|Title of Invention||
"A COMPOSITION COMPRISING A DOUBLE STRANDED RIBONUCLEIC ACID (DSRNA) AND DELIVERY ENHANCING PEPTIDES"
|Abstract||The present invention relates to a composition comprising a double stranded ribonucleic acid (dsRNA) and a delivery enhancing peptide, wherein the dsRNA comprises an antisense strand that is complementary to a tumor necrosis factor-α (TNF-α) mRNA that comprises a nucleic acid sequence of CCGACUCAGCGCUGAGAUCAA (SEQ ID NO: 132)|
|Full Text||OF TREATING AN INFLAMMATORY DISEASE BY DOUBLE STRANDED RIBONUCLEIC
The invention relates to methods and compositions for delivering nucleic acids into cells. More specifically,
the invention relates to procedures and preparations for delivering double-stranded polynucleotides into cells to modify
expression of target genes to alter a phenotype, such as a disease state or potential, of the cells.
A central role of tumor necrosis factor (TNF-a) has been identified or implicated in the initiation and/or
persistence of the inflammatory process(es) leading to a variety of diseases. These include rheumatoid arthritis (RA),
Crohn's disease (CD), psoriasis, ankylosing spondylitis, Still's disease, polymyositis and dematomyositis, and
vasculitis (including Behcet's disease and Wegener's granulomatosis) (Lorenz and Kalden (2002) Arthritis Research
4(suppl 3):S17-24). TNF-a production by adipose tissue has also been implicated in diabetes and obesity (Ruan and
Lodish (2003) Cytokine Growth Factor Rev. 14:447-55).
Limitations of established therapies (including methotrexate) have led to identification of certain inflammatory
mediators as therapeutic targets for alternative therapies. In this connection novel therapeutic agents have been
developed that are currently being testing, including monoclonal antibodies, cytokine receptor-human Ig constructs,
and recombinant human proteins. There continues to be an unmet demand for effective therapeutic modalities for
treating this class of diseases.
Various stimuli are known to induce TNF-a production, including endotoxin, tumor cells, several viruses
(including HIV), other cytokines, and various stress related responses. An animal model has been developed for
nonclinical studies that were accepted by the U.S. Food and Drug Administration (FDA) as providing support for
labeling of Remicade® for treatment of Crohn's Disease patients. The murine model (Tg 197, Tg211 and Tg5453
mice) came from the development of transgenic mice that constitutively express human TNF-a (Georgopoulos et al.
(1996) J. Inflammation
46:86-97). Several other anti-TNF-a antibody therapies successfully applied this animal model to assess
potency and prove efficacy (HUMIRA™, Infliximab, Adalimunab, Etanercept, Abgenix ABX10131).
Delivering nucleic acids into animal and plant cells has long been an important object of molecular biology
research and development. Recent developments in the areas of gene therapy, antisense therapy and RNA interference
(RNAi) therapy have created a need to develop more efficient means for introducing nucleic acids into cells,
A diverse array of plasmids and other nucleic acid "vectors" have been developed for delivering large
polynucleotide molecules into cells. Typically these vectors incorporate large DNA molecules comprising intact genes
for the purpose of transforming target cells to express a gene of scientific or therapeutic interest.
The process by which exogenous nucleic acids are delivered artificially into cells is generally referred to as
transfection. Cells can be transfected to uptake a functional nucleic acid from an exogenous source using a variety of
techniques and materials. The most commonly used transfection methods are calcium phosphate transfection, and
electroporation. A variety of other methods for tranducing cells to deliver exogenous DNA or RNA molecules have
been developed, including viral-mediated transduction, cationic lipid or liposomal delivery, and numerous methods
that target mechanical or biochemical membrane disruption/penetration (e.g., using detergents, microinjection, or
RNA interference is a process of sequence-specific post transcriptional gene silencing in cells initiated by a
double-stranded (ds) polynucleotide, usually a dsRNA, that is homologous in sequence to a portion of a targeted
messenger RNA (mRNA). Introduction of a suitable dsRNA into cells leads to destruction of endogenous, cognate
mRNAs (i.e., mRNAs that share substantial sequence identity with the introduced dsRNA). The dsRNA molecules are
cleaved by an RNase III family nuclease called dicer into short-interfering RNAs (siRNAs), which are 19-23
nucleotides (nt) in length. The siRNAs are then incorporated into a multicomponent nuclease complex known as the
RNA-induced silencing complex or "RISC". The RISC identifies mRNA substrates through their homology to the
siRNA, and effectuates silencing of gene expression by binding to and destroying the targeted mRNA.
RNA interference is emerging a promising technology for modifying expression of specific genes in plant and
animal cells, and is therefore expected to provide useful tools to treat a wide range of diseases and disorders amenable
to treatment by modification of endogenous gene expression.
There remains a long-standing need in the art for better tools and methods to deliver siRNAs and other small
miiibitory nucleic acids (siNAs) into cells, particularly in view of the fact that existing techniques for delivering
nucleic acids to cells are limited by poor efficiency and/or high toxicity of the delivery reagents. Related needs exist
for improved methods and formulations to deliver siNAs in an effective amount, in an active and enduring state, and
using non-toxic delivery vehicles, to selected cells, tissues, or compartments to mediate regulation of gene expression
in a manner that will alter a phenotype or disease state of the targeted cells.
SUMMARY OF THE INVENTION
One aspect of the invention is the use of a formulation comprising double stranded ribonucleic acid (dsRNA)
in the manufacture of a medicament for treating an inflammatory disease in a mammal and inhibiting production of
tumor necrosis factor-a (TNF-a) in the mammal. Preferably, the inflammatory disease is a systemic disease. Most
prefereably, the inflammatory disease is rheumatoid arthritis. In an embodiment of the invention, the formulation is
administered to the circulation of the mammal, preferably intravenously. In another embodiment, the siRNA is
delivered to blood leucocytes, preferably monocytes. In another embodiment, administration of the formulation
decreases the levels of TNF-a in the circulation of the mammal. In a preferered embodiment the mammal is a human.
