U.S. patent application number 14/819095 was filed with the patent office on 2015-11-19 for gene silencing by single-stranded polynucleotides.
The applicant listed for this patent is GeneSegues, Inc.. Invention is credited to Gretchen M. Unger.
Application Number | 20150329862 14/819095 |
Document ID | / |
Family ID | 39674821 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150329862 |
Kind Code |
A1 |
Unger; Gretchen M. |
November 19, 2015 |
Gene Silencing by Single-Stranded Polynucleotides
Abstract
The present invention relates to compositions and methods for
concurrently activating antisense and double-stranded RNase
(dsRNase) mechanisms for inhibiting expression of a targeted gene,
by delivering a single stranded bifunctional chimeric DNA/RNA
oligonucleotide optimized for siRNA activity as well as antisense
activity, into the nucleus of a target cell.
Inventors: |
Unger; Gretchen M.; (Chaska,
MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GeneSegues, Inc. |
Chaska |
MN |
US |
|
|
Family ID: |
39674821 |
Appl. No.: |
14/819095 |
Filed: |
August 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13848249 |
Mar 21, 2013 |
9132148 |
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14819095 |
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12525652 |
Aug 3, 2009 |
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PCT/US08/52863 |
Feb 1, 2008 |
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13848249 |
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60898674 |
Feb 1, 2007 |
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Current U.S.
Class: |
514/44A ;
435/6.13 |
Current CPC
Class: |
C12N 2310/11 20130101;
C12N 2310/51 20130101; C12N 2310/315 20130101; C12N 2310/3521
20130101; C12N 15/113 20130101; C12N 2320/32 20130101; C12N
2310/321 20130101; C12N 15/111 20130101; C12N 2310/14 20130101;
G01N 33/5308 20130101; A61K 31/7125 20130101; C12N 2310/321
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; G01N 33/53 20060101 G01N033/53 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in part with government support from
the National Institutes of Health under Grant No. NIH GRANT R43
CA119556. The United States Government may have certain rights in
the invention.
Claims
1. A method for preparing a formulation comprising a single
stranded polynucleotide for inhibiting gene expression in a
mammalian cell, comprising: employing a suitable siRNA design
algorithm to identify a candidate functional double stranded siRNA
to a target RNA; and synthesizing a single stranded polynucleotide
sequence comprising at least a portion of the guidestrand of the
candidate functional double stranded siRNA, wherein a functional
single stranded polynucleotide consisting of 15-25 linked
nucleosides without a self-complementary sequence region is formed;
wherein the functional single stranded polynucleotide sequence
consists of a 3' RNA portion and a 5' DNA portion where the RNA
portion comprises at least three ribonucleotides and the DNA
portion comprises at least 50% of said polynucleotide; wherein each
of the internucleoside linkages is a phosphodiester linkage; and
formulating the resulting single stranded polynucleotide, in the
absence of a passenger strand, with a pharmaceutically acceptable
carrier for delivery to the perinuclear region or the nucleus of
the mammalian cell.
2. The method of claim 1, wherein the number of 3' ribonucleotides
of the single stranded polynucleotide is determined by assaying
gene inhibition in cells plated on extracellular matrix
protein.
3. The method of claim 2, wherein the cells are on a tissue
scaffold.
4. The method of claim 1, wherein the number of 3' ribonucleotides
of the single stranded polynucleotide is determined by assaying
gene inhibition in cells in multicellular spheroids.
5. The method of claim 1, wherein at least one of the nucleotides
of the polynucleotide is a modified nucleotide.
6. The method of claim 1, wherein the pharmaceutically acceptable
carrier is non-viral.
7. The method of claim 1, wherein the pharmaceutically acceptable
carrier further comprises a ligand that directs said carrier to the
mammalian cell.
8. A method of treating a patient having a disease characterized by
the undesired production of a protein, comprising: a. determining
the levels of Argonaute genes or RNAseH genes or both in a tissue
sample of the patient; and b. selecting, based on the level of
Argonaute genes or RNAseH genes or both, a dosage level for
administering a formulation prepared according to the method of
claim 1; and c. administering the formulation to said patient.
9. The method of claim 8, wherein said administration of the
formulation concurrently activates antisense and dsRNAse
mechanisms.
10. The method of claim 8, wherein the disease is cancer.
11. A method for identifying a single stranded polynucleotide that
interferes with function of target RNA in a cell, comprising:
identifying one or more candidate functional double stranded siRNAs
to the target RNA; synthesizing a single stranded polynucleotide
sequence comprising at least a portion of the guidestrand of the
candidate functional double stranded siRNA; and determining whether
said candidate single stranded polynucleotide inhibits said target
RNA, wherein the single stranded polynucleotide sequence comprises
a 3' RNA portion and a 5' DNA portion, and wherein the DNA portion
constitutes at least 50% of the functional single stranded
polynucleotide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application continuation of U.S. application Ser. No.
13/848,249, filed Mar. 21, 2013, which is a continuation of U.S.
application Ser. No. 12/525,652, filed Aug. 3, 2009, which is a 35
U.S.C. .sctn.371 national phase application of PCT/US2008/052863
(WO 2008/095192), filed on Feb. 1, 2008, each entitled "Gene
Silencing by Single-Stranded Polynucleotides," which application
claims the benefit of U.S. Provisional Application Ser. No.
60/898,674, filed Feb. 1, 2007, each of which are incorporated
herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to the field of gene silencing
and concurrent activation of RNAi and antisense pathways in a cell,
via a single-stranded polynucleotide delivered to the nucleus of
the cell.
BACKGROUND
[0004] Oligonucleotide compounds have important therapeutic
applications in medicine. Oligonucleotides can be used to silence
genes that are responsible for a particular disease. Gene silencing
prevents formation of a protein by inhibiting translation.
Importantly, gene-silencing agents are a promising alternative to
traditional small, organic compounds that inhibit the function of
the protein linked to the disease. Antisense, small interfering
RNAs (siRNAs), microRNAs (miRNAs), small hairpin RNAs (shRNAs) are
examples of oligonucleotides that prevent the formation of proteins
by gene silencing.
[0005] The most studied antisense molecules are
oligodeoxynucleotides. Antisense oligodeoxynucleotides are believed
to cause a reduction in target RNA levels principally through the
action of RNase H, an endonuclease that cleaves the RNA strand of
DNA:RNA duplexes. This enzyme, thought to play a role in DNA
replication, has been shown to be capable of cleaving the RNA
component of the DNA:RNA duplexes in cell free systems as well as
in Xenopus oocytes.
[0006] RNA interference (RNAi) is a powerful and specific method
for silencing or reducing the expression of a target gene, mediated
by small single- or double-stranded RNA molecules. These molecules,
such as siRNAs, miRNAs and shRNAs, are important intermediaries in
the RNAi pathway that lead to degradation of specific mRNAs through
the RNA-induced silencing complex (RISC). During assembly of RISC,
a single strand of the RNA molecules binds to the protein Argonaute
2 (Ago2), a key component of RISC. This strand then guides RISC to
its complementary target mRNA, which is finally cleaved by the
RNase activity located in the Ago2 protein, triggering its
destruction.
[0007] It is known in the art that RNase H activity is quite
variable between cell types. Thus a given disease state may not be
a candidate for antisense therapy because the target tissue has
insufficient RNase H activity. Similarly, Ago2 levels can vary
significantly across cell lines. Therefore it is clear there is a
need for methods and compositions to achieve gene silencing across
tissue types, at clinically relevant dosages. Such methods and
compositions would be useful for therapeutic purposes both in vivo
and ex vivo, as well as for diagnostic reagents and research
reagents, including reagents for the study of both cellular and in
vitro events.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows single-stranded chimeric polyoligonucleotides
bind nuclear Ago2 in cells plated on relevant protein matrices".
The figures describe in a) color confocal microscopy displaying
nuclear colocalization as merged yellow to orange signal from Ago2
(red signal) with nucleic acid-associated anti-Fitc signal (green).
Single-stranded chimeric oligos (denoted as "mi CK2") colocalize
with Ago2 in the nucleus following primary or effective nuclear
delivery while single-stranded oligos delivered to the cytosol via
Dotap liposomal complexes do not colocalized with Ago2 in the
nucleus. 100,000 SCC-15 cells were plated on flamed coverslips
precoated with 0.5 .mu.g/sq. cm of model tumor matrix (2:1
Tenascin: Fibronectin). Cultures were fixed and processed for
microscopy 24 hours after treatment as described in the text. Panel
b describes western blotting of lysates prepared from cells treated
as for microscopy and fractionated into nuclear and cytosolic
lysates. Results support nuclear enhancement, enrichment or
accumulation of Ago2 from baseline at 24 hours after treatment.
Nuclear accumulation of Ago2 is also increased on protein matrices
for standard siRNA delivered to nucleus relative to cytosolic
delivery. Blot is representative of 3 independent experiments.
Panel c described immunoprecipitation of nucleic acid-protein
complexes from nuclear lysates of cells grown on protein matrices
or plastic to confirm colocalization studies as true nuclear
binding between Ago2 and Fitc-labeled RNAi molecules. Cells for
lysates were grown in wells coated with model matrix as for
microscopy and NP-40 A lysates were prepared. Lysates were
incubated for 3 hours at 4.degree. C. with 200 nM of various
Fitc-labeled oligos and pulled down using Millipore "Catch and
Release" columns with goat anti-Fitc antibodies. See text and axes
legend for details. Bands indicate that true binding occurs in
nuclear lysates between Ago2 and Fitc-labeled drug in cells grown
on protein but not on plastic.
[0009] FIG. 2 shows Summary Results from FaDu Lowest Effective Dose
study. Using the FaDu model of hypopharangeal cancer, a novel
composition of mi CK2 was assayed for evidence of dose response. In
this model, mice are flank-injected with 4 million cells and
assayed with a regimen consisting of two intravenous doses started
5 days after inoculation when tumors reach 4-6 mm in diameter. A
standard dosing interval of 2 days or "q2D" was used; any
deviations are denoted on the figure axis. Doses are expressed on a
weight basis, i.e. so that "10 pg q2D" represents two doses of 10
pg/kg of body weight over a two day interval. A similar bar pattern
is used throughout bar charts for particular dosing levels. Tumor
tissue and blood were collected at .about.Day 30 when tumors
reached 15 mm in at least one dimension and assayed for molecular
changes. Tumor tissue was fractionated into cytosol and a chromatin
fraction as per (Tawfic et. al, J. Biol. Chem. 269(10): 7489-7493,
1994). Group sizes varied from 3-6 animals and animals were tested
in multiple cohorts with at least untreated controls because of the
number of test conditions. Panel a) shows the effect of dose level
on Day 30 levels of chromatin-bound CK2 (first bar) and
chromatin-bound CK2 as a percentage of total cellular CK2 (2nd bar)
from western blotting. Actin and the transcription factor SP-3 were
used as loading controls. Panel b) shows the effect of dose levels
on serum human IL-6 levels (a surrogate tumor marker) at Day 30
(1rst bar) and Day 30 tumor weight (2nd bar). Tumor weight was
recorded in a final cohort as it was realized that surface area of
resolving wounds was not representative of tumor burden. The
composition used in these studies, "Formula G" was optimized for
uniform particle size distribution and unexpectedly potent. Panel
c) shows the effect of dose level on cytosolic levels of CK2 enzyme
in Day 30 tumors. Panel d) shows the effect of dose level on
cytosolic levels of Caspase 7, an apoptotic marker, in tumor
tissue. Band intensities were normalized to quantities from
equivalent cell lysates per membrane. Quantitation of band
intensities represents two independent electrophoresis runs. Bars
represent means.+-.SE. The statistical significance of mean
differences are represented as follows: "**" p<0.01, "*"
p<0.05, "#" p<0.1
SUMMARY OF THE INVENTION
[0010] Disclosed are methods for inhibiting expression of a
targeted gene, as well as methods for activating both antisense and
double-stranded RNase activity. The methods include the steps of
providing a bifunctional single stranded chimeric polynucleotide
comprising a 3' RNA portion and a 5' DNA portion, where the
bifunctional single stranded chimeric polynucleotide is capable of
specifically hybridizing to the RNA sequence of the target gene,
and delivering the bifunctional single stranded chimeric
polynucleotide into the nucleus of the cell in an amount sufficient
for degradation or inhibition of the target RNA to occur.
Bifunctional single stranded chimeric polynucleotides capable of
activating dsRNase as a guide strand and capable of activating
RNase H are disclosed. Methods of selecting tissues and/or patients
for treatment and enhancing treatment regimens with instant
bifunctional single stranded chimeric polynucleotides are also
disclosed.
[0011] All patents and patent applications referenced herein are
incorporated by reference herein in their entireties.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention provides compositions for gene
silencing and methods of use thereof. In particular, the invention
provides bifunctional single stranded chimeric polynucleotides and
methods for delivering these molecules to cells, for effective and
efficient inhibition of gene expression. Additionally, the
invention provides means for in vitro and in vivo applications.
[0013] The compositions and methods provided herein further expand
the utility of oligonucleotide-based gene silencing technology as a
tool for therapeutic applications as well as functional genomics.
This discovery increases the understanding of cellular properties
with respect to gene silencing function, and in view of this
discovery, the present invention provides important tools to
improve inhibition of gene expression in vivo and in vitro.
[0014] The present invention is based, at least in part, upon the
surprising discovery that antisense and dsRNase enzyme activity can
be concurrently activated by single-stranded polynucleotides
comprising both RNA and DNA. The invention is further based, in
part, upon the discovery that nuclear delivery of these molecules
is important to effect the dual enzyme activity.
[0015] Surprisingly, we have found that RISC complexes comprised of
at least Ago2 and single-stranded chimeric polynucleotides assemble
in the nucleus of cells plated on relevant protein matrices (e.g.,
"three dimensional cell culture") following nuclear but not
cytosolic delivery. Over time, a portion of the Ago2-bifunctional
single stranded chimeric polynucleotide complexes shuttle to the
cytosol. These findings are very surprising in light of current
thinking derived from studies of cells cultured on plastic.
Although structural studies of bacterial Ago proteins identify a
preference or capacity in these proteins for DNA guide strands,
current thinking strongly supports that i) in mammalian cells
single-stranded DNA guide strands are very poorly utilized by Ago2,
and ii) that cytosolic delivery of the guide polynucleotide is
sufficient for Ago2 complex loading and RNAi-related gene
silencing.
[0016] The instant invention is also surprising in view of the
belief in the current art that extrinsic or cellular 5'
phosphorylation of the guide polynucleotide is necessary for gene
silencing activity and that 5' labeling will destroy RISC complex
stability. Our data also highlight the usefulness of the instant in
vitro methods for characterizing polynucleotide and enzyme binding.
Further, the data exemplify the need for efficient bifunctional
molecules given variation in level and activity of critical
gene-silencing enzymes across tissues.
