a mammal. The mammal can be a human,
non-human primate, mouse, rat, dog, cat, horse, or cow, but is not
limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
cancer. A subject can be male or female.
A subject can be one who has been previously diagnosed with or
identified as suffering from or having a condition in need of
treatment (e.g. cancer) or one or more complications related to
such a condition, and optionally, have already undergone treatment
for cancer or the one or more complications related to cancer.
Alternatively, a subject can also be one who has not been
previously diagnosed as having cancer or one or more complications
related to cancer. For example, a subject can be one who exhibits
one or more risk factors for cancer or one or more complications
related to cancer or a subject who does not exhibit risk
factors.
A "subject in need" of treatment for a particular condition can be
a subject having that condition, diagnosed as having that
condition, or at risk of developing that condition.
As used herein, the terms "protein" and "polypeptide" are used
interchangeably herein to designate a series of amino acid
residues, connected to each other by peptide bonds between the
alpha-amino and carboxy groups of adjacent residues. The terms
"protein", and "polypeptide" refer to a polymer of amino acids,
including modified amino acids (e.g., phosphorylated, glycated,
glycosylated, etc.) and amino acid analogs, regardless of its size
or function. "Protein" and "polypeptide" are often used in
reference to relatively large polypeptides, whereas the term
"peptide" is often used in reference to small polypeptides, but
usage of these terms in the art overlaps. The terms "protein" and
"polypeptide" are used interchangeably herein when referring to a
gene product and fragments thereof. Thus, exemplary polypeptides or
proteins include gene products, naturally occurring proteins,
homologs, orthologs, paralogs, fragments and other equivalents,
variants, fragments, and analogs of the foregoing.
As used herein, the term "nucleic acid" or "nucleic acid sequence"
refers to any molecule, preferably a polymeric molecule,
incorporating units of ribonucleic acid, deoxyribonucleic acid or
an analog thereof. The nucleic acid can be either single-stranded
or double-stranded. A single-stranded nucleic acid can be one
nucleic acid strand of a denatured double-stranded DNA.
Alternatively, it can be a single-stranded nucleic acid not derived
from any double-stranded DNA. In one aspect, the nucleic acid can
be DNA. In another aspect, the nucleic acid can be RNA. Suitable
nucleic acid molecules are DNA, including genomic DNA or cDNA.
Other suitable nucleic acid molecules are RNA, including mRNA.
Inhibitors of the expression of a given gene can be an inhibitory
nucleic acid or inhibitory oligonucleotide. In some embodiments,
the inhibitory nucleic acid is an inhibitory RNA (iRNA). In some
embodiments, the inhibitory nucleic acid is an inhibitory DNA
(iDNA). Double-stranded RNA molecules (dsRNA) have been shown to
block gene expression in a highly conserved regulatory mechanism
known as RNA interference (RNAi). The inhibitory nucleic acids
described herein can include an RNA or DNA strand (the antisense
strand) having a region which is 30 nucleotides or less in length,
i.e., 8-30 nucleotides in length, generally 19-24 nucleotides in
length, which region is substantially complementary to at least
part of a precursor or mature form of a target gene's transcript.
The use of these inhibitory oligonucleotides enables the targeted
degradation of the target gene, resulting in decreased expression
and/or activity of the target gene.
As used herein, the term "inhibitory oligonucleotide," "inhibitory
nucleic acid," or "antisense oligonucleotide" (ASO) refers to an
agent that contains an oligonucleotide, e.g. a DNA or RNA molecule
which mediates the targeted cleavage of an RNA transcript. In one
embodiment, an inhibitory oligonucleotide as described herein
effects inhibition of the expression and/or activity of a target
gene. Inhibitory nucleic acids useful in the present methods and
compositions include antisense oligonucleotides, ribozymes,
external guide sequence (EGS) oligonucleotides, siRNA compounds,
single- or double-stranded RNA interference (RNAi) compounds such
as siRNA compounds, modified bases/locked nucleic acids (LNAs),
antagomirs, peptide nucleic acids (PNAs), and other oligomeric
compounds or oligonucleotide mimetics which hybridize to at least a
portion of the target nucleic acid and modulate its function. In
some embodiments, the inhibitory nucleic acids include antisense
RNA, antisense DNA, chimeric antisense oligonucleotides, antisense
oligonucleotides comprising modified linkages, interference RNA
(RNAi), short interfering RNA (siRNA); a micro, interfering RNA
(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA
(shRNA); small RNA-induced gene activation (RNAa); small activating
RNAs (saRNAs), or combinations thereof. For further disclosure
regarding inhibitory nucleic acids, please see US2010/0317718
(antisense oligos); US2010/0249052 (double-stranded ribonucleic
acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs);
US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA);
and WO2010/129746 and WO2010/040112 (inhibitory nucleic acids).
In certain embodiments, contacting a cell with the inhibitor (e.g.
an inhibitory oligonucleotide) results in a decrease in the target
RNA level in a cell by at least about 5%, about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, about 99%, up to and including 100% of the
target mRNA level found in the cell without the presence of the
inhibitory oligonucleotide.
As used herein, the term "iRNA" refers to an agent that contains
RNA as that term is defined herein, and which mediates the targeted
cleavage of an RNA transcript via an RNA-induced silencing complex
(RISC) pathway. In one embodiment, an iRNA as described herein
effects inhibition of the expression and/or activity of the target
gene. In one aspect, an RNA interference agent includes a single
stranded RNA that interacts with a target RNA sequence to direct
the cleavage of the target RNA. Without wishing to be bound by
theory, long double stranded RNA introduced into plants and
invertebrate cells is broken down into siRNA by a Type III
endonuclease known as Dicer (Sharp et al., Genes Dev. 2001,
15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA
into 19-23 base pair short interfering RNAs with characteristic two
base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The
siRNAs are then incorporated into an RNA-induced silencing complex
(RISC) where one or more helicases unwind the siRNA duplex,
enabling the complementary antisense strand to guide target
recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to
the appropriate target mRNA, one or more endonucleases within the
RISC cleaves the target to induce silencing (Elbashir, et al.,
(2001) Genes Dev. 15:188). Thus, in one aspect, an RNA interference
agent relates to a double stranded RNA that promotes the formation
of a RISC complex comprising a single strand of RNA that guides the
complex for cleavage at the target region of a target transcript to
effect silencing of the target gene.
In some embodiments, the inhibitory oligonucleotide can be a
double-stranded nucleic acid (e.g. a dsRNA). A double-stranded
nucleic acid includes two nucleic acid strands that are
sufficiently complementary to hybridize to form a duplex structure
under conditions in which the double-stranded nucleic acid will be
used. One strand of a double-stranded nucleic acid (the antisense
strand) includes a region of complementarity that is substantially
complementary, and generally fully complementary, to a target
sequence. The target sequence can be derived from the sequence of
an mRNA and/or the mature miRNA formed during the expression of the
target gene. The other strand (the sense strand) includes a region
that is complementary to the antisense strand, such that the two
strands hybridize and form a duplex structure when combined under
suitable conditions. Generally, the duplex structure is between 8
and 30 inclusive, more generally between 18 and 25 inclusive, yet
more generally between 19 and 24 inclusive, and most generally
between 19 and 21 base pairs in length, inclusive. Similarly, the
region of complementarity to the target sequence is between 8 and
30 inclusive, more generally between 18 and 25 inclusive, yet more
generally between 19 and 24 inclusive, and most generally between
19 and 21 nucleotides in length, inclusive. In some embodiments,
the dsRNA is between 15 and 20 nucleotides in length, inclusive,
and in other embodiments, the dsRNA is between 25 and 30
nucleotides in length, inclusive. As the ordinarily skilled person
will recognize, the targeted region of an RNA targeted for cleavage
will most often be part of a larger RNA molecule, often an mRNA
molecule. Where relevant, a "part" of an mRNA and/or miRNA target
is a contiguous sequence of an mRNA target of sufficient length to
be a substrate for antisense-directed cleavage (e.g., cleavage
through a RISC pathway). Double-stranded nucleic acids having
duplexes as short as 8 base pairs can, under some circumstances,
mediate antisense-directed RNA cleavage. Most often a target will
be at least 15 nucleotides in length, preferably 15-30 nucleotides
in length.
One of skill in the art will also recognize that the duplex region
is a primary functional portion of a double-stranded inhibitory
nucleic acid, e.g., a duplex region of 8 to 36, e.g., 15-30 base
pairs. Thus, in one embodiment, to the extent that it becomes
processed to a functional duplex of e.g., 15-30 base pairs that
targets a desired RNA for cleavage, an inhibitory nucleic acid
molecule or complex of inhibitory nucleic acid molecules having a
duplex region greater than 30 base pairs is a double-stranded
nucleic acid. Thus, an ordinarily skilled artisan will recognize
that in one embodiment, then, a miRNA is a dsRNA. In another
embodiment, a dsRNA is not a naturally occurring miRNA. In another
embodiment, an inhibitory nucleic acid agent useful to target the
target gene expression is not generated in the target cell by
cleavage of a larger double-stranded nucleic acid molecule.
While a target sequence is generally 15-30 nucleotides in length,
there is wide variation in the suitability of particular sequences
in this range for directing cleavage of any given target RNA. When
miRNAs are targeted, the target sequence can be as short as 8
nucleotides, including the "seed" region (e.g. nucleotides 2-8)).
Various software packages and the guidelines set out herein provide
guidance for the identification of optimal target sequences for any
given gene target, but an empirical approach can also be taken in
which a "window" or "mask" of a given size (as a non-limiting
example, 21 nucleotides) is literally or figuratively (including,
e.g., in silico) placed on the target RNA sequence to identify
sequences in the size range that may serve as target sequences. By
moving the sequence "window" progressively one nucleotide upstream
or downstream of an initial target sequence location, the next
potential target sequence can be identified, until the complete set
of possible sequences is identified for any given target size
selected. This process, coupled with systematic synthesis and
testing of the identified sequences (using assays as described
herein or as known in the art) to identify those sequences that
perform optimally can identify those RNA sequences that, when
targeted with an inhibitory nucleic acid agent, mediate the best
inhibition of target gene expression.
A double-stranded inhibitory nucleic acid as described herein can
further include one or more single-stranded nucleotide overhangs.
The double-stranded inhibitory nucleic acid can be synthesized by
standard methods known in the art as further discussed below, e.g.,
by use of an automated DNA synthesizer, such as are commercially
available from, for example, Biosearch, Applied Biosystems, Inc. In
one embodiment, the antisense strand of a double-stranded
inhibitory nucleic acid has a 1-10 nucleotide overhang at the 3'
end and/or the 5' end. In one embodiment, the sense strand of a
double-stranded inhibitory nucleic acid has a 1-10 nucleotide
overhang at the 3' end and/or the 5' end. In one embodiment, at
least one end of a double-stranded inhibitory nucleic acid has a
single-stranded nucleotide overhang of 1 to 4, generally 1 or 2
nucleotides. Double-stranded inhibitory nucleic acids having at
least one nucleotide overhang have unexpectedly superior inhibitory
properties relative to their blunt-ended counterparts.
In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate.
As used herein, the term "nucleotide overhang" refers to at least
one unpaired nucleotide that protrudes from the duplex structure of
an inhibitory nucleic acid, e.g., a dsRNA. For example, when a
3'-end of one strand of a double-stranded inhibitory nucleic acid
extends beyond the 5'-end of the other strand, or vice versa, there
is a nucleotide overhang. A double-stranded inhibitory nucleic acid
can comprise an overhang of at least one nucleotide; alternatively
the overhang can comprise at least two nucleotides, at least three
nucleotides, at least four nucleotides, at least five nucleotides
or more. A nucleotide overhang can comprise or consist of a
nucleotide/nucleoside analog, including a
deoxynucleotide/nucleoside. The overhang(s) may be on the sense
strand, the antisense strand or any combination thereof.
Furthermore, the nucleotide(s) of an overhang can be present on the
5' end, 3' end or both ends of either an antisense or sense strand
of a double-stranded inhibitory nucleic acid.
The terms "blunt" or "blunt ended" as used herein in reference to a
double-stranded inhibitory nucleic acid mean that there are no
unpaired nucleotides or nucleotide analogs at a given terminal end
of a dsRNA, i.e., no nucleotide overhang. One or both ends of a
double-stranded inhibitory nucleic acid can be blunt. Where both
ends of a double-stranded inhibitory nucleic acid are blunt, the
double-stranded inhibitory nucleic acid is said to be blunt ended.
To be clear, a "blunt ended" double-stranded inhibitory nucleic
acid is a double-stranded inhibitory nucleic acid that is blunt at
both ends, i.e., no nucleotide overhang at either end of the
molecule. Most often such a molecule will be double-stranded over
its entire length.
In this aspect, one of the two strands is complementary to the
other of the two strands, with one of the strands being
substantially complementary to a sequence of a the target gene
precursor or mature miRNA. As such, in this aspect, a
double-stranded inhibitory nucleic acid will include two
oligonucleotides, where one oligonucleotide is described as the
sense strand and the second oligonucleotide is described as the
corresponding antisense strand of the sense strand. As described
elsewhere herein and as known in the art, the complementary
sequences of a double-stranded inhibitory nucleic acid can also be
contained as self-complementary regions of a single nucleic acid
molecule, as opposed to being on separate oligonucleotides.
The skilled person is well aware that inhibitory nucleic acid
having a duplex structure of between 20 and 23, but specifically
21, base pairs have been hailed as particularly effective in
inducing antisense-mediated inhibition (Elbashir et al., EMBO 2001,
20:6877-6888). However, others have found that shorter or longer
inhibitory nucleic acids can be effective as well.
Further, it is contemplated that for any sequence identified,
further optimization could be achieved by systematically either
adding or removing nucleotides to generate longer or shorter
sequences and testing those and sequences generated by walking a
window of the longer or shorter size up or down the target RNA from
that point. Again, coupling this approach to generating new
candidate targets with testing for effectiveness of inhibitory
nucleic acids based on those target sequences in an inhibition
assay as known in the art or as described herein can lead to
further improvements in the efficiency of inhibition. Further
still, such optimized sequences can be adjusted by, e.g., the
introduction of modified nucleotides as described herein or as
known in the art, addition or changes in overhang, or other
modifications as known in the art and/or discussed herein to
further optimize the molecule (e.g., increasing serum stability or
circulating half-life, increasing thermal stability, enhancing
transmembrane delivery, targeting to a particular location or cell
type, increasing interaction with silencing pathway enzymes,
increasing release from endosomes, etc.) as an expression
inhibitor.
An inhibitory nucleic acid as described herein can contain one or
more mismatches to the target sequence. In one embodiment, an
inhibitory nucleic acid as described herein contains no more than 3
mismatches. If the antisense strand of the inhibitory nucleic acid
contains mismatches to a target sequence, it is preferable that the
area of mismatch not be located in the center of the region of
complementarity. If the antisense strand of the inhibitory nucleic
acid contains mismatches to the target sequence, it is preferable
that the mismatch be restricted to be within the last 5 nucleotides
from either the 5' or 3' end of the region of complementarity. For
example, for a 23 nucleotide inhibitory nucleic acid agent strand
which is complementary to a region of the target gene or a
precursor thereof, the strand generally does not contain any
mismatch within the central 13 nucleotides. The methods described
herein or methods known in the art can be used to determine whether
an inhibitory nucleic acid containing a mismatch to a target
sequence is effective in inhibiting the expression of the target
gene. Consideration of the efficacy of inhibitory nucleic acids
with mismatches in inhibiting expression of the target gene is
important, especially if the particular region of complementarity
in the target gene is known to have polymorphic sequence variation
within the population.
In yet another embodiment, the nucleic acid of an inhibitory
nucleic acid, e.g., a dsRNA, is chemically modified to enhance
stability or other beneficial characteristics. The nucleic acids
featured in the invention may be synthesized and/or modified by
methods well established in the art, such as those described in
"Current protocols in nucleic acid chemistry," Beaucage, S. L. et
al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA,
which is hereby incorporated herein by reference. Modifications
include, for example, (a) end modifications, e.g., 5' end
modifications (phosphorylation, conjugation, inverted linkages,
etc.) 3' end modifications (conjugation, DNA nucleotides, inverted
linkages, etc.), (b) base modifications, e.g., replacement with
stabilizing bases, destabilizing bases, or bases that base pair
with an expanded repertoire of partners, removal of bases (abasic
nucleotides), or conjugated bases, (c) sugar modifications (e.g.,
at the 2' position or 4' position) or replacement of the sugar, as
well as (d) backbone modifications, including modification or
replacement of the phosphodiester linkages. Specific examples of
nucleic acid compounds useful in the embodiments described herein
include, but are not limited to nucleic acids containing modified
backbones or no natural internucleoside linkages. Nucleic acids
having modified backbones include, among others, those that do not
have a phosphorus atom in the backbone. For the purposes of this
specification, and as sometimes referenced in the art, modified
nucleic acids that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
oligonucleosides. In particular embodiments, the modified nucleic
acid will have a phosphorus atom in its internucleoside
backbone.
Modified backbones can include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those) having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included.
Representative U.S. patents that teach the preparation of the above
phosphorus-containing linkages include, but are not limited to,
U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;
5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;
5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;
5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253;
5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109;
6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614;
6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715;
6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029;
and U.S. Pat. No. RE39464, each of which is herein incorporated by
reference.
Modified backbones that do not include a phosphorus atom therein
have backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. These include those
having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH2 component
parts.
Representative U.S. patents that teach the preparation of the above
oligonucleosides include, but are not limited to, U.S. Pat. Nos.
5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;
5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;
5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437;
and, 5,677,439, each of which is herein incorporated by
reference.
In other nucleic acid mimetics suitable or contemplated for use in
inhibitory nucleic acids, both the sugar and the internucleoside
linkage, i.e., the backbone, of the nucleotide units are replaced
with novel groups. The base units are maintained for hybridization
with an appropriate nucleic acid target compound. One such
oligomeric compound, a nucleic acid mimetic that has been shown to
have excellent hybridization properties, is referred to as a
peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of
a nucleic acid is replaced with an amide containing backbone, in
particular an aminoethylglycine backbone. The nucleobases are
retained and are bound directly or indirectly to aza nitrogen atoms
of the amide portion of the backbone. Representative U.S. patents
that teach the preparation of PNA compounds include, but are not
limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262,
each of which is herein incorporated by reference. Further teaching
of PNA compounds can be found, for example, in Nielsen et al.,
Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include nucleic acids
with phosphorothioate backbones and oligonucleosides with
heteroatom backbones, and in particular --CH2-NH--CH2-,
--CH2-N(CH3)-O--CH2-[known as a methylene (methylimino) or MMI
backbone], --CH2-O--N(CH3)-CH2-, --CH2-N(CH3)-N(CH3)-CH2- and
--N(CH3)-CH2-CH2-[wherein the native phosphodiester backbone is
represented as --O--P--O--CH2-] of the above-referenced U.S. Pat.
No. 5,489,677, and the amide backbones of the above-referenced U.S.
Pat. No. 5,602,240. In some embodiments, the inhibitory nucleic
acids featured herein have morpholino backbone structures of the
above-referenced U.S. Pat. No. 5,034,506.
Modified nucleic acids can also contain one or more substituted
sugar moieties. The inhibitory nucleic acids featured herein can
include one of the following at the 2' position: OH; F; O-, S-, or
N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or
O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl
and alkynyl. Exemplary suitable modifications include O[(CH2)nO]
mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and
O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In
other embodiments, dsRNAs include one of the following at the 2'
position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl,
aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3,
OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an inhibitory
nucleic acid, or a group for improving the pharmacodynamic
properties of an inhibitory nucleic acid, and other substituents
having similar properties. In some embodiments, the modification
includes a 2' methoxyethoxy (2'-O--CH2CH2OCH3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim Acta,
1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary
modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2
group, also known as 2'-DMAOE, as described in examples herein
below, and 2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH2-O--CH2-N(CH2)2, also described in examples herein
below.
Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy
(2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can
also be made at other positions on the nucleic acid of an
inhibitory nucleic acid, particularly the 3' position of the sugar
on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5'
position of 5' terminal nucleotide. Inhibitory nucleic acids may
also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl sugar. Representative U.S. patents that teach
the preparation of such modified sugar structures include, but are
not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain
of which are commonly owned with the instant application, and each
of which is herein incorporated by reference.
An inhibitory nucleic acid can also include nucleobase (often
referred to in the art simply as "base") modifications or
substitutions. As used herein, "unmodified" or "natural"
nucleobases include the purine bases adenine (A) and guanine (G),
and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
Modified nucleobases include other synthetic and natural
nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and
guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in Modified Nucleosides in Biochemistry,
Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008;
those disclosed in The Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley &
Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages
289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of the oligomeric compounds featured in the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research
and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are
exemplary base substitutions, even more particularly when combined
with 2'-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain
of the above noted modified nucleobases as well as other modified
nucleobases include, but are not limited to, the above noted U.S.
Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30;
5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;
5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,
5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197;
6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438;
7,045,610; 7,427,672; and 7,495,088, each of which is herein
incorporated by reference, and U.S. Pat. No. 5,750,692, also herein
incorporated by reference.
The nucleic acid of an inhibitory nucleic acid can also be modified
to include one or more locked nucleic acids (LNA). A locked nucleic
acid is a nucleotide having a modified ribose moiety in which the
ribose moiety comprises an extra bridge connecting the 2' and 4'
carbons. This structure effectively "locks" the ribose in the
3'-endo structural conformation. The addition of locked nucleic
acids to siRNAs has been shown to increase siRNA stability in
serum, and to reduce off-target effects (Elmen, J. et al., (2005)
Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol
Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids
Research 31(12):3185-3193).
Representative U.S. Patents that teach the preparation of locked
nucleic acid nucleotides include, but are not limited to, the
following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;
6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is
herein incorporated by reference in its entirety.
Another modification of the nucleic acid of an inhibitory nucleic
acid featured in the invention involves chemically linking to the
nucleic acid one or more ligands, moieties or conjugates that
enhance the activity, cellular distribution, pharmacokinetic
properties, or cellular uptake of the inhibitory nucleic acid. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA,
1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med.
Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,
beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,
3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids
Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,
10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330;
Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995,
14:969-973), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra
et al., Biochim Biophys. Acta, 1995, 1264:229-237), or an
octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke
et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In one embodiment, a ligand alters the distribution, targeting or
lifetime of an inhibitory nucleic acid agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g, molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand. Preferred ligands will not take part
in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a
protein (e.g., human serum albumin (HSA), low-density lipoprotein
(LDL), or globulin); carbohydrate (e.g., a dextran, pullulan,
chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a
lipid. The ligand may also be a recombinant or synthetic molecule,
such as a synthetic polymer, e.g., a synthetic polyamino acid.
Examples of polyamino acids include polylysine (PLL), poly L
aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride
copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl
ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide
copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol
(PVA), polyurethane, poly(2-ethylacryllic acid),
N-isopropylacrylamide polymers, or polyphosphazine. Example of
polyamines include: polyethylenimine, polylysine (PLL), spermine,
spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic
polyamine, dendrimer polyamine, arginine, amidine, protamine,
cationic lipid, cationic porphyrin, quaternary salt of a polyamine,
or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue
targeting agent, e.g., a lectin, glycoprotein, lipid or protein,
e.g., an antibody, that binds to a specified cell type such as an
hepatopcyte or a macrophage, among others. A targeting group can be
a thyrotropin, melanotropin, lectin, glycoprotein, surfactant
protein A, Mucin carbohydrate, multivalent lactose, multivalent
galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine
multivalent mannose, multivalent fucose, glycosylated
polyaminoacids, multivalent galactose, transferrin, bisphosphonate,
polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile
acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or
RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g.
acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins
(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons
(e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
EDTA), lipophilic molecules, e.g, cholesterol, cholic acid,
adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and
peptide conjugates (e.g., antennapedia peptide, Tat peptide),
alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),
MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled
markers, enzymes, haptens (e.g. biotin), transport/absorption
facilitators (e.g., aspirin, vitamin E, folic acid), synthetic
ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole
clusters, acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, or AP
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,
molecules having a specific affinity for a co-ligand, or antibodies
e.g., an antibody, that binds to a specified cell type such as a
hepatocyte or macrophage. Ligands may also include hormones and
hormone receptors. They can also include non-peptidic species, such
as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent
lactose, multivalent galactose, N-acetyl-galactosamine,
N-acetyl-gulucosamine multivalent mannose, or multivalent
fucose.
The ligand can be a substance, e.g, a drug, which can increase the
uptake of the inhibitory nucleic acid agent into the cell, for
example, by disrupting the cell's cytoskeleton, e.g., by disrupting
the cell's microtubules, microfilaments, and/or intermediate
filaments. The drug can be, for example, taxon, vincristine,
vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin
A, phalloidin, swinholide A, indanocine, or myoservin.
In some embodiments, a ligand attached to an inhibitory nucleic
acid as described herein acts as a pharmacokinetic (PK) modulator.
As used herein, a "PK modulator" refers to a pharmacokinetic
modulator. PK modulators include lipophiles, bile acids, steroids,
phospholipid analogues, peptides, protein binding agents, PEG,
vitamins etc. Examplary PK modulators include, but are not limited
to, cholesterol, fatty acids, cholic acid, lithocholic acid,
dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,
naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that
comprise a number of phosphorothioate linkages are also known to
bind to serum protein, thus short oligonucleotides, e.g.,
oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,
comprising multiple of phosphorothioate linkages in the backbaone
are also amenable to the present invention as ligands (e.g. as PK
modulating ligands). In addition, aptamers that bind serum
components (e.g. serum proteins) are also suitable for use as PK
modulating ligands in the embodiments described herein.
For macromolecular drugs and hydrophilic drug molecules, which
cannot easily cross bilayer membranes, entrapment in
endosomal/lysosomal compartments of the cell is thought to be the
biggest hurdle for effective delivery to their site of action. A
number of approaches and strategies have been devised to address
this problem. For liposomal formulations, the use of fusogenic
lipids in the formulation have been the most common approach
(Singh, R. S., Goncalves, C. et al. (2004). On the Gene Delivery
Efficacies of pH-Sensitive Cationic Lipids via Endosomal
Protonation. A Chemical Biology Investigation. Chem. Biol. 11,
713-723.). Other components, which exhibit pH-sensitive
endosomolytic activity through protonation and/or pH-induced
conformational changes, include charged polymers and peptides.