Another aspect of the invention is a pharmaceutical formulation for treating an inflammatory disease in a
mammal comprising double stranded ribonucleic acid (dsRNA), wherein the dsRNA is capable of modifying
expression of tumor necrosis factor-a (TNF-a) in cells of the mammal. In a preferred embodiment, the peptide
comprises an amino acid sequence is selected from the group consisting of:
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ED NO: 59);
KKA VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 165);
VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 166); AQKKDGKKRKRSRKESYSVYVYKVLKQ
(SEQ ID NO: 167); KDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 168); KKRKRSRKESYSVYVYKVLKQ
(SEQ ID NO: 169); KRSRKESYSVYVYKVLKQ (SEQ ID NO: 170); RKESYSVYVYKVLKQ (SEQ ID NO: 171);
SYSVYVYKVLKQ (SEQ ID NO: 172); VYVYKVLKQ (SEQ ID NO: 173) and YKVLKQ (SEQ ID NO: 174). In a
related preferred embodiment, the dsRNA comprises a ribonucleic acid sequence selected from the group consisting
of: GCGUGGAGCUGAGAGAUAA (SEQ ED NO: 109); GCCUGUAGCCCAUGUUGUA (SEQ ID NO: 110);
GGUAUGAGCCCAUCUAUCU (SEQ ID NO: 111); CCAGGGACCUCUCUCUAAU (SEQ ID NO: 112);
GCCCGACUAUCUCGACUUU (SEQ ID NO: 113); UGACAAGCCUGUAGCCCAU (SEQ ID NO: 114);
GGUCUACUUUGGGAUCAUU (SEQ ID NO: 115); CCCAGGGACCUCUCUCUAA (SEQ ID NO: 116);
AAUCGGCCCGACUAUCUCGACUU (SEQ ID NO: 117); AAUGGCGUGGAGCUGAGAGAU (SEQ ED NO:
118); AACCUCCUCUCUGCCAUCAAG (SEQ ID NO: 119); AACUGAAAGCAUGAUCCGGGA (SEQ ID NO:
120); AAUCUCGACUUUGCCGAGUCU (SEQ ID NO: 121); AAGGGUGACCGACUCAGCGCU (SEQ ED NO:
122); AAUCAGCCGCAUCGCCGUCUC (SEQ ED NO: 123); AACCCAUGUGCUCCUCACCCA (SEQ ID NO:
124); AAGCUCCAGUGGCUGAACCGC (SEQ ED NO: 125); AAGUCAGAUCAUCUUCUCGAA (SEQ ID NO:
126); AAGGGACCUCUCUCUAAUCAG (SEQ ID NO: 127); CCUCAGCCUCUUCUCCUUCCUGA (SEQ ID
NO: 128); AAUCCUCAGCCUCUUCUCCUU (SEQ ID NO: 129); AACCAAUGCCCUCCUGGCCAA (SEQ ID
NO: 130); CUGAUUAAGUUGUCUAAACAA (SEQ ID NO: 131); CCGACUCAGCGCUGAGAUCAA (SEQ ID
NO: 132); CUUGUGAUUAUUUAUUAUUUA (SEQ ID NO: 133); AAGCCUGUAGCCCAUGUUGUA (SEQ ID
NO: 134); UAGGGUCGGAACCCAAGCUUA (SEQ ED NO: 135); CUGAAAGCAUGAUCCGGGA (SEQ DO NO:
136); AGGCGGUGCUUGUUCCUCA (SEQ ID NO: 137); CCACCACGCUCUUCUGCCU (SEQ ID NO: 138);
AGGGACCUCUCUCUAAUCA (SEQ ED NO: 139); UGACAAGCCUGUAGCCCAU (SEQ ED NO: 140);
GCCUGUAGCCCAUGUUGUA (SEQ ID NO: 141); UAGCCCAUGUUGUAGCAAA (SEQ ED NO: 142);
CCAAUGCCCUCCUGGCCAA (SEQ ID NO: 143); CCAAUGGCGUGGAGCUGAG (SEQ ID NO: 144);
GGCGUGGAGCUGAGAGAUA (SEQ ID NO: 145); GCGUGGAGCUGAGAGAUAA (SEQ ID NO: 146);
GCCUGUACCUCAUCUACUC (SEQ ID NO: 147); CCUCCUCUCUGCCAUCAAG (SEQ ID NO: 148);
GGUAUGAGCCCAUCUAUCU (SEQ ED NO: 149); GCUGGAGAAGGGUGACCGA (SEQ ED NO: 150);
GAGAAGGGUGACCGACUCA (SEQ ID NO: 151); GCCCGACUAUCUCGACUUU (SEQ ID NO: 152);
GCAGGUCUACUUUGGGAUC (SEQ ID NO: 153); GGUCUACUUUGGGAUCAUU (SEQ ID NO: 154);
UGGGAUCAUUGCCCUGUGA (SEQ ID NO: 155); GGUCGGAACCCAAGCUUAG (SEQ ED NO: 156);
CCAGAAUGCUGCAGGACUU (SEQ ID NO: 157); GAGAAGACCUCACCUAGAA (SEQ ED NO: 158);
GAAGACCUCACCUAGAAAU (SEQ ED NO: 159); CCAGAUGUUUCCAGACUUC (SEQ ED NO: 160);
CUAUUUAUGUUUGCACUUG (SEQ ID NO: 161); UCUAAACAAUGCUGAUUUG (SEQ ID NO: 162); and
GACCAACUGUCACUCAUU (SEQ ED NO: 163). Most preferably, the dsRNA comprises a ribonucleic acid
sequence selected from the group consisting of: AAUCGGCCCGACUAUCUCGACUU (SEQ ID NO: 117);
AAUGGCGUGGAGCUGAGAGAU (SEQ ID NO: 118); AACCUCCUCUCUGCCAUCAAG (SEQ ED NO: 119);
AACUGAAAGCAUGAUCCGGGA (SEQ ED NO: 120); AAUCUCGACUUUGCCGAGUCU (SEQ ED NO: 121);
AAGGGUGACCGACUCAGCGCU (SEQ ID NO: 122); AAUCAGCCGCAUCGCCGUCUC (SEQ ID NO: 123);
IACCCAUGUGCUCCUCACCCA (SEQ ID NO: 124); AAGCUCCAGUGGCUGAACCGC (SEQ ID NO.- 125);
AAGUCAGAUCAUCUUCUCGAA (SEQ ID NO: 126); AAGGGACCUCUCUCUAAUCAG (SEQ ID NO: 127);
CCUCAGCCUCUUCUCCUUCCUGA (SEQ ID NO: 128); AAUCCUCAGCCUCUUCUCCUU (SEQ ID NO: 129);
AACCAAUGCCCUCCUGGCCAA (SEQ ID NO: 130); CUGAUUAAGUUGUCUAAACAA (SEQ ID NO: 131);
CCGACUCAGCGCUGAGAUCAA (SEQ ID NO: 132); CUUGUGAUUAUUUAUUAUUUA (SEQ ID NO: 133);
AAGCCUGUAGCCCAUGUUGUA (SEQ ID NO: 134); and UAGGGUCGGAACCCAAGCUUA (SEQ ID NO:
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates peptide-mediated uptake and the effect on cell viability of siRNAs complexed or
conjugated to a polynucleotide delivery-enhancing polypeptide of the invention (SEQ ID NO: 35). Cell uptake and
cell viability are expressed in percent.