[0017] A bifunctional nucleic acid drug offers advantages over one
with single functionality in being better able to maintain potency
across tissue variations. A single-stranded polynucleic acid drug
offers advantages over double-stranded polynucleic acids in terms
of lower molecular weight, lower production cost, and potentially
lower potential for toxicity due to the absence of potential
off-targeting through the "passenger strand" or the activation of
immune responses by double-stranded nucleic acids. Additionally,
the low molecular weight of the nucleic acids used, and efficient
nuclear delivery of them as provided by the present inventive
method, allows efficacious therapy with very low dosing, resulting
not only in reduced cost and toxicity, but also new treatment
strategies (taking advantage of the bifunctional efficacy) such as
route of administration (for example, eye drops rather than
injections for treatment of diseases of the eye with nucleic acid
drugs, or oral administration rather than intravenous infusion for
cholesterol depletion regimens in treating cardiovascular
disease).
[0018] It is reported in the art that single-stranded RNAi
treatments require 10-100.times. higher dosages than
double-stranded RNAi to achieve the same potency. Beyond medicinal
chemistry and sequence efficacy, a portion of this disadvantage is
attributable to the instability of the single strand moiety, an
issue that may be compounded by delivery into the harsh cytosol
environment. Potentially delivery of single stranded molecules to
the nucleus, particularly in a capsule-type of formulation,
provides a significant advantage in terms of retained potency.
[0019] So the invention may be better understood, the following
terms are defined.
[0020] "Specifically hybridizable" and "complementary" are terms
that are used to indicate a sufficient degree of complementarity
such that stable and specific binding occurs between a single
stranded chimeric polynucleotide of the present invention, and a
target RNA molecule. It is understood in the art that the sequence
of a polynucleotide need not be 100% complementary to that of its
target RNA molecule to be specifically hybridizable. A
polynucleotide is specifically hybridizable when (a) binding of the
polynucleotide to the target RNA molecule interferes with the
normal function of the target RNA molecule, and (b) there is
sufficient complementarity so that binding of the polynucleotide to
the target RNA molecule is highly selective and largely avoids
non-specific binding of the polynucleotide to non-target sequences
under conditions in which specific binding is desired, i.e., under
conditions in which in vitro assays are performed or under
physiological conditions for in vivo assays or therapeutic
uses.
[0021] The term "RNA" or "RNA molecule" or "ribonucleic acid
molecule" refers to a polymer of ribonucleotides. "Target RNA"
refers to any RNA that can hybridize with a sufficiently
complementary bifunctional single stranded chimeric polynucleotide
of the present invention. Target RNA can be a pre-mRNA, pre-miRNA,
pri-miRNA, mRNA, miRNA, small nuclear or cytosolic non-coding
regulatory RNAs, ribosomal RNA, transfer RNAs, an hnRNA at any
stage in the mRNA processing pathway, or mitochondrial RNAs. "mRNA"
or "messenger RNA" is single-stranded RNA that specifies the amino
acid sequence of one or more polypeptide chains. In one embodiment,
the target mRNA of the invention specifies the amino acid sequence
of a cellular protein (e.g., a nuclear, cytoplasmic, mitochondrial,
transmembrane, or membrane-associated protein). In another
embodiment, the target mRNA of the invention specifies the amino
acid sequence of an extracellular protein (e.g., an extracellular
matrix protein or secreted protein). The term "DNA" or "DNA
molecule" or deoxyribonucleic acid molecule" refers to a polymer of
deoxyribonucleotides.
[0022] The term "nucleic acid molecule" or "polynucleotide" refers
to any nucleic acid containing molecule, including but not limited
to, DNA or RNA. The term encompasses sequences that include any of
the known base analogs of DNA and RNA
[0023] The term "small interfering RNA" ("siRNA") (also referred to
in the art as "short interfering RNAs") refers to an RNA (or RNA
analog) comprising between about 10-50 nucleotides (or nucleotide
analogs) which is capable of directing or mediating RNA
interference. As used herein, the term "RNA interference" ("RNAi")
refers to a selective intracellular degradation of RNA.
[0024] "Concurrent activation" and/or "bifunctional" refers to
initiating, resuming or elevating two or more gene inhibition
mechanisms in a given cell through the introduction of the
single-stranded chimeric polynucleotide of the present invention.
Those mechanisms include antisense mechanism ("antisense") and
either double-stranded RNase enzymes or double-stranded
RNase-related gene silencing mechanisms ("dsRNase"), or a
combination thereof. The period of activation of antisense and
dsRNase mechanisms within said cell may overlap in entirety, or in
part, or not at all. The activation of antisense and dsRNase (or
dsRNase related) mechanisms within said cell may be the result of
one or more delivered bifunctional single stranded chimeric
polynucleotides of the present invention affecting both mechanisms,
or may be the result of a subpopulation of the delivered single
stranded chimeric polynucleotides separately affecting one
mechanism type, and a separate subpopulation affecting the other
mechanism type, or a combination thereof. It is understood
concurrent activation also contemplates potentially not activating
antisense mechanism in a given cell, when enzymes or other
molecules necessary for such activation, such as, for example in
certain cases, RNaseH enzymes, are not present or are present in
very low amounts in said cell. It is also understood concurrent
activation contemplates potentially not activating dsRNase activity
in a given cell, when enzymes or other molecules necessary for such
activation, such as, for example in certain cases, Argonaute 2
proteins, are not present or are present in very low amounts in
said cell.
[0025] "Antisense mechanism activation" refers to initiating,
resuming, or elevating mechanisms of inhibiting gene expression
through cleavage of complementary target RNA by RNase H enzyme
activation, or by inhibition of transcription or translation, or a
combination thereof. "RNase H enzyme activation" refers to
initiating, resuming, or elevating silencing activity of target RNA
by RNase H, an endonuclease that cleaves the RNA strand in
antisense DNA:RNA hybrid duplexes. In one embodiment, RNase H is
RNase H1, NCBI accession number NM.sub.--002936. In another
embodiment, RNase H is RNase H2, NCBI accession numbers
NM.sub.--006397, NM.sub.--024570, and NM.sub.--032192. Selective
RNA degradation is induced by creating duplexes of antisense
DNA:RNA that serve as substrates for cellular RNase H enzymes. The
antisense strand of the DNA:RNA duplex may be comprised of DNA or,
as in the present invention, be part of a DNA:RNA chimeric, and
without limitations include nucleic acid modifications thereof.
"Transcription inhibition" refers to mechanisms including binding
of pre-mRNA leading to its degradation, or inhibition of its
processing such as 5' end-capping, pre-mRNA splicing,
polyadenylation, and transcriptional termination, or mechanisms
leading to changes in the chromatin state of the target gene of
interest, or a combination thereof. "Translation inhibition" refers
to mechanisms including prevention of mRNA transport and steric
hindrance of ribosomal subunit binding or other factors involved in
the progression of translation complexes.
[0026] "Double-stranded RNase enzyme activation" refers to
initiating, resuming, or elevating mechanisms of gene inhibition
involving RNA-induced silencing complex (RISC) and related
mechanisms. Methods for RISC-based gene inhibition include, but are
not limited to, degradation of mRNA targets by binding of the RISC
complex to the target RNA and subsequent cleavage. Mechanisms
related to double-stranded RNase enzyme activation include, but are
not limited to, initiating, resuming, or elevating the silencing
activity of RISC by binding of RISC to target RNA and subsequent
translational repression and other types of post-transcriptional
gene regulation, or subsequent chromatin modification. The RISC
includes at least an Argonaute family member and antisense guide
strand. The present invention includes guide strand composition.
"Double-stranded RNase" (dsRNase) refers to the RNA cleavage
resulting from the RISC-mediated binding of the antisense RNA to
target RNA.
[0027] Without wishing to be bound by theory, the term "Argonaute"
refers to the mammalian Argonaute protein family currently known in
the art to consist of eight members, four of which are ubiquitously
expressed (Ago subfamily), with the remaining four (Piwi subfamily)
being expressed in germ cells. While Ago2 has been shown to be at
the core of the RISC complex that carries out
oligonucleotide-guided target RNA cleavage in the region of
complementarity, Ago1, 3, and 4 are thought to lack this cleavage
activity and may therefore function in related
oligonucleotide-guided gene silencing pathways that do not involve
target RNA cleavage in the region of complementarity. Similarly,
Ago2 may function in gene silencing independent of such cleavage
activity, such as in translational repression. The proteins
referred to herein may also be identified by their NCBI accession
numbers; Ago1, NP.sub.--036331; Ago2, NP.sub.--036286, Ago3,
NP.sub.--079128, and Ago4, NP.sub.--060099.
[0028] The term "nucleoside" refers to a molecule having a purine
or pyrimidine base covalently linked to a ribose or deoxyribose
sugar. Exemplary nucleosides include adenosine, guanosine,
cytidine, uridine and thymidine. The term "nucleotide" refers to a
nucleoside having one or more phosphate groups joined in ester
linkages to the sugar moiety. Exemplary nucleotides include
nucleoside monophosphates, diphosphates and triphosphates. The
terms "polynucleotide" and "nucleic acid molecule" are used
interchangeably herein and refer to a polymer of nucleotides, and
in one embodiment of the present invention, are joined together by
a phosphodiester linkage between 5' and 3' carbon atoms of the
sugar moiety.
[0029] The term "nucleotide analog" or "altered nucleotide" or
"modified nucleotide" refers to a less commonly occurring
nucleotide, including natural and non-naturally occurring
ribonucleotides or deoxyribonucleotides. Nucleotide analogs may be
modified at any position so as to alter certain chemical properties
of the nucleotide yet retain the ability of the nucleotide analog
to perform its intended function. Nucleotide analogs may also
comprise modifications to the sugar portion of the nucleotides. The
phosphate group of the nucleotide may also be modified, e.g., by
substituting one or more of the oxygens of the phosphate group with
sulfur (e.g., phosphorothioates), or by making other substitutions
which allow the nucleotide to perform its intended function. For
use in preparing the nucleoside structural subunits of the
compounds of the invention, suitable nucleobases for incorporation
in these nucleoside subunits include purines and pyrimidines such
as adenine, guanine, cytosine, uridine, and thymine, as well as
other synthetic and natural nucleobases such as xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil
and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl,
hydroxyl and other 8-substituted adenines and guanines,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine. Further purines and pyrimidines include those
disclosed in U.S. Pat. No. 3,687,808, those disclosed in the
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and
those disclosed by Englisch et: al., Angewandte Chemie,
International Edition, 1991, 30, 613.
[0030] "Phosphodiester" refers to a polynucleotide with an oxygen
atom linking consecutive nucleotides. "Phosphorothioate" refers to
a polynucleotide in which the oxygen atom normally linking two
consecutive nucleotides has been replaced with sulfur and which
resists degradation by cellular enzymes. Polynucleotides of the
present invention have their nucleoside subunits connected by
phosphorus linkages from a list including phosphodiester,
phosphorothioate, 3'-(or -5')deoxy-3'-(or
-5')thio-phosphorothioate, phosphorodithioate, phosphoroselenates,
3'-(or -5')deoxy phosphinates, borano phosphates, 3'-(or
-5')deoxy-3'-(or 5')amino phosphoramidates, hydrogen phosphonates,
borano phosphate esters, phosphoramidates, alkyl or aryl
phosphonates and phosphotriester phosphorus linkages.
[0031] The term "phosphorylated" means that at least one phosphate
group is attached to a chemical (e.g., organic) compound. Phosphate
groups can be attached, for example, to proteins or to sugar
moieties via the following reaction: free hydroxyl
group+phosphate.fwdarw.donor phosphate ester linkage. Also intended
to be included within the scope of the instant invention are
phosphate group analogs which function in the same or similar
manner as the mono-, di-, or triphosphate groups found in
nature.
[0032] The term "polynucleotide" refers to RNA or DNA sequences of
more than 1 nucleotide in either single chain, duplex or multiple
chain form. The term "polynucleotide" is also meant to encompass
polydeoxyribonucleotides containing 2'-deoxy-D-ribose or modified
forms thereof, RNA and any other type of polynucleotide which is an
N-glycoside or C-glycoside of a purine or pyrimidine base, or
modified purine or pyrimidine base or basic nucleotide. The
polynucleotide may encode promoter regions, operator regions,
structural regions, termination regions, combinations thereof or
any other genetically relevant material that regulates or modifies
chromatin or other polynucleotides. Similarly, the term "genetic"
as used herein, refers to any material capable of modifying gene
expression.
[0033] "Chimeric" refers but is not limited to a molecule that is
composed of both RNA and DNA moieties that are naturally occurring
or nucleotide analogs, linked by phosphodiester, phosphorothioate,
and/or any other naturally occurring or synthetic linkage that
permits the nucleotides or analogs to retain their intended
function. The oligomeric or polynucleotide can be referred to as
having at least a first segment and a second segment. The first
segment is defined as the portion beginning at the 3' end of the
polynucleotide and is the ribonucleic acid segment ad should
include at least about three consecutive ribonucleotides, and the
second segment is defined as the portion ending at the 5' end of
the polynucleotide and is the deoxyribonucleic portion, and
comprises at least about 10 consecutive deoxyribonucleotides. In
one embodiment of the polynucleotide chimeric is not more than 50%
ribonucleic acid. Preferred bifunctional single stranded chimeric
polynucleotides in accordance with this invention preferably
comprise from about 8 to about 50 nucleoside subunits. In the
context of this invention it is understood that this encompasses
non-naturally occurring oligomers as hereinbefore described, having
8 to 50 nucleoside subunits. It is more preferred that the
bifunctional single stranded chimeric polynucleotides of the
present invention comprise from about 15 to about 25 nucleoside
subunits. Accordingly, bifunctional single stranded chimeric
polynucleotides can be 8 nucleotides in length, 9 nucleotides in
length, 10 nucleotides in length, 11 nucleotides in length, 12
nucleotides in length, 13 nucleotides in length, 14 nucleotides in
length, 15 nucleotides in length, 16 nucleotides in length, 17
nucleotides in length, 18 nucleotides in length, 19 nucleotides in
length, 20 nucleotides in length, 21 nucleotides in length, 22
nucleotides in length, 23 nucleotides in length, 24 nucleotides in
length, 25 nucleotides in length, 26 nucleotides in length, 27
nucleotides in length, 28 nucleotides in length, 29 nucleotides in
length, 30 nucleotides in length, 31 nucleotides in length, 32
nucleotides in length, 33 nucleotides in length, 34 nucleotides in
length, 35 nucleotides in length, 36 nucleotides in length, 37
nucleotides in length, 38 nucleotides in length, 39 nucleotides in
length, 40 nucleotides in length, 41 nucleotides in length, 42
nucleotides in length, 43 nucleotides in length, 44 nucleotides in
length, 45 nucleotides in length, 46 nucleotides in length, 47
nucleotides in length, 48 nucleotides in length, 49 nucleotides in
length, or 50 nucleotides in length. As will be appreciated, a
"nucleoside subunit" is a nucleobase and sugar or sugar surrogate
combination suitably bound to adjacent subunits through phosphorus
linkages in oligoribonucleotides and through non-phosphorus
linkages in oligoribonucleosides. In this context, the term
"nucleoside subunit" is used interchangeably with the term
"nucleoside unit" or "nucleoside." More preferably, the chimeric
oligonucleotides of the invention will have nucleosides linked by
naturally occurring phosphodiester linkages.