Examples may be found in Hoffman, A. S., Stayton, P. S. et al.
(2002). Design of "smart" polymers that can direct intracellular
drug delivery. Polymers Adv. Technol. 13, 992-999; Kakudo, Chaki,
T., S. et al. (2004). Transferrin-Modified Liposomes Equipped with
a pH-Sensitive Fusogenic Peptide: An Artificial Viral-like Delivery
System. Biochemistry 436, 5618-5628; Yessine, M. A. and Leroux, J.
C. (2004). Membrane-destabilizing polyanions: interaction with
lipid bilayers and endosomal escape of biomacromolecules. Adv. Drug
Deliv. Rev. 56, 999-1021; Oliveira, S., van Rooy, I. et al. (2007).
Fusogenic peptides enhance endosomal escape improving inhibitory
nucleic acid-induced silencing of oncogenes. Int. J. Pharm. 331,
211-4. They have generally been used in the context of drug
delivery systems, such as liposomes or lipoplexes. For folate
receptor-mediated delivery using liposomal formulations, for
instance, a pH-sensitive fusogenic peptide has been incorporated
into the liposomes and shown to enhance the activity through
improving the unloading of drug during the uptake process (Turk, M.
J., Reddy, J. A. et al. (2002). Characterization of a novel
pH-sensitive peptide that enhances drug release from
folate-targeted liposomes at endosomal pHs is described in Biochim
Biophys. Acta 1559, 56-68).
In certain embodiments, the endosomolytic components can be
polyanionic peptides or peptidomimetics which show pH-dependent
membrane activity and/or fusogenicity. A peptidomimetic can be a
small protein-like chain designed to mimic a peptide. A
peptidomimetic can arise from modification of an existing peptide
in order to alter the molecule's properties, or the synthesis of a
peptide-like molecule using unnatural amino acids or their analogs.
In certain embodiments, they have improved stability and/or
biological activity when compared to a peptide. In certain
embodiments, the endosomolytic component assumes its active
conformation at endosomal pH (e.g., pH 5-6). The "active"
conformation is that conformation in which the endosomolytic
component promotes lysis of the endosome and/or transport of the
modular composition of the invention, or its any of its components
(e.g., a nucleic acid), from the endosome to the cytoplasm of the
cell.
Exemplary endosomolytic components include the GALA peptide
(Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA
peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586),
and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002,
1559: 56-68). In certain embodiments, the endosomolytic component
can contain a chemical group (e.g., an amino acid) which will
undergo a change in charge or protonation in response to a change
in pH. The endosomolytic component may be linear or branched.
Exemplary primary sequences of endosomolytic components include
H2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H (SEQ ID NO: 16);
H2N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO2H (SEQ ID NO: 17); and
H2N-(ALEALAEALEALAEA)-CONH2 (SEQ ID NO: 18).
In certain embodiments, more than one endosomolytic component can
be incorporated into the inhibitory nucleic acid agent of the
invention. In some embodiments, this will entail incorporating more
than one of the same endosomolytic component into the inhibitory
nucleic acid agent. In other embodiments, this will entail
incorporating two or more different endosomolytic components into
inhibitory nucleic acid agent.
These endosomolytic components can mediate endosomal escape by, for
example, changing conformation at endosomal pH. In certain
embodiments, the endosomolytic components can exist in a random
coil conformation at neutral pH and rearrange to an amphipathic
helix at endosomal pH. As a consequence of this conformational
transition, these peptides may insert into the lipid membrane of
the endosome, causing leakage of the endosomal contents into the
cytoplasm. Because the conformational transition is pH-dependent,
the endosomolytic components can display little or no fusogenic
activity while circulating in the blood (pH .about.7.4). "Fusogenic
activity," as used herein, is defined as that activity which
results in disruption of a lipid membrane by the endosomolytic
component. One example of fusogenic activity is the disruption of
the endosomal membrane by the endosomolytic component, leading to
endosomal lysis or leakage and transport of one or more components
of the modular composition of the invention (e.g., the nucleic
acid) from the endosome into the cytoplasm.
Suitable endosomolytic components can be tested and identified by a
skilled artisan. For example, the ability of a compound to respond
to, e.g., change charge depending on, the pH environment can be
tested by routine methods, e.g., in a cellular assay. In certain
embodiments, a test compound is combined with or contacted with a
cell, and the cell is allowed to internalize the test compound,
e.g., by endocytosis. An endosome preparation can then be made from
the contacted cells and the endosome preparation compared to an
endosome preparation from control cells. A change, e.g., a
decrease, in the endosome fraction from the contacted cell vs. the
control cell indicates that the test compound can function as a
fusogenic agent. Alternatively, the contacted cell and control cell
can be evaluated, e.g., by microscopy, e.g., by light or electron
microscopy, to determine a difference in the endosome population in
the cells. The test compound and/or the endosomes can labeled,
e.g., to quantify endosomal leakage.
In another type of assay, an inhibitory nucleic acid agent
described herein is constructed using one or more test or putative
fusogenic agents. The inhibitory nucleic acid agent can be labeled
for easy visulization. The ability of the endosomolytic component
to promote endosomal escape, once the inhibitory nucleic acid agent
is taken up by the cell, can be evaluated, e.g., by preparation of
an endosome preparation, or by microscopy techniques, which enable
visualization of the labeled inhibitory nucleic acid agent in the
cytoplasm of the cell. In certain other embodiments, the inhibition
of gene expression, or any other physiological parameter, may be
used as a surrogate marker for endosomal escape.
In other embodiments, circular dichroism spectroscopy can be used
to identify compounds that exhibit a pH-dependent structural
transition. A two-step assay can also be performed, wherein a first
assay evaluates the ability of a test compound alone to respond to
changes in pH, and a second assay evaluates the ability of a
modular composition that includes the test compound to respond to
changes in pH.
In one embodiment of the aspects described herein, a ligand or
conjugate is a lipid or lipid-based molecule. Such a lipid or
lipid-based molecule preferably binds a serum protein, e.g., human
serum albumin (HSA). An HSA binding ligand allows for distribution
of the conjugate to a target tissue, e.g., a non-kidney target
tissue of the body. Other molecules that can bind HSA can also be
used as ligands. For example, neproxin or aspirin can be used. A
lipid or lipid-based ligand can (a) increase resistance to
degradation of the conjugate, (b) increase targeting or transport
into a target cell or cell membrane, and/or (c) can be used to
adjust binding to a serum protein, e.g., HSA.
In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, such agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
Peptides suitable for use with the present invention can be a
natural peptide, e.g., tat or antennopedia peptide, a synthetic
peptide, or a peptidomimetic. Furthermore, the peptide can be a
modified peptide, for example peptide can comprise non-peptide or
pseudo-peptide linkages, and D-amino acids. A peptidomimetic (also
referred to herein as an oligopeptidomimetic) is a molecule capable
of folding into a defined three-dimensional structure similar to a
natural peptide. The attachment of peptide and peptidomimetics to
inhibitory nucleic acid agents can affect pharmacokinetic
distribution of the inhibitory nucleic acid, such as by enhancing
cellular recognition and absorption. The peptide or peptidomimetic
moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15,
20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation
peptide, cationic peptide, amphipathic peptide, or hydrophobic
peptide (e.g., consisting primarily of Tyr, Trp or Phe). The
peptide moiety can be a dendrimer peptide, constrained peptide or
crosslinked peptide. In another alternative, the peptide moiety can
include a hydrophobic membrane translocation sequence (MTS). An
exemplary hydrophobic MTS-containing peptide is RFGF having the
amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 19). An RFGF
analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 20))
containing a hydrophobic MTS can also be a targeting moiety. The
peptide moiety can be a "delivery" peptide, which can carry large
polar molecules including peptides, oligonucleotides, and protein
across cell membranes. For example, sequences from the HIV Tat
protein (GRKKRRQRRRPPQ (SEQ ID NO: 21)) and the Drosophila
Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 22)) have been
found to be capable of functioning as delivery peptides. A peptide
or peptidomimetic can be encoded by a random sequence of DNA, such
as a peptide identified from a phage-display library, or
one-bead-one-compound (OBOC) combinatorial library (Lam et al.,
Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic
tethered to a dsRNA agent via an incorporated monomer unit is a
cell targeting peptide such as an arginine-glycine-aspartic acid
(RGD)-peptide, or RGD mimic A peptide moiety can range in length
from about 5 amino acids to about 40 amino acids. The peptide
moieties can have a structural modification, such as to increase
stability or direct conformational properties. Any of the
structural modifications described below can be utilized.
A "cell permeation peptide" is capable of permeating a cell, e.g.,
a microbial cell, such as a bacterial or fungal cell, or a
mammalian cell, such as a human cell. A microbial cell-permeating
peptide can be, for example, an .alpha.-helical linear peptide
(e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide
(e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a
peptide containing only one or two dominating amino acids (e.g.,
PR-39 or indolicidin). A cell permeation peptide can also include a
nuclear localization signal (NLS). For example, a cell permeation
peptide can be a bipartite amphipathic peptide, such as MPG, which
is derived from the fusion peptide domain of HIV-1 gp41 and the NLS
of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.
31:2717-2724, 2003).
In some embodiments, the inhibitory nucleic acid oligonucleotides
described herein further comprise carbohydrate conjugates. The
carbohydrate conjugates are advantageous for the in vivo delivery
of nucleic acids, as well as compositions suitable for in vivo
therapeutic use, as described herein. As used herein,
"carbohydrate" refers to a compound which is either a carbohydrate
per se made up of one or more monosaccharide units having at least
6 carbon atoms (which may be linear, branched or cyclic) with an
oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a
compound having as a part thereof a carbohydrate moiety made up of
one or more monosaccharide units each having at least six carbon
atoms (which may be linear, branched or cyclic), with an oxygen,
nitrogen or sulfur atom bonded to each carbon atom. Representative
carbohydrates include the sugars (mono-, di-, tri- and
oligosaccharides containing from about 4-9 monosaccharide units),
and polysaccharides such as starches, glycogen, cellulose and
polysaccharide gums. Specific monosaccharides include C5 and above
(preferably C5-C8) sugars; di- and trisaccharides include sugars
having two or three monosaccharide units (preferably C5-C8). In
some embodiments, the carbohydrate conjugate further comprises
other ligand such as, but not limited to, PK modulator,
endosomolytic ligand, and cell permeation peptide.
In some embodiments, the conjugates described herein can be
attached to the inhibitory nucleic acid oligonucleotide with
various linkers that can be cleavable or non cleavable. The term
"linker" or "linking group" means an organic moiety that connects
two parts of a compound. Linkers typically comprise a direct bond
or an atom such as oxygen or sulfur, a unit such as NR8, C(O),
C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not
limited to, substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,
heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,
heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,
heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,
alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,
alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
alkylheteroarylalkenyl, alkylheteroarylalkynyl,
alkenylheteroarylalkyl, alkenylheteroarylalkenyl,
alkenylheteroarylalkynyl, alkynylheteroarylalkyl,
alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,
alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or
more methylenes can be interrupted or terminated by O, S, S(O),
SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted
aliphatic. In one embodiment, the linker is between 1-24 atoms,
preferably 4-24 atoms, preferably 6-18 atoms, more preferably 8-18
atoms, and most preferably 8-16 atoms.
A cleavable linking group is one which is sufficiently stable
outside the cell, but which upon entry into a target cell is
cleaved to release the two parts the linker is holding together. In
a preferred embodiment, the cleavable linking group is cleaved at
least 10 times or more, preferably at least 100 times faster in the
target cell or under a first reference condition (which can, e.g.,
be selected to mimic or represent intracellular conditions) than in
the blood of a subject, or under a second reference condition
(which can, e.g., be selected to mimic or represent conditions
found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g.,
pH, redox potential or the presence of degradative molecules.
Generally, cleavage agents are more prevalent or found at higher
levels or activities inside cells than in serum or blood. Examples
of such degradative agents include: redox agents which are selected
for particular substrates or which have no substrate specificity,
including, e.g., oxidative or reductive enzymes or reductive agents
such as mercaptans, present in cells, that can degrade a redox
cleavable linking group by reduction; esterases; endosomes or
agents that can create an acidic environment, e.g., those that
result in a pH of five or lower; enzymes that can hydrolyze or
degrade an acid cleavable linking group by acting as a general
acid, peptidases (which can be substrate specific), and
phosphatases.
A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The pH of human serum is 7.4, while the average
intracellular pH is slightly lower, ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and
lysosomes have an even more acidic pH at around 5.0. Some linkers
will have a cleavable linking group that is cleaved at a preferred
pH, thereby releasing the cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by
a particular enzyme. The type of cleavable linking group
incorporated into a linker can depend on the cell to be targeted.
Further examples of cleavable linking groups include but are not
limited to, redox-cleavable linking groups (e.g. a disulphide
linking group (--S--S--)), phosphate-based cleavable linkage
groups, ester-based cleavable linking groups, and peptide-based
cleavable linking groups. Representative U.S. patents that teach
the preparation of RNA conjugates include, but are not limited to,
U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584;
5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;
5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;
4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;
5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;
5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;
5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297;
7,037,646; each of which is herein incorporated by reference.
In general, the suitability of a candidate cleavable linking group
can be evaluated by testing the ability of a degradative agent (or
condition) to cleave the candidate linking group. It will also be
desirable to also test the candidate cleavable linking group for
the ability to resist cleavage in the blood or when in contact with
other non-target tissue. Thus one can determine the relative
susceptibility to cleavage between a first and a second condition,
where the first is selected to be indicative of cleavage in a
target cell and the second is selected to be indicative of cleavage
in other tissues or biological fluids, e.g., blood or serum. The
evaluations can be carried out in cell free systems, in cells, in
cell culture, in organ or tissue culture, or in whole animals. It
may be useful to make initial evaluations in cell-free or culture
conditions and to confirm by further evaluations in whole animals.
In preferred embodiments, useful candidate compounds are cleaved at
least 2, 4, 10 or 100 times faster in the cell (or under in vitro
conditions selected to mimic intracellular conditions) as compared
to blood or serum (or under in vitro conditions selected to mimic
extracellular conditions).
It is not necessary for all positions in a given compound to be
uniformly modified, and in fact more than one of the aforementioned
modifications can be incorporated in a single compound or even at a
single nucleoside within an inhibitory nucleic acid. The present
invention also includes inhibitory nucleic acid compounds that are
chimeric compounds. "Chimeric" inhibitory nucleic acid compounds or
"chimeras," in the context of this invention, are inhibitory
nucleic acid compounds, e.g. dsRNAs, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of a dsRNA compound. These
inhibitory nucleic acid typically contain at least one region
wherein the nucleic acid is modified so as to confer upon the
inhibitory nucleic acid increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
inhibitory nucleic acid may serve as a substrate for enzymes
capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example,
RNase H is a cellular endonuclease which cleaves the RNA strand of
an RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of inhibitory nucleic acid inhibition of gene
expression. Consequently, comparable results can often be obtained
with shorter inhibitory nucleic acids when chimeric inhibitory
nucleic acids are used, compared to, e.g., phosphorothioate deoxy
dsRNAs hybridizing to the same target region. Cleavage of the RNA
target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
In certain instances, the nucleic acid of an inhibitory nucleic
acid can be modified by a non-ligand group. A number of non-ligand
molecules have been conjugated to inhibitory nucleic acids in order
to enhance the activity, cellular distribution or cellular uptake
of the inhibitory nucleic acid, and procedures for performing such
conjugations are available in the scientific literature. Such
non-ligand moieties have included lipid moieties, such as
cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007,
365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,
86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett.,
1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et
al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.
Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,
1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk
et al., Biochimie, 1993, 75:49), a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res.,
1990, 18:3777), a polyamine or a polyethylene glycol chain
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or
adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36:3651), a palmityl moiety (Mishra et al., Biochim Biophys. Acta,
1995, 1264:229), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such nucleic acid conjugates
have been listed above. Typical conjugation protocols involve the
synthesis of an nucleic acid bearing an aminolinker at one or more
positions of the sequence. The amino group is then reacted with the
molecule being conjugated using appropriate coupling or activating
reagents. The conjugation reaction may be performed either with the
nucleic acid still bound to the solid support or following cleavage
of the nucleic acid, in solution phase. Purification of the nucleic
acid conjugate by HPLC typically affords the pure conjugate.
The term "aptamer" refers to a nucleic acid molecule that is
capable of binding to a target molecule, such as a polypeptide. For
example, an aptamer of the invention can specifically bind to a
target molecule, or to a molecule in a signaling pathway that
modulates the expression and/or activity of a target molecule. The
generation and therapeutic use of aptamers are well established in
the art. See, e.g., U.S. Pat. No. 5,475,096.
As used herein, the term "specific binding" refers to a chemical
interaction between two molecules, compounds, cells and/or
particles wherein the first entity binds to the second, target
entity with greater specificity and affinity than it binds to a
third entity which is a non-target. In some embodiments, specific
binding can refer to an affinity of the first entity for the second
target entity which is at least 10 times, at least 50 times, at
least 100 times, at least 500 times, at least 1000 times or greater
than the affinity for the third nontarget entity. A reagent
specific for a given target is one that exhibits specific binding
for that target under the conditions of the assay being
utilized.
As used herein, the terms "treat," "treatment" "treating," or
"amelioration" refer to therapeutic treatments, wherein the object
is to reverse, alleviate, ameliorate, inhibit, slow down or stop
the progression or severity of a condition associated with a
disease or disorder, e.g. cancer. The term "treating" includes
reducing or alleviating at least one adverse effect or symptom of a
condition, disease or disorder associated with a cancer. Treatment
is generally "effective" if one or more symptoms or clinical
markers are reduced. Alternatively, treatment is "effective" if the
progression of a disease is reduced or halted. That is, "treatment"
includes not just the improvement of symptoms or markers, but also
a cessation of, or at least slowing of, progress or worsening of
symptoms compared to what would be expected in the absence of
treatment. Beneficial or desired clinical results include, but are
not limited to, alleviation of one or more symptom(s), diminishment
of extent of disease, stabilized (i.e., not worsening) state of
disease, delay or slowing of disease progression, amelioration or
palliation of the disease state, remission (whether partial or
total), and/or decreased mortality, whether detectable or
undetectable. The term "treatment" of a disease also includes
providing relief from the symptoms or side-effects of the disease
(including palliative treatment).
As used herein, the term "pharmaceutical composition" refers to the
active agent in combination with a pharmaceutically acceptable
carrier e.g. a carrier commonly used in the pharmaceutical
industry. The phrase "pharmaceutically acceptable" is employed
herein to refer to those compounds, materials, compositions, and/or
dosage forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
As used herein, the term "administering," refers to the placement
of a compound as disclosed herein into a subject by a method or
route which results in at least partial delivery of the agent at a
desired site. Pharmaceutical compositions comprising the compounds
disclosed herein can be administered by any appropriate route which
results in an effective treatment in the subject.
The term "statistically significant" or "significantly" refers to
statistical significance and generally means a two standard
deviation (2SD) or greater difference.
Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or reaction
conditions used herein should be understood as modified in all
instances by the term "about." The term "about" when used in
connection with percentages can mean.+-.1%.
As used herein the term "comprising" or "comprises" is used in
reference to compositions, methods, and respective component(s)
thereof, that are essential to the method or composition, yet open
to the inclusion of unspecified elements, whether essential or
not.
The term "consisting of" refers to compositions, methods, and
respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
As used herein the term "consisting essentially of" refers to those
elements required for a given embodiment. The term permits the
presence of elements that do not materially affect the basic and
novel or functional characteristic(s) of that embodiment.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of this disclosure, suitable methods and materials are
described below. The abbreviation, "e.g." is derived from the Latin
exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
Definitions of common terms in cell biology and molecular biology
can be found in "The Merck Manual of Diagnosis and Therapy", 19th
Edition, published by Merck Research Laboratories, 2006 (ISBN
0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); Benjamin Lewin, Genes X, published by Jones &
Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al.
(eds.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8) and Current Protocols in Protein Sciences 2009,
Wiley Intersciences, Coligan et al., eds.
Unless otherwise stated, the present invention was performed using
standard procedures, as described, for example in Sambrook et al.,
Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et
al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1995); or Methods in Enzymology:
Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A.
R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current
Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed.,
John Wiley and Sons, Inc.), Current Protocols in Cell Biology
(CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.),
and Culture of Animal Cells: A Manual of Basic Technique by R. Ian
Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell
Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather
and David Barnes editors, Academic Press, 1st edition, 1998) which
are all incorporated by reference herein in their entireties.
Other terms are defined herein within the description of the
various aspects of the invention.
All patents and other publications; including literature
references, issued patents, published patent applications, and
co-pending patent applications; cited throughout this application
are expressly incorporated herein by reference for the purpose of
describing and disclosing, for example, the methodologies described
in such publications that might be used in connection with the
technology described herein. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to
be exhaustive or to limit the disclosure to the precise form
disclosed. While specific embodiments of, and examples for, the
disclosure are described herein for illustrative purposes, various
equivalent modifications are possible within the scope of the
disclosure, as those skilled in the relevant art will recognize.
For example, while method steps or functions are presented in a
given order, alternative embodiments may perform functions in a
different order, or functions may be performed substantially
concurrently. The teachings of the disclosure provided herein can
be applied to other procedures or methods as appropriate. The
various embodiments described herein can be combined to provide
further embodiments. Aspects of the disclosure can be modified, if
necessary, to employ the compositions, functions and concepts of
the above references and application to provide yet further
embodiments of the disclosure. These and other changes can be made
to the disclosure in light of the detailed description. All such
modifications are intended to be included within the scope of the
appended claims.
Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
The technology described herein is further illustrated by the
following examples which in no way should be construed as being
further limiting.
Some embodiments of the technology described herein can be defined
according to any of the following numbered paragraphs: 1. A
chimeric molecule comprising a cancer marker-binding aptamer domain
and an inhibitory nucleic acid domain. 2. The molecule of paragraph
1, wherein the cancer marker is EpCAM or EphA2. 3. The molecule of
any of paragraphs 1-2, wherein the molecule is an aptamer-siRNA
chimera (AsiC). 4. The molecule of any of paragraphs 1-3, wherein
the inhibitory nucleic acid specifically binds to a gene product
upregulated in a cancer cell. 5. The molecule of any of paragraphs
1-4, wherein the inhibitory nucleic acid inhibits the expression of
a gene selected from the group consisting of: Plk1; MCL1; EphA2;
PsmA2; MSI1; BMI1; XBP1; PRPF8; PFPF38A; RBM22; USP39; RAN; NUP205;
and NDC80. 6. The molecule of any of paragraphs 1-5, wherein the
cancer marker is EpCAM and the inhibitory nucleic acid domain
inhibits the expression of Plk1. 7. The molecule of any of
paragraphs 1-6, wherein the cancer marker-binding aptamer domain
comprises the sequence of SEQ ID NO: 33. 8. The molecule of any of
paragraphs 1-6, wherein the cancer marker-binding aptamer domain
consists essentially of the sequence of SEQ ID NO: 33. 9. The
molecule of any of paragraphs 1-8, wherein the inhibitory nucleic
acid domain comprises the sequence of SEQ ID NO: 2. 10. The
molecule of any of paragraphs 1-8, wherein the inhibitory nucleic
acid domain consists essentially of the sequence of SEQ ID NO: 2.
11. The molecule of any of paragraphs 1-10, comprising the sequence
of one of SEQ ID NOs: 1-3. 12. The molecule of any of paragraphs
1-11, consisting essentially of the sequence of one of SEQ ID NOs:
1-3. 13. The molecule of any of paragraphs 1-12, wherein the 3' end
of the molecule comprises dTdT. 14. The molecule of any of
paragraphs 1-13, wherein the molecule comprises at least one 2'-F
pyrimidine. 15. A pharmaceutical composition comprising the
molecule of any of paragraphs 1-14 and a pharmaceutically
acceptable carrier. 16. The composition of paragraph 15, comprising
at least two chimeric molecules of any of paragraphs 1-14, wherein
the chimeric molecules have different aptamer domains or inhibitory
nucleic acid domains. 17. The composition of paragraph 16, wherein
different apatmer or inhibitory nucleic acid domains recognize
different targets. 18. The composition of paragraph 16, wherein
different apatmer or inhibitory nucleic acid domains have sequences
and recognize the same target. 19. A method of treating cancer, the
method comprising administering a molecule or composition of any of
paragraphs 1-18. 20. The method of paragraph 19, wherein the cancer
is an epithelial cancer or breast cancer 21. The method of
paragraph 20, wherein the breast cancer is triple-negative breast
cancer. 22. The method of any of paragraphs 19-21, wherein the
administration is subcutaneous. 23. The method of any of paragraphs
19-22, wherein the subject is further administered an additional
cancer treatment. 24. The method of paragraph 23, wherein the
cancer treatment is paclitaxel.
EXAMPLES
Example 1: Gene Knockdown by EpCAM Aptamer-siRNA Chimeras Inhibits
Basal-Like Triple Negative Breast Cancers and their
Tumor-Initiating Cells
Effective therapeutic strategies for in vivo siRNA delivery to
knockdown genes in cells outside the liver are needed to harness
RNA interference for treating cancer. EpCAM is a tumor-associated
antigen highly expressed on common epithelial cancers and their
tumor-initiating cells (T-IC, also known as cancer stem cells). It
is demonstrated herein that aptamer-siRNA chimeras (AsiC, an EpCAM
aptamer linked to an siRNA sense strand and annealed to the siRNA
antisense strand) are selectively taken up and knockdown gene
expression in EpCAM+ cancer cells in vitro and in human cancer
biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere
formation (in vitro T-IC assays) and tumor initiation by EpCAM+
luminal and basal-A triple negative breast cancer (TNBC) cell
lines, but not EpCAM- mesenchymal basal-B TNBCs, in nude mice.
Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM+ Her2+
and TNBC tumors and suppress their growth. Thus EpCAM-AsiCs provide
an attractive approach for treating epithelial cancer.