Figure 2 further illustrates peptide-mediated uptake of siRNAs complexed or conjugated to a polynucleotide
delivery-enhancing polypeptide of the invention (SEQ ID NO: 35). Cell uptake is expressed as mean fluorescent
Figure 3 shows peptide-mediated uptake of siRNAs in human monocytes with several different polynucleotide
Figure 4 shows that siRNA/peptide injected mice have a delayed RA progression comparable to that exhibited
by Ramicade-treated subjects. RA progression was measured by a paw scoring index.
Figure 5 provides results of uptake efficacy and viability studies in mouse tail fibroblast cells for PN73
rationally-designed derivative polynucleotide delivery-enhancing polypeptides of the invention.
Figure 6 shows that siNAs conjugated to a polynucleotide delivery-enhancing polypeptide have greater
knockdown activity in vitro than siRNAs complexed with a polynucleotide delivery-enhancing polypeptide.
DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
The present invention satisfies these needs and fulfills additional objects and advantages by providing novel
compositions and methods that employ a short interfering nucleic acid (siNA), or a precursor thereof, in combination
with a polynucleotide delivery-enhancing polypeptide. The polynucleotide delivery-enhancing polypeptide is a natural
or artificial polypeptide selected for its ability to enhance intracellular delivery or uptake of polynucleotides, including
siNAs and their precursors.
Within the novel compositions of the invention, the siNA may be admixed or complexed with, or conjugated
to, the polynucleotide delivery-enhancing polypeptide to form a composition that enhances intracellular delivery of the
siNA as compared to delivery resulting from contacting the target cells with a naked siNA (i.e., siNA without the
delivery-enhancing polypeptide present).
As used herein "cell" is used in its usual biological sense, and does not refer to an entire multicellular
organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and
mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,
bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent
or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem
cell, or a fully differentiated cell.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
By "comprising" is meant including, but not limited to, whatever follows the word "comprising." Thus, use of
the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional
and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase
"consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that
no other elements may be present. By "consisting essentially of is meant including any elements listed after the
phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the
disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are
required or mandatory, but that other elements are optional and may or may not be present depending upon whether or
not they affect the activity or action of the listed elements.
The term "biodegradable" as used herein, refers to degradation in a biological system, for example enzymatic
degradation or chemical degradation.
The term "biologically active molecule" as used herein, refers to compounds or molecules that are capable of
fciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules
either alone or in combination with other molecules contemplated by the instant invention include therapeutically
active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small
molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic
acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs
thereof. Biologically active molecules of the invention also include molecules capable of modulating the
pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers
such as polyamines, polyamides, polyethylene glycol and other polyethers.
The term "phospholipid" as used herein, refers to a hydrophobic molecule comprising at least one phosphorus
group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl
group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
The term "ligand" refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter,
that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that
interacts with a ligand can be present on the surface of a cell or can alternately be an intracellular receptor. Interaction
of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or
By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid
chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the
remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a
commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a
base at the 1 '-position.
By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified
bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety.
Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified
at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified
nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen,
supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication
No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several
examples of modified nucleic acid bases known in the art as summarized by Limbach et al, 1994, Nucleic Acids Res.
22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules
include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl
uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,
ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine),
propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified bases" in
this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.
By "target site" is meant a sequence within a target RNA that is "targeted" for cleavage mediated by a siNA
construct which contains sequences within its antisense region that are complementary to the target sequence.
By "detectable level of cleavage" is meant cleavage of target RNA (and formation of cleaved product RNAs)
to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation
of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the
background for most methods of detection.
By "biological system" is meant, material, in a purified or unpurified form, from biological sources, including
but not limited to human, animal, plant, insect, bacterial, viral or other sources, wherein the system comprises the
components required for RNAi activity. The term "biological system" includes, for example, a cell, tissue, or
organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in
an in vitro setting.
The term "biodegradable linker" as used herein, refers to a nucleic acid or non-nucleic acid linker molecule
that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically
active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the
invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such
delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can
lie modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and
chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other
2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer,
tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based
linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule
can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
In certain embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is a histone
protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof. Within these embodiments, the
siNA is admixed, complexed or conjugated with one or more full length histone proteins or polypeptides
corresponding at least in part to a partial sequence of a histone protein, for example of one or more of the following
histones: histone HI, histone H2A, histone H2B, histone H3 or histone H4, or one or more polypeptide fragments or
derivatives thereof comprising at least a partial sequence of a histone protein, typically at least 5-10 or 10-20
contiguous residues of a native histone protein. In more detailed embodiments, the siRNA/histone mixture, complex
or conjugate is substantially free of amphipathic compounds. In other detailed embodiments, the siNA that is admixed,
complexed, or conjugated with the histone protein or polypeptide will comprise a double-stranded double-stranded
RNA, for example a double-stranded RNA that has 30 or fewer nucleotides, and is a short interfering RNA (siRNA).