[0034] As used herein, the term "guide strand" refers to the
single-stranded polynucleotide chimeric that is incorporated into
RISC complex and/or related mechanisms for degradation or
inhibition of target RNA.
[0035] In one embodiment, the present invention includes a nonviral
carrier for delivery to the nucleus. The term "non-viral carrier"
refers to any molecule or mechanical process that is able to
transfer polynucleotides across the plasma membrane into the
cytosol and/or nucleus of a cell that is not dependent on a virus,
naturally occurring or engineered.
[0036] The bifunctional single stranded chimeric polynucleotides of
the present invention may be conveniently and routinely made
through the well-known technique of solid phase synthesis, see, for
example, "Oligonucleotide synthesis, a practical approach," Ed. M.
J. Gait, IRL Press, 1984.
[0037] In one embodiment, the method of the invention is practiced
with a polynucleotide that inhibits a gene other than a Casein
Kinase 2 (CK2) gene, including CK2 alpha (csnk2a1), CK2 alpha prime
(csnk2a2), and/or CK2 beta (csnk2b), to treat prostate cancer and
head neck cancer.
[0038] In another embodiment, the method of the invention is
practiced with a polynucleotide that inhibits a gene other than a
Casein Kinase 2 (CK2) gene, including CK2 alpha, CK2 alpha prime,
and/or CK2 beta, to treat solid tumors. In another embodiment, the
method of the invention is practiced with a polynucleotide that
inhibits a gene other than a Casein Kinase 2 (CK2) gene, including
CK2 alpha, CK2 alpha prime, and/or CK2 beta
[0039] The following provides guidance for designing the
bifunctional single stranded chimeric polynucleotides of the
present invention. While not wishing to be bound by theory, it is
believed that a requirement exists for a DNA end in the
single-stranded bifunctional chimeric polynucleotide with a segment
of RNA. This suggest a potential model where the 5' DNA end
contacts the PIWI domain of Ago2 as has been described for DNA
guide strand-preferring Ago2 bacterial proteins. The RNA segment,
located, in one embodiment, on the 3' end, provides a higher
affinity contact, potentially necessary for the additional
mechanical stress that chimeric polynucleotides which are greater
than 50% DNA could encounter when acting as a guide strand for
dsRNase in contacting the target RNA.
[0040] Conventional antisense design typically optimizes for
uniform hybridization energies across sequences at sites of low
target mRNA secondary structure while siRNA design is more focused
on optimizing a hybridization profile across the sequence within
the context of sequence "rules". Design algorithms such as Soligo
for antisense and SiRNA for siRNA are publicly available (see
http:/sfold.wadsworth.org and supplier websites). Surprisingly, the
inventor's data indicates sequences validated as active guide
strands by derivation from functional double-stranded siRNAs as
currently designed are more suitable for incorporation into this
methodology than are sequences designed using tools for
conventional antisense sequences acting through RNAseH or steric
effects. However, an antisense sequence demonstrated successful by
in vivo studies, may benefit from translation into a hybrid guide
strand strategy and the capacity for binding Ago2 may be readily
confirmed in vitro as described in Example 2. The sequence "LZas4"
described herein provides an example of an optimized antisense
validated as a species of bifunctional single stranded chimeric
polynucleotide with in vivo data.
[0041] Sequence selection then consists of routine optimization in
currently available double-stranded siRNA formats, followed by
chemistry transfer and in vivo or in vitro assessment in target
cells plated on relevant protein or 3-D matrices in combination
with nuclear delivery. Peptide delivery vehicles incorporating a
nuclear localization signal have been shown to function effectively
to promote minimal RISC binding of single-stranded
polyoligonucleotides.
[0042] A discussion of conventional siRNA sequence selection is
incorporated below:
[0043] The target RNA cleavage reaction guided by siRNAs is highly
sequence specific. In general, siRNA containing a nucleotide
sequences identical to a portion of the target gene are preferred
for inhibition. However, 100% sequence identity between the siRNA
and the target gene is not required to practice the present
invention. Thus the invention has the advantage of being able to
tolerate sequence variations that might be expected due to genetic
mutation, strain polymorphism, or evolutionary divergence. For
example, siRNA sequences with insertions, deletions, and single
point mutations relative to the target sequence have also been
found to be effective for inhibition. Alternatively, siRNA
sequences with nucleotide analog substitutions or insertions can be
effective for inhibition.
[0044] Moreover, not all positions of a siRNA contribute equally to
target recognition. Mismatches in the center of the siRNA are most
critical and essentially abolish target RNA cleavage. In contrast,
the 3' nucleotides of the siRNA do not contribute significantly to
specificity of the target recognition. In particular, residues 3'
of the siRNA sequence which is complementary to the target RNA
(e.g., the guide sequence) are not critical for target RNA
cleavage.
[0045] Sequence identity may be determined by sequence comparison
and alignment algorithms known in the art. To determine the percent
identity of two nucleic acid sequences (or of two amino acid
sequences), the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the first sequence or
second sequence for optimal alignment). The nucleotides (or amino
acid residues) at corresponding nucleotide (or amino acid)
positions are then compared. When a position in the first sequence
is occupied by the same residue as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=of identical positions/total # of
positions.times.100), optionally penalizing the score for the
number of gaps introduced and/or length of gaps introduced.
[0046] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In one embodiment, the alignment generated
over a certain portion of the sequence aligned having sufficient
identity but not over portions having low degree of identity (i.e.,
a local alignment). A preferred, non-limiting example of a local
alignment algorithm utilized for the comparison of sequences is the
algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into
the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10.
[0047] In another embodiment, the alignment is optimized by
introducing appropriate gaps and percent identity is determined
over the length of the aligned sequences (i.e., a gapped
alignment). To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment,
the alignment is optimized by introducing appropriate gaps and
percent identity is determined over the entire length of the
sequences aligned (i.e., a global alignment). A preferred,
non-limiting example of a mathematical algorithm utilized for the
global comparison of sequences is the algorithm of Myers and
Miller, CABIOS (1989). Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
[0048] Greater than 90% sequence identity, e.g., 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity,
between the ss-siRNA and the portion of the target gene is
preferred. Alternatively, the siRNA may be defined functionally as
a nucleotide sequence (or oligonucleotide sequence) that is capable
of hybridizing with a portion of the target gene transcript (e.g.,
400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 degree C. or 70
degree C. hybridization for 12-16 hours; followed by washing).
Additional preferred hybridization conditions include hybridization
at 70 degree C. in 1.times.SSC or 50 degree C. in 1.times.SSC, 50%
formamide followed by washing at 70 degree C. in 0.3.times.SSC or
hybridization at 70 degree C. in 4.times.SSC or 50 degree C. in
4.times.SSC, 50% formamide followed by washing at 67 degree C. in
1.times.SSC. The hybridization temperature for hybrids anticipated
to be less than 50 base pairs in length should be 5-10 degree C.
less than the melting temperature (Tm) of the hybrid, where Tm is
determined according to the following equations. For hybrids less
than 18 base pairs in length, Tm (degree C.)=2(# of A+T bases)+4(#
of G+C bases). For hybrids between 18 and 49 base pairs in length,
Tm ((degree C.)=81.5+16.6(log 10[Na+])+0.41(% G+C)-(600/N), where N
is the number of bases in the hybrid, and [Na+] is the
concentration of sodium ions in the hybridization buffer ([Na+] for
1.times.SSC=0.165 M). Additional examples of stringency conditions
for polynucleotide hybridization are provided in Sambrook, J., E.
F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., chapters 9 and 11, and Current Protocols in Molecular
Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons,
Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
The length of the identical nucleotide sequences may be at least
about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45,
47 or 50 bases.
[0049] Phosphorothioate modification of nucleoside linkages for
increased stability has been reported to minimally effect silencing
activity (chiu+rana, Prydz). Full substitution of PS modifications
into an active single-stranded, bifunctional oligo abrogates Ago2
binding (rniLCK vs. PSLCK). While Ago2 loading of the mi (see
Example 2) version of the sequence "LZas4" was poor, it was
improved over the classic 2-O-Me/PS/2-O-Me gapmer design with a PS
center region. A PS/2-O-Me organization may be of value in
situations where PO/2-O-Me seems limited.
[0050] This analysis identifies a useful method where
oligonucleotide candidate, preferably a guide strand, garnered from
standard optimization techniques can be further improved in potency
by an additional optimization strategy consisting of i) confirming
candidate loading of Ago2 and RNAseH binding by microscopy in
vitro, preliminarily with a DNA/RNA chimeric containing six 3' RNA
nucleotides, ii) an optional subsequent determination of a
potentially more optimal number of 3' RNA nucleotides and iii)
utilization of nuclear delivery to promote Ago2 loading of the now
bifunctional single-stranded oligonucleotide.
[0051] The term "oligonucleotide" refers to a short length of
single-stranded polynucleotide chain. Oligonucleotides are
typically less than 200 residues long (e.g., between 8 and 100.
[0052] "Polynucleotide having a nucleotide sequence encoding a
gene," means a nucleic acid sequence comprising the coding region
of a gene, i.e., the nucleic acid sequence that encodes a gene
product.
[0053] The term "completely complementary," for example when used
in reference to a bifunctional single stranded chimeric
polynucleotide of the present invention refers to a single stranded
polynucleotide chimeric where all of the nucleotides are
complementary to a target sequence (e.g., a gene). It is understood
that a single stranded polynucleotide chimeric as described in the
present invention need not be 100% complementary to its target RNA
sequence to be specifically hybridizable.
[0054] The term "partially complementary," for example when used in
reference to a single stranded polynucleotide chimeric of the
present invention, refers to a single stranded polynucleotide
chimeric where at least one nucleotide is not complementary to the
target sequence. The bifunctional single stranded chimeric
polynucleotides of the invention, in one embodiment, will comprise
at least a portion that is completely complementary to a target
RNA. The remainder of the polynucleotide may be partially
complementary or noncomplementary or a combination. Described
elsewhere are methods for designing oligos that are sufficiently
complementary to be useful in the present invention and such design
skills are within the purview of one of skill in the art.
[0055] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA,
and other ncRNAs). The polypeptide can be encoded by a full length
coding sequence or by any portion of the coding sequence so long as
the desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, immunogenicity,
etc.) of the full-length or fragment is retained.
[0056] The term "gene expression" refers to the process of
converting genetic information encoded in a gene into RNA (e.g.,
mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene
(i.e., via the enzymatic action of an RNA polymerase), and for
protein encoding genes, into protein through "translation" of mRNA.
Gene expression can be regulated at many stages in the process.
"Up-regulation" or "activation" refers to regulation that increases
the production of gene expression products (i.e., RNA or protein),
while "down-regulation" or "repression" refers to regulation that
decrease production. Molecules (e.g., transcription factors) that
are involved in up-regulation or down-regulation are often called
"activators" and "repressors," respectively.
[0057] The term "inhibition of gene expression" refers to
conditions where a bifunctional chimeric single stranded
polynucleotide of the present invention hybridizes to a gene and
provides partial or completed loss of function of said gene. It is
understood that an oligomeric compound of the invention need not be
100% complementary to its target RNA sequence to be specifically
hybridizable. A reduction of target gene expression by at least
10%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% is desired relative
to the level of expression in the absence of the bifunctional
chimeric single stranded polynucleotides of the present invention.
The present invention is not limited to the inhibition of
expression of a particular gene.
[0058] As used herein, the term "delivering to the nucleus of a
cell" refers to the single stranded polynucleotide chimeric of the
present invention localizing in the nucleus after introduction into
the cell. In instances where a plurality of single stranded
polynucleotide chimerics are introduced into the cell, this term
refers to about ten percent or more, about twenty percent or more,
about thirty percent or more, about forty percent or more, about
fifty percent or more, of the molecules localizing in the nucleus.
It is understood that this term includes instances where initial
delivery of the single stranded polynucleotide chimeric is to
subcellular areas other than the nucleus, such as the perinuclear
region, wherein substantial cargo thereafter migrates to the
nucleus prior to activation of gene inhibition mechanisms. It is
also understood that this term includes the cellular pathway used
for trafficking by the single stranded polynucleotide to the
nucleus or perinuclear region prior to activation of the gene
inhibition mechanisms. It is also understood that delivery to the
nucleus of the cell includes methods that delivery a plurality, but
not all of, a cargo to the nucleus and that once delivered to the
nucleus, the bifunctional chimeric single stranded polynucleotides
of the invention may then leave the nucleus, either by passive
diffusion or active transport by natural systems.
[0059] Methods by which to deliver oligonucleotides to the nucleus
of the cell are known in the art and all such methods are
potentially adaptable to the instant invention by those of skill in
the art. In one embodiment, a method by which to deliver
oligonucleotides, including oligonucleotides of the present
invention, into the nucleus includes delivery by sub-50 nm
nanocapsules known in the art. Such capsules are described in, for
example, the following U.S. patents and patent applications and are
incorporated herein by reference in their entireties: Unger, U.S.
Pat. No. 6,632,671, issued Oct. 14, 2003; as well as U.S. Patent
Publication Nos. 20070098713, 20060018826, 20040137071,
20040038406, 2004038303, 20040023855, and 20030170893. Examples of
such nanocapsule manufacture are disclosed herein in the Examples
section.
[0060] Other nuclear delivery methods include, for example, use of
the cell penetrating peptide MPG for nuclear delivery as described
in U.S. Pat. No. 6,841,535 and used as known in the art, e.g., as
described in Simeoni et al., (Nucleic Acids Research, 2003, Vol.
31, 2717-2724). There are at least 20 peptides that increase the
delivery of oligonucleotides to cells and many of these direct
nuclear delivery. See U.S. Pat. No. 6,867,043, which is
incorporated herein by reference, for more details on known peptide
delivery vehicles. Fusion peptides comprising regions for nucleic
acid condensation, cellular targeting and nuclear localization are
readily assembled using common techniques of protein engineering
for targeted, nuclear delivery of polynucleotides in nanoparticles
complexes. See Kumar et. al, Nature 448 39-43, 2007 for effective
example of in vivo application, readily transferable to other organ
systems.
[0061] Delivery vehicles useful for efficient nuclear delivery of
single-stranded chimeric oligos, including both peptide
transduction domains and s50 protein nanocapsules traffic via
caveolae aka lipid raft vesicles in cells grown on relevant protein
matrices or 3-D culture. A relevant protein matrices refers to
proteins that primarily comprise the extracellular matrix contacted
by the target cell in vivo. This will vary by target cell type, but
matrices for tumor cells are provided in this application. Plating
cells on relevant protein or protein-coated scaffold induces many
changes in cells, including architectural and spatial changes as
well changes in the proteins expressed and relative levels of
expression. Caveolae are small (<60 nm) invaginations in the
cell membrane that can pinch off and traffic through the cell.