Introduction
RNA interference (RNAi) offers the opportunity to treat disease by
knocking down disease-causing genes..sup.1 Recent early phase
clinical trials have shown vigorous (75-95%), sustained (lasting
for several weeks or up to several months) and safe knockdown of a
handful of gene targets in the liver using lipid
nanoparticle-encapsulated or GalNAc-conjugated siRNAs..sup.2-5 The
liver, the body's major filtering organ, traps particles and,
hence, is relatively easy to transfect. The major obstacle to
harnessing RNAi for treating most diseases however has yet to be
solved--namely efficient delivery of small RNAs and gene knockdown
in cells beyond the liver. In particular, the delivery roadblock is
a major obstacle to harnessing RNAi to treat cancer..sup.6
Triple negative breast cancers (TNBC), a heterogeneous group of
poorly differentiated cancers defined by the lack of estrogen,
progesterone and Her2 receptor expression, has the worst prognosis
of any breast cancer subtype..sup.7-9 Most TNBCs have epithelial
properties and are classified as basal-like or belong to the
basal-A subtype, although a sizable minority are mesenchymal
(basal-B subtype). TNBC afflicts younger women and is the subtype
associated with BRCA1 genetic mutations. No targeted therapy is
available. Although most TNBC patients respond to chemotherapy,
within 3 years about a third develop metastases and eventually die.
Thus new approaches are needed.
Described herein is a flexible, targeted platform for gene
knockdown and treatment of basal-like TNBCs that might also be
suitable for therapy against most of the common (epithelial)
cancers. We deliver small interfering RNAs (siRNA) into epithelial
cancer cells by linking them to an RNA aptamer that binds to EpCAM,
the first described tumor antigen, a cell surface receptor
over-expressed on epithelial cancers, including basal-like TNBCs.
Aptamer-linked siRNAs, known as aptamer-siRNA chimeras (AsiC), have
been used in small animal models to treat prostate cancer and
prevent HIV infection..sup.10-18 We chose EpCAM for targeting
basal-like TNBC because EpCAM is highly expressed on epithelial
cancers. A high affinity EpCAM aptamer was previously identified.
{Shigdar, 2011 #17903} EpCAM also marks tumor-initiating cells
(T-ICs, also known as cancer stem cells)..sup.20-27 These cells are
thought responsible not only for initiating tumors, but are also
relatively resistant to conventional cytotoxic therapy and are
thought responsible for tumor relapse and metastasis. Devising
therapies to eliminate T-ICs is an important unmet goal of cancer
research..sup.28
In normal epithelia, EpCAM is only weakly expressed on basolateral
gap junctions, where it may not be accessible to drugs..sup.29 In
epithelial cancers it is not only more abundant (by orders of
magnitude), but is also distributed along the cell membrane.
Ligation of EpCAM promotes adhesion and enhances cell proliferation
and invasivity. Proteolytic cleavage of EpCAM releases an
intracellular fragment that increases stem cell factor
transcription..sup.30,31 EpCAM's oncogenic properties may make it
difficult for tumor cells to develop resistance by down-modulating
EpCAM. In one study about 2/3 of TNBCs, presumably the basal-A
subtype, stained strongly for EpCAM..sup.25 The number of EpCAM+
circulating cells is linked to poor prognosis in breast
cancer..sup.32-36 An EpCAM antibody has been evaluated clinically
for epithelial cancers, but had limited effectiveness on its
own..sup.37-39 EpCAM expression identifies circulating tumor cells
in an FDA-approved method for monitoring metastatic breast, colon
and prostate cancer treatment.sup.32-36. Moreover, about 97% of
human breast cancers and virtually 100% of other common epithelial
cancers, including lung, colon, pancreas and prostate, stain
brightly for EpCAM, suggesting that the platform developed here
could be adapted for RNAi-based therapy of common cancers.
It is demonstrated herein that all epithelial breast cancer cell
lines tested stained brightly for EpCAM, while immortalized normal
breast epithelial cells, fibroblasts and mesenchymal tumor cell
lines did not. EpCAM-AsiCs caused targeted gene knockdown in
luminal and basal-A TNBC cancer cells and human breast cancer
tissues in vitro, but not in normal epithelial cells, mesenchymal
tumor cells or normal human breast tissues. Knockdown was
proportional to EpCAM expression. Moreover EpCAM-AsiC-mediated
knockdown of PLK1, a gene required for mitosis, suppressed in vitro
T-IC functional assays (colony and mammosphere formation) of
epithelial breast cancer lines. Ex vivo treatment specifically
abrogated tumor initiation. Subcutaneously injected PLK1
EpCAM-AsiCs were taken up specifically by EpCAM+ basal-A triple
negative breast cancer (TNBC) orthotopic xenografts of poor
prognosis basal-A and Her2 breast cancers and caused rapid tumor
regression.
EpCAM is Highly Expressed on Epithelial Breast Cancer Cell
Lines
First, EpCAM expression was examined in breast cancer cell lines.
Based on gene expression data in the Cancer Cell Line
Encyclopedia40,EpCAM mRNA is highly expressed in basal-A TNBC and
luminal breast cancer cell lines, but poorly in basal-B
(mesenchymal) TNBCs (FIG. 1A). Surface EpCAM staining, assessed by
flow cytometry, was 2-3 logs brighter in all luminal and basal-like
cell lines tested, than in normal epithelia immortalized with hTERT
(BPE).sup.41, fibroblasts or mesenchymal TNBCs (FIG. 1B). Thus
EpCAM is highly expressed in epithelial breast cancer cell lines
compared to normal cells or mesenchymal tumors.
EpCAM-AsiCs Selectively Knock Down Gene Expression in EpCAM+ Breast
Cancer Cells
A 19 nucleotide (nt) aptamer that binds to human EpCAM with 12 nM
affinity.sup.19 was identified by SELEX..sup.42,43 It does not bind
to mouse EpCAM (data not shown). A handful of EpCAM-AsiCs that
linked either the sense or antisense strand of the siRNA to the
3'-end of the aptamer by several linkers were designed and
synthesized with 2'-fluoropyrimidine substitutions and 3'-dTdT
overhangs to enhance in vivo stability, avoid off-target knockdown
of partially complementary genes bearing similar sequences, and
limit innate immune receptor stimulation. To test RNA delivery,
gene knockdown and anti-tumor effects, siRNAs were incorporated to
knockdown eGFP (as a useful marker gene); AKT1, an endogenous gene
expressed in all the cell lines studied, whose knockdown is not
lethal; and PLK1, a kinase required for mitosis, whose knockdown is
lethal (FIG. 9). The AsiC that performed best in dose response
studies of gene knockdown joined the 19 nt EpCAM aptamer to the
sense (inactive) strand of the siRNA via a U-U-U linker (FIG. 1C).
The EpCAM-AsiC was produced by annealing the chemically synthesized
.about.42-44 nt long strand (19 nt aptamer+linker+20-22 nt siRNA
sense strand) to a 20-22 nt antisense siRNA strand. Commercially
synthesized with 2'-fluoropyrimidines {Jackson, 2003 #11353;
Scacheri, 2004 #11912; Jackson, 2006 #13758; Wheeler, 2011 #17906},
these are RNase resistant and very stable in human serum
(T.sub.1/2>>36 hr, FIG. 7) and do not trigger innate immunity
when injected in vivo into tumor-bearing mice (FIG. 7).
To verify selective uptake by EpCAM+ tumor cells, confocal
fluorescence microscopy was used to compare internalization of the
EpCAM aptamer, fluorescently labeled at the 5'-end with Cy3, in
EpCAM+ MDA-MB-468 TNBC cells and BPE, EpCAM.sup.dim immortalized
breast epithelial cells (data not shown). Without wishing to be
bound by theory, because AsiCs contain only one aptamer, they do
not crosslink the receptor they recognize. As a consequence,
cellular internalization is slow since it likely occurs via
receptor recycling, rather than the more rapid process of
activation-induced endocytosis.
Only MDA-MB-468 cells took up the aptamer. Uptake was clearly
detected at 22 hr, but increased greatly after 43 hr. To test
whether EpCAM-AsiCs are specifically taken up by EpCAM bright cell
lines, the 3' end of the antisense strand of the AsiC was
fluorescently labeled. EpCAM+ BPLER, a basal-A TNBC cell line
transformed from BPE by transfection with human TERT, SV40 early
region and H-RASV12, took up Alexa-647 EpCAM-AsiCs when analyzed
after a 24 hr incubation, but BPE cells did not (FIG. 1D). Previous
studies have shown that AsiCs are processed within cells by Dicer
to release the siRNA from the aptamer (10, 12, 15). To verify that
the released siRNA was taken up by the RNA induced silencing
complex (RISC), qRT-PCR was utilized to amplify that PLK1 siRNA
immunoprecipitated with Ago when MDA-MB-468 cells were incubated
with PLK1 EpCAM-AsiCs (FIG. 33). No PLK1 siRNA bound to Ago when
the same cells were incubated with PLK1 siRNAs.
TNBC cells took up Alexa-467 EpCAM-AsiCs, but no uptake was
detectable in BPE cells (FIG. 1E). Next to assess whether gene
knockdown was specific to EpCAM+ tumors, eGFP knockdown was
compared in these same cell lines, which stably express eGFP, by
eGFP EpCAM-AsiCs and lipid transfection of eGFP siRNAs (FIG. 1D).
Although transfection of eGFP siRNAs knocked down gene expression
equivalently in BPE and BPLER, Incubation with EpCAM-AsiCs in the
absence of any transfection lipid selectively knocked down
expression only in BPLER. AsiC knockdown was uniform and comparable
to that achieved with lipid transfection. Next we compared the
specific knockdown of the endogenous AKT1 gene by AKT1 AsiCs and
transfected AKT1 siRNAs in 6 breast cancer cell lines compared to
normal human fibroblasts (FIG. 1E). AKT1 was selectively knocked
down by EpCAM-AsiCs targeting AKT1 only in EpCAM.sup.bright luminal
and basal-A TNBCs, but not in mesenchymal basal-B TNBCs,
fibroblasts or BPE ells (data not shown). As expected, AsiCs
targeting eGFP had no effect on AKT1 levels and transfection of
AKT1 siRNAs comparably knocked down expression in all the cell
lines studied. Moreover, EpCAM-AsiC knockdown of AKT1 strongly
correlated with EpCAM expression (FIG. 1G). Similar results were
obtained when AKT1 protein was analyzed by flow cytometry in
stained transfected cells (FIG. 1G, 1H). Thus in vitro knockdown by
EpCAM-AsiCs is effective and specific for EpCAM.sup.bright tumor
cells.
PLK1 EpCAM-AsiCs Selectively Kill EpCAM.sup.bright Tumor Cells In
Vitro
To explore whether EpCAM-AsiCs could be used as anti-tumor agents
in breast cancer, we examined by CellTiterGlo assay the effect of
AsiCs directed against PLK1, a kinase required for mitosis, on
survival of 10 breast cancer cell lines that included 5 basal-A
TNBCs, 2 luminal cell lines, and 3 basal-B TNBCs. EpCAM-AsiCs
targeting PLK1, but not control AsiCs directed against eGFP,
decreased cell proliferation in the basal-A and luminal cell lines,
but did not inhibit basal-B cells (FIG. 2A). Lipid transfection of
PLK1 siRNAs suppressed the growth of all the cell lines. The
anti-proliferative effect strongly correlated with EpCAM expression
(FIG. 2B). The reduction in viable EpCAM+ cells after knockdown was
due to induction of apoptosis, assessed by annexin V-propidium
iodide staining and caspase activation (data not shown). To
determine whether ligation of the EpCAM aptamer contributed to the
anti-proliferative effect of the EpCAM-AsiC, we compared survival
of cells that were treated with the PLK1 EpCAM-AsiC with cells
treated with the aptamer on its own (FIG. 2C). The aptamer by
itself did not have a reproducible effect on survival of any breast
cancer cell lines, possibly because as a monomeric agent it does
not cross-link the EpCAM receptor to alter EpCAM signaling. Thus
the PLK1 EpCAM-AsiC asserts its specific anti-tumor effect on
EpCAM+ breast cancer cells by gene knockdown.
To determine whether EpCAM-AsiCs specifically target EpCAM+ cells
when mixed with EpCAM.sup.dim non-transformed epithelial cells, we
incubated co-cultures of GFP- TNBC cells and GFP+ BPE cells with
PLK1 EpCAM-AsiCs or medium and used GFP fluorescence to measure
their relative survival by flow cytometry 3 days later (FIG. 2D,
2E). EpCAM-AsiCs targeting PLK1 greatly reduced the proportion of
surviving EpCAM+ basal-A tumor cells, but had no effect on survival
of an EpCAM- basal-B cell line. Thus PLK1 EpCAM-AsiCs are
selectively cytotoxic for EpCAM+ tumor cells when mixed with normal
cells.
EpCAM-AsiCs Concentrate in EpCAM+ Breast Tumor Biopsy Specimens
It was next examined whether EpCAM-AsiCs concentrate in human
breast tumors relative to normal breast samples within intact
tissues. Paired normal tissue and breast tumor biopsies from 3
breast cancer patients were cut into cubes with .about.3 mm edges
and placed in Petri dishes. The tumor sample cells were all
EpCAM.sup.bright and the normal tissue cells were EpCAM.sup.dim
(FIG. 3A). Fluorescently labeled Alexa647-siRNAs (not expected to
be taken up by either normal tissue or tumor),
Alexa647-cholesterol-conjugated siRNAs (chol-siRNAs, expected to be
taken up by both), or Cy3-EpCAM-AsiCs were added to the culture
medium and the tissues were incubated for 24 hr before harvest. The
Cy3 signal of the AsiC, which could be visualized by the naked eye,
concentrated only in the tumor specimens and was not detected in
normal tissue (FIG. 3B). To quantify RNA uptake, flow cytometry
analysis was performed on washed single cell suspensions of the
tissue specimens (representative tumor-normal tissue pair (FIG.
3C), mean.+-.SD of triplicate biospies from 3 EpCAM.sup.bright
paired breast tumor-normal tissue samples (FIG. 3D)). The
EpCAM-AsiC was significantly taken up by the tumor, but not normal
tissue, while neither took up the unconjugated siRNA and both took
up the chol-siRNA to some extent. Thus, within intact tissue,
EpCAM-AsiCs are selectively delivered to EpCAM.sup.bright tumors
relative to normal tissue.
PLK1 EpCAM-AsiCs Inhibit T-ICs of EpCAM+ Tumors
EpCAM was chosen for targeting in part because EpCAM marks T-ICs
and metastasis-initiating cells (M-IC)..sup.20,22,26,27,31 To
investigate whether EpCAM-AsiCs inhibit T-ICs, we compared colony
and mammosphere formation (T-IC functional surrogate assays) after
mock treatment, treatment with paclitaxel or with EpCAM-AsiCs
against eGFP or PLK1. PLK1 EpCAM-AsiCs more strongly inhibited
colony and mammosphere formation of EpCAM+ basal-A TNBCs and
luminal cell lines than paclitaxel, but were inactive against
EpCAM- basal-B TNBCs (FIGS. 4A-C). T-IC inhibition was specific,
since eGFP AsiCs had no effect. Incubation with PLK1 EpCAM-AsiCs,
but not eGFP AsiCs, also reduced the proportion of cells with the
phenotype of T-ICs, namely the numbers of CD44.sup.+CD24.sup.low/-
and ALDH+ cells specifically in basal-A and luminal breast cancer
cell lines (data not shown). To evaluate the effect of EpCAM-AsiCs
on tumor initiation, EpCAM+ MB468 cells stably expressing
luciferase were treated overnight with medium or PLK1 or eGFP
EpCAM-AsiCs and equal numbers of viable cells were then implanted
sc in nude mice. PLK1 EpCAM-AsiCs completely blocked tumor
formation assessed by in vivo tumor cell luminescence (data not
shown). In contrast similar treatment of basal-B MB436 cells had no
effect on tumor initiation (data not shown). Thus PLK1 EpCAM-AsiCs
inhibit in vitro T-IC assays and tumor initiation selectively for
EpCAM+ breast cancers.
Subcutaneously Administered EpCAM-AsiCs are Selectively Taken Up by
Distant EpCAM+ TNBCs
To be clinically useful, EpCAM-AsiCs need to be taken up by
disseminated tumor cells. Intravenous injection of fluorescent
EpCAM-AsiCs in the tail vein of mice did not lead to significant
AsiC accumulation within subcutaneous tumors implanted in the
flanks of nude mice (data not shown), probably because their size
(.about.25 kDa) is below the threshold for kidney filtration and
they are rapidly excreted. Linkage to polyethylene glycol greatly
enhanced the circulating half-life, tumor accumulation and
antitumor therapeutic effect of PSMA-AsiCs in a mouse xenograft
model of prostate cancer..sup.11 However, to see if this
modification could be bypassed, we examined by live animal
epifluorescence imaging whether sc injection of Alexa750-labeled
eGFP EpCAM-AsiCs in the scruff of the neck of 7 mice led to
accumulation in distant EpCAM+ MB468 and EpCAM- MB231 TNBCs
implanted sc in each flank (FIG. 5A, 5B). Within a day of
injection, EpCAM-AsiCs concentrated only in the EpCAM+ tumor and
persisted there for at least 4 days. The EpCAM-AsiCs were detected
around the injection site on day 2, but were only found within the
EpCAM+ tumor on day 4.
PLK1 EpCAM AsiCs Cause Regression of Basal-A TNBC and her2 Breast
Cancer Xenografts
Because sc injected EpCAM-AsiCs concentrated in distant EpCAM+
tumors, we next looked at whether sc injection of PLK1 EpCAM-AsiCs
could selectively inhibit the growth of an EpCAM+ TNBC xenografted
tumor. EpCAM+ MB468-luc cells were implanted in Matrigel in one
flank of a nude mouse and EpCAM- MB231-luc-mCherry cells were
implanted on the opposite flank. Once the luciferase signal of both
tumors was clearly detected above background, groups of 5-6 mice
were mock treated or injected sc with 5 mg/kg of EpCAM-AsiCs
targeting PLK1 or eGFP every 3 d for 2 wks. Tumor growth was
followed by luminescence. All the EpCAM+ tumors rapidly completely
regressed only in mice that received the PLK1-targeting AsiCs (FIG.
6A, 6B). The EpCAM+ tumors in mice treated with eGFP-targeting
AsiCs and all the EpCAM- tumors continued to grow. This experiment
was repeated with similar results after injection of PLK1 AsiCs.
Tumors also continued to grow without significant change in
additional groups of control mice treated with just the EpCAM
aptamer or the PLK1 siRNA (data not shown) and into mice bearing
Her2+ MCF10A-CA1a (FIG. 34). Thus sc injected PLK1 EpCAM-AsiCs show
specific antitumor activity against basal-A TNBCs and EpCAM+ human
xenografts.
Discussion
Targeted therapy so far has relied on using tumor-specific
antibodies or inhibitors to oncogenic kinases. No one before has
shown that an unconjugated AsiC can have potent antitumor effects
or that AsiCs could be administered sc. There is currently no
targeted therapy for TNBC or for T-ICs. Developing targeted therapy
for TNBC and developing ways of eliminating T-ICs are important
unmet goals of cancer research.
It is demonstrated herein that EpCAM-AsiCs can be used to knockdown
genes selectively in epithelial breast cancer cells and their stem
cells, sparing normal epithelial cells and stroma, to cause tumor
regression and suppress tumor initiation. In one very aggressive
TNBC xenograft model, the EpCAM-AsiCs caused complete tumor
regression after only 3 injections. This is a flexible platform for
targeted therapy, potentially for all the common epithelial
cancers, which uniformly express high levels of EpCAM.
Although EpCAM-AsiCs targeting PLK1 was used herein, the siRNA can
be varied to knockdown any tumor dependency gene that would be
customized to the tumor subtype or the molecular characteristics of
an individual patient's tumor. AsiC cocktails targeting more than
one gene would be ideal for cancer therapeutics to lessen the
chances of developing drug resistance. Targeted cancer therapy so
far has relied on using tumor-specific antibodies or small molecule
inhibitors to oncogenic kinases. Using EpCAM as an AsiC ligand and
developing RNAi therapy to target cancer stem cells is novel. No
one before has shown that an unconjugated AsiC can have potent
antitumor effects or that AsiCs could be administered sc. Moreover,
preliminary studies of sc administered CD4-AsiCs in humanized mice
showed strong knockdown in CD4 cells in the spleen and distant
lymph nodes, suggesting that AsiCs targeting receptors on cells
located elsewhere in the body could also be administered sc. There
is currently no targeted therapy for TNBC or for T-ICs. Targeted
delivery has the advantage of reduced dosing and reduced toxicity
to bystander cells.
The major obstacle to harnessing RNAi for cancer is delivering
small RNAs into disseminated cells. Described herein is the use of
AsiCs to overcome this obstacle. Described herein is a new class of
potent anticancer drugs. AsiCs are a flexible platform that can
target different cell surface receptors and knockdown any gene or
combination of genes. {Burnett, 2012 #18447; Zhou, 2011 #18448;
Thiel, 2010 #18445} By changing the aptamer, the AsiC platform can
tackle the delivery roadblock that has thwarted the application of
RNAi-based therapy to most diseases. This approach is ideal for
personalized cancer therapy, since the choice of genes to target
can be adjusted depending on a tumor's molecular characteristics.
Moreover RNA cocktails can knockdown multiple genes at once to
anticipate and overcome drug resistance. AsiCs are the most
attractive method for gene knockdown outside the liver. They are
better than complicated liposomal, nanoparticle or conjugated
methods of delivering RNAs because they are a single chemical
entity that is stable in the blood, easy to manufacture,
nonimmunogenic, able to readily penetrate tissues and are not
trapped in the filtering organs.
An important cancer research goal is to eliminate T-ICs (cancer
stem cells). T-ICs are relatively resistant to chemotherapy and are
thought responsible for tumor relapse and metastasis. {Federici,
2011 #19371} The AsiCs described herein target (epithelial) T-ICs
with high efficiency. As such they may eliminate this aggressive
subpopulation within tumors at risk for progressive disease (see
FIG. 6A, 6B).
The small size of the EpCAM aptamer used here is ideal for an AsiC
drug, since RNAs <60 nt can be efficiently synthesized.
In addition to their potential therapeutic use, EpCAM-AsiCs are
also a powerful in vivo research tool for identifying the
dependency genes of tumors and T-ICs to define novel drug targets.
In principle, aptamer chimeras could be designed to deliver not
only siRNAs but also miRNA mimics or antagomirs, antisense
oligonucleotides that function by other mechanisms besides RNAi, or
even longer mRNAs or noncoding RNAs (50, 51). They could also be
designed to incorporate more than one aptamer, multiple siRNAs, or
even toxins or small molecule anticancer drugs.
Its small size is ideal for an AsiC drug, since RNAs <60 nt can
be efficiently synthesized. Not only is the siRNA targeted to the
tumor, but the drug targets can also be chosen to attack the
tumor's Achilles' heels by knocking down tumor dependency genes.
This flexibility can be used for personalized cancer therapy that
targets the molecular vulnerabilities of an individual patient's
cancer.
Material and Methods
Cell Culture.
Human BPE and BPLER cells were grown in WIT medium (Stemgent).
MB468 were transduced with a luciferase reporter. All other human
cell lines were obtained from ATCC and grown in MEM (MCF7, BT474),
McCoy's 5A (SKBR3), RPMI1640 (HCC1806, HCC1143, HCC1937, HCC1954,
HCC1187, MB468, T47D) or DMEM (MB231, BT549, MB436) all
supplemented with 10% FBS, 1 mM L-glutamine and
penicillin/streptomycin (Gibco) unless otherwise indicated. 4T1
mouse breast cancer cells were grown in 10% FBS DMEM. For in vivo
imaging, MB468 cells stably expressing Firefly luciferase
(MB468-luc) were used and MB231 cells stably expressing Firefly
luciferase and mCherry (MB231-luc-mCherry) were selected after
infection with pLV-Fluc-mCherry-Puro lentivirus (provided by Andrew
Kung, Columbia University). MB231 Cells were selected with
puromycin.
For uptake and silencing treatment, cells were plated at low
density (10,000 cells/well in 96-well plates) and treated
immediately. All AsiC and siRNA treatments were performed in either
OptiMEM or WIT medium. Cell viability was assessed by CellTiter-Glo
(Promega) or by Trypan-Blue staining in 96-well plates.
For colony formation assay, 1,000 viable cells were treated for 6 h
in round bottom 96-well plates and then transferred to 10-cm plates
in serum-containing medium. Medium was replaced every 3 d. After
8-14 d, cells were fixed in methanol (-20 C) and stained with
crystal violet. For sphere formation assay, 1,000/ml viable cells
were treated for 6 h in round bottom 96-well plates and then
cultured in suspension in serum-free DMEM/F12 1:1 (Invitrogen),
supplemented with EGF (20 ng/ml, BD Biosciences), B27 (1:50,
Invitrogen), 0.4% bovine serum albumin (Sigma) and 4 .mu.g/ml
insulin (Sigma). Spheres were counted after 1 or 2 weeks.
siRNA Transfection.
Cells were transfected with Dharmafect I per the manufacturer's
protocol. See FIG. 9 for all siRNA sequences.
Flow Cytometry.
For flow cytometry, cells were stained as previously described (Yu,
F. et al (2007). let-7 Regulates Self Renewal and Tumorigenicity of
Breast Cancer Cells. Cell 131, 1109-1123.), briefly, direct
immunostaining of EpCAM and AKT1 was performed using 1:50 dilutions
of hAb for 30-60 minutes at 4.degree. C. (BioLegend/BD). Cells were
stained in PBS containing 0.5% FCS, 1 mM EDTA, and 25 mM HEPES.
Samples were washed twice in the same buffer. Data was acquired
using FACS-Canto II (BD Biosciences). Analyses were performed in
triplicate and 10,000 gated events/sample were counted. All data
analysis was performed using FlowJo (Treestar Inc.).
RNA Analysis.
qRT-PCR analysis was performed as described (Petrocca, F., et al.
(2008). E2F1-regulated microRNAs impair TGFbeta-dependent
cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 13,
272-286). Briefly, total RNA was extracted with Trizol (Invitrogen)
and cDNA prepared from 1000 ng total RNA using Thermoscript RT kit
(Invitrogen) as per the manufacturer's SYBR Green Master Mix
(Applied Biosystems) and a BioRad C1000 Thermal Cycler (Biorad).
Relative CT values were normalized to GAPDH and converted to a
linear scale.
Collagenase Digestion of Human Breast Tissue.