In exemplary embodiments, the histone polynucleotide delivery-enhancing polypeptide comprises a fragment of
histone H2B, as exemplified by the polynucleotide delivery-enhancing polypeptide designated PN73 described herein
below. In yet additional detailed embodiments, the polynucleotide delivery-enhancing polypeptide may be pegylated
to improve stability and/or efficacy, particularly in the context of in vivo administration.
Within additional embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is selected
or rationally designed to comprise an amphipathic amino acid sequence. For example, useful polynucleotide deliveryenhancing
polypeptides may be selected which comprise a plurality of non-polar or hydrophobic amino acid residues
that form a hydrophobic sequence domain or motif, linked to a plurality of charged amino acid residues that form a
charged sequence domain or motif, yielding an amphipathic peptide.
In other embodiments, the polynucleotide delivery-enhancing polypeptide is selected to comprise a protein
transduction domain or motif, and a fusogenic peptide domain or motif. A protein transduction domain is a peptide
sequence that is able to insert into and preferably transit through the membrane of cells. A fusogenic peptide is a
peptide that destabilizes a lipid membrane, for example a plasma membrane or membrane surrounding an endosome,
which may be enhanced at low pH. Exemplary fusogenic domains or motifs are found in a broad diversity of viral
fusion proteins and in other proteins, for example fibroblast growth factor 4 (FGF4).
To rationally design polynucleotide delivery-enhancing polypeptides of the invention, a protein transduction
domain is employed as a motif that will facilitate entry of the nucleic acid into a cell through the plasma membrane. In
certain embodiments, the transported nucleic acid will be encapsulated in an endosome. The interior of endosomes has
a low pH resulting in the fusogenic peptide motif destabilizing the membrane of the endosome. The destabilization
and breakdown of the endosome membrane allows for the release of the siNA into the cytoplasm where the siNA can
associate with a RISC complex and be directed to its target mRNA.
Exemplary polynucleotide delivery-enhancing polypeptides within the invention may be selected from the
WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27); GKINLKALAALAKKIL (SEQ ID
NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29),
GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK
(SEQ ID NO: 31), Poly Lys-Trp, 4:1, MW 20,000-50,000; and Poly Orn-Trp, 4:1, MW 20,000-50,000. Additional
polynucleotide delivery-enhancing polypeptides that are useful within the compositions and methods herein comprise
all or part of the mellitin protein sequence.
Still other exemplary polynucleotide delivery-enhancing polypeptides are identified in the examples below.
Any one or combination of these peptides may be selected or combined to yield effective polynucleotide deliveryenhancing
polypeptide reagents to induce or facilitate intracellular delivery of siNAs within the methods and
compositions of the invention.
In more detailed aspects of the invention, the mixture, complex or conjugate comprising a siRNA and a
'lynucleotide delivery-enhancing polypeptide can be optionally combined with (e.g., admixed or complexed with) a
cationic lipid, such as LIPOFECTIN* In this context it is unexpectedly disclosed herein that polynucleotide deliveryenhancing
polypeptides complexed or conjugated to a siRNA alone will effectuate delivery of the siNA sufficient to
mediate gene silencing by RNAi. However, it is further unexpectedly disclosed herein that a siRNA/polynucleotide
delivery-enhancing polypeptide complex or conjugate will exhibit even greater activity for mediating siNA delivery
and gene silencing when admixed or complexed with a cationic lipid, such as lipofectin. To produce these
compositions comprised of a polynucleotide delivery-enhancing polypeptide, siRNA and a cationic lipid, the siRNA
and peptide may be mixed together first in a suitable medium such as a cell culture medium, after which the cationic
lipid is added to the mixture to form a siRNA/delivery peptide/cationic lipid composition. Optionally, the peptide and
cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, where after the siRNA
can be added to form the siRNA/delivery peptide/cationic lipid composition.
Examples of useful cationic lipids within these aspects of the invention include N-[l-(2,3-dioleoyloxy)propyl]-
N,N,N-trimethylammonium chloride, 1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane, 1,2-dimyristyloxypropyl-
3-dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N-
[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l -propanaminiu m trifluoracetate, 1,3-dioleoyloxy-2-(6-
carboxyspermyl)-propylamid, 5-carboxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine,
tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and
tetramethyldioleyl spermine. DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium chloride), DOTAP
(l,2-bis(oleoyloxy)-3,3-(trimethylammonium)propane), DMRIE (l,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl
ammonium bromide) or DDAB (dimethyl dioctadecyl ammonium bromide). Polyvalent cationic lipids include
lipospermines, specifically DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l -propanamini
um trifluoro-acetate) and DOSPER (l,3-dioleoyloxy-2-(6carboxy spermyl)-propyl-amid, and the di- and tetra-alkyltetra-
methyl spermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS
(tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristyl
spermine) and TMDOS (tetramethyldioleyl spermine) DOGS (dioctadecyl-amidoglycylspermine (TRANSFECTAM1.
Other useful cationic lipids are described, for example, in U.S. Patent No. 6,733,777; U.S. Patent No. 6,376,248; U.S.
Patent No. 5,736,392; U.S. Patent No. 5,686,958; U.S. Patent No. 5,334,761 and U.S. Patent No. 5,459,127.
Cationic lipids are optionally combined with non-cationic lipids, particularly neutral lipids, for example lipids
such as DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol. A
cationic lipid composition composed of a 3:1 (w/w) mixture of DOSPA and DOPE or a 1:1 (w/w) mixture of DOTMA
and DOPE (LIPOFECTIN*, Invitrogen) are generally useful in transfecting compositions of this invention. Preferred
transfection compositions are those which induce substantial transfection of a higher eukaryotic cell line.