Caveolar transport is distinctly different from that of
clathrin-coated pits in that it is a recycling pathway and not
destructive. Caveolae are believed to proceed both from existing
invaginations and binding events in receptors segregated into
cholesterol domains known as lipid rafts (hence the alternate name
of lipid raft vesicle). Caveolae are identified at the EM level and
also by the presence of characteristic proteins from the
caveolin/cavatellin family of proteins, including such members as
flotillin and Mal. While all cells do not strictly speaking have
caveolae, all cells have caveolae-like structures or lipid raft
vesicles for recycling purposes. While not wishing to be bound by
theory, we have observed colocalization of Fitc-labeled ss chimeric
oligonucleotides, Ago2 and caveolin in cells treated with both s50
protein nanocapsules and protein transduction domain nanoparticles
suggesting that lipid raft-based delivery systems will be preferred
for execution of effective single-stranded guide strand loading
into Risc. These delivery systems will necessarily be somewhat
size-restricted because of the small size of caveolae, but several
chemistries are well-known in the art to provide execution. Because
of caveolae and lipid raft are downregulated tremendously by
multiple logs in cells plated on plastic, partial restoration of
cellular architecture by 3-D plating practice enables more
effective in vitro modeling and methods to accomplish are provided
in this application. Because, even 3-dimensional plating practice
cannot completely reconstitute cellular membranes, we show that in
vitro results from 3D culture can provide rank order guidance to in
vivo studies.
[0062] Because of the focus on nanoparticles delivery to larger
vesicles, e.g. cationic liposomes via clathrin-coated pits, the
importance of ultra-small particle size has not been fully
recognized in the art. In the present application, we provide
surprising in vivo results for a new nanoparticle composition of
highly uniform, ultra-small particles. These data teach the
importance of uniform particle size and effects on activity.
[0063] In one embodiment, the stabilization solution during
manufacturing of the above-identified nanocapsules include
Sr.sup.+3 in the stabilization solution 7.5 nM and the Mg.sup.2+ is
2.3 nM before incubating the capsules with nominal rotation for 48
hours before centrifugation was used. Sequences manufactured as
chimeric polynucleotides were optionally not propyl 3'
end-blocked.
[0064] The term "in vitro" has its art recognized meaning, e.g.,
involving purified reagents or extracts, e.g., cell extracts. The
term "in vivo" also has its art recognized meaning, e.g., involving
living cells, e.g., immortalized cells, primary cells, cell lines,
and/or cells in an organism.
[0065] "Cells," "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0066] As used, the term "eukaryote" refers to organisms
distinguishable from "prokaryotes." It is intended that the term
encompass all organisms with cells that exhibit the usual
characteristics of eukaryotes, such as the presence of a true
nucleus bounded by a nuclear membrane, within which lie the
chromosomes, the presence of membrane-bound organelles, and other
characteristics commonly observed in eukaryotic organisms. Thus,
the term includes, but is not limited to such organisms as fungi,
protozoa, and animals (e.g., humans).
[0067] The phrase "therapeutically-effective amount" as used herein
means that amount of a compound, material, or composition
comprising a compound of the present invention which is effective
for producing some desired therapeutic effect in at least a
sub-population of cells in an animal at a reasonable benefit/risk
ratio applicable to any medical treatment.
[0068] 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.
[0069] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting the subject compound from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
induce adverse events in the treated host.
[0070] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0071] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates
such as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, ayes,
etc. and non-vertebrate animals such as drosophila and nematode. In
some embodiments, "non-human animals" further refers to prokaryotes
and viruses such as bacterial pathogens, viral pathogens.
[0072] 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. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0073] The discovery of methods and compositions capable of
concurrently activating more than one mechanism of gene inhibition
in a cell, with a single nucleic acid molecule, creates significant
opportunities for new uses of nucleic acid therapy. The present
invention provides, for example, means for characterizing the gene
silencing capacity of a given cell type with regard to, for
example, Ago2 and RNase H1 activity levels, and implementing
optimized nucleic acid designs and application regimens
incorporating the polynucleotide of the present invention in
proportion to the measured activity levels. This is an advantageous
and attractive approach, for several reasons. First, the resulting
regimen would reflect the cell's "disease fighting" capacity, not
just the cell's "disease protein signals" or "disease load" (such
as, for example, tumor size in a cancer patient). Second, there
would be significant advantages in incorporating a single-stranded
molecule rather than a double-stranded molecule in such a regimen,
including lower molecular weight, lower production cost, and lower
potential for toxicity due to the absence of potential
off-targeting through the "passenger strand," or the activation of
immune responses by double-stranded nucleic acids. Third, the low
molecular weight of the nucleic acids used, and efficient nuclear
delivery of them as provided by the present inventive method,
allows efficacious therapy with very low dosing, resulting not only
in reduced cost and toxicity, but also new treatment strategies
(taking advantage of the bifunctional efficacy) such as route of
administration (for example, eye drops rather than injections for
treatment of diseases of the eye with nucleic acid drugs, or oral
administration rather than intravenous infusion for cholesterol
depletion regimens in treating cardiovascular disease). Fourth, a
given disease state (and patient) that today may not be a candidate
for nucleic acid therapy, because the target tissue has relatively
low RNase H activity or relatively low dsRNase activity, would have
a greater chance of becoming treatment eligible if the regimen
included a bifunctional polynucleotide delivered in accordance with
the methods of the invention.
[0074] Similarly, given that antisense and dsRNase or
dsRNase-related activities can be rate limiting and may have
adverse effects on the corresponding endogenous biological pathways
that these nucleic acid tools make use of, the use of
polynucleotides of the present invention potentially can be used as
a synergistic strategy, silencing a given gene target by harnessing
two distinct mechanisms or, alternatively, could be used to target
two different genes with just one polynucleotide of the present
invention. Potential synergistic effects could be realized in, for
example, antiviral applications.
[0075] In one embodiment, the invention includes a method of
activating antisense and double-stranded RNase activity toward a
target RNA. This includes the following steps, in any order. In one
step, a bifunctional single stranded chimeric polynucleotide
comprising a 3' RNA portion and a 5' DNA portion, wherein the
bifunctional single stranded chimeric polynucleotide is capable of
specifically hybridizing to the target RNA, is provided. In another
step, the bifunctional single stranded chimeric polynucleotide is
delivered into the nucleus of the cell. The bifunctional single
stranded chimeric polynucleotide is capable of activating dsRNase
as a guide strand and also, the bifunctional single stranded
chimeric polynucleotide, when duplexed with the target RNA, is
capable of activating RNase H.
[0076] In another embodiment of the instant invention, provided is
a method for inhibiting expression of a targeted gene. This
includes the following steps, in any order. In one step, a
bifunctional single stranded chimeric polynucleotide comprising a
3' RNA portion and a 5' DNA portion, wherein the bifunctional
single stranded chimeric polynucleotide is capable of specifically
hybridizing to the target RNA, is provided. In another step, the
bifunctional single stranded chimeric polynucleotide is delivered
into the nucleus of the cell. The bifunctional single stranded
chimeric polynucleotide is capable of activating dsRNase as a guide
strand and also, the bifunctional single stranded chimeric
polynucleotide, when duplexed with the target RNA, is capable of
activating RNase H.
[0077] Compounds of the invention can be utilized as diagnostics,
therapeutics and as research reagents and kits. They can be
utilized in pharmaceutical compositions by adding an effective
amount of a compound of the invention to a suitable
pharmaceutically acceptable diluent or carrier. They further can be
used for treating organisms having a disease characterized by the
undesired production of a protein. The organism can be contacted
with a compound of the invention having a sequence that is capable
of specifically hybridizing with a strand of target nucleic acid
that codes for the undesirable protein.
[0078] In one embodiment, the cell is in a 3-D cell culture.
Conventional 2-D tissue cultures grow in monolayers on the bottom
or sides of vessels in a two-dimensional architecture resulting in
a flat, two-dimensional (2-D) sheet of cultured cells, or as cells
growing individually suspended in media. Three-dimensional tissue
culture of complex cells in three dimensions may be achieved by
methods known in the art, e.g., substrate of sponge matrices,
collagen gels and what are known as organ culture systems on
filters or meshes. In addition, cell suspensions can be converted
into multicellular spheroids, another form of three-dimensional
culture. The resulting system provides an "in vivo" environment for
the full differentiation of the tissue.
[0079] In one embodiment, the cell is in vivo or ex vivo.
[0080] The formulation of therapeutic compositions of the present
invention, and their subsequent administration is believed to be
within the skill of those in the art. In general, for therapeutics,
a patient in need of such therapy is administered a compound in
accordance with the invention, commonly in a pharmaceutically
acceptable carrier, in dosages and novel regimen strategies as
described elsewhere herein. In one embodiment of the present
invention, administration is determined per kg of body weight
depending on the age of the patient and the severity of the disease
state being treated.
[0081] In one embodiment, the bifunctional single stranded chimeric
polynucleotide is delivered at a dose of less than about 10
.mu.g/kg body weight. In other embodiments, the single stranded
chimeric polynucleotide is delivered at a dose of less than about 1
.mu.g/kg body weight, less than about 100 nanogram (ng)/kg body
weight, less than about 10 ng/kg body weight, less than about 1
ng/kg body weight less than about 100 picogram (pg)/kg body weight,
less than about 10 pg/kg body weight, less than about 1 pg/kg body
weight less than about 100 femtogram (fg)/kg body weight, less than
about 10 fg/kg body weight, less than about 1 fg/kg body weight
less than about 100 attogram (ag)/kg body weight, less than about
10 ag/kg body weight, less than about 1 ag/kg body weight.
[0082] In another embodiment, the bifunctional single stranded
chimeric polynucleotide is delivered at a dose of between about 1
.mu.g/kg body weight and about 1 attog/kg body weight. In other
embodiments, dosages are between 100 ng/kg body weight and about 10
ag/kg body weight. In other embodiments, dosages are between about
10 nanogram (ng)/kg body weight and about 100 attogram (ag)/kg body
weight. In other embodiments, dosages are between about 1 ng/kg
body weight and about 1 fg/kg body weight. In another embodiment,
dosages are between about 100 picogram (pg)/kg body weight and
about 10 femtogram (fg)/kg body weight. In other embodiments,
dosages are between about 10 pg/kg body weight and about 100 fg/kg
body weight. In other embodiments, dosages are between about 1
pg/kg body weight and about 10 fg/kg body weight.
[0083] In some embodiments, the bifunctional single stranded
chimeric polynucleotide is formulated as a pharmaceutical
composition which contains a pharmaceutically acceptable carrier.
Such a pharmaceutically acceptable carrier includes, for example,
the sub-50 nanocapsules as described elsewhere herein.
[0084] Further, the treatment regimen may last for a period of time
which will vary depending upon the nature of the particular
disease, its severity and the overall condition of the patient, and
may extend from once daily to once every 20 years. Following
treatment, the patient is monitored for changes in his/her
condition and for alleviation of the symptoms of the disease state.
The dosage of the compound may either be increased in the event the
patient does not respond significantly to current dosage levels, or
the dose may be decreased if an alleviation of the symptoms of the
disease state is observed, or if the disease state has been
ablated.
[0085] In some cases it may be more effective to treat a patient
with a compound of the invention in conjunction with other
traditional therapeutic modalities. For example, a patient being
treated for a viral disease may be administered a compound of the
invention in conjunction with a known antiviral agent, or a patient
with atherosclerosis may be treated with a compound of the
invention following angioplasty to prevent reocclusion of the
treated arteries.
[0086] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the compound of the invention is
administered in maintenance doses, ranging from 0.01 .mu.g to 100 g
per kg of body weight, once or more daily, to once every 20
years.
[0087] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic, vaginal,
rectal, intranasal, transdermal), oral or parenteral. Parenteral
administration includes intravenous administration, subcutaneous,
intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
[0088] Formulations for topical administration may include
transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders. Conventional
pharmaceutical carriers, aqueous, powder or oily bases, thickeners
and the like may be necessary or desirable. Coated condoms, gloves
and the like may also be useful. Compositions for oral
administration include powders or granules, suspensions or
solutions in water or non-aqueous media, capsules, sachets or
tablets. Thickeners, flavoring agents, diluents, emulsifiers,
dispersing aids or binders may be desirable.
[0089] Compositions for intrathecal or intraventricular
administration may include sterile aqueous solutions which may also
contain buffers, diluents and other suitable additives.
[0090] Formulations for parenteral administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives.
[0091] Dosing is dependent on severity and responsiveness of the
disease condition to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual compounds, and can generally be
estimated based on EC50 found to be effective in in vitro and in
vivo animal models. Dosages may be given once or more daily,
weekly, monthly or yearly, or even once every 2 to 20 years.
[0092] In one embodiment, the present invention is useful for
treating any condition in which inhibiting a target gene is
potentially of use. In one embodiment, the present invention may be
used for treating a proliferative disease. By "proliferative
disease" is meant any human or animal disease or disorder,
affecting any one or any combination of organs, cavities, or body
parts, which is characterized by single or multiple local abnormal
proliferations of cells, groups of cells, or tissues, whether
benign or malignant. There are many disorders associated with a
dysregulation of cellular proliferation. The conditions of interest
include, but are not limited to, the following conditions. In one
embodiment, proliferative disease includes proliferation and/or
migration of smooth muscle cells, and/or inflammatory cells into
the intimal layer of a vessel, resulting in restricted blood flow
through that vessel, i.e. neointimal occlusive lesions. Occlusive
vascular conditions of interest include atherosclerosis, graft
coronary vascular disease after transplantation, vein graft
stenosis, peri-anastomatic prosthetic graft stenosis, restenosis
after angioplasty or stent placement, and the like. Other
proliferative diseases include abnormal angiogenesis, notably tumor
growth (including tumor nests) and metastasis, and other conditions
in which blood vessel proliferation is increased, such as diabetic
retinopathy, psoriasis and arthropathies. Other proliferative
diseases include those where there is hyperproliferation and tissue
remodeling or repair of reproductive tissue, e.g. uterine,
testicular and ovarian carcinomas, endometriosis, squamous and
glandular epithelial carcinomas of the cervix, etc. Proliferative
diseases include cirrhosis of the liver (a condition in which
scarring has overtaken normal liver regeneration processes),
treatment or inhibition of keloid (hypertrophic scar) formation
(disfiguring of the skin in which the scarring process interferes
with normal renewal), psoriasis (a common skin condition
characterized by excessive proliferation of the skin and delay in
proper cell fate determination), benign tumors, fibrocystic
conditions, and tissue hypertrophy (e.g., prostatic
hyperplasia).
[0093] Examples of proliferative diseases, disorders, and/or
conditions that can be treated, prevented, and/or diagnosed by the
particles of the present invention include, but are not limited to
neoplasms located in the: colon, abdomen, bone, breast, digestive
system, liver, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye,
head and neck, nervous (central and peripheral), lymphatic system,
pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative diseases, disorders, and/or
conditions can also be treated, prevented, and/or diagnosed by
particles of the present invention. Examples of such
hyperproliferative diseases, disorders, and/or conditions include,
but are not limited to: hypergammaglobulinemia, lymphoproliferative
diseases, disorders, and/or conditions, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0094] In one embodiment, the method of the invention is practiced
to treat a disease other than solid tumor cancer.