Fresh breast or colon cancer and control biopsies were received
from the UMASS Tissue Bank, samples were cut into 3.times.3.times.3
mm samples and placed in a 96 well plate with 100 ul RPMI. Samples
were treated with either Alexa647-siRNA-GFP,
Alexa647-chol-siRNA-GFP or Cy3-AsiC-GFP for 24 hr. Samples were
photographed and digested. Three samples from each treatment were
pooled and put in 10 ml RPMI containing 1 mg/ml collagenase II
(Sigma-Aldrich) for 30 minutes at 37.degree. C. with shaking.
Samples were disrupted in a gentleMACS dissociator (Miltenyi) using
the spleen program for 30 minutes at 37.degree. C. both before and
after collagenase digestion. Cell suspensions were passed through a
70-.mu.m cell strainer (BD Falcon), washed with 30 ml RPMI, and
stained for flow cytometry.
Animal Experiments.
All animal procedures were performed with Harvard Medical School
and Boston Children's Hospital Animal Care and Use Committee
approval. Nude mice were purchased from the Jackson Laboratory.
In Vivo Experiments.
For tumor initiation studies 8-week old female Nu/J mice (Stock
#002019, Jackson Laboratories) were injected subcutaneously with
MB468-luc (5.times.10.sup.6) cells pretreated for 24 h with
EpCAM-AsiC-GFP, EpCAM-AsiC-PLK1 or untreated. Cells were
trypsinized with Tryple Express (Invitrogen), resuspended in WIT
media and injected subcutaneously in the flank. Following
intraperitoneal injection of 150 mg/kg D-luciferin (Caliper Life
Sciences) luminescent images of the whole body were taken every 5
days for a total of 20 days using the IVIS Spectra system (Caliper
Life Sciences).
For AsiC uptake experiments MB468-luc (5.times.10.sup.6) and
MB231-luc-mCherry (5.times.10.sup.5) cells trypsinized with Tryple
Express (Invitrogen), were resuspended in a 1:1 WIT-Matrigel
solution and injected subcutaneously in the flank of 8-week old
female Nu/J mice (Stock #002019, Jackson Laboratories). Tumors size
was analyzed daily using the IVIS Spectra system (Caliper Life
Sciences). After 5 days tumors were clearly visible and mice were
injected subcutaneously in the neck area with
Alexa750-EpCAM-AsiC-GFP (0.5 mg/kg). Localization of the AsiC
compared to the tumor was tested every 48 h for 7 days.
For tumor inhibition studies, MB468-luc (5.times.10.sup.6) and
MB231-luc-mCherry (5.times.10.sup.5) cells trypsinized with Tryple
Express (Invitrogen), resuspended in a 1:1 WIT-Matrigel solution
and injected subcutaneously in the flank of 8-week old female Nu/J
mice (Stock #002019, Jackson Laboratories). Tumors size was
analyzed daily using the IVIS Spectra, after 5 days tumors were
clearly visible. Mice bearing tumors of comparable size were
randomized into 5 groups and treated with 5 mg/kg of
EpCAM-AsiC-PLK1, EpCAM-AsiC-GFP, EpCAM-Aptamer, siRNA-PLK1 or
untreated. Mice were treated every 72 h for 14 days.
All Images were analyzed using Living Image.RTM. software (Caliper
Life Sciences).
Statistical Analysis.
Student's t-tests, computed using Microsoft Excel, were used to
analyze the significance between the treated samples and the
controls where the test type was set to one-tail distribution and
two-sample equal variance. To assess innate immune stimulation,
one-way analysis of variance (ANOVA) with Bonferroni's Multiple
comparison test was performed using GraphPad Prizm 4 software
(GraphPad Software, San Diego, Calif.). P<0.05 was considered
significant.
Measurement of Innate Immune Stimulation.
Mice were injected sc with eGFP EpCAM-AsiCs (5 mg/kg) or ip with
Poly(I:C) (5 or 50 mg/kg). Serum samples, collected at baseline and
6 and 16 hr after treatment were stored at -80.degree. C. before
measuring IFN.beta., IL-6 and IP-10 using the ProcartaPlex
Multiplex Immunoassay (Affymetrix/eBioscience, San Diego, Calif.).
Spleens, harvested at sacrifice 16 hr post treatment, were stored
in RNAlater (Qiagen) before extracting RNA using TRIZOL
(Invitrogen) with the gentleMACS Dissociator (MACS Miltenyi Biotec,
San Diego, Calif.). cDNA was synthesized using Superscript III and
random hexamers (Invitrogen) and PCR was performed using SsoFast
EvaGreen Supermix and a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad
Laboratories, Hercules, Calif.) using the following primers:
TABLE-US-00001 (SEQ ID NO: 4) Gapdh forward: 5'-
TTCACCACCATGGAGAAGGC-3', (SEQ ID NO: 5) Gapdh reverse: 5'-
GGCATGGACTGTGGTCATGA-3', (SEQ ID NO: 6) ifnb forward:
5'-CTGGAGCAGCTGAATGGAAAG-3', (SEQ ID NO: 7) ifnb reverse: 5'-
CTTGAAGTCCGCCCTGTAGGT-3', (SEQ ID NO: 8) il-6 forward:
5'-TGCCTTCATTTATCCCTTGAA-3', (SEQ ID NO: 9) il-6 reverse:
5'-TTACTACATTCAGCCAAAAAGCAC-3', (SEQ ID NO: 10) ip-10 forward:
5'-GCTGCCGTCATTTTCTGC-3', (SEQ ID NO: 11) ip-10 reverse:
5'-TCTCACTGGCCCGTCATC-3', (SEQ ID NO: 12) oas-1 forward:
5'-GGAGGTTGCAGTGCCAACGAAG-3', (SEQ ID NO: 13) oas-1 reverse:
5'-TGGAAGGGAGGCAGGGCATAAC-3', (SEQ ID NO: 14) stat1 forward:
5'-TTTGCCCAGACTCGAGCTCCTG-3', (SEQ ID NO: 15) stat1 reverse:
5'-GGGTGCAGGTTCGGGATTCAAC-3'.
TABLE-US-00002 EpCAM PLK1 sense SEQ ID NO: 1 GCG ACU GGU UAC CCG
GUC GUU UUG AAG AAG AUC ACC CUC CUU AdTdT EpCAM PLK1 anti-sense SEQ
ID NO: 2 UAA GGA GGG UGA UCU UCU UCA dTdT EpCAM PLK1 anti-sense SEQ
ID NO: 3 GCG ACU GGU UAC CCG GUC GUU UUAA GGA GGG UGA UCU UCU UCA
dTdT EpCAM aptamer SEQ ID NO: 33 GCG ACU GGU UAC CCG GUC GUU U
EpCAM is over expressed in basal A and luminal but not basal B
breast cancer cell lines (data not shown). FACS was performed with
8 different breast cancer cell lines, testing EpCAM expression
levels by flow cytometery using a hEpCAM Antibody. EpCAM is over
expressed in all basal A and luminal cells lines and not in basal
B.
Specific decrease in cell viability in Basal A breast cancer cell
lines is PLK1 dependent. Ten different breast cancer cell lines
representing basal A, B and luminal cells were treated with either
EpCAM-AsiC targeting PLK1 or just the EpCAM-aptamer and compared to
untreated controls. None of the cell lines treated with
EpCAM-aptamer displayed decrease in cell viability, while basal A
and luminal cell lines displayed a decrease in cell viability
following treatment with EpCAM-AsiC targeting PLK1 (data not
shown).
EpCAM-AsiC is taken up by both healthy and colon cancer biopsies.
Cy3-EpCAM-AsiC targeting GFP, Alexa647-siRNA-GFP or
Alexa647-chol-siRNA-GFP (2 .mu.M of each) were added to colon
cancer and control explants and incubated for 24 h before tissues
were digested with collagenase to a single cell suspension and
analyzed by flow cytometry. EpCAM-AsiC, siRNA and chol-siRNA
penetrated both tumor and healthy tissue with similar efficacy. At
day 5 the tumors were removed and visualized to validate that the
Alexa750 labeled EpCAM-AsiC targeting GFP indeed entered the
tumors. Increased level of Alexa750 is negatively correlated with
mCherry levels (n=8, *P<0.05, t-test EpCAM+ versus EpCAM- cells)
(data not shown).
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Example 2
Described herein is the development of targeted siRNA delivery
(aptamer-siRNA chimeras (AsiC)) that use chimeric RNAs composed of
a structured RNA, called an aptamer, selected for high affinity
binding to a cell surface protein, that is covalently linked to an
siRNA. These AsiCs are taken up by cells expressing a receptor that
the aptamer recognizes and are processed within cells to release
the active siRNA. This is a flexible platform that can be modified
to target different cells by targeting specific cell surface
receptors and can be designed to knockdown any gene or combination
of genes.
The aptamer, was selected for high affinity binding to human EpCAM
(CD326 or ESA) which is expressed on all epithelial cells, but is
much more highly expressed on epithelial cancers including poorly
differentiated breast cancers, such as basal-like TNBC. All the
common cancers (lung, pancrease, prostate, breast and colon) have
high EpCAM expression and can potentially be targeted.
It is demonstrated herein that epithelial breast cancer cells, but
not mesenchymal or normal epithelial cells, selectively take up
EpCAM-AsiCs and undergo gene knockdown in vitro. Moreover, the
extent of knockdown strongly correlates with EpCAM levels.
Knockdown of PLK1, a gene needed for mitosis, using EpCAM-AsiCs
eliminates cancer cell line growth and stem cell properties
including colony and mammosphere formation and tumor initiation in
xenografts. This platform can be used to eliminate cancer cells and
the malignant cancer stem cells within epithelial tumors.
EpCAM AsiCs can be delivered specifically to basal-like tumors and
inhibit tumor growth. These AsiCs can also be a powerful research
tool for identifying the genes that T-IC cells depend on, which
could be good targets for either conventional drugs or RNAi-based
drugs.
Example 3
A ubiquitous mechanism for regulating gene expression is called RNA
interference. It uses small RNAs bearing a short complementary
sequence to block the translation of genetic information into
proteins. Harnessing this endogenous process offers the exciting
possibility to treat disease by knocking down expression of
disease-causing genes. The major obstacle is delivering small RNAs
into cells, where the RNA interference machinery lies. In the past
year, preliminary clinical studies have shown very promising
results without significant toxicity in a few diseases caused by
aberrant gene expression in the liver. However, delivery to the
liver, an organ that traps particles in the blood, is easier to
accomplish than delivering drugs to metastatic tumor cells.
Described herein is a strategy for targeting RNAs into epithelial
cancer cells that is especially good at targeting the most
aggressive type of breast cancer, triple negative breast cancer
(TNBC). Moreover, it also targets the most malignant subpopulation
in most breast cancers, which are called cancer stem cells. These
cells are resistant to chemotherapy drugs and are thought
responsible for tumor recurrence and metastasis. An important goal
of current cancer research is to replace cytotoxic chemotherapy
drugs that are toxic for both cancer cells and normally dividing
cells (such as the blood forming cells and cells lining the gut)
with agents that have selective activity against the tumor,
especially against the cancer stem cells within the tumor.
Targeted therapy for one type of breast cancer (Her2+) has
revolutionized treatment and significantly improved survival. There
is currently no targeted therapy for TNBC or for breast cancer stem
cells.
Described herein in are data demonstrating that RNAs that link an
interfering RNA to a structured RNA (aptamer) that recognizes a
cell surface protein can knockdown gene expression in aggressive
breast cancer cells. Aptamers that bind to proteins highly
expressed on breast cancer stem cells and most TNBC cells can knock
down proteins required for cancer cell division or survival
specifically in the most common subtype of TNBC. These RNAs can be
tested, e.g., in both tissue culture and in mouse models of TNBC.
Described herein is a platform for harnessing RNA-based drugs to
treat poor prognosis breast cancer and demonstration in a mouse
model of its efficacy.
Ultimate applicability for treating breast cancer (which patients,
how will it help them, clinical applications/benefits/risks,
projected time to patient-related outcome) The proteins that this
therapy can target are expressed on all epithelial cancer cells,
but are more strongly expressed on the least differentiated, and
hence most malignant, cancer cells. This approach could be used to
treat not only most epithelial breast cancers (and most breast
cancer cells are epithelial), but also has the potential to treat
the common cancers, including colon, lung, pancreas, and prostate.
Our focus is on the most aggressive and poorest prognosis breast
cancer, TNBC, which preferentially strikes down young women and
women from minority populations. This approach permits a new
platform for breast cancer therapy. Any cancer-causing or promoting
gene, or combinations of genes, could be knocked down, making this
strategy ideal for the coming era of personalized cancer therapy in
which each patient's therapy will be customized according to the
molecular characteristics of her individual tumor.
Moreover, if a tumor is nonresponsive or becomes resistant, the
cocktail of target genes could be nimbly adjusted. Because normal
epithelial cells express low levels of the proteins used for
targeting, there may be some uptake and toxicity to normal
epithelial cells, which is evaluated herein. However, the platform
is flexible so that the therapeutic siRNA cargo can be chosen to
kill tumor cells with minimal toxicity to normal cells.
Described herein are the design and testing in mouse TNBC models of
several molecules capable of causing tumor-specific gene knockdown
and tumor suppression.
There is no targeted therapy for TNBC or for highly malignant
tumor-initiating cell subpopulations within breast cancers.
Triple negative breast cancer (TNBC) has the worst breast cancer
prognosis. 1-4 There is no targeted therapy, and TNBCs often
relapse. Described herein is the development of small RNA-based
drugs that knockdown tumor dependency genes in basal-like (or
basal-A) TNBCs. In principle RNA interference (RNAi) can be
harnessed to knockdown disease-causing genes to treat any disease.
5-9 However, converting small RNAs into drugs is challenging.
Recent Phase I and II clinical trials have shown dramatic and
durable gene knockdown in the liver (.about.80-95%, lasting for
almost a month after a single injection) with no significant
toxicity. 10-16 Realizing the potential of gene knockdown for
treating cancer, however, requires a robust method to deliver RNAs
into disseminated cancer cells, which the liver-targeting RNAs are
unable to do. 7 An ideal therapy would selectively knockdown genes
in cancer cells, sparing normal cells to minimize toxicity. 17
AsiCs are composed of an RNA aptamer (a structured RNA with high
affinity for a receptor)18,19 covalently linked to an siRNA (FIGS.
10A-10B).
Described herein is the use of an AsiC to knockdown genes in
epithelial cancers using an EpCAM aptamer. 37 EpCAM, the first
described tumor antigen, is highly expressed on all common
epithelial cancers. 38-45 On epithelial breast cancers, EpCAM is
.about.400-fold more abundant than on normal breast tissue. 46
EpCAM39,45,47-53 is also highly expressed on most epithelial cancer
tumor-initiating cells (T-IC, also known as cancer stem cells).
39,45,47-53
The EpCAM aptamer has high affinity (12 nM) and is short (19 nt),
which is ideal for an AsiC drug, since RNAs <60 nt can be
cheaply and efficiently synthesized. The EpCAM-AsiCs consist of a
long 42-44 nt strand (19 nt aptamer+3 nt linker+20-22 nt siRNA
sense strand) annealed to a 20-22 nt antisense (active) siRNA
strand (FIG. 10B). They are commercially synthesized with
2'-fluoropyrimidines, which enhance serum stability (T1/2>3d)
and block innate immune recognition. 28,54-56
EpCAM targeting can cause selective gene knockdown in basal-like
TNBCs, relative to normal epithelia. Selective knockdown will
reduce both the drug dose and normal tissue toxicity. In normal
epithelia, EpCAM is only expressed on basolateral gap junctions,
where it may not be accessible. In epithelial cancers, it's both
more abundant and distributed along the whole cell membrane. EpCAM
promotes adhesion, and also enhances proliferation and
invasiveness. Proteolytic cleavage of EpCAM releases an
intracellular fragment that increases transcription of stem cell
factors. The oncogenic properties of EpCAM may make it difficult
for tumor cells to develop resistance by down-modulating EpCAM. The
number of EpCAM+ circulating cells is linked to poor prognosis in
breast cancer. In fact, enumerating circulating EpCAM+ cells is the
basis of an FDA-approved method for monitoring metastatic breast,
colon and prostate cancer treatment. In our studies, 9 of 9 basal-A
TNBC and luminal breast cancer cell lines were strongly EpCAM+,
while a normal breast cancer epithelial line and mesenchymal TNBCs
had close to background levels (FIG. 1B). Thus most basal-like
TNBCs and luminal breast cancers will likely be targeted by
EpCAM-AsiCs. In preliminary data, EpCAM-AsiCs selectively knocked
down expression in EpCAM+ breast and colon cancer cell lines but
not in normal epithelial cells or mesenchymal tumor cells;
knockdown was uniform and comparable to lipid transfection, but
lipid transfection uniformly knocked down gene expression in all
the lines. (FIG. 3A-3C)
AKT1 knockdown and inhibition of cell proliferation by EpCAM-AsiCs
against PLK1, a kinase required for mitosis, correlated with EpCAM
levels. When normal transformed epithelial cells (BPE) 57 were
mixed with epithelial TNBC cell lines, EpCAM-AsiCs caused
PLK1-sensitive cell death only in the tumor cells, sparing BPE
cells (not shown). Moreover when tumor biopsies and normal tissue
biopsies were coincubated with fluorescent AsiCs, only the tumors
took up the AsiCs and fluoresced (not shown). These results suggest
that EpCAM-AsiCs are specific for epithelial tumor cells compared
to normal epithelia.
EpCAM also marks T-ICs. 40,45,58 An important goal of cancer
research is to develop a way to target T-ICs. Although the stem
cell hypothesis is controversial and may not apply to all cancers,
there is good evidence that breast cancers contain a T-IC
subpopulation. 59-82 T-Ics are relatively resistant to chemotherapy
and are also thought responsible for tumor relapse and metastasis.
The AsiCs described herein are designed to target (epithelial)
T-ICs with high efficiency. As such they may be suitable for
eliminating this aggressive subpopulation within patients at risk
for relapse. To investigate whether EpCAM-AsiCs inhibit TNBC T-ICs,
we compared mammosphere and colony formation (in vitro surrogates
of T-IC function) of breast tumor cells that were mocktreated or
treated with EpCAM-AsiCs against eGFP or PLK1. PLK1 EpCAM-AsiCs,
but not control GFP AsiCs, eliminated mammosphere and colony
formation of breast luminal and basal-like TNBC cell lines (FIG.
11D). PLK1 EpCAM-AsiCs also reduced CD44+ CD24low and Aldefluor+
cells (not shown). Importantly, treatment with PLK1 EpCAM-AsiCs
eliminated tumor initiation by basal-like TNBCs, but, as expected,
had no effect on basal-B TNBC tumor initiation (data not shown).
Luciferase-expressing cell lines were mock-treated or treated
overnight with AsiCs before orthotopic implantation in the mammary
fatpad.
AsiCs targeting EphA2, important in EGF receptor signaling. 83-92
are also contemplated herein. EphA2 is expressed on epithelial and
mesenchymal (basal-A and basal-B, respectively) TNBC cell lines,
including their T-ICs, but less than EpCAM and only weakly on other
breast cancers. Inhibiting EphA2 reduces tumor growth and
angiogenesis in multiple cancer models. Furthermore, EphA2 is
selectively accessible on cancer cells, but not normal cells.
Also contemplated herein are mouse-human cross-reactive AsiCs,
which will be valuable for future drug development, since they will
enable us to evaluate toxicity and effectiveness in spontaneous
mouse tumor models.
AsiCs targeting EphA2 can produce dual functioning RNAs that both
inhibit EphA2 signaling and cell proliferation and knockdown
genes.
AsiCs are ideal for personalized cancer therapy, since the genes
targeted for knockdown can be adjusted to the molecular
characteristics of a tumor. Moreover cocktails of RNAs can be
assembled to knockdown multiple genes at once for combinatorial
therapy to anticipate and overcome drug resistance. AsiCs not only
target the drug to the tumor, but the siRNAs can also be chosen to
attack the specific Achilles' heels of the tumor. siRNAs also
provide a unique opportunity to target "undruggable" genes. AsiCs
that knock down tumor dependency genes, required for tumor, but not
normal cell, survival, should have reduced toxicity. To identify
genetic dependencies of basal-like TNBCs that we could knockdown,
we performed a genomewide siRNA lethality screen comparing 2 TNBC
cell lines--basal-like BPLER and myoepithelial HMLER cells, human
10 breast epithelial cells transformed with the same oncogenes in
different media. 57,93
Although essentially isogenic, BPLER are highly malignant and
enriched for T-ICs, forming tumors in nude mice with only 50 cells,
while HMLER require >105 cells to initiate tumors. The screen
identified 154 genes on which BPLER, but not HMLER, depended.
Proteasome genes were highly enriched (P<10-14). BPLER
dependency gene expression correlated with poor prognosis in
breast, but not lung or colon, cancer. Because TNBCs are
heterogeneous1,3,4,94, to identify shared dependencies in
basal-like TNBCs, we did another screen to test 17 breast cancer
cell lines for their dependency on the 154 BPLER dependency genes
(unpublished). Although many of the BPLER dependencies were shared
with only a subset of basal-like TNBC cell lines, the proteasome,
MCL1, some splicing genes, and a few other novel genes stood out
because virtually all (at least 8 of 9) basal-like TNBC lines were
dependent on these genes, but normal cells were not. As the screen
predicted, the proteasome inhibitor bortezomib both killed basal-A
TNBCs and also blocked T-IC function, assessed by colony and
mammosphere formation, again mostly selectively in basal-like
TNBCs. Brief exposure to bortezomib also inhibited colony formation
and tumor inhibition of a mouse epithelial TNBC line. Bortezomib
strongly inhibited tumor growth of multiple human basal-A lines and
primary TNBCs that arose spontaneously in Tp53+/- mice, but not
basal-B or luminal cell lines. Bortezomib also blocked metastatic
lung colonization of IV-injected TNBC cells. However, bortezomib
does not penetrate well into solid tumors. The maximum tolerated
dose was needed to inhibit proteasome activity and suppress tumors.
Although tumor penetration may improve with proteasome inhibitors
in development, proteasome gene knockdown might provide more
sustained and efficient proteasome inhibition.
EpCAM- and EphA2-AsiCs can be used for targeted gene knockdown to
treat basal-like TNBC cancers, sparing normal cells, and eliminate
the T-Ics within them. There may be some uptake in normal
epithelial cells that weakly express EpCAM or EphA2, but gene
knockdown will be concentrated in aptamer ligandbright tumor
cells.
It can be determined which breast cancer subtypes EpCAM- and
EphA2-AsiCs target and determine how aptamer ligand level affects
gene silencing. uptake/knockdown in cancer tissues vs normal
epithelium can also be evaluated. EpCAM-AsiCs can be compared with
EphA2-AsiCs for effectiveness in causing knockdown in basal-like
TNBCs. It can be determined whether EpCAM-AsiCs and EphA2-AsiCs can
target T-ICs to inhibit tumor initiation.
Pharmacokinetics (PK)/pharmacodynamics (PD) studies of EpCAM- and
EphA2-AsiCs can be performed using live animal imaging of
orthotopic TNBC xenografted mice. Treated tissue samples and
animals can be examined for toxicity and innate immune activation,
and AsiCs will be chemically modified if needed to improve PK/PD or
reduce toxicity. As proof of principle, the antitumor effect of
knockdown of PLK1 will be assessed. Suppression of recently
identified basal-A TNBC dependency genes, such as MCL1 and
proteasome genes can be accomplished according to the methods
described herein.
Contemplated herein are: cross-species reactive aptamers that
recognize EpCAM and EphA2 and are internalized selectively into
basal-A TNBCs vs normal epithelial cells verification of selective
uptake, gene silencing and cytotoxic effect in vitro of
TNBC-targeting AsiCs in breast cancer cell lines vs normal
epithelial cells, determination of the subtypes of breast cancer
cell lines they transfect and evaluation of their potential to
transfect and eliminate breast T-Ics Evaluation of systemic
delivery and tumor concentration in vivo, definition of PK and PD
and maximally tolerated dose of TNBC-targeting AsiCs, and
evaluation of the antitumor effect of optimized TNBC-targeting
AsiCs that knockdown PLK1 and dependency genes of basal-like TNBC
in human TNBC cell line models of primary and metastatic cancer in
mice
Selection of TNBC-targeting aptamers. Aptamers that bind to a
chosen target are identified by iterative screening of
combinatorial nucleic acid sequence libraries of vast complexity
(typically 1012-1014 distinct sequences) by a process termed SELEX
(Systematic Evolution of Ligands by Exponential enrichment). 95,96
In the classic method, the RNA library is incubated with the
protein target and the RNAs that bind are separated and amplified
to generate a pool of binding RNAs. These are again applied in
multiple cycles to generate increasingly enriched high affinity RNA
pools. Identification of the sequences that emerge after multiple
rounds of SELEX was previously accomplished by cloning and
sequencing <100 individual sequences.
While this often provided a sufficient number of winning sequences
to identify aptamers, the number of sequences that were analyzed
was quite small in comparison with the sequence complexity of
evolved oligonucleotide pools. With many selection cycles, some
effective aptamer sequences that are not efficiently amplified may
be depleted and lost. Next generation deep sequencing methods and
bioinformatics can permit evaluation of more sequences within early
cycle SELEX sequence pools to identify winning aptamer sequences at
earlier selection rounds, thus reducing the time and resources
needed to complete identification of high affinity aptamers.
30,97-104
An important property of aptamers useful for incorporation into
AsiCs is efficient internalization into cells. Some ligands of
cell-surface proteins are efficiently internalized after binding
their cell surface protein targets, while others are not. Another
strategy ("toggle SELEX") selects for cross-reactive aptamers that
recognize the same ligand from different species, a useful
attribute for preclinical development. By toggling cycles between
selection with orthologous protein ligands (e.g., mouse and human
forms), it is possible to enrich for cross-species reactive
aptamers. 105
These SELEX techniques can permit identification of high affinity
cross-species reactive aptamers for EpCAM and EphA2 that are
internalized into human (and mouse) basal-like TNBCs, but not into
a normal immortalized epithelial cell line. To select additional
EpCAM and EphA2 aptamers that have antagonistic activity and/or
cross-recognize the corresponding mouse antigen (the published
EpCAM aptamer does not recognize mouse EpCAM (data not shown)), we
can toggle between commercially available mouse and human purified,
recombinant target proteins, starting with a library of 1012 RNA
sequences containing 2'-fluoropyrimidines. This library of 51 nt
long oligonucleotides is designed with a random region of 20
nucleotides flanked by constant regions of known sequence for PCR
amplification at each selection round. Previously described methods
will be used to select for high affinity RNAs that bind to
immobilized C-terminal tagged proteins.37 (This leaves the
N-terminal region exposed to facilitate selection of aptamers that
recognize the extracellular domain.) A tagged control protein can
be used to pre-clear the RNA aptamer library to remove non-specific
binders. 7-10 iterative rounds of SELEX can be performed to enrich
for specific aptamers. Enrichment after each round can be monitored
by Surface Plasmon Resonance. Enriched pools that show specific
binding can be sequenced using high-throughput sequencing.