Double stranded siRNA molecules
By "asymmetric hairpin" as used herein is meant a linear siNA molecule comprising an antisense
region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer
nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base
pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the
invention can comprise an antisense region having length sufficient to mediate RNAi in a T-cell (e.g. about 19 to about
22 (e.g., about 19, 20, 21, or 22) nucleotides) and a loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 7, or
8) nucleotides, and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, or 18) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also
comprise a 5'-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin
siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described
By "asymmetric duplex" as used herein is meant a siNA molecule having two separate strands comprising a
sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region
to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and
form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region
having length sufficient to mediate RNAi in a T-cell (e.g. about 19 to about 22 (e.g. about 19, 20, 21, or 22)
nucleotides) and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
or 18) nucleotides that are complementary to the antisense region.
By "modulate gene expression" is meant that the expression of a target gene is upregulated or downregulated,
which can include upregulation or downregulation of mRNA levels present in a cell, or of mRNA translation, or of
synthesis of protein or protein subunits, encoded by the target gene. Modulation of gene expression can be determined
also be the presence, quantity, or activity of one or more proteins or protein subunits encoded by the target gene that is
up regulated or down regulated, such that expression, level, or activity of the subject protein or subunit is greater than
or less than that which is observed in the absence of the modulator (e.g., a siRNA). For example, the term "modulate"
can mean "inhibit," but the use of the word "modulate" is not limited to this definition.
By "inhibit", "down-regulate", or "reduce" expression, it is meant that the expression of the gene, or level of
RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or level or activity of
one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the
nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction
with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another
embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the
presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment,
inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is
greater in the presence of the nucleic acid molecule than in its absence.
Gene "silencing" refers to partial or complete loss-of-function through targeted inhibition of gene expression
in a cell and may also be referred to as "knock down". Depending on the circumstances and the biological problem to
be addressed, it may be preferable to partially reduce gene expression. Alternatively, it might be desirable to reduce
gene expression as much as possible. The extent of silencing may be determined by methods known in the art, some
of which are summarized in International Publication No. WO 99/32619. Depending on the assay, quantification of
gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of the
invention, including prophylactic and therapeutic methods, which will be capable of knocking down target gene
expression, in terms of mRNA levels or protein levels or activity, for example, by equal to or greater than 10%, 30%,
50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as
may be associated with particular disease states or other conditions targeted for therapy.
The phrase "inhibiting expression of a target gene" refers to the ability of a siNA of the invention to initiate
gene silencing of the target gene. To examine the extent of gene silencing, samples or assays of the organism of
interest or cells in culture expressing a particular construct are compared to control samples lacking expression of the
construct. Control samples (lacking construct expression) are assigned a relative value of 100%. Inhibition of
expression of a target gene is achieved when the test value relative to the control is about 90%, often 50%, and in
certain embodiments 25-0%. Suitable assays include, e.g., examination of protein or mRNA levels using techniques
known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation,
enzyme function, as well as phenotypic assays known to those of skill in die art.
By "subject" is meant an organism, tissue, or cell, which may include an organism as the subject or as a donor
or recipient of explanted cells or the cells that are themselves subjects for siNA delivery. "Subject" therefore may
refers to an organism, organ, tissue, or cell, including in vitro or ex vivo organ, tissue or cellular subjects, to which the
nucleic acid molecules of the invention can be administered and enhanced by polynucleotide delivery-enhancing
polypeptides described herein. Exemplary subjects include mammalian individuals or cells, for example human
patients or cells.
By "RNA" is meant a molecule comprising at least one ribonucleotide residue.
By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a .beta.-D-ribo-furanose
moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from
naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such
alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example
at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise
non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or
deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
By "highly conserved sequence region" is meant, a nucleotide sequence of one or more regions in a target gene
does not vary significantly from one generation to the other or from one biological system to the other.
By "sense region" is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense
region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence
having homology with a target nucleic acid sequence.
By "antisense region" is meant a nucleotide sequence of a siNA molecule having complementarity to a target
aeicleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid
sequence having complementarity to a sense region of the siNA molecule.
By "target nucleic acid" is meant any nucleic acid sequence whose expression or activity is to be modulated.
The target nucleic acid can be DNA or RNA.
By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid
sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the
present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to
allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies
for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-
133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-
3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can
form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10
nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid
sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
"Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with
the same number of contiguous residues in a second nucleic acid sequence.
The term "universal base" as used herein refers to nucleotide base analogs that form base pairs with each of the
natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include Cphenyl,
C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-
nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic
Acids Research, 29, 2437-2447).
The term "acyclic nucleotide" as used herein refers to any nucleotide having an acyclic ribose sugar, for
example where any of the ribose carbons (Cl, C2, C3, C4, or C5), are independently or in combination absent from the
By "abasic" is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1'
position, see for example Adamic et al., U.S. Pat. No. 5,998,203.
By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to
the 1'carbon of beta.-D-ribo-furanose.
By "modified nucleoside" is meant any nucleotide base which contains a modification in the chemical
structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides
are shown by Formulae I-VII and/or other modifications described herein.
By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the
oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These
terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery
and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may
be present on both termini. In non-limiting examples, the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy
abasic residue (moiety); 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-
2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or nonbridging
Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue
(moiety), 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic
nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl
phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco
nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-
inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or
non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging
methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Lyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
In connection with 2'-modified nucleotides as described for the present invention, by "amino" is meant 2'-NH2
or 2'-O--NH2, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et
al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Patent. No. 6,248,878.
The siNA molecules can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered
to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to through injection,
infusion pump or stent, with or without their incorporation in biopolymers. In another embodiment, polyethylene
glycol (PEG) can be covalently attached to siNA compounds of the present invention, to the polynucleotide deliveryenhancing
polypeptide, or both. The attached PEG can be any molecular weight, preferably from about 2,000 to about
50,000 daltons (Da).
The sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide
linker or a non-nucleotide linker.
"Inverted repeat" refers to a nucleic acid sequence comprising a sense and an antisense element positioned so
that they are able to form a double stranded siRNA when the repeat is transcribed. The inverted repeat may optionally
include a linker or a heterologous sequence such as a self-cleaving ribozyme between the two elements of the repeat.