[0095] In another embodiment, the present invention includes a
method of selecting a dosage level for a tissue selected for
inhibiting expression of a targeted gene with a bifunctional single
stranded chimeric polynucleotide, comprising the steps of:
characterizing the tissue for the amount of any of the following
genes: Argonaute 2, RNase H1, or a combination; and selecting a
dosage level of bifunctional single stranded chimeric
polynucleotide treatment based on the amount of Argonaute 2, RNase
H1, or a combination present in the tissue. In addition to
Argonaute 2 or RNase H1, any other protein including RNase H2
involved in gene silencing can be characterized and used in this
embodiment.
[0096] In another embodiment, the method includes wherein the
dosage of bifunctional single stranded chimeric polynucleotide is
adjusted to an amount sufficient for degradation of the target gene
to occur by an RNAi mechanism where the tissue has an amount of
Argonaute2 that indicates functional RNAi activity.
[0097] In another embodiment, the method includes wherein the
dosage of bifunctional single stranded chimeric polynucleotide is
adjusted to an amount sufficient for degradation of the target gene
to occur by an antisense mechanism where the tissue has an amount
of Argonaute2 that does not indicate functional RNAi activity or
where the tissue has an amount of RNase H1 that indicates
functional antisense activity.
[0098] In yet another aspect, the invention provides a method for
deriving information about the function of a gene in a cell or
organism. The method includes the steps of: (a) introducing into
said cell or organism a single-stranded small interfering RNA
molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule
is sufficiently complementary to a target mRNA sequence to direct
target-specific RNA interference (RNAi) and wherein the 5'
nucleotide is 5' phosphorylated or is capable of being 5'
phosphorylated in situ or in vivo; (b) maintaining the cell or
organism under conditions such that target-specific RNAi can occur;
(c) determining a characteristic or property of said cell or
organism; and (d) comparing said characteristic or property to a
suitable control, the comparison yielding information about the
function of the gene.
[0099] Various methodologies of the instant invention include step
that involves comparing a value, level, feature, characteristic,
property, etc. to a "suitable control", referred to interchangeably
herein as an "appropriate control". A "suitable control" or
"appropriate control" is any control or standard familiar to one of
ordinary skill in the art useful for comparison purposes. In one
embodiment, a "suitable control" or "appropriate control" is a
value, level, feature, characteristic, property, etc. determined
prior to performing an RNAi methodology, as described herein. For
example, a transcription rate, mRNA level, translation rate,
protein level, biological activity, cellular characteristic or
property, genotype, phenotype, etc. can be determined prior to
introducing a ss-siRNA of the invention into a cell or organism. In
another embodiment, a "suitable control" or "appropriate control"
is a value, level, feature, characteristic, property, etc.
determined in a cell or organism, e.g., a control or normal cell or
organism, exhibiting, for example, normal traits. In yet another
embodiment, a "suitable control" or "appropriate control" is a
predefined value, level, feature, characteristic, property,
etc.
[0100] Physical methods of introducing nucleic acids include
injection of a solution containing the RNA, bombardment by
particles covered by the RNA, soaking the cell or organism in a
solution of the RNA, or electroporation of cell membranes in the
presence of the RNA.
[0101] RNA may be directly introduced into the cell (i.e.,
intracellularly); or introduced extracellularly into a cavity,
interstitial space, into the circulation of an organism, introduced
orally, or may be introduced by bathing a cell or organism in a
solution containing the RNA. Vascular or extravascular circulation,
the blood or lymph system, and the cerebrospinal fluid are sites
where the RNA may be introduced.
[0102] The polynucleotide may be introduced in an amount which
allows delivery of at least 1, 5, 10, 50, 100 or 500
polynucleotides per cell.
[0103] In another embodiment, the dosage of bifunctional single
stranded chimeric polynucleotide is decreased where the tissue has
an amount of dsRNase and RNAseH that indicates above average levels
of both functional double stranded RNase activity and RNAseH
activity. One skilled in the art will understand that the situation
where higher levels of dual key mechanistic enzyme are present may
result in synergy such that dose reduction is required to
proactively manage subject responses.
[0104] In another embodiment, the dosage of bifunctional single
stranded chimeric polynucleotide is increased where the tissue has
either an amount of dsRNase that does not indicate functional
double stranded RNase activity but an amount of RNase H that does
indicate functional antisense activity or the reverse situation.
One skilled in the art will understand that dosage may be used to
compensate for loss of standard enzymatic activity but this is an
advantage of this invention.
[0105] In another embodiment, the dosage of bifunctional single
stranded chimeric polynucleotide is increased even more where the
tissue has decreased amounts of both dsRNase and RNAseH that does
not indicate either functional double stranded RNase activity or
functional antisense activity. We have shown in the examples that
the hybrid guide strands of the instant invention are capable of
surprising enhancement of nuclear dsRNAse levels relative to
baseline so that low baseline characteristic activity is not
necessarily an indicator of unsuitability for treatment by this
method but suggests that a dosage adjustment may be required.
[0106] While the invention has been particularly shown and
described with reference to a number of embodiments, it would be
understood by those skilled in the art that changes in the form and
details may be made to the various embodiments disclosed herein
without departing from the spirit and scope of the invention and
that the various embodiments disclosed herein are not intended to
act as limitations on the scope of the claims.
EXAMPLES
[0107] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
Formulation of Single-Stranded, Bifunctional Nucleic Acid Molecules
for In Vitro and In Vivo Nuclear Delivery to Target Cells
[0108] This example describes how illustrative colloidal
formulations of diverse cargos may be generated to practice the
present inventions. Nanocapsules for uptake/binding, expression and
imaging studies were prepared by the "dispersion atomization"
method described in U.S. Pat. No. 6,632,671, which is incorporated
herein by reference in its entirety, with modifications as
described herein. These particles are used for in vitro
transfection studies in full serum-containing media. The cell
penetrating peptide MPG for nuclear delivery is a 27-mer peptide,
composed of the N-terminal domain of the HIV gp41 fusion sequence
fused to the C-terminal domain derived from the nuclear
localization signal derived from the SV40 large-T antigen and is
more fully described in U.S. Pat. No. 6,841,535 and used here
according to Simeoni et al., (Nucleic Acids Research, 2003, Vol.
31, 2717-2724) which is incorporated herein by reference. MPG is
representative of a class of useful delivery vehicles called
"protein transduction agents" or peptide delivery vehicles which
take advantage of highly cationic groups of amino acids to interact
with cell surface receptors. There are at least 20 peptides that
increase the delivery of oligonucleotides to cells and many of
these direct nuclear delivery. See U.S. Pat. No. 6,867,043, which
is incorporated herein by reference, for more details on known
peptide delivery vehicles. Fusion peptides comprising regions for
nucleic acid condensation, cellular targeting and nuclear
localization are readily assembled using common techniques of
protein engineering for targeted, nuclear delivery of
polynucleotides.
[0109] The cell penetrating peptide MPG for nuclear delivery is a
27-mer peptide, composed of the N-terminal domain of the HIV gp41
fusion sequence fused to the C-terminal domain derived from the
nuclear localization signal derived from the SV40 large-T antigen
and is more fully described in U.S. Pat. No. 6,841,535 and used
here according to Simeoni et. al, (Nucleic Acids Research, 2003,
Vol. 31, 2717-2724) which is incorporated herein by reference. MPG
is representative of a class of useful delivery vehicles called
"protein transduction agents" or peptide delivery vehicles which
take advantage of highly cationic groups of amino acids to interact
with cell surface receptors. There are at least 20 peptides that
increase the delivery of oligonucleotides to cells and many of
these direct nuclear delivery. See U.S. Pat. No. 6,867,043, which
is incorporated herein by reference, for more details on known
peptide delivery vehicles. Fusion peptides comprising regions for
nucleic acid condensation, cellular targeting and nuclear
localization are readily assembled using common techniques of
protein engineering for targeted, nuclear delivery of
polynucleotides in nanoparticles complexes.
[0110] For example, Kumar has developed and demonstrated the
noncovalent siRNA packaging properties of protein transduction
domains by synthesizing a cell-specific targeting ligand.
[0111] RVG with a poly-arginine motif for siRNA complexing (RVG-9R)
(see Kumar et al., Nature 448 39-43, 2007). Systemic administration
of RVG-9R/siRNA complexes to GFP transgenic mice resulted in siRNA
passage across the blood brain barrier and knockdown of GFP within
the brains of adult mice while not affecting GFP expression in
other organ systems. This simple yet specific siRNA delivery
approach is readily applicable to other organ systems.
[0112] Briefly, to prepare each formula below, the following
procedures were used:
[0113] Formula A, 250 .mu.g of plasmid DNA was first complexed with
36.5 .mu.g of 25 kDa polyethyleneimine (PEI; Sigma Chemical Co.,
St. Louis, Mo.), a branched cationic polymer, and dispersed into
150 .mu.l of sterile water using a water-insoluble surfactant
system (TM-diol, 10 .mu.g in DMSO or SE-30 (Air Products)). The DNA
used in these experiments was a reporter plasmid containing the
luciferase gene driven by the caags promoter (pultimateLuc, Nature
Technology). Following emulsification with a water-miscible solvent
(DMSO), the complexes were then inverted and diluted by the
addition of 750 .mu.l of PBS.
[0114] The resultant hydrophobic micelles were coated
(non-covalently) by the addition of 6.3 mcg of recombinant
fibrinogen fragment of tenascin (TBG; prepared by the method of
Aukhill et al. (1993, J. Biol Chem., 268:2542-53, with
modifications as described herein) then atomized into a LiCl and
CaCl2 salt receiving solution (135 mM Li.sup.+, 9 mM Ca.sup.2+,
27.7 nM Sr.sup.2+, 10 nM Mg.sup.2+ (all ultrapure)). Following
cold-room incubation (4.degree. C.) with nominal rotation in 50 ml
round-bottomed tubes for 48 hours, which stabilizes the coated
micelles in the salt solution, the sub-50 nm nanocapsules were
recovered by centrifugation at 20,000.times.g for 2 hrs and
resuspended in PBS+10% lactitol (at a concentration of 0.5
.mu.g/.mu.l) for filter sterilization through a 0.2 .mu.m filter.
Alternatively, this formulation may also be prepared in a
stabilization solution where the Sr.sup.+3 in the stabilization
solution is modified to 13.75 nM and the Mg.sup.2+ is modified to 5
nM before incubating the capsules with nominal rotation for 48
hours before centrifugation. In all formulations described except
Formula F, a small amount (1% of coating weight) of Syrian Hamster
IgG was "spiked" into the ligand coat to enable immunodetection of
nanocapsules uptake by anti-Syrian Hamster antibodies
[0115] Average capsule size was less than 50 nm as measured by
tapping mode atomic force microscopy using elliptical diameters of
a 1 ng/ml sample dried down on a mica sheet. Most typically the
average capsule size ranged from about 8 nanometers to about 30
nanometers.
[0116] Tenfibgen (TBG) preparation: For all formulas, TBG was
prepared by the method of Aukhil with modifications, i.e. TBG was
isolated and refolded from bacterial lysate by washing the
insoluble pellet once with lysis buffer (50 mM Tris-HCl, 1.0 mM
EDTA, 0.1 M NaCl, 0.2 mg/ml lysozyme, 0.1% Triton X-100, 0.1 mM
PMSF, pH 8.0), containing 2 M urea and resuspending in 4M GuCL, 5
mM DTT in 0.02 M Tris-HCl, pH 8.0. After additional centrifugation,
the clarified TBG solution was diluted with 2 M Guanidine-HCl, 20
mM Tris-HCl, pH 8.0 to make a final OD280 of about 1 and diluted
dropwise about 10-fold into 20 mM Tris-HCl, 0.2 M NaCl, 0.5 M
Arginine-HCl, 10 .mu.M CuCl2 pH 8.0 for overnight stirred
incubation (4.degree. C.). After diafiltration against 20 mM
Tris-HCl, pH 8.0 with an approximate 4-5 fold reduction in
concentration and 0.45 .mu.M filtration, a final purification was
performed on heparan sepharose in 20 mM Tris-HCl, pH 8.0, with
elution by bringing the NaCl concentration to 0.6 M.
[0117] Formula B: sub-50 nm nanocapsules coated with TBG were
generated as described in Formula A except that 12.5 mcg of TBG was
added to 500 mcg of a standard unmodified siRNA oligo designed to
inhibit beta-galactosidase (si.beta.gal-1, Dharmacon) and condensed
with 175 mcg of 300 MW spermine (Sigma). When generating these
nanocapsules, the Tbg-coated micelles were atomized into a modified
LiCl salt receiving solution (135 mM Lit, 9 mM Ca.sup.2+, 41.25 nM
Sr.sup.2+, 13.75 nM Mg.sup.2+ (all ultrapure)) before incubating
the capsules for 48 hours before centrifugation with nominal
rotation. Average capsule size was less than 50 nm as measured by
tapping mode atomic force microscopy using elliptical diameters of
a 1 ng/ml sample dried down on a mica sheet.
[0118] Formula C: sub-50 nm nanocapsules coated with TBG were
generated as described in Formula A except that 3.1 mcg of TBG was
added to 250 mcg of various sequences condensed with either 87.5
mcg 300 MW spermine (Sigma) for double-stranded variants or 63 mcg
of 10 kD polyornithine (Sigma) for single-stranded sequence
variants. When generating these nanocapsules, the Tbg-coated
micelles were atomized into a modified LiCl salt receiving solution
(70 mM Li.sup.+, 14 mM Ca.sup.2+, 39 nM Sr.sup.2+, 17.5 nM
Mg.sup.2+ (all ultrapure)) and capsules were incubated with nominal
rotation for 14.5 hours before centrifugation. Optionally, 39 nM
Sr.sup.2+ may be substituted in for single-stranded sequence
variants. Capsules were resuspended following centrifugation in
PBS+10% Lactitol. Average capsule size was less than 50 nm as
measured by tapping mode atomic force microscopy using elliptical
diameters of a 1 ng/ml sample dried down on a mica sheet.
TABLE-US-00001 TABLE 1a SEQ ID Molecular NO. Seq. Name Target
Sequence (5'-3')* Citation 1 si.beta.gal-1 .beta.gal CUA CAC AAA
UCA Mau et al., Bioconjugate GCG AUU UUU Chem. 2006, 17, 1209-1218
2 siZ7 .beta.gal AA GCC AAU AUU GAA Griesenbach et al., ACC CAC GG
(P) Respiratory Research, 7:26 2006 3 LZas4 .beta.gal AAC AGg tat
tcg ctg Supra. GUC AC 4 siLamin lamin UGU UCU UCU GGA Martinez et
al., Cell AGU CCA GUU CCU 110:563-742002, CCU UC (p) 5 RFP3 RFP CCA
AGA AGC CCG This application UGC AGC U (p) *p denotes passenger
strand, sense strand or mRNA target region. Lowercase denotes DNA,
upper case RNA. Sequences were synthesized in formats other than
listed as described in the text.