Sequences can be chosen for experimental validation using
bioinformatics analysis of the enriched library sequences as
described. 97,98,106
The top 10-15 sequences from the high throughput sequencing and
bioinformatics analysis can be evaluated by Surface Plasmon
Resonance to assess relative binding affinities as described,
99,106 using the previously characterized human aptamers for
comparison.
An alternative approach to dentify high affinity cross-reactive
aptamers, is cellinternalization SELEX, positively selecting on
293T cells transfected to expression human or mouse EpCAM and
preclearing on cells expressing a control protein. The ability of
the 5 highest affinity aptamers to be internalized into
EpCAM/EphA2+ cells will be compared to the previously selected
aptamers by qRT-PCR and flow cytometry (using fluorescently tagged
aptamers) as previously described. 37
These aptamers can also be evaluated for their ability to inhibit
tumor cell line proliferation specifically. Aptamers with this
property may be receptor antagonists, which will be verified by
examining their effect on cell signaling. Given the high homology
between the human and mouse EphA2 extracellular domains (>90%
identity; >90% structural homology), identifying aptamers that
cross-react with human and mouse EphA2 can be as simple as testing
the already selected aptamers for cross-reactivity against mouse.
The existing set of 20 human EphA2 aptamers can therefore first be
evaluated for the ability to bind mouse EphA2. Alternatively, the
approach described above can be followed. For a few of the top
aptamers, truncated sequences (lacking either or both of the
library adapter sequences) can be synthesized to define the minimal
sequence required for binding.
Aptamers of .about.20-35 nt in length can be identified for each
ligand, which can be designed into AsiCs amenable for chemical
synthesis.
In vitro assessment of TNBC-targeting AsiCs and their activity
against T-Ics. It can be defined which breast cancer subtypes are
efficiently transfected with TNBC-targeting AsiCs and evaluated
whether tumor knockdown is specific relative to normal tissue
cells, first in cell lines and then in 10 tumor specimens to verify
that the results for cell lines translate to 10 tissues. We can
also evaluate the potential of TNBC-targeting AsiCs to transfect
and target breast T-ICs.
AsiC design and initial testing The most attractive aptamers
identified above (prioritized based on considerations of affinity,
selectivity of binding and expression in poor prognosis cancer vs
normal cells, truncation to shorter length, the importance of the
ligand in oncogenesis and stem cell behavior, receptor antagonism
and cross-species reactivity) can be designed into AsiCs by linkage
to siRNAs targeting eGFP, AKT1 and PLK1 (vs control scrambled
siRNAs) that have been used for the initial EpCAM-AsiCs as
described above herein.
Basal-like NBC cell lines stably expressing destabilized (d1)EGFP
(protein T1/2 of .about.1 hr) were previously generated using
lentiviruses. GFP expression can be readily quantified by flow and
imaging, and its knockdown has no biological consequences. The
short T1/2 allows for rapid and sensitive detection of knockdown.
AKT1, which is expressed in all cells, is a good endogenous gene to
study, since its knockdown does not much affect cell viability.
PLK1 is used for its antitumor effect because its knockdown is
cytotoxic to dividing cells. Described herein is robust and
reproducible gene knockdown with EpCAM-AsiCs targeting each of
these genes. AsiCs will be chemically synthesized with
2'-fluoropyrimidines for stability and inhibition of innate immune
recognition and dT residues at their 3'-ends to protect against
exonuclease digestion. The 2 strands will be annealed to generate
the final RNA (FIG. 10A-10B). These AsiCs can be evaluated and
compared to the original EpCAM-AsiC (as positive control) and CD4-
or PSMA- AsiCs (as negative control) in in vitro dose response
experiments for AsiC uptake (using fluorophores such as AF-647
(which doesn't affect AsiC activity) conjugated to the 3'end of the
short strand), gene knockdown and reduced tumor cell line growth
and survival. Selective uptake, gene knockdown and antitumor effect
in a few human basal-A TNBC cell lines (MB468, HCC1937, BPLER vs
immortalized epithelial cells) can be quantified by flow cytometry;
flow cytometry and qRT-PCR; and Cell-TiterGlo and annexin-PI
staining, respectively. These experiments can permit the selection
of a handful of the best performing AsiCs that recognize EpCAM and
EphA2.
Types of breast cancer responsive to TNBC-targeting AsiCs. It can
be determined which types of breast cancer can be transfected with
the selected AsiCs and how specific gene knockdown is in tumors
relative to normal epithelial cells. In vitro knockdown by the
selected AsiCs in 20 human breast cancer cell lines that represent
the common breast cancer subtypes, but are weighted towards TNBC
(14 TNBC lines, plus a sampling of luminal and Her2+ cell lines)
can be evaluated. 93 Aptamer ligand expression, uptake of
fluorescent-labeled AsiC and gene silencing can be compared to
BPE57 and fibroblast lines as negative controls. This large panel
of cell lines can permit evaluation of how cell surface EpCAM and
EphA2 levels influence RNA uptake and gene silencing and whether
there is an expression threshold needed for efficient knockdown. A
dose response experiment can permit verification that the high
affinity of the aptamers is preserved in the AsiC. Specificity of
uptake (versus nonspecific "sticking") will be verified by using
acid washing to remove loosely adhered aptamers and showing that
binding is competed by unlabeled aptamers and eliminated when cells
are trypsinized prior to treatment. AsiC-mediated transfection will
be compared to lipid transfection as positive control and to naked
siRNA as negative control. Knockdown will be assessed by flow
cytometry and qRT-PCR after 5 d, the optimal time for AsiC-mediated
knockdown. It is expected that uptake and gene silencing will
correlate with aptamer ligand levels. To verify that specificity
for tumor cells is maintained in mixtures of ligand+ and
liganddim/- untransformed breast epithelial cells, we can compare
fluorescent AsiC uptake, gene knockdown and survival when PLK1 is
the gene target in mixtures of tumor cells expressing different
aptamer ligand levels with different numbers of GFP+ BPE cells.
Do epithelial primary breast cancer cells preferentially take up
TNBC-targeting AsiCs and show knockdown relative to normal
epithelial cells in tissue explants? To assess primary tumor uptake
and knockdown and anticipate potential toxicity to normal tissue
cells, we can next assess in situ transfection and gene knockdown
in explants of 10 luminal, Her2+ and TNBC breast cancers and
surrounding normal tissue. We can analyze samples from .about.25
tumors to provide a comprehensive look at common tumor subtypes.
Tumor typing can be confirmed by histology and immunohistochemistry
(IHC) staining for ER, PR, Her2 and E-cadherin. If the aptamer
recognizes the mouse ligand, we can also assess potential toxicity
to normal epithelia using mouse tumor/normal tissues. We can
compare normal tissues that have no large competing source of tumor
cells to tissues that contain tumor cells. This might be important
for anticipating toxicity in situations where AsiCs are given to
patients with low/undetectable tumor burden following therapy or
surgery. These experiments can also permit assessment of whether
knockdown by 10 tumors is comparable to that in cell lines, whether
tissue architecture affects uptake/knockdown in tumor cells and how
well different tumor subtypes are transfected. It is contemplated
herein that epithelial breast cancers will undergo efficient gene
knockdown, but normal epithelial cells will not.
Biopsies, cut into .about.3.times.3.times.3 mm3 pieces, can be
transfected in microtiter wells, which should mimic in vivo uptake
after SQ or IV infusion. Lipofectamine encapsulated siRNAs and
cholesterol-conjugated siRNAs are both effective at gene knockdown
of normal epithelial cells in polarized columnar and squamous
genital tract mucosa108,109, while naked siRNAs are not taken up.
Similar results are expected with these controls in normal breast
epithelial tissue. In parallel we can analyze knockdown of
collagenase-digested 10 cells to compare knockdown with what is
achieved in tissue and with cancer cell lines. We can first verify
these controls using siRNAs to target epithelial genes, which we
have previously knocked down (such as E-cadherin, cytokeratin
(CK)-5 (a good marker of basal cells) and 14, and nectin-1)
93,108,109, whose expression can be readily followed by IHC,
fluorescence microscopy (FM) or flow cytometry of isolated cells.
Staining of the target gene can be correlated with staining for
phenotypic markers and fluorescently labeled siRNAs to determine
which cell types are targeted. Pan-CK antibody can distinguish
epithelial cells (normal and tumor) from stroma. Of particular
interest is delivery and CK5 knockdown in rare basal tissue stem
cells, since EpCAM-AsiCs can target these cells and potentially
lead to depletion of normal tissue stem cells. Tissue toxicity and
inflammation will be assessed by H&E staining of tissue
sections and qRT-PCR assays for Type I interferons and inflammatory
cytokines (IL-1, IL-6, TNF-!). Additional chemical modifications of
the RNA sequence (besides 2'-fluoropryrimidines) will be introduced
to eliminate potentially harmful inflammation if it's detected.
Can TNBC-targeting AsiCs target breast tumor-initiating cells? We
chose EpCAM and EphA2 as aptamer targets partly because of their
potential to transfect T-ICs. Breast T-ICs are not uniquely defined
by phenotypic markers (and they may in fact be
heterogeneous93,110-113), making experiments challenging, since
T-ICs are defined functionally by their ability to initiate tumors
in small numbers that can be serially transplanted. Staining for
CD44, CD24, EpCAM, CD133, CD49f or ALDH1 in different combinations
enriches for T-ICs.59,72,78,114-121 Different protocols define
overlapping, but not identical, subsets of potential T-ICs. Without
wishing to be bound by theory, it is contemplated herein that
EpCAM- and EphA2-AsiCs will be taken up by and cause gene silencing
in T-ICs and can be used for targeted therapy to eliminate or
cripple T-IC capability within tumors.
To analyze AsiC uptake and gene silencing in T-IC subpopulations,
multicolor flow cytometry of EpCAM, EphA2, CD44 and CD24 in a panel
of breast cancer lines (luminal, Her2+, basal-A and B TNBCs) can be
used to identify which breast cell lines have putative T-IC
populations that contain cells that stain brightly for EpCAM and/or
EphA2. We can also examine EpCAM/EphA2 staining of mammospheres and
Aldefluor+ cells121,123-125 generated from these cell lines. We can
select .about.4-5 lines with the brightest/most uniform EpCAM/EphA2
expression within T-ICs as the most attractive cell lines to study
in this subaim and can produce stable (d1)GFP-expressing variants.
These cell lines, as well as their mammospheres and Aldefluor+
subpopulation, can be incubated with AF647-labeled AsiCs (and as a
negative control, nontargeting PSMA-AsiCs) bearing GFP siRNAs. AsiC
uptake will be assessed by AF-647 fluorescence together with EpCAM
or EphA2, CD44 and CD24 and Aldefluor staining. AsiCs can be taken
up by EpCAM+ or EphA2+ CD44+ CD24-/dim Aldefluor+ cells. To assess
gene knockdown in T-IC phenotype cells, we can monitor GFP
expression in the T-IC population and remaining cells by flow
cytometry and qRT-PCR after treatment with eGFP or control
siRNA-bearing AsiCs. We can also assess knockdown of endogenous
PLK1 and AKT1.
These experiments can indicate whether T-Ics in different subtypes
of breast cancer are targeted by EpCAM/EphA2-AsiCs. In subsequent
experiments we will focus on the cell lines in which we have
>80% knockdown in T-IC-enriched populations. If knockdown is
inefficient, we can modify the transfection conditions (amount of
AsiC, number of cells, volume, etc). Next, we can assess whether
AsiCs inhibit mammosphere and colony formation, reduce CD44 and
ALDH1-expressing subpopulations, and the size of the side
population. In addition to knocking down PLK1, we can design and
evaluate AsiCs against a few additional genes that breast T-ICs
depend on for self-renewal or maintaining multipotency. Basal-like
TNBC T-ICs are selectively sensitive to proteasome inhibition.
93
We can therefore evaluate knockdown of a proteasome component
(PSMA2) and potentially other selective T-IC dependency genes (such
as MSI1 (Musashi), an RNA binding protein in breast T-ICs that
regulates Wnt and Notch signaling126-130 or BMI1, a polycomb
component required for stem cell self-renewal131-134). After
verifying that these genes are expressed and knocked down in
mammosphere cells, we can treat both adherent cells and
mammospheres with AsiCs targeting PLK1, MSI1, BMI1 or PSMA2 or with
AsiCs targeting eGFP as a negative control and measure the size of
T-IC subpopulations after 5-7 d by staining with CD44, CD24, EpCAM,
CD133, CD49f and ALDH1. We can also measure the proportion of cells
that efflux small molecule dyes (the "side population"). These
experiments can be complemented by functional assays quantifying
the frequency of colony forming cells and mammospheres. Serial
replating can assess whether the ability to continuously propagate
T-ICs as spheres is inhibited. It is contemplated herein that
knocking down PLK1, MSI1, BMI1 or PSMA2 can reduce T-IC numbers,
proliferation and function in the T-ICs from some cell lines, but
different genes may be more active for different breast cell lines.
For example proteasome inhibition eliminated T-ICs in basal-like
TNBCs, but only in 1 of 3 mesenchymal TNBC cell lines and not in
more differentiated non-TNBC tumors. 93
The knockdown approaches that suppress T-IC can be further
investigated by experiments using chemical inhibitors where
available (such as bortezomib) or by examining whether knocking
down other genes in the same pathway (such as NOTCH1,
.beta.-catenin or WNT1 for MSI1) also has anti-T-IC activity. Next,
we can determine whether short-term ex vivo exposure of basal-like
TNBC lines to AsiCs inhibits TNBC tumor initiation as the ultimate
measure of inhibition of T-IC capacity, using AsiCs that look
promising in vitro. Cell lines, treated overnight with the chosen
AsiCs (and as negative controls AsiCs that use PSMA aptamer or
contain eGFP siRNA), will be assessed for viability. After
verifying that short-term siRNA exposure does not affect viability,
ex vivo treated cells will be injected in a range of cell numbers
orthotopically into NOD/scid/"c-/- (NSG) mice (these mice have the
highest take for tumor implantation). Bortezomib treatment for 24
hr (at this time .about.40% of cells are still viable) can serve as
a positive control.
In vivo evaluation of TNBC-targeting AsiCs
A few of the AsiCs that perform best can next be evaluated in vivo
using nude mice bearing mammary fatpad xenografts of an aptamer
ligand+ basal-A TNBC line, such as MB468 or HCC1187, on one side
compared to ligand-breast cancer cell line, such as basal-B MB231,
on the other (.about.5-8 mice/gp to obtain reproducible statistics
based on our experience with these models). For in vivo imaging, we
have already made stable luminescent/fluorescent cell lines by
infection with luciferase- and mCherry-expressing lentivirus.
Systemic delivery and knockdown in tumor cells Because unmodified
AsiCs are small (.about.30 kDa), when injected IV or IP they are
rapidly eliminated by kidney filtration. 20 kDa polyethylene glycol
(PEG) can be attached to the 5'-end of the inactive (passenger)
strand of the siRNA. 21 IV injected PEGylated PSMA-AsiCs
concentrated in subcutaneous tumors; PEGylation extended the
circulating T1/2 of Ipinjected AsiCs from <35 min to >>30
hr, increased the durability of gene silencing to .about.5 d and
reduced the effective tumor-inhibitory dose 8-fold to 250
pmol.times.5 injections. We have also found (nto shown) that SQ
injection of 5 mg/kg unmodified CD4-AsiCs caused systemic specific
knockdown in CD4+ cells in the spleen and proximal and distal lymph
nodes of humanized mice. Therefore we can compare AsiC levels after
IV and SQ administration of the original AsiC constructs and
PEG-AsiCs by in vivo imaging using AF-790-coupled AsiCs and the
IVIS Spectrum and by Taqman assay of the active strand in blood,
urine, liver and tumor samples. Samples can be analyzed over 5 d
with frequent sample collection the first day. Tissue sections can
be assessed for tissue damage and the blood can be analyzed for
hematological, liver and kidney toxicity by blood counts and serum
chemistries. Toxicity associated with induction of innate immunity
or inflammation can be assessed by ELISA assays of serum
interferons and inflammatory cytokines. The circulating T1/2 and
proportion of the injected drug that localizes to the EpCAM+ tumor
can be calculated. Based on our preliminary experiments with SQ and
IV administration of the CD4-AsiCs and in vivo experience with the
PSMA-AsiC9,21,25, it is contemplated herein, without wishing to be
bound by theory, that unPEGylated AsiCs will be rapidly excreted
after IV administration, but that SQ EpCAM-AsiC and IV PEG-AsiCs
will have more favorable localization to tumor xenografts.
Knockdown of mCherry and PLK1 following a single AsiC injection in
a range of concentrations can be assessed by in vivo imaging and by
flow cytometry, FM, and qRT-PCR of tumor specimens harvested 4, 7
and 12 d post-treatment. These experiments can provide estimates of
the effective dose required for peak tumor gene knockdown of 50, 75
and 90% (ED50, ED75, ED90) and for the durability of knockdown in
the tumor (quantified as T-KD50=time for tumor expression to return
halfway to control from the peak knockdown). These parameters can
be determined for each construct. We can also determine the
maximally tolerated dose (MTD) for the PLK1 constructs. Inadequate
PK/PD or signs of innate immune stimulation will lead us to adjust
chemical modifications (adding 2'-OMe riboses to some residues) or
add longer PEG polymers to improve these parameters using
straightforward.
Antitumor effect. It can be tested by in vivo imaging how effective
the best TNBC-targeting AsiCs are against basal-A tumors implanted
in the mammary fat pads or injected IV (as a metastasis model) in
nude mice. We can begin by targeting PLK1 as proof of principle.
21,107 PLK1-AsiCs can be injected SQ and/or IV in groups of 8 mice
(group size chosen for statistical significance based on previous
experiments) bearing a basal-A TNBC fatpad tumor using dosing
schedules chosen based on the PK/PD results. Mice can be treated as
soon as tumors become palpable. Effects on a representative ligand+
and ligand- tumor will be compared. Control mice can be treated
with PBS or naked siRNAs, AsiCs bearing a scrambled siRNA and PLK1
PSMA-AsiCs. Tumor size can be quantified by imaging and calipers.
If the antitumor effect is suboptimal, the dosing regimen can be
adjusted to the maximally tolerated regimen.
We can also compare the effect of PLK1 knockdown and
standard-of-care chemotherapy, administered on their own and in
combination to anticipate potential clinical studies. If there is
complete tumor regression, we can evaluate decreased doses.
Effective regimens can also be evaluated in mice implanted with a
few other basal-A TNBC lines to verify the generality of the
antitumor response. We can also evaluate AsiC treatment after tumor
cells are injected IV to determine effectiveness against distal
metastases. At the time of sacrifice, mice can be sacrificed and
mammary fatpads can be inspected for residual microscopic or
macroscopic tumor by FM, H&E and IHC. Residual tumor cells can
also be assessed for EpCAM/EphA2 expression to determine whether
tumor resistance may have developed as a consequence of
down-regulating the aptamers ligand. Treated mice can also be
observed for clinical signs of toxicity and at time of sacrifice
can be carefully examined for gut and bone marrow toxicity, by
blood counts and pathological examination of gut, bone marrow and
spleen. AsiCs designed with the cross-reacting aptamers can be used
to evaluate normal epithelial toxicity. Using our best AsiC design,
we can next begin to compare PLK1 knockdown with knockdown of TNBC
dependency genes (such as PSMA2 or MCL1) identified in our siRNA
screen93 tested alone or in combination with PLK1.
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Example 4
RNA interference (RNAi) offers the exciting opportunity to treat
disease by knocking down disease-causing genes. Recent early phase
clinical trials have shown promising and sustained gene knockdown
and/or clinical benefit in a handful of diseases caused by aberrant
gene expression in the liver. The major obstacle to harnessing RNAi
for cancer treatment is delivery of small RNAs to disseminated
cancer cells. Most epithelial cancer cells and the tumor-initiating
cells (T-IC) within them highly express EpCAM, the first described
tumor antigen. All epithelial breast cancer cell lines we tested
stain brightly for EpCAM, while immortalized normal breast
epithelial cells and fibroblasts do not. Targeted gene knockdown in
epithelial cancer cells in vitro can be achieved using chimeric
RNAs composed of a structured RNA, called an aptamer, selected for
high affinity binding to EpCAM, that is covalently linked to an
siRNA. These EpCAM aptamer-siRNA chimeras (AsiC) are taken up by
EpCAM+ cells and selectively cause gene knockdown in epithelial
breast cancer cells, but not normal epithelial cells. Moreover
knockdown of PLK1 with EpCAM-AsiCs suppresses colony and
mammosphere formation of epithelial breast cancer lines, in vitro
assays of tumor-initiating potential, and tumor initiation.
Subcutaneously injected PLK1 EpCAM-AsiCs are taken up specifically
by EpCAM+ basal-A triple negative breast cancer (TNBC) orthotopic
xenografts and cause rapid tumor regression. TNBC has the worst
prognosis of any breast cancer and there is no targeted therapy for
it. It is specifically contemplated herein that EpCAM-AsiCs can be
used for targeted gene knockdown to treat epithelial (basal-like)
TNBC cancers, sparing normal cells, and eliminate the T-ICs within
them. It can be defined which breast cancer subtypes can be
targeted by EpCAM-AsiCs and determine how EpCAM level affects
uptake and gene silencing. Relative uptake/knockdown in cancer
cells expressing EpCAM and normal epithelium can be evaluated in
human breast cancer tissue explants. It can also be determined
whether EpCAM-AsiCs can target breast T-ICs to disrupt tumor
initiation.
The drug-like features of EpCAM-AsiCs can be optimized EpCAM-AsiCs
can be optimized for cell uptake, endosomal release, systemic
delivery and in vivo gene knockdown. Pharmacokinetics (PK) and
pharmacodynamics (PD) of EpCAM-AsiC uptake and gene silencing and
tumor suppression can be evaluated using live animal imaging in
TNBC cell line xenograft models. As proof of principle, the
antitumor effect of knockdown of PLK1, which is needed for cell
proliferation can be evaluated. In addition knockdown of novel gene
targets identified in a genome-wide siRNA screen for TNBC genetic
dependencies will be evaluated in mouse xenograft models. An
optimized EpCAM-AsiC and knowledge of its PK, PD and possible
toxicity, can be used in experiments for further toxicity and other
preclinical studies.
Described herein is the development of EpCAM aptamer-siRNA chimeras
as a method for targeted gene knockdown in basal-like triple
negative breast cancer and other epithelial cancers and the
tumor-initiating cells within them. There is currently no targeted
therapy for triple negative breast cancers, which frequently
relapse, or for highly malignant tumor-initiating cell
subpopulations within breast cancers, which may be responsible for
some cases of drug resistance and relapse. These RNAs provide a
versatile and flexible platform for RNA-based drugs to treat poor
prognosis breast cancers.
Example 5
It is demonstrated herein that (1) the EpCAM aptamer on its own
does not affect cell growth or viability of EpCAM+ breast tumor
cell lines (not shown); (2) when normal breast biopsies are mixed
with EpCAM+ TNBC human breast tumor tissues in vitro, fluorescent
EpCAM-AsiCs only concentrate in the tumor (FIG. 14); (3) treatment
of EpCAM+ luminal and basal-A TNBC cells, but not mesenchymal
TNBCs, with PLK1 EpCAM-AsiCs blocks in vitro assays of
tumor-initiating cells (T-IC, colony and mammosphere formation) and
in vivo tumor initiation (FIGS. 15A-15C and 16); (4) subcutaneously
(sc) injected EpCAM-AsiCs concentrate in EpCAM+ tumors in mice
bearing EpCAM+ and EpCAM- TNBCs on either flank, distantly located
from the injection site (FIG. 17A-18B); and (5) most importantly,
sc injection of PLK1 EpCAM-AsiCs leads to complete regression of
palpable basal-A TNBC xenografts (FIG. 18A-18B). In addition (6) a
new siRNA screen identified novel shared genetic dependencies of
basal-A TNBCs for EpCAM-AsiC knockdown (FIG. 19).
Without wishing to be bound by theory, T-ICs are heterogeneous and
plastic in epithelial/mesenchymal gene expression. Although
mesenchymal traits may facilitate initial tissue invasion,
formation of clinically significant metastases (colonization) may
require epithelial properties. EpCAM-mediated delivery of siRNA
effectively blocks tumor initiation, but only for epithelial
(basal-A TNBC, luminal) breast cancers.
The high affinity of the EpCAM aptamer and our uptake, gene
knockdown, and proliferation experiments in uniform and mixed
populations of cells show specific targeting to EpCAM+ cells.
Normal epithelial cells and fibroblasts are not targeted. New data
showing that EpCAM-AsiCs are not taken up by normal human breast
biopsies are compelling.
Triple negative breast cancer (TNBC), a diverse group of highly
malignant cancers that don't express the estrogen, progesterone and
Her2 receptors, has the worst breast cancer prognosis. There is no
targeted therapy for TNBCs, which often relapse after cytotoxic
therapy. Described herein is a platform for gene knockdown
therapeutics for basal-like TNBC, using specifically targeted RNA
interference (RNAi). RNAi can selectively knockdown disease-causing
genes. Realizing the therapeutic potential of gene knockdown for
treating cancer, however, requires a robust method to deliver RNAs
into disseminated cancer cells. There are 2 bottlenecks--getting
RNAs across the cell membrane and from endosomes to the target cell
cytoplasm where the RNAi machinery sits. An ideal=therapy would
selectively knockdown genes in cancer cells, while sparing most
normal cells to minimize toxicity.
Described herein is the knockdown of genes in basal-like TNBCs (the
majority of TNBCs) with chimeric RNAs that use an aptamer (a
structured nucleic acid selected for high affinity binding to a
target molecule against EpCAM (also known as CD326 or ESA)"+, the
first described tumor antigen. EpCAM is highly expressed on
epithelial breast cancers (including basal-like TNBC)--on average
400-fold more than on normal breast tissue. It is also highly
expressed on other epithelial cancers and is a marker of "cancer
stem cells" (also called tumor-initiating cells (T-IC)).