The elements of the inverted repeat have a length sufficient to form a double stranded RNA. Typically, each element
of the inverted repeat is about 15 to about 100 nucleotides in length, preferably about 20-30 base nucleotides,
preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or doublestranded
form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone
residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
"Large double-stranded RNA" refers to any double-stranded RNA having a size greater than about 40 base
pairs (bp) for example, larger than 100 bp or more particularly larger than 300 bp. The sequence of a large dsRNA may
represent a segment of a mRNA or the entire mRNA. The maximum size of the large dsRNA is not limited herein. The
double-stranded RNA may include modified bases where the modification may be to the phosphate sugar backbone or
to the nucleoside. Such modifications may include a nitrogen or sulfur heteroatom or any other modification known in
The double-stranded structure may be formed by self-complementary RNA strand such as occurs for a hairpin
or a micro RNA or by annealing of two distinct complementary RNA strands.
"Overlapping" refers to when two RNA fragments have sequences which overlap by a plurality of nucleotides
on one strand, for example, where the plurality of nucleotides (nt) numbers as few as 2-5 nucleotides or by 5-10
nucleotides or more.
"One or more dsRNAs" refers to dsRNAs that differ from each other on the basis of sequence.
"Target gene or mRNA" refers to any gene or mRNA of interest. Indeed any of the genes previously identified
by genetics or by sequencing may represent a target. Target genes or mRNA may include developmental genes and
regulatory genes as well as metabolic or structural genes or genes encoding enzymes. The target gene may be
expressed in those cells in which a phenotype is being investigated or in an organism in a manner that directly or
indirectly impacts a phenotypic characteristic. The target gene may be endogenous or exogenous. Such cells include
any cell in the body of an adult or embryonic animal or plant including gamete or any isolated cell such as occurs in an
immortal cell line or primary cell culture.
hi exemplary embodiments, the instant invention features compositions comprising a small nucleic acid
molecule, such as short interfering nucleic acid (siNA), a short interfering RNA (siRNA), a double-stranded RNA
micro-RNA (mRNA), or a short hairpin RNA (shRNA), admixed or complexed with, or conjugated to, a
j%rynucleotide delivery-enhancing polypeptide.
As used herein, the term "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short
interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically-modified short
interfering nucleic acid molecule", refers to any nucleic acid molecule capable of inhibiting or down regulating gene
expression or viral replication, for example by mediating RNA interference "RNAi" or gene silencing in a sequencespecific
manner. Within exemplary embodiments, the siNA is a double-stranded polynucleotide molecule comprising
self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence in a target nucleic acid molecule for down regulating expression, or a portion
thereof, and the sense region comprises a nucleotide sequence corresponding to (i.e., which is substantially identical in
sequence to) the target nucleic acid sequence or portion thereof.
"siNA" means a small interfering nucleic acid, for example a siRNA, that is a short-length double-stranded
nucleic acid (or optionally a longer precursor thereof), and which is not unacceptably toxic in target cells. The length
of useful siNAs within the invention will in certain embodiments be optimized at a length of approximately 21 to 23 bp
long. However, there is no particular limitation in the length of useful siNAs, including siRNAs. For example, siNAs
can initially be presented to cells in a precursor form that is substantially different than a final or processed form of the
siNA that will exist and exert gene silencing activity upon delivery, or after delivery, to the target cell. Precursor
forms of siNAs may, for example, include precursor sequence elements that are processed, degraded, altered, or
cleaved at or following the time of delivery to yield a siNA that is active within the cell to mediate gene silencing.
Thus, in certain embodiments, useful siNAs within the invention will have a precursor length, for example, of
approximately 100-200 base pairs, 50-100 base pairs, or less than about 50 base pairs, which will yield an active,
processed siNA within the target cell. In other embodiments, a useful siNA or siNA precursor will be
approximately 10 to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length.
In certain embodiments of the invention, as noted above, polynucleotide delivery-enhancing polypeptides are
used to facilitate delivery of larger nucleic acid molecules than conventional siNAs, including large nucleic acid
precursors of siNAs. For example, the methods and compositions herein may be employed for enhancing delivery of
larger nucleic acids that represent "precursors" to desired siNAs, wherein the precursor amino acids may be cleaved or
otherwise processed before, during or after delivery to a target cell to form an active siNA for modulating gene
expression within the target cell. For example, a siNA precursor polynucleotide may be selected as a circular, singlestranded
polynucleotide, having two or more loop structures and a stem comprising self-complementary sense and
antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide
sequence in a target nucleic acid molecule or a portion thereof, and the sense region having nucleotide sequence
corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be
processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
In mammalian cells, dsRNAs longer than 30 base pairs can activate the dsRNA-dependent kinase PKR and 2'-
5'-oligoadenylate synthetase, normally induced by interferon. The activated PKR inhibits general translation by
phosphorylation of the translation factor eukaryotic initiation factor 2x (eIF2a), while 2'-5'-oligoadenylate synthetase
causes nonspecific mRNA degradation via activation of RNase L. By virtue of their small size (referring particularly
to non-precursor forms), usually less than 30 base pairs, and most commonly between about 17-19, 19-21, or 21-23
base pairs, the siNAs of the present invention avoid activation of the interferon response.
In contrast to the nonspecific effect of long dsRNA, siRNA can mediate selective gene silencing in the
mammalian system. Hairpin RNAs, with a short loop and 19 to 27 base pairs in the stem, also selectively silence
expression of genes that are homologous to the sequence in the double-stranded stem. Mammalian cells can convert
short hairpin RNA into siRNA to mediate selective gene silencing.
RISC mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of
the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense
strand of the siRNA duplex. Studies have shown that 21 nucleotide siRNA duplexes are most active when containing
two nucleotide 3'-overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'-deoxy (2'-H) or
2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3'-terminal siRNA overhang nucleotides
with deoxy nucleotides (2'-H) has been reported to be tolerated.
Studies have shown that replacing the 3'-overhanging segments of a 21-mer siRNA duplex having 2 nucleotide
3' overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4
nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated whereas
(Iffhplete substitution with deoxyribonucleotides results in no RNAi activity.