[0119] Formula D: MPG nanoparticles were prepared using the peptide
vector MPG:
Gly-Ala-Leu-Phe-Leu-Gly-Phe-Leu-Gly-Ala-Ala-Gly-Ser-Thr-Met
Gly-Ala-Trp-Ser-Gln-Pro-Lys-Ser-Lys-Arg-Lys-Val [SEQ ID NO:12] as
described in in Simeoni et al., (Nucleic Acids Research, 2003, Vol.
31, No. 11 2717-2724). Briefly, nanoparticles were prepared at a
charge ratio of 10:1 (MPG: nucleic acid) by incubating 0.375 .mu.g
of ss chimeric oligonucleotides and 14 .mu.l of 10 .mu.M MPG in 250
.mu.l of serum-free media for 30 minutes at 37.degree. C. with
gentle rotation. 35 mm cultures were incubated in nanoparticles
diluted 1:2 in serum-free media for 30 minutes before the addition
of 500 .mu.l of 2.times. serum-containing media.
[0120] Formula E: sub-50 nm nanocapsules coated with TBG were
generated as described in Formula C except that 3.1 mcg of TBG
(reprecipitated in ultra-pure 40% ammonium sulfate containing 250
ppb As.sup.+3, 25 ppm Se.sup.+4, 2.5 ppm Hg.sup.+2 and 25 ppm
Mo.sup.+5 for about 16 hours) was used to coat micelles.
TABLE-US-00002 TABLE 1b SEQ ID Molecular NO. Seq. Name Target
Sequence (5'-3') Citation 6 LCK CK2 ata caa ccc aaa ctC
PCT/US2005/0458 CAC AT 20 7 AsCK2 CK2 gtc ccg aca tgt caG
Pepperkok, Exp ACA GG Cell Res. 197:245- 253, 1991 8 siLuc,
luciferase CGU ACG CGG AAU Ohrt et al., NAR a.k.a. CUU CGA (p)
34(5), 1369, 2006 siGL2 *p denotes passenger strand, sense strand
or mRNA target region. Lowercase denotes DNA, upper case RNA.
Sequences were synthesized in formats other than listed as
described in the text.
[0121] Formula F: sub-50 nm nanocapsules coated with TBG were
generated as described in Formula A except that 31.25 mcg of TBG
(reprecipitated in ultra-pure 40% ammonium sulfate containing 250
ppb As.sup.+3, 25 ppm Se.sup.+4, 2.5 ppm Hg.sup.+2 and 25 ppm
Mo.sup.+5 for about 16 hours) was added to 500 mcg of an antisense
oligo to CK2 (phosphodiester 3' and propyl-end-blocked-2O'ME RNA
chimeric, "LCK-6", PCT/US 2005/045820) and condensed with 125 mcg
of 10 kD polyornithine (Sigma). When generating these nanocapsules,
the Tbg-coated micelles were atomized into a modified LiCl salt
receiving solution (135 mM Li.sup.+, 9 mM Ca.sup.2+, 10 nM
Sr.sup.2+, 2.3 nM Mg.sup.2+ (all ultrapure)) and capsules were
incubated with nominal rotation for 48 hours before centrifugation.
Capsules were resuspended following centrifugation in PBS+10%
Lactitol. Average capsule size was less than 50 nm as measured by
tapping mode atomic force microscopy using elliptical diameters of
a 1 ng/ml sample dried down on a mica sheet and a surface charge of
-2.7.+-.3 mev was measured on Zetasizer 4 dynamic light scattering
device at a potential of 20 volts with a 2-second pause between
measurements in 1 mM KCl at 2 .mu.g/ml.
[0122] Formula G: sub-50 nm nanocapsules coated with TBG were
generated as described in Formula A except that a stabilization
solution where the Sr.sup.+3 in the stabilization solution is
modified to 7.5 nM and the Mg.sup.2+ is modified to 2.3 nM before
incubating the capsules with nominal rotation for 48 hours before
centrifugation was used. Sequences manufactured as chimeric
polynucleotides were optionally not propyl 3' end-blocked.
Example 2
Nuclear Delivery Creates Significant Utility for Single-Stranded
Nucleic Acid Drug Species
[0123] Cultures were prepared for confocal microscopy by precoating
flame-sterilized glass coverslips, with a model tumor matrix (2:1
Tenascin:Fibronectin, Millipore CC065: Sigma F0895) at a
concentration of 0.5 .mu.g per sq. cm. in six-well plates overnight
at room temperature. Cells were plated at 100,000 per well and
treated 2-3 days later. Cultures were treated with 200 nM
Fitc-labeled ds siRNA or oligos formulated as s50 or MPG protein
nanoparticles at 50 nM as described in Example 1, Formula C-E or
lipid complexes using DOTAP (Roche) per the manufacturer's
instructions. Protein nanoparticles formulation utilizing
nonendosomal uptake mechanisms are used to illustrate impact of
nuclear delivery relative to conventional cytosolic delivery via
endosomal escape following uptake by lipid complexes into
clathrin-coated pits. Following treatment with either nanoparticles
or lipid complexes, cultures were washed with PBS and fixed for 30
minutes in 2% paraformaldehyde after 2-36 hours of incubation. The
following antibodies were used to localize Fitc-labeled nucleic
acids with respect to human Ago2 and RNAseH1 in various double and
triple-labeling strategies: anti-Fitc (Meridian K59098G, Genetex
263445); anti-h. Ago2 (Abnova h00027161, Proteintech 10686);
anti-RNAseH1 (BD 611356, Santa Cruz 30318, 30319). Slides were
examined, described and ranked on a conventional Olympus
microscope, before four channel imaging on a spectral confocal
Nikon C1si.
[0124] Cellular trafficking was examined in three different
carcinoma cell lines, SCC-15 (derived from human tongue), Fat-1
(derived from rat nasal mucosa) and PC3-LN4 (derived from human
prostate xenograft metastases) were utilized. Standard unmodified
ds siRNAs against luciferase, betagal and lamin were used as
positive controls and compared to ss guide strands synthesized in a
3'-(2-O-Me) RNA chimeric format using a phosphodiester backbone.
For simplicity, a standard configuration of 14 DNAs linked to six
2'-O-Me RNAs was used (henceforth referred to as "mi" or RNAi
oligos) as a representative species of the instant single stranded
chimeric polynucleotides. Guide strands (in the mi format) were
targeted against Red Fluorescent Protein (rniRFP), betagal
(rnibgal, rniZ7) and a therapeutic target, the kinase CK2 (rniCK2).
Key timepoints (24 hours for SCC-15s, 8 hours for PC3-LN4s) were
repeated 1-2 times.
[0125] Nanocapsule delivery: In SCC-15s, we observed for
s50-delivered (s50 is an alternative term for sub-50 nm
nanocapsule) cargo a uniform, initial nuclear colocalization of
Ago2 with both RNAi oligos (i.e., single stranded chimeric
polynucleotides) and siRNAs at 8 and 24 hours post-treatment. By 36
hours, these Ago2 complexes were relocated to the cytosol. RNAi
oligos also colocalized with RNAseH1, which siRNAs did not.
[0126] Baseline levels of both Ago2 and RNAseH were generally
poorly detectable in untreated SCC-15 and Fat-1 cells, making
shuttling more apparent than in PC3-LN4s which had much higher
baselines of both enzymes. Colocalization was similar for all three
cell lines, but the kinetics of cytosolic shuttling were faster in
the PC3-LN4s in that complexes had moved from the nucleus by 24
hours. FIG. 1a shows nuclear Ago2 colocalization with ds siRNA and
chimeric polyoligonucletide following nuclear delivery but not
cytosolic delivery for the chimeric oligo.
[0127] Delivery via MPG peptide: MPG nanoparticles bearing
5'-labeled rnibgal and 3'-labeled siLamin were examined at 8 and 24
hours. At 24 hours in SCC-15 cells, nuclear colocalization of RNAi
oligos with both Ago2 and RNaseH1 did not appear different from
that of RNAi oligos formulated into s50 protein nanoparticles. At 8
hours in PC3-LN4s, uptake was not complete, however, in those cells
where uptake had occurred, nuclear colocalization was extant
between either RNAi oligos and the two ribonucleases or siRNA and
Ago2 alone. Triple-labeling of Fitc-drug label, Ago2 and caveolin
showed colocalization for both s50 and MPG nanoparticles in cells
plated on relevant protein matrices. These data suggest that s50
protein nanoparticles and peptide transduction domain delivery
vehicles may use similar approaches for in vivo nuclear
delivery.
[0128] Interestingly, in SCC-15s and Fat-1s, RNAi oligos delivered
by DOTAP complexes (representative for cytosolic delivery through
endosomal escape) did not show recruitment of Ago2 to the drug in
the nucleus (and thus nuclear enrichment of Ago2), and only poor
recruitment of RNAseH. Using DOTAP complexes, however, good
recruitment of RNAseH (but not Ago2) to the drug in the nucleus was
observed in PC3-LN4s with higher baseline levels of RNaseH. RNaseH
is known to show tissue-based variation in levels and substrate
sensitivity. siRNA did not colocalize with RNAseH. These data
suggest i) single stranded chimeric polynucleotides according to
the instant invention can efficiently bind Ago2 in cells plated on
relevant matrix and ii) direct or effective nuclear delivery is
required for RNAi oligos to bind Ago2 forming minimal Risc
complexes (see Rivas et al., Nature SMB 12(4):340-9, 2005)
consistent with a concentration dependency.
[0129] To confirm and extend microscopy observations by another
method, nuclear and cytosolic fractions of SCC-15s cultured on
either plastic or model tumor matrix were western blotted 24 after
treatment with nanoparticles and lipid complexes or
immunoprecipitated following incubation of lysates with
unformulated nucleic acids. Lysate preparation was similar to that
used for nuclear run-on reactions using an NP-40-based lysate
buffer in a procedure from (see Weinberg et al., RNA. 12(2):256-62,
2006) with the addition of a dounce separation. For western
blotting, lysates were heated at 95 C and clarified. Nuclear
pellets were resuspended in an SDS Lysis buffer (10 mM Tris-HCl [pH
7.5], 1% SDS, 100 mM DTT). Membranes were detected for Ago2,
RNAseH, using the transcription factor SP-3 and actin as loading
controls. Fractions were free of contaminants as indicated by
appropriate location of loading control bands. RNAseH levels in
SCC-15 nuclear fractions did not appear manipulated by treatments
unlike Ago2 bands. Ago 2 bands were located or enriched in the
nuclear fraction at 24 hours, consistent with microscopy of cells
plated on relevant matrices. FIG. 1c shows a representative blot
from one of three independent experiments. By densitometry, Ago2
levels at 24 hours, expressed as Fold Sp-3, were 1.1 (s50 rniCK2);
1.7 (s50siLuc); 0.5 (buffer); 0.5 (Dotap CK2); 1.0 (Dotap siLuc);
0.7 (Dotap). Nucleic acids were 5'-Fitc labeled. As Ago2 levels are
not detected in heated (or unheated) SCC-15 cytosolic lysates, the
increase in nuclear levels of Ago2 may more likely result from
either de novo protein production or post-binding stabilization
rather than cytosolic redistribution. This experiment suggests
nuclear delivery is optimal for both ds siRNA and ss chimeric
polynucleotides of the present invention.
[0130] Putative nucleic acid-protein complexes were prepared by 3
hour incubation of nuclear lysates with 200 nM nucleic acid at
4.degree. C. then immunoprecipitated using goat anti-Fitc
antibodies and 6-his capture reagent and nickel columns (Millipore,
Catch and Release) per the manufacturer instructions. Subsequent
western blot membranes were detected for Ago2 using the Abnova
antibody. IP from goat serum was used as a negative control.
Equivalent density bands were detected from both ss chimeric
polynucleotides against multiple targets and siRNAs using both 5'
and 3' Fitc labeling strategies from lysates prepared from cell
cultured on relevant protein matrices. Only barely detectable bands
were observed from IP prepared using nuclear lysates from cells
cultured on plastic or mixed nuclear/cytosolic lysates. These data
support that colocalization results represent true binding events
between ss chimeric polynucleotides, siRNA and Ago2. They also
suggest that additional factors (either protein, nucleic acid,
spatial or combination), present in cells cultured on matrix, but
not in cells cultured on plastic, are required for efficient
single-stranded guide strand binding of Ago2. FIG. 1b shows a
representative western blot from these experiments.
[0131] These findings are very surprising in light of current
thinking derived from studies of cells cultured on plastic.
Although, structural studies of bacterial Ago proteins identify a
preference or capacity in these proteins for DNA guide strands (see
Yuan et al., Mol. Cell, 19:405-29, 2005), current thinking strongly
supports, in mammalian cells, i) poor utilization of
single-stranded guide strands by Ago2, ii) an absolute requirement
for majority A-helical or RNA-like character in guide strands to
facilitate cleavage, and iii) a requirement for cytosolic, rather
than nuclear delivery to facilitate Ago2 complex loading.
Example 3
Incorporation of Functional Guide Strand Sequences into
Nuclear-Delivered Bifunctional ss Chimeric Polynucleotides Provides
Method for Executing Potent Protein Inhibition
[0132] Having observed that unmodified ss chimeric polynucleotides
combined with nuclear delivery to target cells creates a
bifunctional RNAseH and RNAi molecule (interacts with type III
dsRNAses), we sought to identify a useful procedure for application
of this potentially more potent strategy to the problem of variable
gene silencing and protein inhibition. A bifunctional nucleic acid
drug offers advantages over one with single functionality in being
better able to maintain potency across tissue variations. We were
particularly interested in identifying guidelines for sequence
selection and medicinal chemistry constraints. Using the
experimental microscopy paradigm described in Example 2 in
combination with different timepoints and the three cell lines,
with formulation as described in Example 1, Formulas C-E, a series
of validated antisense and siRNA sequences against multiple
molecular targets were compared for their ability to bind both Ago2
and RNAseH when prepared using different chemistries. A subset of
studies was performed with MPG nanoparticles and results were
similar across cell lines as discussed in Example 2. The results
from these colocalization studies are summarized in the following
table:
TABLE-US-00003 TABLE 2 Colocalization with Cargo in Nucleus s50/MPG
Dotap Nuclear Cytosolic Sequence Medicinal delivery* delivery
Target Name Type Chemistry Ago2 RNaseH Ago2 RNaseH In vivo
results** CK2 rni LCK gs 5'-labeled +++/ ++ 0 + @ 8 hr Anti-tumor
activity at 2 .times. 10 atg/kg hybrid +++ not 24 by s50 capsules
PSLCK gs 5'-labeled 0/0 + to 0 0 to Anti-tumor activity to 2
.times. 1 .mu.g PS oligo +++ +++ without formulation. rni asCK2 as
5'-labeled 0 + 0 ND No anti-tumor activity at 2 .times. hybrid 10
.mu.g/kg by s50 capsules. Bgal rni sibgal gs 5'-hybrid ++/ ++/ 0 0
0.5 mg/kg for siRNA, 10 ng/kg ++ ++ for rni by s50 in tumors for
protein inhibition. rni Z7 gs 5'-hybrid ND ND ND ND 100 ng/kg by
s50 in tumor model for protein inhibition. rni LZas4 as 5'-hybrid
+/+ ND ND ND ND LZas4 as PS-2OMe- 0/0 Drug ND ND Inhibits mrna but
not Gapmer uptake# protein@160 .mu.g via intranasal (5/10/5) ++/0
GL67 liposomes. RFP rni RFP3 gs 5'-hybrid ++ +++ 0 0 ND gs
Hybrid-3' +++ +++ 0 0 ND Lamin siLamin ds 3'-siRNA ++/ 0/0 ++ 0 ND
+++ Luc siLuc ds 5'-siRNA + 0 + 0 ND Abbreviations: gs (guide
strand or antisense strand from ds siRNA), as (conventional
antisense), ds (double-stranded species), hybrid denotes
phosphodiester 2'-O--Me chimeric with six 2'-O--Me RNAs on 3' end
and 5' or 3' denotes position of Fitc labeling, ND, not determined,
Scale: +++ (very intense), ++ (above bkg), + (barely above bkg), 0
(signal not above bkg). PSLCK scores shown as range due to
variances observed between cell lines. *Nuclear delivery scores are
shown as s50/MPG respectively. If only one score is shown,
represents s50 delivery. **Lowest dose at which specified activity,
either at a gross level or at a molecular level has been observed.