Aptamer-siRNA chimeras (AsiC) covalently link a targeting aptamer
to an siRNA (FIG. 10B). Dicer cleaves the siRNA from the aptamer
inside cells.
Epithelial breast cancer cells, but not mesenchymal or normal
epithelial cells, selectively take up EpCAM-AsiCs and undergo gene
knockdown in vitro. Moreover, knockdown strongly correlates with
EpCAM levels. Knockdown of PLK1, a gene needed for mitosis, using
EpCAM-AsiCs eliminates colony and mammosphere formation (in vitro
assays that correlate with self renewal and tumor initiation) and
tumor initiation in vivo, suggesting that EpCAM-AsiCs might be used
to target T-ICs. Sc injection of PLK1 EpCAM-AsiCs caused complete
regression of EpCAM+ TNBC xenografts, but had no effect on EpCAM-
mesenchymal TNBCs.
It is described herein that EpCAM-AsiCs can be used for targeted
gene knockdown to treat basallike TNBC cancers, sparing normal
cells, and eliminate the T-ICs within them. Aside from their
selective delivery to target cells, AsiCs have important advantages
for cancer treatment compared to RNA delivery by nanoparticles,
liposomes or RNA-binding proteins--(1) they bypass liver and lung
trapping and concentrate in tumors; (2) as a single RNA molecule
they are simpler and cheaper to manufacture than multicomponent
drugs; (3) they have virtually no toxicity and do not stimulate
innate immunity or inflammation or cause significant off-target
effects; (4) because they do not elicit antibodies, they can be
used repeatedly; (5) they are stable in serum and other body
fluids.
It can be defined which breast cancer subtypes can be targeted by
EpCAM-AsiCs and determine how EpCAM level affects uptake and gene
silencing. The relative uptake/knockdown in cancer tissues vs
normal epithelium can be evaluated. It can also be determined
whether EpCAM-AsiCs can target breast T-ICs to inhibit tumor
initiation. An important aim is to optimize EpCAM-AsiCs for uptake,
endosomal release, systemic delivery and in vivo knockdown.
Pharmacokinetics (PK) and pharmacodynamics (PD) of EpCAM-AsiC
uptake, gene silencing and tumor suppression will be evaluated by
live animal imaging in TNBC orthotopic xenografts. As proof of
principle, the antitumor effect of knockdown of PLK1, which is
needed for cell proliferation can be evaluated. Knockdown of other
genes we identified in a genome-wide RNAi screen as genetic
dependencies of basal-like TNBC can be evaluated. Described herein
is the development of optimized EpCAM-AsiC and knowledge of its PK,
PD and possible toxicity and identification of novel basallike TNBC
dependency genes to target
Described herein is: the verification of selective EpCAM-AsiC
activity in epithelial breast cancers compared with normal
epithelia and evaluate the potential of EpCAM-AsiCs to transfect
and eliminate breast T-ICs (i.e., cancer stem cells); optimization
of EpCAM-AsiCs to transfect and knockdown genes in epithelial TNBC
cells in vitro and for systemic delivery and tumor concentration in
vivo, and define PK and PD and maximally tolerated dose; evaluation
of the antitumor effect of optimized EpCAM-AsiCs targeting PLK1 and
novel dependency genes of basal-like TNBC in human epithelial TNBC
models of primary and metastatic cancer in mice
Although most TNBC patients respond to chemotherapy, within 3 yr
about a third develop metastases and eventually die. Thus we need
new approaches. TNBCs are heterogeneous, poorly differentiated
tumors that may need to be treated by subtype or with
individualized therapy. 1,3,4,72 Most TNBCs are basal-like or
belong to the basal-A subtype. Described herein is a flexible,
targeted platform for treating basal-like TNBCs that is suitable
for personalized therapy. Not only will the drug be targeted to the
tumor, but the drug targets can also be chosen to attack the
tumor's Achilles' heels by knocking down tumor dependency genes.
This present approach delivers small interfering RNAs (siRNA) into
epithelial cancer cells by linking them to an RNA aptamer that
binds to EpCAM (FIG. 10B), a cell surface receptor over-expressed
on epithelial cancers, including basal-like TNBCs. EpCAM is highly
expressed on epithelial cancers and their T-Ics.
EpCAM targeting can cause selective gene knockdown in basal-like
TNBCs, but not normal epithelia. Selective knockdown will both
reduce the drug dose and reduce tissue toxicity.
As described herein, 9 of 9 basal-A TNBC and luminal breast cancer
lines were strongly EpCAM+, while a normal breast epithelial cell
line, fibroblasts and mesenchymal TNBCs had close to background
EpCAM (FIG. 1B). Thus virtually all basal-like TNBCs (and probably
luminal breast cancers) will be targeted by EpCAM-AsiCs. Moreover,
since .about.100% of epithelial cancers, including lung, colon,
pancreas and prostate, stain brightly for EpCAM, this platform
could also be used for RNAi-based therapy of common cancers.
When RNAi was found in mammals, small RNAs were hailed as the next
new drug class. Soon investigators realized that getting RNAi to
work as a drug was not simple., However, after addressing the main
obstacle to RNA therapy (cellular uptake), there is now optimism
about RNAi-based drugs. Recent phase I/II studies have shown 80-95%
gene knockdown in hypercholesterolemia, transthyretin-related
amyloidosis, hepatitis C, hemophilia and liver metastasis, caused
by aberrant liver gene expression. However, applying RNAi for
cancer therapy is still a dream. The major obstacle to harnessing
RNAi for cancer is delivering small RNAs into disseminated cells.
Described herein are methods and compositions that overcome this
problem, e.g., by the use of AsiCs.
AsiCs are a flexible platform that can target different cell
surface receptors and knockdown any gene or combination of genes.
By changing the aptamer, the AsiC platform can tackle the delivery
roadblock that has thwarted the application of RNAi-based therapy
to most diseases. This approach is ideal for personalized cancer
therapy, since the choice of genes to target can be adjusted
depending on a tumor's molecular characteristics. Moreover RNA
cocktails can knockdown multiple genes at once to anticipate and
overcome drug resistance.
Described herein is the development of an optimized EpCAM-AsiC with
well defined PK/PD.
An important cancer research goal is to eliminate T-ICs (cancer
stem cells). T-ICs are relatively resistant to chemotherapy and are
thought responsible for tumor relapse and metastasis The AsiCs
described herein are designed to target (epithelial) T-ICs with
high efficiency. As such they can eliminate this aggressive
subpopulation within tumors at risk for progressive disease (see
FIG. 16).
In addition to their potential therapeutic use, EpCAM-AsiCs can
also be a powerful in vivo research tool for identifying the
dependency genes of tumors and T-ICs to define novel drug
targets.
Described herein is a novel targeted therapy for epithelial
cancers, and the T-ICs within them by targeting EpCAM, a tumor
antigen widely over-expressed in epithelial cancers and their
T-ICs. Targeted therapy so far has relied on using tumor-specific
antibodies or inhibitors to oncogenic kinases. No one before has
shown that an unconjugated AsiC can have potent antitumor effects
or that AsiCs could be administered sc. There is currently no
targeted therapy for TNBC or for T-ICs. Developing targeted therapy
for TNBC and developing ways of eliminating T-ICs are important
unmet goals of cancer research.
The methods described herein are targeted in 2 ways--the aptamer
specifically delivers the therapeutic RNA to tumor cells, while the
genes chosen for knockdown can be selected based on the specific
molecular dependencies of the targeted tumor. By testing in vivo
knockdown, it can be demonstrated that basal-like TNBCs and their
T-ICs are selectively dependent on the proteasome, MCL1 and the
U4/U6-U5 tri-snRNP splicing complex. This work can identify a new
set of drug targets, suitable for both conventional and RNAi-based
drugs.
The trafficking of siRNAs in transfected cells can be examined and
each step of RNA processing in cells be systematically optimized to
improve the drug features of an siRNA.
CD4-AsiCs durably knockdown gene expression in CD4+ T lymphocytes
and macrophages and inhibit HIV transmission to humanized mice.
CD4-AsiCs specifically suppressed gene expression in CD4+ T cells
and macrophages in polarized human cervicovaginal tissue explants
and in the female genital tract of humanized mice. Because they are
monomeric and don't cross-link the receptor, CD4-AsiCs did not
activate the targeted cells. They also did not stimulate innate
immunity Intravaginal application of only 80 pmol of CD4-AsiCs
directed against HIV genes and/or CCR5 to humanized mice completely
blocked HIV sexual transmission. RNAi-mediated gene knockdown in
vivo lasted several weeks. Transmission was blocked by CCR5
CD4-AsiCs applied 2 d before challenge. Significant, but
incomplete, protection also occurred when exposure was delayed for
4 or 6 d. CD4-AsiCs targeting gag/vif provided protection when
administered post-exposure. Thus CD4-AsiCs are promising for use in
an HIV microbicide.
Protection against HIV transmission requires local knockdown in the
genital tract. However, systemic delivery is more challenging and
is needed for cancer. Because AsiCs are small enough to be filtered
by the kidney, they are rapidly eliminated and do not efficiently
cause gene silencing. In some embodiments, polyethylene glycol
(PEG) can be attached to the 5'-end of the inactive (passenger)
strand of the siRNA. iv injected PEG-AsiCs concentrated in sc
tumors. PEGylation extended the circulating T1/2 of ip injected
AsiC from <35 min to >>30 hr, increased the durability of
gene silencing to .about.5 d and reduced the needed dose 8-fold. sc
injection of unmodified CD4-AsiCs caused .about.80% gene knockdown
specifically in CD4+ cells in the spleen, proximal and distal lymph
nodes of humanized mice (not shown). Sc injection of EpCAM-AsiCs
similarly led to specific concentration/knockdown in EpCAM+ tumors
(see below).
EpCAM-AsiCs selectively knockdown gene expression in EpCAM+ cancer
cells The EpCAM-AsiCs have a .about.42-44 nt long strand (19 nt
aptamer+linker+20-22 nt siRNA strand) annealed to a 20-22 nt
complementary siRNA strand (FIG. 10B). Commercially synthesized
with 2'-fluoropyrimidines, they are RNase resistant (T1/2>3 d in
serum, data not shown) and do not trigger innate immunity
37,91-93
Surface EpCAM was high in all luminal and basal-like cell lines
tested, but close to background in normal epithelia immortalized
with hTERT (BPE) 94, fibroblasts and mesenchymal TNBCs (FIG. 1B).
Several of a handful of designs tested (with the sense and
antisense strands exchanged and several linkers) knocked down gene
expression specifically in EpCAM+ cell lines, but the most
effective design is shown in FIG. 10B. Gene knockdown of eGFP and
AKT1 by EpCAM-AsiCs was uniform and selective for EpCAM+ cells and
as effective as siRNA lipid transfection, which was not selective
(FIG. 13A-13C). In 8 breast cancer cell lines, AKT1 knockdown and
inhibition of cell proliferation by PLK1 EpCAM-AsiCs strongly
correlated with EpCAM levels (FIG. 11B-11C). The EpCAM aptamer on
its own had no effect on cell proliferation (not shown). When
EpCAM- BPE cells were mixed with epithelial TNBC cell lines,
EpCAM-AsiCs knocked down AKT1 and caused PLK1-sensitive cell death
only in tumor cells, sparing the normal epithelial cells (not
shown). The proportion of surviving tumor cells decreased 7-fold
after 3 d. When we added fluorescent AsiCs, cholesterol-conjugated
siRNAs (chol-siRNA, taken up by normal epithelia) or naked siRNAs
to normal breast and tumor biopsy samples, EpCAM-AsiCs concentrated
only in the tumors (FIG. 14). Thus EpCAM-AsiCs are specific for
epithelial tumor cells.
EpCAM-AsiCs inhibit T-ICs of EpCAM+ tumors. EpCAM was chosen for
targeting partly because EpCAM marks T-ICs and
metastasis-initiating cells (M-IC). To investigate whether
EpCAM-AsiCs inhibit T-ICs, we compared colony and mammosphere
formation (T-IC functional surrogates) after mock treatment,
treatment with paclitaxel or with EpCAM-AsiCs against eGFP or PLK1.
PLK1 EpCAM-AsiCs more strongly inhibited colony and mammosphere
formation of multiple EpCAM+ basal-like TNBCs and a luminal cell
line than paclitaxel, but was inactive against EpCAM- basal-B TNBCs
(FIG. 15A-15C). To evaluate EpCAM-AsiC's effect on tumor
initiation, viable luc+ EpCAM+ MB468 and EpCAM- MB231 cells,
treated overnight with medium or PLK1 or GFP EpCAM-AsiCs, were
implanted sc in nude mice. PLK1 EpCAM-AsiCs blocked tumor
formation, but only in EpCAM+ tumors (FIG. 16 and data not shown).
Thus EpCAM-AsiCs inhibit tumor initiation in EpCAM+ breast
cancers.
EpCAM-AsiCs are selectively taken up by EpCAM+ TNBCs and cause
tumor regression To investigate the potential clinical usefulness
of EpCAM-AsiCs, we first examined delivery of Alexa750-labeled
EpCAM-AsiCs injected sc in the scruff of the neck of mice bearing
EpCAM+ and EpCAM- TNBCs in each flank (FIG. 17A-17B). EpCAM-AsiCs
concentrated only in the EpCAM+ tumor. Mice bearing bilateral
tumors were mock treated or injected biweekly with PLK1 or GFP
EpCAM-AsiCs and tumor growth was followed by luminescence. The
EpCAM+ tumors rapidly completely regressed only in mice that
received the PLK1-targeting AsiCs (FIG. 18A-18B). This experiment
was repeated with additional control groups, the EpCAM aptamer on
its own or PLK1 siRNA, neither of which had any anti-tumor activity
(data not shown). Thus sc injected EpCAM-AsiCs show specific
antitumor activity against basal-A TNBCs.
Live cell imaging of siRNA uptake, endosomal release and gene
silencing An optimized spinning disk confocal microscope capable of
single molecule detection was used to detect the weak cytosolic
signal of released fluorescent RNAs, which was not before possible.
HeLa cells incubated with Alexa647-siRNA lipoplexes were imaged
every 3 s. RNA-containing late endosomes released a small fraction
of their cargo RNA, which diffused rapidly to fill the cytosol
(data not shown). Release occurred during a narrow time frame,
.about.15-20 min after endocytosis. .about.104 siRNAs were released
in a typical event. In HeLa cells, stably expressing eGFP-dl, GFP
siRNAs caused GFP expression to decrease rapidly after endosomal
release with a T1/2 of .about.2.5 h. Only 1000 cytosolic siRNAs
were needed for efficient gene silencing. Release triggered
autophagy, which sequestered the RNA-containing endosome within a
double autophagic membrane. No release occurred after that.
We applied this method to study uptake/release of Cy3-labeled
EpCAM-AsiCs, comparing EpCAM+ MB468 TNBCs with EpCAM- BPE cells.
Uptake and release were negligible in BPE, but clear cut in MB468.
This imaging method and our understanding of siRNA trafficking can
be used to optimize EpCAM-AsiC design to improve endosomal release
and knockdown.
Identification of basal-like TNBC dependency genes (BDGs). To
identify genetic dependencies of basal-like TNBCs that EpCAM-AsiCs
could target, a genomewide siRNA lethality screen was performed
comparing basal-like BPLER and myoepithelial HMLER cells, human
primary breast epithelial cells transformed with the same oncogenes
in different media. Although essentially isogenic, BPLER are highly
malignant and enriched for T-ICs, forming tumors in nude mice with
only 50 cells, while HMLER require >105 cells to initiate
tumors. The screen identified 154 genes on which BPLER, but not
HMLER, depended. Proteasome genes were highly enriched
(P<10-14). Expression of BPLER dependency genes correlated with
poor prognosis in breast, but not lung or colon, cancer. Proteasome
inhibitor sensitivity was a shared feature of basal-A TNBCs and
correlated with MCL1 dependency. Normal breast epithelial cells,
luminal breast cancer lines and mesenchymal TNBC lines did not
depend on the proteasome or MCL1. Proteasome inhibition not only
killed basal-A TNBCs, it also blocked T-IC function by colony and
mammosphere assays, again mostly selectively in basal-like TNBCs.
Brief exposure to bortezomib also inhibited tumor initiation of a
mouse basallike TNBC line.
We next tested whether proteasome inhibition inhibited the growth
of basal-like TNBC tumors in mice. Bortezomib does not penetrate
well into solid tumors, which has limited its clinical use. The
maximum tolerated iv dose (MTD) was needed to inhibit proteasome
activity in sc tumors. Treatment with the MTD strongly inhibited
tumor growth of 3 human and 1 mouse basal-A TNBC cell lines and 10
TNBCs that arose spontaneously in Tp53+/- mice, but was not active
against basal-B or luminal cell lines. Similar results were
obtained with carfilzomib. Bortezomib also blocked lung
colonization of iv-injected mouse TNBC cells. Thus the proteasome
is selectively required for epithelial TNBC growth, tumor
initiation and metastasis. Although tumor penetration and PD may
improve with newer proteasome inhibitors, proteasome gene knockdown
might provide more effective proteasome inhibition.
Because TNBCs are heterogeneous1,3,4,72, we rescreened the 154
BPLER dependency genes in 4 basal-A TNBC and 3 luminal human cancer
lines. Our goal was to identify additional shared dependencies of
basal-like TNBC cell lines as potential EpCAM-AsiC targets. Only 21
of the 154 BPLER dependency genes reduced viability by at least
2-fold in 3 of 4 basal-A cell lines tested. These putative BDGs
clustered in 4 functional groups--4 proteasome genes and MCL1
(previously validated), 10 genes implicated in RNA splicing, 2
genes implicated in mitosis and 2 genes required for nuclear
export. 20 of the 21 BDGs genes were retested using a new set of
siRNAs and 14 genes reconfirmed (the other "hits" may have been
secondary to off-target effects or their knockdown could have been
insufficient to cause lethality). Of note, 9 of 10 splicing genes
reconfirmed. They included 4 members of the U4/U6-U5 tri-snRNP
complex, PRPF8, PFPF38A, RBM22, USP39. Other interesting shared
hits were the RAN nuclear export G protein and the nucleoporin
NUP205, and NDC80, a kinetochore component that anchors the
kinetochore to the mitotic spindle. (USP39 is also required for the
mitotic spindle checkpoint).
TNBCs are known to be particularly susceptible to antimitotic
agents. USP39 is overexpressed in breast cancer cells vs normal
breast tissue and USP39 knockdown inhibited proliferation and
colony formation of luminal MCF7 cells. Moreover in zebrafish,
USP39 mutation leads to splicing defects of tumor suppressor genes
like rb1 and p21. To explore the therapeutic effect of inhibiting
splicing in basal-like TNBCs, we silenced the 4 spliceosome
tri-snRNP complex BDGs (PRPF8, PRPF38A, RBM22, USP39) in 6
basallike cell lines and in luminal MCF7 cells (FIG. 19). Knock
down of PRPF8, PRPF38A or RBM22 activated caspase-3 and was lethal
for 6 of 6 basal-like cell lines, but not for MCF7; USP39 knockdown
killed 3 of 6 basal-like cell lines. Spliceosome proteins were
frequently up regulated in breast cancer cell lines of all
subtypes. The viability of all 6 basal-like cells lines, but not
MCF7 cells, was reduced at least 2-fold by knockdown of the mitotic
kinetochore gene NDC80 or of nuclear export genes RAN or NUP205.
Moreover, knockdown of each of the tri-snRNP complex genes, RAN,
NUP205 or NDC80 blocked colony formation (a surrogate of T-IC
potential) in 3 of 3 basal-like TNBC cell lines
EpCAM-AsiCs can cause targeted gene knockdown in EpCAM+ tumors and
the T-ICs within them. Although there may be some uptake in normal
epithelial cells that weakly express EpCAM, gene knockdown will be
concentrated in EpCAMbright tumor cells, especially in T-ICs.
EpCAM-AsiCs can be optimized, as described herein, for favorable
PK/PD to suppress tumor growth and metastasis of basal-like TNBCs
with acceptable toxicity in mouse models.
EpCAM-AsiCs targeting eGFP, AKT1 and PLK1 are used herein as models
for assessing gene knockdown and optimizing AsiC design. Cell lines
stably expressing destabilized (d1)EGFP, with a protein T1/2 of
.about.1 hr, can be generated using lentiviruses. GFP expression
can be readily quantified by flow and imaging, and its knockdown
has no biological consequences. The short T1/2 allows for rapid and
sensitive detection of knockdown. AKT1, which is expressed in all
the cells we test, is a good endogenous gene to study, since its
knockdown in TNBCs doesn't affect cell viability much. PLK1 is used
as proof-of-concept for its antitumor effect because its knockdown
is cytotoxic to all dividing cells. We previously showed that PLK1
knockdown using a different delivery strategy dramatically
suppressed Her2+ breast cancer in mice. In a recent screen, PLK1
was unique amongst kinase genes because its knockdown eliminated
breast T-ICs. We have achieved robust and reproducible gene
knockdown with EpCAM-AsiCs targeting each of these genes.
EpCAM-AsiCs can be be purchased, e.g., as non-GMP RNAs from TriLink
or NITTO Avecia. Each strand of the EpCAM-AsiC was synthesized with
2'-fluoropyrimidines and dT residues at their 3'-ends to protect
against exonuclease digestion and then annealed to generate the
final RNA (FIG. 10B). As we optimize the AsiC, other chemical
modifications can be substituted and tested to determine if they
confer improved activity. The aptamer alone and AsiCs bearing a
nontargeting siRNA can serve as controls. Some of the eGFP
EpCAM-AsiCs can also be annealed to an antisense strand modified at
the 3'-end with a fluorophore (which doesn't affect AsiC activity
(not shown)) to quantify AsiC uptake and trafficking within cells
and in vivo.
Specific EpCAM-AsiC knockdown in epithelial breast cancers and
breast cancer T-ICs vs normal epithelial cells. It can be
determined which breast cancer subtypes are transfected with
EpCAM-AsiCs and evaluate whether tumor knockdown is specific to
cancer cells, first in cell lines and then in 10 tumor tissues to
verify that the results for cell lines translate to tissues in
situ. Because EpCAM-AsiCs might also transfect normal tissue stem
cells, knockdown and toxicity to these rare basal cells will be
assessed in the tissue experiments. We can also evaluate the
potential of EpCAM-AsiCs to transfect and target breast T-ICs.
Types of breast cancer responsive to EpCAM-AsiCs We first need to
know which types of breast cancer can be transfected with
EpCAM-AsiCs and how specific gene knockdown is in tumors relative
to normal epithelial cells. We extend our prelim. studies (FIGS.
13A-13C and 11B-11C) by evaluating in vitro knockdown in a panel of
20 human breast cancer cell lines that represent the common breast
cancer subtypes, but are weighted towards TNBC (14 TNBC lines, plus
a sampling of luminal and Her2+ cell lines).95 EpCAM expression,
uptake of Cy3-labeled AsiC and gene silencing in tumor lines can be
compared to that in BPE94 and fibroblasts. This large tumor panel
will enable us to evaluate how cell surface EpCAM levels influence
gene silencing and whether there is an EpCAM expression threshold
for efficient knockdown. We can also verify in a dose response
experiment using a few EpCAM+ cell lines that the reported high
binding affinity of the EpCAM aptamer is preserved in the AsiC.
Specificity of uptake (versus nonspecific "sticking") can be
verified by using acid washing to remove loosely adhered aptamers
and showing that binding is competed by unlabeled aptamers and
eliminated when cells are trypsinized before treatment.
EpCAM-AsiC-mediated transfection can be compared to lipid
transfection and naked siRNAs as controls. Knockdown will be
assessed by flow cytometry and qRT-PCR after 5 d, the optimal time
for AsiC-mediated knockdown. We expect that uptake and gene
silencing will correlate with EpCAM levels. To verify that
specificity for EpCAM+ cells is maintained in mixtures of EpCAM+
and EpCAMdim untransformed breast epithelial cells, we can compare
fluorescent EpCAM-AsiC uptake, gene knockdown and survival when
PLK1 is the gene target in mixtures of tumor cells expressing
different levels of EpCAM (MFI ranging between 100-1000) with
different numbers of GFP+ BPE cells.
Do epithelial breast cancer cells preferentially take up
EpCAM-AsiCs and show knockdown relative to normal epithelial cells
in tissue explants? To assess primary tumor knockdown and
anticipate potential toxicity to normal tissue cells, we can assess
in situ transfection and gene knockdown in explants of 10 luminal,
Her2+ and TNBC breast cancers and surrounding normal tissue from
mastectomy specimens. Samples from .about.25 tumors can be analyzed
to provide a comprehensive look at tumor subtypes. Tumor typing can
be confirmed by histology and IHC staining for ER, PR, Her2,
E-cadherin. We can compare normal tissues that have no large
competing source of EpCAM+ cells to tissues that contain tumor
cells. This might be important for anticipating toxicity in
situations where AsiCs are given to patients with low/undetectable
tumor burden following therapy or surgery. These experiments can
permit the assessment of whether knockdown by 10 tumors is
comparable to that in cell lines, whether tissue architecture
affects uptake/knockdown in tumor cells and how well different
tumor subtypes are transfected.
Based on the data presented herein, e.g., FIG. 14, it is
contemplated herein that epithelial breast cancers, but not normal
epithelial cells, can undergo efficient gene knockdown. Tissues cut
into 3.times.3.times.3 mm3 samples can be transfected in Optimem
solution in microtiter wells. Lipoplexed siRNA and chol-siRNAs both
knockdown genes in normal columnar and squamous genital tract
epithelia, while naked siRNAs are not taken up. We can first verify
these controls using siRNAs to target epithelial genes, which we
have previously knocked down (such as E-cadherin, claudin3,
cytokeratin (CK)-5 (a good marker of basal cells), and nectin-1),
whose expression can be readily followed by IHC, fluorescence
microscopy (FM) or flow cytometry of separated cells. Staining of
the target gene product can be correlated with staining for
phenotypic markers and fluorescent siRNAs to determine which cell
types within the tissue are targeted. Pan-CK antibody can be used
to distinguish epithelial cells (normal and tumor) from stroma. We
can also compare knockdown of collagenase-digested 10 cells to
tissue knockdown. Without wishing to be bound by theory, delivery
and CK5 knockdown in rare basal tissue stem cells can be assessed,
since EpCAM-AsiCs may target these cells and potentially lead to
toxicity. Because toxicity to the GI tract is often dose limiting
for cancer drugs, we can repeat these studies using colon tumor
specimens to determine whether colon cancer cells, normal gut
epithelia and crypt stem cells are transfected. These experiments
can provide useful data regarding clinical toxicity and the choice
of genes to knockdown, i.e. we might knockdown cancer dependency
genes that are not essential for normal stem cells, if tissue stem
cells are efficiently transfected. (Hematopoietic cells don't
express EpCAM, so hematological toxicity is not expected.)