Alternatively, the siNAs can be delivered as single or multiple transcription products expressed by a
polynucleotide vector encoding the single or multiple siNAs and directing their expression within target cells. In these
embodiments the double-stranded portion of a final transcription product of the siRNAs to be expressed within the
target cell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp long. Within exemplary embodiments,
double-stranded portions of siNAs, in which two strands pair up, are not limited to completely paired nucleotide
segments, and may contain nonpairing portions due to mismatch (the corresponding nucleotides are not
complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), overhang, and the like.
Nonpairing portions can be contained to the extent that they do not interfere with siNA formation. In more detailed
embodiments, a "bulge" may comprise 1 to 2 nonpairing nucleotides, and the double-stranded region of siNAs in
which two strands pair up may contain from about 1 to 7, or about 1 to 5 bulges. In addition, "mismatch" portions
contained in the double-stranded region of siNAs may be present in numbers from about 1 to 7, or about 1 to 5. Most
often in the case of mismatches, one of the nucleotides is guanine, and the other is uracil. Such mismatching may be
attributable, for example, to a mutation from C to T, G to A, or mixtures thereof, in a corresponding DNA coding for
sense RNA, but other cause are also contemplated. Furthermore, in the present invention the double-stranded region of
siNAs in which two strands pair up may contain both bulge and mismatched portions in the approximate numerical
The terminal structure of siNAs of the invention may be either blunt or cohesive (overhanging) as long as the
siNA retains its activity to silence expression of target genes. The cohesive (overhanging) end structure is not limited
only to the 3' overhang as reported by others. On the contrary, the 5' overhanging structure may be included as long as
it is capable of inducing a gene silencing effect such as by RNAi. In addition, the number of overhanging nucleotides
is not limited to reported limits of 2 or 3 nucleotides, but can be any number as long as the overhang does not impair
gene silencing activity of the siNA. For example, overhangs may comprise from about 1 to 8 nucleotides, more often
from about 2 to 4 nucleotides. The total length of siNAs having cohesive end structure is expressed as the sum of the
length of the paired double-stranded portion and that of a pair comprising overhanging single-strands at both ends. For
example, in the exemplary case of a 19 bp double-stranded RNA with 4 nucleotide overhangs at both ends, the total
length is expressed as 23 bp. Furthermore, since the overhanging sequence may have low specificity to a target gene,
it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence. Furthermore, as long
as the siNA is able to maintain its gene silencing effect on the target gene, it may contain low molecular weight
structure (for example a natural RNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNA molecule), for
example, in the overhanging portion at one end.
In addition, the terminal structure of the siNAs may have a stem-loop structure in which ends of one side of
the double-stranded nucleic acid are connected by a linker nucleic acid, e.g., a linker RNA. The length of the doublestranded
region (stem-loop portion) can be, for example, 15 to 49 bp, often 15 to 35 bp, and more commonly about 21
to 30 bp long. Alternatively, the length of the double-stranded region that is a final transcription product of siNAs to
be expressed in a target cell may be, for example, approximately 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp long.
When linker segments are employed, there is no particular limitation in the length of the linker as long as it does not
hinder pairing of the stem portion. For example, for stable pairing of the stem portion and suppression of
recombination between DNAs coding for this portion, the linker portion may have a clover-leaf tRNA structure. Even
if the linker has a length that would hinder pairing of the stem portion, it is possible, for example, to construct the
linker portion to include introns so that the introns are excised during processing of a precursor RNA into mature
RNA, thereby allowing pairing of the stem portion. In the case of a stem-loop siRNA, either end (head or tail) of RNA
with no loop structure may have a low molecular weight RNA. As described above, these low molecular weight
RNAs may include a natural RNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNA molecule.
The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to
nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule
does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate
group, such as a 5'-phosphate (see for example Martinez et al.,Cell., 110: 563-574 (2002) and Schwarz et al.,
Molecular Cell, 10: 537-568(2002), or 5',3'-diphosphate.
As used herein, the term siNA molecule is not limited to molecules containing only naturally-occurring RNA
or DNA, but also encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the
short interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides. In certain
- 12. rembodiments
short interfering nucleic acids do not require the presence of nucleotides having a 2'-hydroxy group for
mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any
ribonucleotides (e.g., nucleotides having a 2'-OH group). Such siNA molecules that do not require the presence of
ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other
attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. Optionally,
siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules
that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (mRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering
nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene
silencing RNA (ptgsRNA), and others.
In other embodiments, siNA molecules for use within the invention may comprise separate sense and antisense
sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide
linker molecules, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals
interactions, hydrophobic interactions, and/or stacking interactions.
"Antisense RNA" is an RNA strand having a sequence complementary to a target gene mRNA, and thought to
induce RNAi by binding to the target gene mRNA. "Sense RNA" has a sequence complementary to the antisense
RNA, and annealed to its complementary antisense RNA to form siRNA. These antisense and sense RNAs have been
conventionally synthesized with an RNA synthesizer.
As used herein, the term "RNAi construct" is a generic term used throughout the specification to include small
interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs.
RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving
rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
Optionally, the siRNA include single strands or double strands of siRNA.
An siHybrid molecule is a double-stranded nucleic acid that has a similar function to siRNA. Instead of a
double-stranded RNA molecule, an siHybrid is comprised of an RNA strand and a DNA strand. Preferably, the RNA
strand is the antisense strand as that is the strand that binds to the target mRNA. The siHybrid created by the
hybridization of the DNA and RNA strands have a hybridized complementary portion and preferably at least one
siNAs for use within the invention can be assembled from two separate oligonucleotides, where one strand is
the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary
(i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such
as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the
double stranded region is about 19 base pairs). The antisense strand may comprise a nucleotide sequence that is
complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand
may comprise a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
Alternatively, the siNA can be assembled from a single oligonucleotide, where the self-complementary sense and
antisense regions of the siNA are linked by means of a nucleic acid-based or non-nucleic acid-based linker(s).
Within additional embodiments, siNAs for intracellular delivery according to the methods and compositions of
the invention can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary
structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide
sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion
thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a
Non-limiting examples of chemical modifications that can be made in an siNA include without limitation
phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro
ribonucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl
and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA
constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum
stability of these compounds.