#Drug uptake represents anti-Fitc signal detecting presence of
Fitc-labeled oligo.
[0133] Conventional antisense design typically optimizes for
uniform hybridization energies across sequences at sites of low
target mRNA secondary structure while siRNA design is more focused
on optimizing a hybridization profile across the sequence within
the context of sequence "rules". Design algorithms such as Soligo
for antisense and SiRNA for siRNA are publicly available (see
http:/sfold.wadsworth.org and supplier websites). Inspection of the
table indicates sequences validated as active guide strands by
derivation from functional double-stranded siRNAs as currently
designed are more suitable for incorporation into this methodology
rather than sequences designed using tools for conventional
antisense sequences acting through RNAseH or steric effects
(compare rniLCK vs. rniasCK2 and rnisibgal vs. rniLZas4). However,
a highly functional antisense sequence as demonstrated by
successful in vivo studies, may benefit from translation into a
hybrid guide strand strategy and the capacity for binding Ago2 may
be readily confirmed in vitro as described in Example 2. The
sequence "LZas4" provides an example of an optimized antisense
validated with in vivo data. The guide strand/antisense pair of
siZ7 and LZas4 respectively are from a publication describing a
fairly intensive in vitro and in vivo optimization for anti-bgal
sequences using proprietary large company software (Griesenbach et
al., Respiratory Research, 7:26 2006).
[0134] Phosphorothioate modification of nucleoside linkages for
increased stability have been reported to minimally effect
silencing activity (Chiu and Rana, RNA 9:1035-48, 2003), We have
found that full substitution of PS modifications into an active
single-stranded, bifunctional oligo abrogates Ago2 binding (rniLCK
vs. PSLCK). Along these lines, replacement of the classic
PS-centered 2-O-Me/PS/2-O-Me gapmer design with a hybrid guide
strand "rni" strategy increased Ago2 loading. However, a PS/2-O-Me
organization may be of value in situations where PO/2-O-Me seems
limited.
[0135] This analysis identifies a useful method where
oligonucleotide candidate, preferably a guide strand, garnered from
standard optimization techniques can be further improved in potency
by an additional optimization strategy consisting of i) confirming
candidate loading of Ago2 and RNAseH binding by microscopy in
vitro, preliminarily with a DNA/RNA chimeric containing six 3' RNA
nucleotides, ii) an optional subsequent determination of a
potentially more optimal number of 3' RNA nucleotides and iii)
utilization of nuclear delivery to promote Ago2 loading of the now
bifunctional single-stranded oligonucleotide. Confirmation of Ago2
binding may be considered optional for guide strand-derived
candidates, but not antisense candidates.
Example 4
Bifunctional, Ss Chimeric Polynucleotides Dual Binding of RNAseH
and Ago2 Corresponds with Increased Inhibition of Target Protein In
Vitro in Cells Cultured on Relevant Protein Matrices
[0136] The correspondence between ss chimeric
polynucleotides-enzyme binding as indicated by microscopy
colocalization and in vitro inhibition of target protein (betagal)
was examined in mouse CT26 betagal-positive colon carcinoma
cells.
[0137] Cells were plated at 12,000 cells/well with 1% fetal calf
serum into 24 wells trays containing 0.3-0.5 um thick polymeric
spun scaffolds coated with 100 pg/ml of 2:1 human tenascin:
fibronectin protein as a relevant matrix (tumor matrix).
3-dimensional tissue scaffolds such as Ultramax (Corning,
Surmodics, Donaldson Co.) can be useful for making bulk protein,
RNA or enzymatic measurements under more biologically relevant
conditions such as required for careful assessment of the
bifunctional, ss chimeric polynucleotides. Optimization of cell
culture conditions for three-dimensional matrices involves
identifying plating and matrix coating conditions that produce best
results in a functional activity or enzymatic assay. For example,
using in vitro growth inhibition as functional assay, we have
determined that rat cells perform optimally at 0.5% fetal calf
serum on matrices coated with 10 pg/ml of human proteins rather
than the 100 pg/ml preferred by human and feline cell lines. Future
optimization with the murine CT26 line, for example, will involve
consideration of 10 pg/ml coating conditions.
[0138] Cells were treated with nanoparticles of Fitc-labeled oligos
used cellular trafficking studies in Example 2 and unlabeled
double-stranded siRNAs. Relative change in .beta.-galactosidase
enzymatic activity was measured in lysates from cells 24 hours
after 200 nM treatment as described in Simon and Lis (1987, NAR 15:
2971-2988) using the following lysis buffer (25 mM Tris-HCl pH 7.8,
1 mM EDTA, 8 mM magnesium chloride, 1% Triton X-100, 1 mM DTT). We
observed an increase in .beta.-galactosidase activity inhibition in
oligos relative siRNAs in cells plated on relevant matrices and
results are summarized in Table 3. Recall from Example 2 using
nuclear-delivery, ds siRNA molecules were observed to colocalize
with Ago2 but not RNaseH1 while antisense oligos in conventional
chemistries were observed to colocalize with RNAseH1 but not Ago2
and ss chimeric polynucleotides molecules in the 3'-RNA
phosphodiester chimeric format bound both Ago2 and RNaseH1. These
data are consistent with a cell line containing higher RNAseH than
Ago2 levels under these conditions as the conventional antisense
induced increased .beta.-galactosidase inhibition over the siRNAs.
However, in both cases (siRNA to ss chimeric polynucleotides and
antisense to ss chimeric polynucleotides), the dual functionality
induced by the phosphodiester ss chimeric polynucleotides format
increases sequence activity supporting that optimization of
candidate sequence into ss chimeric polynucleotides of the instant
invention will provide a method for enhanced activity across
biological variation. These data are surprising, as it is believed
in the current art, that extrinsic or cellular 5' phosphorylation
is required for single-stranded guide activity and that 5' labeling
will destroy RISC complex stability.
[0139] These data do not distinguish between different modes of
action by a dsRNAse such as Ago2, i.e. cleavage or repression as
both follow from guide strand binding and result in decreased
levels of target protein. Using the model mi molecule, mi CK2, we
have measured significant reduction in CK2 mRNA in vitro in cells
plated on 3D-scaffolds, raising the possibility that either or both
modes of action in combination are feasible for bifunctional
molecules. In conclusion, these data highlight the usefulness of
our in vitro methods for characterizing binding and exemplify the
need for bifunctional molecules given tissue variation in critical
gene-silencing enzymes.
TABLE-US-00004 TABLE 3 s50 5F- Fitc s50 5F-Fitc s50 5F-Fitc s50
siZ7 s50 sibgal rnibgal rniLZas4 LZas4 nucleic acid unlabeled
unlabeled labeled labeled labeled species siRNA siRNA rni oligo rni
oligo antisense Day 1 .beta.gal activity 104 .+-. 5 107 .+-. 4.2 69
.+-. 0.7 51 .+-. 0.7 72 .+-. 0.7 (% control, CPRG, mean .+-. SE)
Table 1 Ago2 n/a n/a ++ + (-) binding Table 1 RNaseH n/a n/a ++ ND
++ binding
Example 5
Evaluation of Importance of 3' Terminal RNA Segment in
Bifunctional, ss Chimeric Polynucleotides
[0140] The impact of chimeric structure on enhanced bifunctional
antisense activity was assessed by comparing different
phosphodiester ss chimeric polynucleotides configurations by 48
hour growth inhibition in vitro. Standard segments of six
2'-O-Me-modified RNAs were shuffled in the 20mer anti-CK2 sequence
"LCK" (Table 1b) and prepared as s50 nanocapsules for in vitro
growth inhibition assay as described in Example 1, Formula E. A
double-stranded siRNA type chemistry was included as a comparison.
This chemistry consisted of an unmodified RNA guide strand annealed
to a heavily modified DNA chimeric passenger strand. The chemistry
is described in Hofgrefe et al., 2006 (Nucleosides, Nucleotides,
and Nucleic Acids, 25:889-907). Results, summarized in Table 4,
indicate the 3' RNA ss chimeric polynucleotide provides the most
effective growth inhibition in the range tested while the 5' RNA ss
chimeric polynucleotides and RNA gapmer are 30-50% higher. The
classic DNA gapmer design, (see "outer"), viewed as a standard in
the art, is significantly worse than the 3' RNA chimeric.
[0141] It should be noted that while cell plating on coated 3D
scaffolds may replace important factors and reconstitute cellular
architecture, e.g. lipid rafts and caveolae important necessary for
modeling a biological phenomenon (e.g. Argo2 cobinding drug and
caveolin), matrix also can provide significant survival advantage
to carcinoma cells. For example, SCC-15 tongue carcinoma cells
exhibit an IC 50 of about 0.5 .mu.M for Docetaxel when plated on
plastic in full serum. No cell death is observed when cells, plated
on uncoated scaffolds in reduced serum, are treated at the highest
feasible stock dilution of 10 .mu.M. It should also be noted that
the model molecular target in this study, Casein Kinase 2, has an
extended half-life in tumor cells (plated on plastic) of .gtoreq.5
days (see Seeber, et al., Apoptosis 10: 875-885, 2005) highlighting
the observation that effective treatment based on protein
inhibition may require consideration of protein half-life when
considering the usefulness of repeat dosing
[0142] While not wishing to be bound by theory, these data suggest
a requirement for a DNA end in the single-stranded bifunctional
oligo with a segment of RNA. This suggest a potential model where
the 5' DNA end of the highly DNA guide strand contacts the PIWI
domain of Ago2 as has been described for DNA guide
strand-preferring Ago2 bacterial proteins (Yuan et al., Mol Cell
19:405-19, 2005). The RNA segment, located preferably on the 3' end
provides a higher affinity contact, potentially necessary for the
additional mechanical stress a highly DNA guide strand could
encounter, together with as yet unknown factors, in contacting the
target RNA. Interestingly, the target CK2 sequence itself, has
self-complementarity in the region from which the sequence, "LCK"
was derived and the true sense is a weak antisense suggesting the
possibility where the 5' chimeric of "LCK" could elicit some
bifunctional activity. In this example, we used a standard run of
six 2'-O-Me-modified RNA for an RNA segment. This was chosen as an
intermediate screening value and was also the determined optimum
for the model sequence "LCK". We have arrived at optimums for
different sequences using activity assays as decision-making tool,
ranging from 4-10, but not 12 or greater from the 3' end for
sequences of a standard 20mer length.
TABLE-US-00005 TABLE 4 Growth inhibition (cell viability) in FaDu
carcinoma cell lines "rni CK2" "Forward" "Inner" "Outer" "sHCK2 +
5P" Dose 3' RNA 5' RNA DNA/RNA/DNA RNA/DNA/RNA ds hybrid (.mu.M)
chimeric chimeric gapmer gapmer siRNA 20 46.7 .+-. 5 68.1 .+-. 10
64.1 .+-. 20 76.5 .+-. 9 72.0 .+-. 16 10 64.1 .+-. 17 81.5 .+-. 10
88.5 .+-. 38 90.0 .+-. 14 85.1 .+-. 30 5 64.4 .+-. 18 99.7 .+-. 16
118.8 .+-. 63 85.5 .+-. 20 94.2 .+-. 31 2.5 86.2 .+-. 24 87.6 .+-.
15 117.9 .+-. 60 96.1 .+-. 15 89.4 .+-. 12 1.25 71.2 .+-. 22 96.6
.+-. 11 138.1 .+-. 66 86.4 .+-. 12 79.8 .+-. 21 0 100 100 100 100
100
[0143] Oligonucleotides administered to 3,000 FaDu cells plated
with 0.5% FCS media on matrix-coated 3-D scaffolds in 96 wells,
formulated into s50 protein nanocapsules as described in Example 1,
Formula E. Proliferation rates were calculated relative to sugar
capsule by thymidine incorporation in the last 16 hours of the
growth period. Experiment performed 2-4 times in duplicate. Values
reported as Mean.+-.SE.
[0144] Besides the impact of chemistry organization on activity of
dual function hybrid guide strands, we considered the impact of
biological variation in critical enzymes on ss chimeric
polynucleotide activity. While not wanting to be bound by theory,
it is known in the art that enzymatic processes are rate-limited by
concentration, activity levels and availability of key enzymes. We
noticed considerable variation in levels of RNAseH and Ago2 across
cell lines by microscopy in Example 2 and asked whether there might
be correspondence between intrinsic enzyme activity and sensitivity
and potentially benefit from the dual function chimeric format.
Using the rni CK2 sequence as a model dual function chimeric oligo,
we compared enzyme rankings with chimeric activity in growth
inhibition (see Table 5). The data suggests that cells containing
higher baseline levels of key enzymes will require less dual
function oligo for the same amount of activity. While this is an in
vitro comparison, it is known that PC3s respond to the LCK sequence
in vivo at levels 4 logs below those tested in head neck cancers
xenograft tumors arguing a basis for synergy for dual function
oligos.
TABLE-US-00006 TABLE 5 Growth inhibition (cell viability) in
carcinoma cell lines Cell Line Fat-1 SCC-15 PC3-LN4 Cell Viability
following challenge 47.5 .+-. 42 68.0 .+-. 15 42.7 .+-. 17 with 20
.mu.M rni CK2 Cell Viability following challenge 74.62 .+-. 8 78.0
.+-. 5 35.8 .+-. 22 with 10 .mu.M rni CK2 Relative baseline Ago2
Rank 0 + +++ Relative baseline RNaseH Rank + 0 +++
[0145] Oligonucleotides administered to cells on 10-100 pg/ml
matrix-coated 3-D scaffolds in 96 well plates, formulated into s50
protein nanocapsules as described in Example 1, Formula E with the
following conditions: Fat-1, 10,000 cells, 0.5% media; SCC-15,
12,000, 1% media and PC3s, 3000 cells, 0.01% media. Proliferation
rates were calculated relative to sugar capsule by thymidine
incorporation in the last 16 hours of the growth period. Experiment
performed 2-4 times. Values reported as Mean.+-.SE.