Can EpCAM-AsiCs be used to target breast tumor-initiating cells?
One reason we chose EpCAM as aptamer target is its potential to
transfect T-ICs ("cancer stem cells"). T-ICs are drug resistant and
thought responsible for tumor initiation, relapse and metastasis.
Breast T-ICs are not uniquely defined by phenotype, making
experiments challenging, since T-ICs are defined functionally by
their ability to initiate tumors that can be serially transplanted.
Staining for CD44, CD24, EpCAM, CD133, CD49f or ALDH1 in different
combinations enriches for T-ICs. 49,61,67,107-111
Different protocols define overlapping, but not identical, subsets
of potential T-ICs. T-ICs are heterogeneous and show plasticity in
their epithelial vs mesenchymal features (and in fact may have some
features of both states). 28,95,112-118 Some breast T-ICs are
mesenchymal and don't express EpCAM. However, there is increasing
evidence that the ability of basal-like TNBCs to colonize distant
tissues and form macroscopic metastases--arguably the most
clinically important function of T-ICs--depends on epithelial
properties. Moreover our new data (FIGS. 15A-15C and 16) on the
effect of EpCAM-AsiCs on T-IC function and tumor initiation
indicate that EpCAM-AsiCs have anti-T-IC activity for basal-A
TNBCs. We hypothesize that EpCAM-AsiCs are taken up by basal-like
TNBC T-ICs and can be used for targeted therapy to cripple T-IC
capability within them.
To analyze EpCAM-AsiC uptake and gene silencing in T-ICs, we can
first stain a panel of breast cancer lines with EpCAM, CD44 and
CD24 to identify breast cell lines whose putative T-IC populations
contain cells that stain brightly for EpCAM. We can also examine
EpCAM staining of mammospheres and Aldefluor+ cells111,123,124
generated from these cell lines. We can select .about.4-5 lines
with the most uniform EpCAM expression within T-ICs as the most
attractive cell lines to study in this subaim (and as controls, 1-2
basal-B cell lines whose T-ICs might lack EpCAM staining) and can
produce stable eGFP-expressing variants. These cell lines, and
their mammospheres and Aldefluor+ subpopulation, can be incubated
with fluorescent eGFP EpCAM-AsiCs (and as control, nontargeting
PSMA-AsiCs). AsiC uptake can be assessed together with EpCAM, CD44
and CD24 and Aldefluor staining. AsiCs should be taken up by EpCAM+
CD44+ CD24-/dim Aldefluor+ cells. To assess gene knockdown in T-IC
phenotype cells, we can monitor GFP in the T-IC population and
remaining cells after treatment with eGFP or control siRNA-bearing
AsiCs by flow cytometry and qRT-PCR (of Aldefluor+ or mammosphere
populations). We can also assess knockdown of endogenous PLK1 and
AKT1. These experiments can tell us whether T-ICs in different
subtypes of breast cancer are targeted by EpCAM-AsiCs. Next we
assess whether AsiCs inhibit mammosphere and colony formation,
reduce phenotypic T-IC subpopulations, or the side population.
We can also design and evaluate AsiCs against additional genes
needed for self-renewal or multipotency. Because basal-like TNBC
T-ICs are sensitive to proteasome inhibition, we can evaluate
knockdown of a proteasome component (PSMA2). Other potential T-IC
dependency genes we will evaluate are MSI1, a gene highly expressed
in breast T-ICs that regulates Wnt and Notch signaling125-129,
BMI1, a polycomb component needed for self-renewal130-133, and
possibly a few novel BDGs identified in our recent siRNA screen
(FIG. 19). MSI1 knockdown decreases stem cell markers and
mammosphere formation in MCF7 and T47D cells.129
After verifying that these genes are expressed and knocked down in
mammosphere cells, we can treat both adherent cells and
mammospheres with AsiCs targeting these genes or eGFP as a negative
control and measure the size of T-IC subpopulations after 5-7 d by
staining for CD44, CD24, EpCAM, CD133, CD49f and ALDH1. We can also
measure the proportion of cells that efflux small molecule dyes
(the "side population"). These experiments can be complemented by
functional assays quantifying colony forming cells and
mammospheres. Serial replating can investigate whether propagation
of T-ICs as spheres is inhibited.
Knocking down PLK1, MSI1, BMI1 or PSMA2 can reduce T-IC numbers,
proliferation and function in some breast cancer subtypes, but
different genes may be more active for different breast cell lines
(i.e. proteasome inhibition eliminated T-ICs in basal-like TNBCs,
but not non-TNBC tumors and in only 1 of 3 basal-B TNBCs95). The
knockdown approaches that suppress T-IC can be further investigated
by experiments using available chemical inhibitors and/or by
knocking down other genes in the same pathway (such as NOTCH1,
.beta.-catenin or WNT1 for MSI1). The effect on T-ICs of
EpCAM-AsiCs can be compared with the EpCAM aptamer on its own and
the EpCAM antibody, adecatumumab (Amgen).
Next we determine whether short-term ex vivo exposure of basal-like
TNBC lines to EpCAM-AsiCs inhibits tumor initiation as the ultimate
measure of T-IC inhibition. The most promising AsiCs can be tested
in vivo. Cell lines, treated overnight with AsiCs (and as negative
controls AsiCs that use PSMA aptamer or contain eGFP siRNA), can be
assessed for viability. After verifying that short-term siRNA
exposure does not affect viability, ex vivo treated cells will be
injected in a range of cell numbers orthotopically into
NOD/scid/!c-/- (NSG) mice (these mice have the highest take for
tumor implantation). Pretreatment with bortezomib, which reduced
tumor initiation in basal-like TNBC"), or adecatumumab will be
controls.
Optimize EpCAM-AsiCs To improve EpCAM-AsiC drug features, we can
optimize each step of in vitro gene knockdown and in vivo delivery.
We can also modify the chemistry of EpCAM-AsiCs (if needed) to
minimize off-target effects.
In prelim. studies and published work, the AsiC concentration
needed for optimal knockdown in vitro is .about.1-4 .mu.M, many
fold higher than the .about.100 nM (or lower) concentrations used
for lipid transfection. For knockdown, EpCAM-AsiCs follow the
following steps: (1) cell receptor binding, (2) endocytosis, (3)
endosomal release, (4) Dicer processing, (5) incorporation into the
RNA-induced silencing complex (RISC), and (6) target mRNA cleavage.
We can systematically optimize each step, focusing on steps (2) and
(3), where we expect we can obtain the largest gains in efficacy.
The AsiC design variables are the EpCAM aptamer, whose affinity
affects steps 1 and 2; the linker sequence between the aptamer and
the siRNA, which controls step 4; the siRNA sequence, which
controls step 6. In addition each residue used for chemical
synthesis from phosphoramidite building blocks can be chemically
modified to reduce nuclease digestion, off-target suppression of
partially complementary sequences, binding and stimulation of
innate immune RNA sensors and improve cell uptake and in vivo PK.
The most common chemical modifications are substituting S for 0 in
the phosphate backbone (to produce RNase-resistant phosphorothioate
(PS) linkages and substituting 2'-F, 2'-O-methyl (2'OMe), or
2'-O-methyoxyethyl (2'MOE) for the 2'-OH in the ribose. PS, 2'-F
and 2'-OMe modifications are well tolerated in clinical trials and
therefore we concentrate on them. 2'-OMe occurs naturally in rRNA
and tRNA and is therefore safe, and 2'-F is also well tolerated;
heavily Psmodified nucleotides are sticky (and cause binding to
serum proteins, which can improve circulating T1/2) and can cause
unwanted side effects; lightly modified PS-RNAs are not toxic.
Chemical modifications can both inhibit and enhance gene silencing
in steps 5 and 6 This can be an iterative process; as modifications
are made at one step, the most attractive modified candidates can
be optimized for other steps, drawing on lessons learned from
previous candidates. We can verify that the modified AsiCs chosen
for further development do not stimulate innate immunity or result
in cellular toxicity. If they do, we can further modify our designs
to avoid these problems.
Optimize In Vitro Knockdown
(1) EpCAM binding The EpCAM aptamer has 12 nM affinity, It can be
verified that that this affinity is preserved in the EpCAM-AsiC. If
the AsiC has lower affinity than the aptamer, we can use bio-layer
interferometry (OctetRED System, ICCB-Longwood Core) with
recombinant EpCAM to compare the affinity of the aptamer and AsiC.
If the AsiC has lower binding affinity, it may not fold properly.
To enhance folding into the desired conformation we can try
changing the type and length of the linker between the aptamer and
the AsiC sense strand (i.e. we can incorporate more 3C linkers or
triethylene or hexaethylene glycol spacers).
(2) Endocytosis The monomeric AsiC is slowly taken up by
constitutive receptor recycling. This step can be optimized by
receptor crosslinking to trigger active endocytosis, which requires
aptamer multimerization. Multimerization of aptamers (with or
without linked siRNAs) can increase binding avidity (by increasing
valency) or convert an aptamer that does not cause signaling into
an agonistic reagent. Aptamers can be multimerized by using
streptavidin (SA) to bind biotinylated (Bi) aptamers and siRNAs;
extending the aptamer with an adapter that binds to an organizing
oligonucleotide that contains multiple complementary sequences
connected by a flexible linker; or extending the aptamer with
complementary adapter sequences to produce a dimer. We focus on
all-RNA designs, which don't induce antibodies. Some of the designs
we can test are shown in FIG. 20 (we can also test constructs with
sense and antisense strands exchanged).
Time course and dose response experiments will compare
fluorescently tagged multimeric constructs with the monomeric AsiC
to assess the extent and rapidity of uptake and GFP knockdown by
flow cytometry and live cell imaging (data not shown). If
endocytosis is enhanced by multimerization, but knockdown does not
improve, we can use Northern blotting to follow Dicer cleavage and
determine whether the expected antisense strand is produced (see
below). If not, we can alter the design of the linkers, for example
by lengthening the duplex region from 21 to 27 nt, so the
multimerized AsiC is a good Dicer substrate(&) and verify that
the 5' end of the Dicer product originates at the intended base.
Multimerization should reduce the AsiC concentration needed for
knockdown many fold. However, multimerization could cause unwanted
EpCAM signaling and promote tumor cell proliferation. We can verify
that this is not the case using multimerized constructs targeting
eGFP. An attractive feature of multimerization is that it could
link multiple different siRNAs into a single RNA molecule for
combinatorial gene knockdown to produce a cancer "cocktail".
If none of these multimers work, we can test monomeric AsiCs
containing complementary sequences that enable RNAs to selfassemble
into small nanoparticles or the SA-Bi strategy, using less
immunostimulatory SA mutants.
(3) Endosomal release Although fewer than 1000 cytosolic siRNA
molecules are estimated to be needed for knockdown (not shown),
only a few percent of siRNAs in endocytosed liposomes are released
into thecytosol. EpCAM-AsiC endosomal release can be assessed by
live cell imaging to measure the efficiency of cytosolic release of
endocytosed AsiCs. If this indicates less than desired endosomal
release, then improving release should reduce the drug dose
substantially. Preincubation and endocytosis of an amphipathic
cationic peptide (mellitin) or polymer (butyl vinyl ether) that is
reversibly masked, can enhance siRNA escape to the cytosol. Masking
means that at neutral pH the peptide or polymer is uncharged and
does not interact with the plasma membrane and damage it, but at
the negative endosomal pH, a cationic molecule is generated that
damages the endosomal membrane and releases coendocytosed
oligonucleotides. Iv injection of these masked polymers within 2 hr
of siRNA delivery potentiated hepatocyte knockdown by chol-siRNAs
as much as 500 fold in mice and nonhuman primates.
We can first determine by live cell videomicroscopy whether prior
transfection of masked cationic polymers facilitates EpCAM-AsiC
(and lipoplexed siRNA) cytosolic delivery and eGFP knockdown in
vitro. We can also investigate whether incubating EpCAM-AsiCs with
basic peptides/polymers can also determine whether inhibition of
endosomal acidification using bafilomycin A or concanamycin alters
EpCAM-AsiC cytoplasmic release and knockdown, as the proton sponge
theory predicts. If these experiments confirm the proton sponge
theory, we can investigate strategies for altering EpCAM-AsiCs.
These include covalent conjugation (via disulfide bonds
spontaneously reversed in the cytosol's reducing environment) of
the sense or antisense strand to cell penetrating peptides,
including polyarginines of different sizes, protamine152, mellitin,
transportan or penetratin and conjugation of the AsiC sense strand
to butyl and amino vinyl ester or linkage of the sense strand to
phosphospermines of different lengths. We can verify that these
modifications do not alter solubility, result in cytotoxicity or
innate immune stimulation or interfere with specific EpCAM
targeting.
Dicer processing, RISC incorporation, target mRNA cleavage We next
take the top 2-3 EpCAM-AsiCs, with the initial design as control,
and examine whether siRNA function can be optimized Northern blots,
probed for the sense, antisense and aptamer parts of the
EpCAM-AsiC, can analyze EpCAM-AsiC products within cells. Their
migration can be compared to that of synthesized sense and
antisense strands, aptamer and full length EpCAM-AsiC. If Dicer
cleaves the AsiC as expected, we can recover RNAs that migrate like
the sense and antisense strands (as well as unprocessed EpCAM-AsiCs
from endosomes and a band the size of the aptamer joined to its
linker). (Dicer dependence can be verified using HCT116 cells
expressing hypomorphic Dicer). If the intracellular RNAs are not
the expected size, we can clone them to determine where Dicer cuts.
If the bands are not cut or are not where we want, we can redesign
the linker and double stranded region to produce the desired
cleavage. We can also investigate replacing the UUU linker with
alternative linkers or combinations of linkers, by substituting or
adding one or more 3C linkers or triethylene or hexaethylene glycol
spacers, to enhance intracellular processing to the siRNA. We can
also investigate whether a Dicer-independent design in which the
aptamer is covalently joined to the sense or antisense strand of
the siRNA by a disulfide bond, spontaneously reduced in the
cytosol, leads to more efficient knockdown.
Once we have shown that the appropriate antisense strand is
produced, we can next compare antisense strand incorporation into
the RISC. Northern blotting and Taqman PCR will quantify how much
of the input active strand in whole cell lysates is pulled down
with pan-Ago antibody (2A8). Ago binding, the T1/2 of the siRNA in
the RISC, and target gene knockdown are influenced by chemical
modifications of the sense and antisense strands. Specific 2'-F and
2'-OMe chemical modifications on both strands arranged in
proprietary positions and sequences can increase knockdown by
50-fold and PS linkages at the ends greatly increase gene knockdown
duration. We can design a small set of AsiCs bearing different
covalent modifications of the siRNA portions of the AsiC and
analyze their effect on knockdown of eGFP, AKT1 and PLK1
EpCAM-AsiCs targeting additional genes that we evaluate in vivo can
be designed with the most active siRNA sequences and best chemical
modifications. A small group of siRNA sequences to test for
knockdown (without aptamers, by transfection) can be identified by
web algorithms. The most efficient siRNAs (pM activity), which also
have low predicted melting temperatures (Tm), can be used, since
these are processed better. If we need to use sequences with higher
Tms, we can add a mismatch at the 3'-end of the sense strand to
promote siRNA unwinding and incorporation of the active strand in
the RISC.
Eliminate off target effects and toxicity These experiments can be
performed with the original and the best optimized AsiCs. The lack
of toxicity of the various AsiCs encoding eGFP siRNA (whose
knockdown should not affect viability) can be formally assessed by
Cell Titer-Glo assay of AsiC-incubated TNBC lines. Based on prior
work, we do not expect significantly reduced viability. Lipid
transfection will be used as a control for cytotoxic RNA delivery.
Finally we can verify that each of the AsiCs is not
immunostimulatory by qRT-PCR, performed 6 and 24 hr post AsiC
incubation, to amplify a panel of inflammatory and innate immune
response genes (IFNB, IFNG, IL1, IL8, IL10, OAS1, STAT1, IP10).
qRT-PCR is the most sensitive assay for immunostimulation and we
chose times that capture the peak response. Cells treated with
poly(I:C) can serve as positive controls and mock-treated cells
will be negative controls. If any AsiC is immunostimulatory (a
sequence and concentration dependent property), we can evaluate
whether additional chemical modifications, which reduce innate
immune sensor binding, eliminate immune stimulation without
compromising gene knockdown. A 2'-F or 2'-OMe modification of the
second residue of either the full AsiC or the Dicer cleavage
product can accomplish this goal.
Since the CD4-AsiC is not immunostimulatory in our prelim. studies
and the optimized AsiCs are active at greatly reduced
concentrations (and off-target effects are concentration
dependent), innate immune stimulation is unlikely, but if detected,
can be easily suppressed by chemical modification. In conjunction
with the tissue explant studies we can also examine tissue
histology carefully for disruption of epithelial tissue
architecture and cell necrosis.
Optimize tumor concentration and define PK/PD, Next we evaluate and
improve systemic T1/2 and tumor targeting in tumor-bearing mice. We
can focus on the original AsiC design and a few of the in vitro
optimized constructs (as they are identified). We can use qRT-PCR
to measure circulating T1/2 and tissue distribution, in vivo
imaging of the fluorescent AsiC to look at tumor localization and
silencing of tumor cell mCherry (GFP is not used because of
background autofluorescence) as a readout of gene silencing.
Studies of EpCAM-AsiC PK/PD can be facilitated by our recent
experience with in vivo imaging (FIGS. 16, 17A-17B, and 18A-18B,
data not shown). These experiments can use nude mice bearing
mammary fatpad xenografts of Luciferase-mCherry stable
transfectants we have generated of EpCAM+ basal-A TNBC lines, such
as MB468 or HCC1187, compared to an EpCAM- mesenchymal basal-B TNBC
cell line, such as MB231. We have an expression plasmid for these
tags and use lentivirus infection to produce stable transfectants.
.about.5-8 mice/gp will be used to obtain statistical significance
based on our prelim. data in these models. We can first compare the
blood and tumor concentration after iv and sc administration of the
original AsiC construct and the constructs optimized for in vitro
knockdown. Mice can be examined frequently for clinical signs of
toxicity. Samples can be analyzed over 5d with frequent sample
collection the first day. At each timepoint, blood and urine can be
harvested and analyzed by Taqman assay for the antisense strand.
Tumor and sample organs can be harvested at fewer timepoints from
euthanized animals. Blood can be analyzed for hematological, liver
and kidney toxicity by blood counts and serum chemistries. The
circulating T1/2 and proportion of the injected drug that localizes
to the EpCAM+ tumor can be calculated. Without wishing to be bound
by theory, based on our preliminary experiments with sc and iv
administration of the CD4-AsiCs and in vivo experience with the
PSMA-AsiC, we expect that most of these EpCAM-AsiCs will be rapidly
excreted after iv administration, but that sc injected EpCAM-AsiCs
will concentrate in tumor xenografts. The larger multimerized
constructs (FIG. 20) might resist kidney filtration and have better
tumor concentration when given iv. The sc and iv PK results will be
compared with mCherry knockdown following a single EpCAM-AsiC
injection in a range of concentrations, assessed both by in vivo
imaging (using the IVIS Spectrum) and by flow cytometry, FM, and
qRT-PCR of tumor specimens harvested 4, 7 and 12 d post-treatment.
These experiments can provide estimates of the effective dose
required for peak tumor gene knockdown of 50, 75 and 90% (ED50,
ED75, ED90) and for the durability of knockdown in the tumor
(quantified as T-KD50=time for tumor expression to return halfway
to control from the peak knockdown). These parameters can be
determined for each chosen construct.
Next we assess ways to improve the circulating T1/2. These include
increasing the size of the AsiC (i.e. by PEG conjugation comparing
a few sizes, such as 10, 20 and 30 kD, avoiding polymers known to
be toxic, such as PEI) and increasing binding to serum proteins to
reduce renal filtration (i.e. by conjugation with cholesterol,
which binds to serum LDL158,159 or by adding a diacyl tail to
promote binding to serum albumin. We avoid strategies that produce
particles or aggregates since these will have poorer tumor
penetration and may be trapped in the liver. Linking PEG to the
5'-end of the aptamer, the 3'-end of the inactive siRNA or the
3'-end of the active strand should not interfere with RNAi. In vivo
PK/PD/toxicity evaluation can be performed as above, using the
unconjugated AsiC as a positive control (and benchmark) and the
conjugated siRNA (without the aptamer) as a negative control. Two
or three of the constructs that have the lowest ED75 or ED90 and
longest T-KD50 for GFP will be retested using a PLK1 EpCAM-AsiC to
determine the corresponding PK/PD parameters, to aid in designing
the dosing regimen for antitumor efficacy experiments. We can also
determine the maximally tolerated dose (MTD) for these PLK1
constructs.
Antitumor Effect of EpCAM AsiCs against basal-like TNBCs Our final
goal is to test the EpCAM-AsiCs against orthotopic mammary fat pad
tumors and metastases. We can use nude mice unless tumors do not
grow or grow slowly, in which case we will switch to NSG mice. Live
animal imaging can be performed using an IVIS Spectrum, sensitive
for multicolor fluorescence and bioluminescence. These experiments
can evaluate 2-3 of the best EpCAM-AsiCs identified.
Activity of PLK1 EpCAM-AsiCs against orthotopic xenografts We can
begin by targeting PLK1/A few PLK1 EpCAM-AsiC designs, optimized as
described above, can be injected sc and/or iv in groups of 5-8 mice
(size chosen from power calculations based on previous experiments
in which this group size gave statistically significant results)
using doses and dosing schedules/injection route chosen based on
the PK/PD results above. For example if the ED90 is well below the
MTD, an initial experiment might investigate administering 2ED90
every T-KD50/2 d. Mice can initially be treated as soon as their
tumors become palpable, but in later experiments we can investigate
whether larger tumors of fixed diameters regress after multiple
administrations. Mice bearing representative EpCAM+ basal-A (MB468,
HC1187, BPLER) and EpCAM- basal-B (MB231) tumors will be compared.
For some experiments, we can treat mice bearing these tumors in
each flank, but these may require more mice because of intra-animal
variations in tumor sizes. Control mice can be treated with PBS or
naked siRNAs, the EpCAM aptamer on its own, EpCAM-AsiCs bearing
scrambled siRNA sequences and PLK1 PSMA-AsiCs. In some experiments
we can compare EpCAM-AsiC treatment with adecatumumab or
paclitaxel. Tumor size will be quantified by luminescence and
caliper measurements q3d. Treated mice can also be weighed and
observed for clinical signs of toxicity and at time of sacrifice
can be carefully examined for gut and bone marrow toxicity by blood
counts and pathological examination of gut, bone marrow and spleen.
Differences between groups can be assessed by one way ANOVA with
corrections for multiple comparisons as needed. For AsiCs that are
effective, we can also examine the immediate effect of treatment to
evaluate the mechanism of antitumor activity and verify that the
AsiCs are not activating innate immune responses. Tumor-bearing
mice can be sacrificed 1-3 d after a single therapeutic or control
injection and the tumors stained for activated caspases to
determine if death is by apoptosis and by H&E to look for
mitotic spindles to follow the expected effect of PLK1 knockdown.
Serum interferons and pro-inflammatory cytokines can be assessed by
multiplexed ELISA, and spleen and tumor cells analyzed by qRT-PCR
for the corresponding mRNAs. If there is no antitumor effect or the
antitumor effect is suboptimal, the dosing regimen can be adjusted
to the MTD. If the antitumor effect is complete (complete tumor
regression), then we can evaluate decreased doses and/or larger
tumors at start of therapy. When control mice are sacrificed
because untreated tumors have reached the allowed size, the treated
mice can be sacrificed and mammary fatpads inspected for residual
microscopic or macroscopic tumor by FM, H&E and IHC. Residual
tumor cells can also be assessed for EpCAM expression to determine
whether tumor resistance, if it occurs, may have developed as a
consequence of down-regulating EpCAM. If no residual tumor cells
are noted, we can perform an additional experiment to determine
whether tumors are eradicated--mice will be treated for 1-2 weeks
after the luciferase measurement has returned to background levels,
and then mice can be observed for 1-2 months off treatment to see
if tumors regrow or metastases appear. The most effective
regimen(s) for basal-A TNBCs can also be evaluated against other
breast cancer subtypes (luminal, Her2+) that we expect EpCAM to
target.
PLK1 EpCAM-AsiC activity against metastatic tumors To evaluate the
effectiveness of EpCAM-AsiCs against metastatic cancer cells, we
can evaluate the PLK1 EpCAM-AsiCs against basal-A TNBC cell lines
injected intravenously in NSG mice, which have the best tumor take.
We can begin to treat mice as soon as lungs become luciferase+
after tail vein injection of basal-A (or basal-B as control) TNBCs.
The treatment dosing can use the effective schedule and mode of
administration determined above for primary tumors. Mice can be
imaged q3d. The controls can be reduced to a mock-treated group and
groups treated with paclitaxel or an EpCAM-AsiC containing a
non-targeting siRNA. When the control mice need to be sacrificed,
all groups can be imaged. Lungs, livers and brains can be
dissected, weighed, imaged to quantify tumor burden, sections can
be analyzed by H&E and staining for EpCAM, and one lung from
each animal will be analyzed by qRT-PCR for relative expression of
human/mouse Gapdh to quantify tumor burden independently. If mice
treated with PLK1 EpCAM-AsiCs are completely protected from
metastases or show a significant advantage compared to control
groups, we can determine if mice with greater metastatic burdens
are also protected by delaying the beginning of treatment until the
tumor burden is greater.
We can also compare the most effective iv regimen with the most
effective sc regimen identified above for treating orthotopic
tumors, since RNA delivery/knockdown at metastatic sites could
differ from primary tumor sites. We can also use this metastasis
model to evaluate in vivo knockdown of our screen's BDG genes and
genes identified above herein as necessary for tumor initiation ex
vivo, since M-IC capability is thought to correlate with T-IC
function.