[0146] We conclude these data provide a basis for treatment
approaches incorporating the dual function chimeric
polyoligonucleotide and assessment of relevant baseline enzymatic
activities in target tissues to define appropriate personal
medicines approaches.
Example 6
Reduction of Metastatic Tumor Burden with Targeted, Low Dosing in
Human Xenograft Tumors Using Model Therapeutic Bifunctional ss
Chimeric Polynucleotides
[0147] In these examples, we have been using a subdomain of the
extracellular matrix protein tenascin, i.e. tenfibgen, as a
nanoparticles ligand for directing s50 particles to tumor cells.
Tenascin has been linked to the vascularization of tumor tissue;
specifically, tenascin (i) has been found in and around tumor
microvessels, (ii) is produced by migrating endothelial cells, and
(iii) when coated on tissue culture plates, stimulates sprouting by
and migration of endothelial cell. Antibodies to tenascin were one
of the earliest anti-angiogenic approaches explored in cancer
treatment, and this therapy continues in active human clinical
development.
[0148] As described below, TBG s50 nanoparticle has been
demonstrated to provide specific whole body transfection, which
illustrates that the strategy of using bifunctional chimeric
molecules with nuclear entry s50 nanoparticles to target both RNAi
and antisense mechanisms (here, Ago2-RNaseH) at low doses to
minimize any side effects and provide a greater therapeutic index
can also be used for therapeutic delivery of biologics to
proliferating tumor cells and associated microvasculature. Also,
for tissues or cancers which express varying amounts of the enzymes
associated with RNAi mechanisms and antisense (such as,
Ago2-RNaseH), a bifunctional molecule can help assure a therapeutic
effect.
[0149] We tested sensitivity of the 3 SCCHN models to lower dose
levels of nanoparticle-delivered therapeutic ss chimeric
polynucleotides, by re-treatment of a small number (n=14) of mice
with larger tumors remaining at the end of a survival study. Mice
were re-treated with repeat doses ranging from 10 .mu.g/kg to 100
ng/kg. Best results were achieved at lower doses in the range 100
ng/kg as evidence of tumor lysis syndrome was observed at .mu.g/kg
dosing (transient dehydration, fistulas from rapidly dying visceral
metastases and organ fibrosis secondary to rapid necrotic tumor
death). These mice had starting tumor dimensions of 15 mm in one
dimension with liver, lung or brain metastases by whole-body
imaging.
[0150] Good efficacy was seen in the lowest dose groups at 100
ng/kg dosing, suggesting that an indicator of dual RNAi and
antisense pathway activation is efficacy at lower doses of
therapeutic in view of the catalytic action of traditional siRNA,
as well as activation of antisense. Best responders were mice that
had been flank-inoculated with the SCC-15 tumor (4 million cells)
and previously treated with short-term chemotherapy
(cisplatin+docetaxel).
[0151] To begin the retreatment study, after 6 months of the short
term chemotherapy treatment, mice were selected for retreatment on
the basis of positive tumor signal by imaging (by whole-body
bioluminescence imaging using the Xenogen.TM. technique--in this
technique, mice are administered plasmid DNA encoding the
luciferase reporter gene nanoencapsulated in TBG using techniques
described previously (particles formulated per Example 1, Formula
A). The purpose was to assess the existence of remaining tumor
burden sensitive to TBG uptake. After waiting 7 days to enable gene
expression, mice were injected with D-luciferin contrast (the
substrate for the expressed luciferin enzyme). Bioluminescence was
then collected from anesthetized mice in the Xenogen.TM. apparatus
to suggest the location of nanocapsule uptake, which in this case,
would be tumor.
[0152] During active treatment, mice were reimaged every two weeks
using exactly the same protocol with plasmid readministered every 4
weeks. Plasmid administration was titered during method development
so as to not negatively affect tumor growth. In both cases of
surviving mice, mice showed strong liver signal and in one mouse,
some lymph node and kidney signal. After two to four weeks of
thrice weekly 100 ng/kg intravenous injection of
nanocapsule-formulated ss chimeric polynucleotides (prepared as in
Example 1, Formula F), both mice were clear of liver, kidney and
lymph node signal.
[0153] Following the clearance of visceral mets, the mice were
treated with repeat i.p. dosing of the nanocapsule-formulated ss
chimeric polynucleotides and 20 .mu.g/kg cisplatin (and surgery for
one mouse) to remove surface lesions. Occasional topical
administration of a 3 .mu.g/ml suspension of nanocapsule-formulated
ss chimeric polynucleotides continued through the survival period.
The two nonsurvivors of the low dose group died of complications
from either surgery or a treatment-induced lung fistula from a
dying met (without apparent lung fibrosis induction. These data
indicate that nanoencapsulated anti-cancer therapeutics such as rni
CK2 will be efficacious for treating metastatic and disseminated
cancer in aggressive, clinically relevant models at ultralow
dosing, suggesting that an indicator of dual RNAi and antisense
pathway activation is efficacy at lower doses of therapeutic in
view of the catalytic action of traditional siRNA, as well as
activation of antisense.
Example 7
Bifunctional Ss Chimeric Polynucleotides Exhibit High Potency In
Vivo with Surprising Persistent Effect in a Model Therapeutic
Sequence
[0154] We further tested the anti-CK2 sequence, "LCK" (Table 1b)
for evidence of a dose response in nude mice bearing FaDu flank
xenograft tumors. Mice were injected with 4 million cells and
treatment commenced intravenously with LCK nanoparticles made as
described in Example 1, Formula G, 5 days following tumor
inoculation when tumor diameters was 4-5 mm. Relative to the
composition Formula F used in Example 6, Formula G was optimized to
improve particle size variation as measured by both AFM. By Dynamic
light scattering, a bulk measurement technique made in water rather
than the dry AFM measurement, 98.4% of Formula G particles was
25.2.+-.4.4 nm.
[0155] For activity testing with the more uniform particle size
composition, Formula G, group sizes ranged from 3-6 animals and the
study was conducted in multiple cohorts due to the large numbers of
test conditions. Tumors were followed with caliper measurements
over a period of about 30 days until tumors reached about 15 mm in
at least one dimension. Then mice were euthanized and blood and
tumor tissue collected for analysis. In the last cohort of animals
residual tumors were additionally weighed.
[0156] Based on the anti-tumor activity demonstrated by the
nanoparticle LCK sequence in the bifunctional ss chimeric
polynucleotides format at repeat doses in 100 ng/kg with mice
bearing large tumor burdens, we used a starting regimen of two
doses with a 2 day interval (q2D) at doses ranging from 10 .mu.g/kg
to 10 attogram/kg. At a gross level, we found evidence of
anti-tumor activity into the attogram range with a decrease in
percentage of responders with dose rather than the pattern of
response. At the molecular level, Day 30 tumor tissue was
fractionated into nuclear and cytosolic fractions and western
blotted for compartmental levels of CK2 and Caspase 7 to assay for
evidence of apoptosis. Terminal blood was also assayed by ELISA for
human IL-6 as a surrogate marker for tumor levels. Results are
summarized in FIG. 2.
[0157] CK2 (Casein Kinase 2) is a ubiquitous enzyme overdriven by
tumor cells to promote survival by multiple pathways and
accumulates in cell nuclei under conditions of stress and in
tumors. Shuttling of CK2 from the nuclear compartment precedes
apoptosis and the inability of the tumor cell to maintain CK2
precedes tumor death. Flank tumors receiving higher doses showed a
pattern of continued growth accompanied by swelling and subsequent
necrosis with wound healing sequalae. FIG. 2b shows that in a later
cohort, not yet analyzed by western blotting, residual tumor
quantified by weight (and not wound surface area) was resolved most
rapidly with 3 lower pg/kg doses rather than the two used in the
standard dose ranging regimen. Residual lesions were followed for
healing capacity in one individual from 4 lower dose groups and all
healed over a period of 3 months without recurrence. Two of the 4
recurred in an additional 3 month period before humane
euthanasia.
[0158] Although unexpected and completely surprising, the extreme
potency showed by particle-optimized Formula G may be partially
understood by the concept of improved particle molarity, i.e.
uniform, smaller particles will the same amount of bifunctional
oligo in an increased number of particles. When combined with the
surprising potency of the bifunctional oligo format, the
synergistic result is extremely low dosing.
[0159] Results suggest significant persistence of molecular effects
in vivo with limited dosing, i.e. molecular changes persisted in
tumor tissue 3 weeks after two small doses. These are very
surprising results, to say the least. FIG. 2a shows that higher
.mu.g/kg dosing is required for persistent reduction in the nuclear
fraction of CK2 enzyme, but inhibition of CK2 in the cytoplasmic
compartment is observed at all dose levels quantified (FIG. 2c).
Reduced levels of IL-6 also occurred at the 10 .mu.g/kg dose level
(FIG. 2b) and corresponded with decreased nuclear CK2 and is
consistent with the more rapid (and problematic tumor necrosis with
associated wound care in mice) observed at .mu.g/kg doses.
[0160] At lower dosing levels of 10 ftg/kg and 10 atg/kg, ongoing
apoptosis appeared to be more prevalent as 3-6 fold increases in 35
kD Caspase 7 with cleavage products were measured (FIG. 2d). This
was consistent with less rapid and difficult-to-manage tumor death.
Flank tumors receiving higher doses showed a pattern of continued
growth accompanied by swelling and subsequent necrosis with wound
healing sequalae. FIG. 2b shows that in a later cohort, not yet
analyzed by western blotting, residual tumor quantified by weight
(and not wound surface area) was resolved most rapidly with 3 lower
pg/kg doses rather than the two used in the standard dose ranging
regimen. Residual lesions were followed for healing capacity in one
individual from 4 lower dose groups and all healed over a period of
3 months without recurrence. Two of the 4 recurred in an additional
3 month period before humane euthanasia.
[0161] These observations on decreased magnitude of dosing levels,
while not fully explainable by current thinking on feasibility for
in vivo single-stranded RNAi, or decreased potency and persistence
of effect believed to be associated with standard single-stranded
RNAi (see Holen et al., NAR 31(9):2401-7, 2003), are consistent
with in vitro work detailing an enhanced protein half-life for CK2
alpha in tumor cells relative to normal cells (.gtoreq.5 days vs.
14 hours) and the capacity for siRNA but not conventional antisense
to inhibit the tetrameric kinase's activity at doses not showing
detectable changes in mRNA or protein levels (see Seeber, et al.,
Apoptosis 10: 875-885, 2005).
[0162] The increased potency of the dual chimeric polynucleotide
strategy is also not consistent with any indirect immunomodulation
as single 10 mg/kg doses of s50 tenfibgen particles bearing LCK
sequence in the "mi" format did not any evidence of early
inflammatory responses in outbred mice. While the LCK sequence
contains no CpG motifs or other known immunomodulatory sequence
motifs, these results still support that increased chimeric
polynucleotide potency derived from dual mechanism activation
rather than immunomodulation.
[0163] The surprising extreme potency showed by particle-optimized
Formula G may be, in retrospect, partially understood by the
concept of improved particle molarity, i.e. uniform, smaller
particles will distribute the same amount of bifunctional oligo in
an increased number of particles. When combined with the surprising
potency of the bifunctional oligo format, the synergistic result is
extremely low dosing.
Example 8
Targeted s50 Nanoparticle Lowers ss Chimeric Polynucleotide
Delivery Requirements for Efficacy
[0164] To investigate the usefulness of our method for enhancing
potency of oligonucleotide molecules for efficacy, i.e. inhibit
production of a target protein in vivo, we performed limited dose
ranging studies of guide strands from published siRNA molecules
formatted into the chimeric chemistry of a dual functional
oligonucleotide, i.e., ss chimeric polynucleotides. We evaluated
two anti-.beta.-galactosidase sequences, "sibgal-1" and "siZ7" as
rnibgal and "rniZ7" respectively (see Table 1a).
[0165] Earlier we had established an in vivo baseline for
anti-betagalactosidase siRNA activity using a model system
consisting of Balb/c mice inoculated with .beta.-galactosidase-(+)
CT26 tumors. The CT26 mouse colon carcinoma line, available from
ATCC, has been retrovirally modified to produce
.beta.-galactosidase. The published "sibgal-1" sequence was
formulated with tenfibgen nanoparticle targeting as described in
Example 1 Formula E for target cell nuclear delivery according to
our method. The baseline study consisted of 18 mice in 6 treatment
groups. Tumors were collected 3 days after i.v. treatment of s50
nanoparticle-target si.beta.gal oligos and processed for microscopy
western blotting and qPCR. Western blotting indicated a significant
70% reduction in .beta.-galactosidase in tumor lysates from
sham-treated at a single dose of 0.5 mg/kg. QPCR indicated a
reduction of more than 98% in mRNA groups. A dose of 50 .mu.g/kg
was not effective in inhibition of target protein.
[0166] Given that dual functional ss chimeric polynucleotides had
exhibited increased target protein inhibition in in vitro testing,
we assayed for persistence of rank order between in vitro and in
vivo studies by treating a number of mice bearing CT-26
.beta.-galactosidase-(+) tumors decreasing doses of dual functional
ss chimeric polynucleotides based on the same sequence and
formulated into nanoparticles for nuclear delivery as described in
Example 1, Formula I-K. Tumors were allowed to grow to 4-6 nm in
diameter before starting treatments. Assay results were scored by
confocal fluorescence microscopy of frozen tumor sections stained
with a rabbit anti-betagalactosidase antibody (Cortex, San
Francisco, Calif.). Results are summarized in Table 6 below:
TABLE-US-00007 TABLE 6 Tissue Collection Result Test (time after
(microscopy Rank Test Regimen Sequence 1rst dose) score for
.beta.gal) Order 2 .times. 10 ng/kg sugar none 2 or 3 days +++ 6
nanoparticles 1 .times. 1 .mu.g/kg rnibgal 3 days 0 1 2 .times. 100
ng/kg q 24 h rnibgal 2 days 0 2 2 .times. 10 ng/kg q 24 h rnibgal 2
days + 4 2 .times. 100 ng/kg q 24 h rniZ7 2 days 0 3 2 .times. 10
ng/kg q 24 h rniZ7 2 days ++ 5 Scale: 0 = no expression; + = poor,
dim, or patchy expression, ++ = moderate or >40% incidence, +++
= good or >70% incidence of expression.
[0167] The data shows that additional optimization as a dual
functional ss chimeric polynucleotide according to the present
invention could provide significant, here .about.3.5 logs, in vivo
efficacy enhancement for siRNA molecules.
Sequence CWU 1
1
12121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1cuacacaaau cagcgauuuu u
21222RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2aagccaauau ugaaacccac gg
22320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3aacaggtatt cgctggucac 20429RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4uguucuucug gaaguccagu uccuccuuc 29519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5ccaagaagcc cgugcagcu 19620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6atacaaccca aactccacat 20720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7gtcccgacat gtcagacagg 20818RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8cguacgcgga aucuucga 18927PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Gly
Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10
15 Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val 20 25
109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5
116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 11His His His His His His 1 5 1227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide vector
12Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 1
5 10 15 Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val 20 25
* * * * *
References