Activity of EpCAM-AsiCs targeting BDF genes We can next compare
PLK1 knockdown with knockdown of TNBC dependency genes identified
in our siRNA screens or in the literature (such as XBP1). These in
vivo experiments for each gene target chosen can involve (1)
identifying active siRNAs for each gene and evaluating the effect
of knockdown on cell proliferation and T-IC function in vitro; (2)
designing and in vitro testing of AsiCs to knockdown the specific
gene; (3) evaluating the effect of gene knockdown on in vitro
proliferation and T-IC function in a variety of breast cancer cell
lines; and (4) verifying the lack of off-target immune stimulation
of the individual AsiC. The genes that behave best in vitro can be
advanced to in vivo testing in orthotopic and metastatic models as
described above for PLK1. In these experiments we can compare
untreated mice with mice treated with EpCAM-AsiCs targeting the
specific gene or PLK1. If there is a specific inhibitor drug for a
particular gene target (i.e. bortezomib/carfilzomib for the
proteasome), a group of control mice can also be treated with the
drug for comparison. Exemplary genes for such experiments are
proteasome genes and MCL1, U4/U6-U5 tri-snRNP complex genes96,97,
XBP1 and the kinetochore gene NDC80. AsiCs that have the best in
vivo activity on their own will also be evaluated in combinations
with PLK1 AsiCs and each other. Since proteasome inhibitor
sensitivity correlates strongly with MCL1 dependency in vitro (not
shown), we hypothesize that proteasome gene and MCL1 knockdown will
be synergistic. The synergy of different AsiC and AsiC/drug
combinations can be formally tested by the isobologram method using
different RNA dose combinations or combinations with relevant
inhibitor drugs. In particular we will determine whether combining
EpCAM-AsiCs with standard of care drugs, such as paclitaxel, is
synergistic with the original construct.
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Example 6
Material and Methods
Cell Culture
Human BPE and BPLER cells were grown in WIT medium (Stemgent).
MB468 were transduced with a luciferase reporter. All other human
cell lines were obtained from ATCC and grown in MEM (MCF7, BT474),
McCoy's 5A (SKBR3), RPMI1640 (HCC1806, HCC1143, HCC1937, HCC1954,
HCC1187, MB468, T47D) or DMEM (MB231, BT549, MB436) all
supplemented with 10% FBS, 1 mM L-glutamine and
penicillin/streptomycin (Gibco) unless otherwise indicated. 4T1
mouse breast cancer cells, were grown in 10% FBS DMEM. For in vivo
imaging, MB468 cells stably expressing Firefly luciferase
(MB468-luc) were used and MB231 cells stably expressing Firefly
luciferase and mCherry (MB231-luc-mCherry) were selected after
infection with pLV-Fluc-mCherry-Puro lentivirus. MB231 Cells were
selected with puromycin.
For uptake and silencing treatment, cells were plated at low
density (10,000 cells/well in 96-well plates) and treated
immediately. All AsiC and siRNA treatments were performed in either
OptiMEM or WIT medium. Cell viability was assessed by CellTiter-Glo
(Promega) or by Trypan-Blue staining in 96-well plates.
For colony formation assay, 1,000 viable cells were treated for 6 h
in round bottom 96-well plates and then transferred to 10-cm plates
in serum-containing medium. Medium was replaced every 3 d. After
8-14 d, cells were fixed in methanol (-20 C) and stained with
crystal violet. For sphere formation assay, 1,000/ml viable cells
were treated for 6 h in round bottom 96-well plates and then
cultured in suspension in serum-free DMEM/F12 1:1 (Invitrogen),
supplemented with EGF (20 ng/ml, BD Biosciences), B27 (1:50,
Invitrogen), 0.4% bovine serum albumin (Sigma) and 4 .mu.g/ml
insulin (Sigma). Spheres were counted after 1 or 2 weeks.
siRNA Transfection
Cells were transfected with Dharmafect I per the manufacturer's
protocol. See below herein for all siRNA sequences.
Flow Cytometry.
For flow cytometry, cells were stained as previously described (Yu,
F. et al (2007). let-7 Regulates Self Renewal and Tumorigenicity of
Breast Cancer Cells. Cell 131, 1109-1123.), briefly, direct
immunostaining of EpCAM and AKT1 was performed using 1:50 dilutions
of hAb for 30-60 minutes at 4.degree. C. (BioLegend/BD). Cells were
stained in PBS containing 0.5% FCS, 1 mM EDTA, and 25 mM HEPES.
Samples were washed twice in the same buffer. Data was acquired
using FACS-Canto II (BD Biosciences). Analyses were performed in
triplicate and 10,000 gated events/sample were counted. All data
analysis was performed using FlowJo (Treestar Inc.).
RNA Analysis.
qRT-PCR analysis was performed as described (Petrocca, F., et al.
(2008). E2F1-regulated microRNAs impair TGFbeta-dependent
cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 13,
272-286). Briefly, total RNA was extracted with Trizol (Invitrogen)
and cDNA prepared from 1000 ng total RNA using Thermoscript RT kit
(Invitrogen) as per the manufacturer's SYBR Green Master Mix
(Applied Biosystems) and a BioRad C1000 Thermal Cycler (Biorad).
Relative CT values were normalized to GAPDH and converted to a
linear scale.
Collagenase Digestion of Human Breast Tissue.
Fresh breast or colon cancer and control biopsies were received
from the UMASS Tissue Bank, samples were cut into 3.times.3.times.3
mm samples and placed in a 96 well plate with 100 ul RPMI. Samples
were treated with either Alexa647-siRNA-GFP,
Alexa647-chol-siRNA-GFP or Cy3-AsiC-GFP for 24 hr. Samples were
photographed and digested. Three samples from each treatment were
pooled and put in 10 ml RPMI containing 1 mg/ml collagenase II
(Sigma-Aldrich) for 30 minutes at 37.degree. C. with shaking.
Samples were disrupted in a gentleMACS dissociator (Miltenyi) using
the spleen program for 30 minutes at 37.degree. C. both before and
after collagenase digestion. Cell suspensions were passed through a
70-.mu.m cell strainer (BD Falcon), washed with 30 ml RPMI, and
stained for flow cytometry.
Animal Experiments
All animal procedures were performed with Harvard Medical School
and Boston Children's Hospital Animal Care and Use Committee
approval. Nude mice were purchased from the Jackson Laboratory.
In Vivo Experiments.
For tumor initiation studies 8-week old female Nu/J mice (Stock
#002019, Jackson Laboratories) were injected subcutaneously with
MB468-luc (5.times.10.sup.6) cells pretreated for 24 h with
EpCAM-AsiC-GFP, EpCAM-AsiC-PLK1 or untreated. Cells were
trypsinized with Tryple Express (Invitrogen), resuspended in WIT
media and injected subcutaneously in the flank. Following
intraperitoneal injection of 150 mg/kg D-luciferin (Caliper Life
Sciences) luminescent images of the whole body were taken every 5
days for a total of 20 days using the IVIS Spectra system (Caliper
Life Sciences).
For AsiC uptake experiments MB468-luc (5.times.10.sup.6) and
MB231-luc-mCherry (5.times.10.sup.5) cells trypsinized with Tryple
Express (Invitrogen), were resuspended in a 1:1 WIT-Matrigel
solution and injected subcutaneously in the flank of 8-week old
female Nu/J mice (Stock #002019, Jackson Laboratories). Tumors size
was analyzed daily using the IVIS Spectra system (Caliper Life
Sciences). After 5 days tumors were clearly visible and mice were
injected subcutaneously in the neck area with
Alexa750-EpCAM-AsiC-GFP (0.5 mg/kg). Localization of the AsiC
compared to the tumor was tested every 48 h for 7 days.
For tumor inhibition studies, MB468-luc (5.times.10.sup.6) and
MB231-luc-mCherry (5.times.10.sup.5) cells trypsinized with Tryple
Express (Invitrogen), resuspended in a 1:1 WIT-Matrigel solution
and injected subcutaneously in the flank of 8-week old female Nu/J
mice (Stock #002019, Jackson Laboratories). Tumors size was
analyzed daily using the IVIS Spectra, after 5 days tumors were
clearly visible. Mice bearing tumors of comparable size were
randomized into 5 groups and treated with 5 mg/kg of
EpCAM-AsiC-PLK1, EpCAM-AsiC-GFP, EpCAM-Aptamer, siRNA-PLK1 or
untreated. Mice were treated every 72 h for 14 days.
All Images were analyzed using Living Image.RTM. software (Caliper
Life Sciences).
Statistical Analysis
Student's t-tests, computed using Microsoft Excel, were used to
analyze the significance between the treated samples and the
controls where the test type was set to one-tail distribution and
two-sample equal variance.
Results:
EpCAM-AsiC Specifically Targets Basal a Breast Cancer Cells
An EpCAM aptamer was selected by Systematic Evolution of Ligands by
Exponential Enrichment (SELEX) for binding to human EpCAM. The
optimized aptamer is only 19 nucleotides (nt) long and binds to
human EpCAM with 12 nM affinity (Shigdar S. et. al. RNA aptamer
against a cancer stem cell marker epithelial cell adhesion molecule
affinity Cancer Sci. 2011 May; 102(5):991-8). It does not bind to
mouse EpCAM (FIG. 22). Its short length is ideal for an AsiC drug,
since RNAs of .about.60 nt or less in length can be cheaply and
efficiently chemically synthesized. The EpCAM-AsiCs we designed
consist of a longer strand of 42-44 nt (19 nt aptamer+3 nt
linker+20-22 nt sense (inactive) strand of the siRNA), which is
annealed to a 20-22 nt antisense (active) siRNA strand (FIG. 21A).
Both strands were commercially synthesized with 2'-fluoropyrimidine
substitutions, which confer enhanced stability in serum and other
bodily fluids (T1/2>>3 d) and prevent stimulation of innate
immune RNA sensors. We first assessed EpCAM cell surface levels by
flow cytometry in a panel of human breast cell lines (Table 2, FIG.
23). EpCAM was highly expressed by all basal A and luminal cancer
cell lines tested, but not by basal B cancer cell lines. EpCAM
staining of normal human epithelial cells (BPE) was close to
background, while its transformed derivative BPLER had bright EpCAM
staining (FIG. 21B). Several of a handful of designs tested (with
the sense and antisense strands exchanged and several linkers)
knocked down gene expression in EpCAM+, but not EpCAM-, cell lines,
but the design that worked best in dose response experiments is
shown in FIG. 21A. To test whether EpCAM-AsiC will be specifically
taken up by EpCAM+ cell lines we labeled the 3' end of the
antisense strand of the AsiC with Alexa647. BPLER basal A TNBC cell
line overexpresses EpCAM, while BPE a control epithelial breast
cell line do not (FIG. 21B). Both BPLER and BPE cell were treated
with the Alexa647-EpCAM-AsiC targeting GFP, only BPLER displayed
uptake of the AsiC (FIG. 21C). We further validated the selective
uptake of EpCAM-AsiC, by treating EpCAM+ MDA-MB-468 cells and BPE
controls with Cy3 labeled EpCAM-Aptamer (the 19 nt aptamer was
labeled with Cy3 at the 5' end). After 22 and 43 hours we clearly
saw selective AsiC uptake in EpCAM+ cells (data not shown). To
understand the ability of EpCAM AsiC to selectively trigger gene
knockdown we chose BPLER and BPE cell lines which stably
overexpress GFP. Cells were treated with either EpCAM-AsiCs
targeting GFP or transfected with GFP-siRNA as a positive control
(FIG. 21D). Although transfection with GFP-siRNAs knocked down gene
expression equivalently in BPE and BPLER, EpCAM-AsiCs selectively
knocked down expression in BPLER without any lipid; knockdown was
uniform and comparable to that achieved with lipid
transfection.
These results clearly indicate that EpCAM-AsiC is selectively
taken-up by EpCAM+ cell and can induce gene knockdown specifically
in these EpCAM+ cells. Also we show that using different
fluorophores (Alexa647 or Cy3) at different locations (5' of
aptamer or 3' of anti-sense strand) did not impact the specific
uptake.
Specific mRNA and protein knockdown was further analyzed on 8
different breast cancer cells lines. Here we show that basal A and
luminal cell lines which overexpress EpCAM displayed decreased AKT1
mRNA and protein levels following treatment with EpCAM-AsiC
targeting AKT1. Transfection with AKT1-siRNA had a similar
knockdown effect on all cell lines, while using EpCAM-AsiC
targeting GFP as a control did not effect any of the cell lines
(FIG. 24A, 24B). There was a clear correlation between EpCAM
expression level and the knockdown effect both at an mRNA and
protein level (FIG. 24D, 24E).
To determine if human epithelial breast cancer tissue can
specifically take up EpCAM-AsiC compared to healthy human tissue.
We tested human epithelial breast cancer biopsies and healthy
control tissue from the same patient. Samples were treated for 24 h
with Alexa647-siRNA-GFP, Alexa647-chol-siRNA-GFP or
Cy3-EpCAM-AsiC-GFP (FIG. 25A). Human tumor samples display higher
EpCAM level as well as higher cytokeratin levels, an epithelial
cell marker (FIG. 25B). Labeled siRNA and chol-siRNA penetrated
both tumor and healthy tissue with similar efficacy while
EpCAM-AsiC was selectively uptaken by the tumor tissue and not by
the healthy control tissue sample (FIG. 25C, 25D). The uptake
experiment was repeated in tumors from three different patients,
each biopsy received was tested 3 times for each treatment. A
summary of all three patients (FIG. 25E). Colon cancer biopsies
were tested and compared to matched healthy samples, both healthy
and tumor colon samples were able to take up Cy3-EpCAM-AsiC-GFP
(FIG. 26)
EpCAM AsiC Targeting PLK1 Specifically Inhibits Cell Proliferation
in Basal a Breast Cancer Cells
To understand whether EpCAM-AsiC can specifically target basal A
and luminal breast cancer cells and inhibit proliferation we
designed an EpCAM-AsiC targeting PLK1. PLK1 is a known trigger for
G2/M transition. The effect of EpCAM-AsiC targeting PLK1 on cell
proliferation was tested on 10 breast cancer cells representative
of basal A, B and luminal cell lines. EpCAM-AsiC targeting PLK1
decreased cell proliferation in both basal A and luminal cell lines
while having no effect on basal B cells (FIG. 27A). A correlation
was seen between EpCAM expression levels and cell viability (FIG.
27B). To understand if EpCAM-AsiC will specifically target EpCAM+
cells in a mix cell population HCC1937 (EpCAM+GFP-) cell were
co-cultured with BPE (EpCAM-GFP+) cells and treated with EpCAM-AsiC
targeting PLK1 or untreated. Untreated co-culture displayed a
similar ration of cells (41% BPE and 59% HCC1937). Following
EpCAM-AsiC targeting PLK1 treatment the ratio of EpCAM+ cells
decreased to 17% and EpCAM- cells increased to 83% indicating that
the EpCAM-AsiC specifically suppresses proliferation in EpCAM+
cells. The co-culture was repeated with other basal A cell lines
(MB468 and HCC1143) similar results were obtained. When BPE cells
were grown in a co-culture with basal B cell (MB231) the ratio
between BPE and MB231 cells stayed the same regardless of the
EpCAM-AsiC treatment (66% BPE and 33% MB231 in untreated co-culture
and 61% BPE and 38% MB231 following EpCAM-AsiC treatment) (FIG.
27C, 27D).
To determine if the suppression effect of EpCAM-AsiC targeting PLK1
on cell viability in basal A cells is triggered by EpCAM-aptamer
binding to the EpCAM receptor or by silencing of PLK1 we treated
cell with the EpCAM-aptamer and compared to EpCAM-AsiC targeting
PLK1. EpCAM-AsiC targeting PLK1 suppressed cell viability in basal
A and luminal cell lines while EpCAM-aptamer didn't effect cell
viability in any of the cell lines (FIG. 28).
One of our goals was to understand if EpCAM-AsiC targeting PLK1
could be utilized to target T-ICs within a tumor. To examine
whether it might be active not only against the bulk of cells
within basal-A and luminal cells, but also against the T-ICs within
them, we treated basal A,B and luminal cell lines with EpCAM-AsiC
targeting PLK1 for 24 hr and tested the effect on in vitro colony
and sphere formation. Basal A and luminal cell lines that form
colonies when plated at clonal density (HCC1937, HCC1954, HCC1806
and MCF7) lost the ability to form colonies after EpCAM-AsiC
targeting PLK1 treatment, whereas resistant clones emerged after
paclitaxel treatment (FIG. FIG. 29A-29B). In contrast, exposure to
EpCAM-AsiC targeting PLK1 did not effect colony formation of basal
B (MB231 and BT549) cells, while paclitaxel had a similar effect to
basal A and luminal cells, reducing colony formation but still
resistant clones invariably emerged. Likewise, among breast cancer
cell lines that form spheres under non-adherent conditions,
paclitaxel, reduced sphere-formation in all (FIG. 29C), while
EpCAM-AsiC targeting PLK1 specifically inhibited sphere formation
in basal A and luminal. To examine whether pretreatment with
EpCAM-AsiC targeting PLK1 will inhibit or delay tumor initiation
in-vivo we treat MB-468-luc cell with EpCAM-AsiC targeting PLK1,
GFP or untreated for 24 h and injected the cells into the flank of
nude mice. Using the IVIS Spectra imaging system we followed tumor
growth every 5 days for 20 days. Cells pretreated with EpCAM-AsiC
targeting PLK1 did not show any sign of a tumor after 20 days while
untreated cells or cells pretreated with EpCAM-AsiC targeting GFP
displayed tumors after 5 days and the tumor size grew during the 20
days (FIG. 29D).
EpCAM AsiC Targeting PLK1 Specifically Inhibits Tumor Initiation
and Growth in Basal a Breast Cancer Cells
We were able to show that EpCAM-AsiC can specifically target EpCAM+
cell in-vitro, to understand whether this ability is retained
in-vivo we first tested the stability of EpCAM-AsiC in mouse and
human serum over time. We saw that EpCAM-AsiC is stable for at
least 36 h in both mouse and human serum (FIG. 30A-30B). We
injected nude mice with both MB468-luc and MB231-luc-mCherry cells
on opposite flanks. After 5 days when tumors were clearly visible
using the IVIS Spectra imaging system, we injected mice s.c. (in
the neck area, as far away as possible from the tumor cells
injection sight) with 0.5 mg/kg of Alexa750 labeled EpCAM-AsiC
targeting GFP. The mice were imaged immediately after injection and
again after 24, 48 hr and 5 days to follow the AsiC localization.
The Alexa750 labeled EpCAM-AsiC targeting GFP was clearly localized
to the MB468-luc tumor (EpCAM+) and not the MB231-luc-mCherry
(EpCAM-) tumor (FIG. 31A). Analysis of 7 mice indicates a
significant increase of Alexa750 in MB468 (EpCAM+) tumors (FIG.
31B). At day 5 the tumors were removed and visualized to validate
that the Alexa750 labeled EpCAM-AsiC targeting GFP indeed entered
the tumors. Increased level of Alexa750 was negatively correlated
with mCherry levels (data not shown)
Our cell viability and tumor initiation data indicates that EpCAM
AsiC targeting PLK1 specifically inhibits tumor growth in Basal A
breast cancer cells. To test this hypothesis we injected nude mice
with ether EpCAM- basal B cells (MB231-luc-mCherry cells) or EpCAM+
basal A cells (MB468-luc cells). Once tumors were clearly visible
by the IVIS imaging system mice were treated with 5 mg/Kg of either
EpCAM AsiC targeting PLK1 or GFP every 72 h for 14 days or left
untreated. Mice were imaged using the IVIS Spectra imaging system
every 72 h for 14 days. MB468-luc tumors treated with EpCAM-AsiC
targeting PLK1 shrunk in size as early as 6 days post treatment and
in many mice completely disappeared after 14 days, while
MB231-luc-mCherry tumors remained unchanged. We believe that
EpCAM-AsiC did have some effect even though it was targeting GFP
since basal A tumor treated with GFP AsiC did not increase in size
as much as control untreated mice. Treatment with EpCAM-Asic
targeting GFP suppress tumor growth in both EpCAM+ and EpCAM-
tumors but didn't eliminate tumors. Untreated tumors both EpCAM+
and EpCAM- increased in size over the 14 days (FIG. 32A-32B).
TABLE-US-00003 TABLE 1 EpCAM-AsiC Sequences SEQ ID AsiC construct
Sequence NO EpCAM PLK1 GCG ACU GGU UAC CCG GUC GUU 1 sense UUG AAG
AAG AUC ACC CUC CUU AdTdT EpCAM PLK1 UAA GGA GGG UGA UCU UCU UCA 2
anti-sense dTdT EpCAM AKT1 GCG ACU GGU UAC CCG GUC GUU 23 sense GCU
GGA GAA CCU CAU GCU GdTdT EpCAM AKT1 CAG CAU GAG GUU CUC CAG CdTdT
24 anti-sense EpCAM GFP GCG ACU GGU UAC CCG GUC GUU 25 sense UGG
CUA CGU CCA GGA GCG CAdTdT EpCAM GFP UGC GCU CCU GGA CGU AGC CdTdT
26 anti-sense siGFP sense UGG CUA CGU CCA GGA GCG 27 siGFP
antisense UGC GCU CCU GGA CGU AGC 28 siAKT1 sense GCU GGA GAA CCU
CAU GCU G 29 siAKT1 antisense CAG CAU GAG GUU CUC CAG C 30 siPLK1
sense UGA AGA AGA UCA CCC UCC UUA 31 siPLK1 antisense UAA GGA GGG
UGA UCU UCU UCA 32
TABLE-US-00004 TABLE 2 EpCAM mean fluorescence intensity (MFI) of
human breast cell lines Cell line Subtype EpCAM MFI BPE
immortalized normal epithelium 2 BPLER basal-A TNBC 109 HMLER
unclassified TNBC (myoepithelial) 72 HCC1143 basal-A TNBC 1068
HCC1937 basal-A TNBC 806 HCC1187 basal-A TNBC 289 HCC1806 basal-A
TNBC 558 HCC70 basal-A TNBC 443 MB468 basal-A TNBC 340 MCF7 luminal
583 T47D luminal 799 BT549 basal-B TNBC 2 MB231 basal-B TNBC 31
MB436 basal-B TNBC 4 Human fibroblast Normal tissue 14
Example 7
Triple negative breast cancers have the worst prognosis of any
breast cancer subtype and there is no targeted TNBC therapy. TNBCs
have the phenotype associated with tumor initiating cells (T-IC),
also known as cancer stem cells. T-IC are resistant to chemotherapy
and thought to be responsible for tumor relapse and metastasis.
EpCAM is expressed at gap junctions at low levels on normal
epithelial cells, but much more highly expressed (100-1000-fold
greater) throughout the membrane of virtually all epithelial
cancers and is a known TI-C marker.
Described herein is a strategy for gene knockdown therapeutics for
basal-like TNBCs. As described herein, the aptamer-siRNA chimera
(AsiC) platform is adapted to transfect epithelial breast cancer
cells while also targeting breast tumor-initiating cells (T-IC).
The aptamer binds to EpCAM, highly expressed on cancer cells and
cancer stem cells. As proof-of-concept, the siRNA is directed at a
kinase required for mitosis in all cells (PLK1).
As demonstrated herein, the EpCAM-AsiC's are stable in human and
mouse. The EpCAM AsiCs can be chemically synthesized with 2'-F
pyrimidines and dTdT at the 3'-ends, which makes them resistant to
RNases and unlikely to stimulate innate immunity.
Cells were treated with 4 mM EpCAM-AsiC for 5 days and specific
AKT1 protein silencing by AKT1-AsiC was detected by flow cytometry
(FIG. 24F).
MB468 tumors regress only after treatment with PLK1 EpCAM-AsiC.
Mice with sc MB468 tumors were treated with 5 mg/kg RNA 2.times./wk
beginning when tumors became palpable. PLK1 EpCAM-AsiC, GFP
SpCAM-AsiC, EpCAM aptamer, PLK1 siRNA, and mock treated samples
were analyzed (FIG. 33)
SEQUENCE LISTINGS
1
33145DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 1gcgacugguu acccggucgu uuugaagaag
aucacccucc uuatt 45223DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 2uaaggagggu
gaucuucuuc att 23345DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 3gcgacugguu acccggucgu
uuuaaggagg gugaucuucu ucatt 45420DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 4ttcaccacca tggagaaggc
20520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5ggcatggact gtggtcatga 20621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6ctggagcagc tgaatggaaa g 21721DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7cttgaagtcc gccctgtagg t
21821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8tgccttcatt tatcccttga a 21924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9ttactacatt cagccaaaaa gcac 241018DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 10gctgccgtca ttttctgc
181118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11tctcactggc ccgtcatc 181222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12ggaggttgca gtgccaacga ag 221322DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 13tggaagggag gcagggcata ac
221422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14tttgcccaga ctcgagctcc tg 221522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15gggtgcaggt tcgggattca ac 221629PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 16Ala Ala Leu Glu Ala Leu
Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala1 5 10 15Leu Glu Ala Leu Ala
Glu Ala Ala Ala Ala Gly Gly Cys 20 251730PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala1
5 10 15Glu Ala Leu Ala Glu Ala Leu Ala Ala Ala Ala Gly Gly Cys 20
25 301815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu
Ala Glu Ala1 5 10 151916PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 152011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Ala
Ala Leu Leu Pro Val Leu Leu Ala Ala Pro1 5 102113PRTHuman
immunodeficiency virus 21Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln1 5 102216PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Arg Gln Ile Lys Ile Trp Phe
Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 152342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 23gcgacugguu acccggucgu ugcuggagaa ccucaugcug tt
422421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 24cagcaugagg uucuccagct t
212543DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 25gcgacugguu acccggucgu uuggcuacgu
ccaggagcgc att 432621DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 26ugcgcuccug
gacguagcct t 212718RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 27uggcuacguc caggagcg
182818RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28ugcgcuccug gacguagc 182919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29gcuggagaac cucaugcug 193019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30cagcaugagg uucuccagc 193121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31ugaagaagau cacccuccuu a 213221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32uaaggagggu gaucuucuuc a 213322RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 33gcgacugguu acccggucgu uu 22
* * * * *