In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules
provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native
RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules
can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified
nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can
improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular
uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is
reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid
molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due
to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can
also minimize the possibility of activating interferon activity in humans.
The siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a
phosphorothioate internucleotide linkage at the 3'-end of said antisense region. In any of the embodiments of siNA
molecules described herein, the antisense region can comprise about one to about five phosphorothioate
internucleotide linkages at the 5'-end of said antisense region. In any of the embodiments of siNA molecules described
herein, the 3'-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or
deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the
embodiments of siNA molecules described herein, the 3'-terminal nucleotide overhangs can comprise one or more
universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3'-terminal
nucleotide overhangs can comprise one or more acyclic nucleotides.
For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic
acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In
yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA)
individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands.
The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA
duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can
comprise one or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both of the 3'- and S'-ends
of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention
can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate
internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands. In another non-limiting
example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both
strands, hi yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand,
the antisense strand, or both strands.
An siNA molecule may be comprised of a circular nucleic acid molecule, wherein the siNA is about 38 to
about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18,
19, 20, 21, 22, or 23) base pairs wherein the circular oligonucleotide forms a dumbbell shaped structure having about
19 base pairs and 2 loops.
A circular siNA molecule contains two loop motifs, wherein one or both loop portions of the siNA molecule is
biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop
portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs, such
as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.
Modified nucleotides present in siNA molecules, preferably in the antisense strand of the siNA molecules, but
also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties
or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules
including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example
Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides
present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the
invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation
while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a
northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl)
nucleotides); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides. 2'-deoxy-2'-
chloro nucleotides, 2'-azido nucleotides, and 2'-O-methyl nucleotides.
The sense strand of a double stranded siNA molecule may have a terminal cap moiety such as an inverted
pfeoxybasic moiety, at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand.
Non-limiting examples of conjugates include conjugates and ligands described in Vargeese et al., U.S.
Application Serial No. 10/427,160, filed April 30, 2003, incorporated by reference herein in its entirety, including the
drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via
a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3'-end of either the sense strand,
the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate
molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemicallymodified
siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3'-end and 5'-end of
either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any
combination thereof, hi one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates
delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the
conjugate molecule attached to the chemically-modified siNA molecule is a poly ethylene glycol, human serum
albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules
contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in
Vargeese et al., U.S. Patent Application Publication No. 20030130186, published July 10, 2003, and U.S. Patent
Application Publication No. 20040110296, published June 10, 2004. The type of conjugates used and the extent of
conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles,
bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to
mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various
conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the
ability to mediate RNAi, for example in animal models as are generally known in the art.
A siNA further may be further comprised of a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide
linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide
linker can be a linker of 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In
another embodiment, the nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as
used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid
molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately,
an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally
bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to
bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the
protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily
generated using techniques generally known in the art. [See, for example, Gold et al, Annu. Rev. Biochem., 64: 763
(1995); Brody and Gold, J. Biotechnol., 74: 5 (2000); Sun, Curr. Opin. Mol. Ther., 2:100 (2000); Kusser, J.
Biotechnol, 74: 27 (2000); Hermann and Patel, Science 287: 820 (2000); and Jayasena, Clinical Chemistry, 45: 1628.
A non-nucleotide linker may be comprised of an abasic nucleotide, polyether, polyamine, polyamide, peptide,
carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having
between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic
Acids Res., 18:6353 (1990) and Nucleic Acids Res., 15:3113 (1987); Cload and Schepartz, J. Am. Chem. Soc.,
113:6324 (1991); Richardson and Schepartz, J. Am. Chem. Soc., 113:5109 (1991); Ma et al., Nucleic Acids Res.,
21:2585 (1993) and Biochemistry 32:1751(1993); Durand et al., Nucleic Acids Res., 18:6353 (1990); McCurdy et al.,
Nucleosides & Nucleotides, 10:287 (1991); Jschke et al., Tetrahedron Lett., 34:301 (1993); Ono et al., Biochemistry,
30:9914 (1991); Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No.
WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem.
Soc., 113:4000 (1991). A "non-nucleotide" further means any group or compound that can be incorporated into a
nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions,
and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does
not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymidine, for
example at the Cl position of the sugar.
The synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a)
synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together
under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two
complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis, hi yet another embodiment,
synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.
Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking
onucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992,
Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al.,
1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998,
Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. Synthesis of RNA, including certain siNA
molecules of the invention, follows general procedures as described, for example, in Usman et al., 1987, J. Am. Chem.
Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23,
2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59.
Supplemental or complementary methods for delivery of nucleic acid molecules for use within then invention
are described, for example, in Akhtar et al., Trends Cell Bio., 2, 139 (1992); Delivery Strategies for Antisense
Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., Mol. Membr. Biol., 16: 129-140 (1999); Hofland and
Huang, Handb. Exp. Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser., 752: 184-192 (2000). Sullivan
et al., International PCT Publication No WO 94/02595, further describes general methods for delivery of enzymatic
nucleic acid molecules. These protocols can be utilized to supplement or complement delivery of virtually any nucleic
acid molecule contemplated within the invention.
Nucleic acid molecules and polynucleotide delivery-enhancing polypeptides can be administered to cells by a
variety of methods known to those of skill in the art, including, but not restricted to, administration within formulations
that comprise the siNA and polynucleotide delivery-enhancing polypeptide alone, or that further comprise one or more
additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer,
stabilizer, preservative, and the like. In certain embodiments, the siNA and/or the polynucleotide delivery-enhancing
polypeptide can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such
as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see e.g.,
O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, a nucleic acid/peptide/vehicle
combination can be locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic
acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard
needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., Clin. Cancer
Res., 5: 2330-2337 (1999) and Barry et al., International PCT Publication No. WO 99/31262.
The compositions of the instant invention can be effectively employed as pharmaceutical agents.
Pharmaceutical agents prevent, modulate the occurrence or severity of, or treat (alleviate one or more symptom(s) to a
detectable or measurable extent) of a disease state or other adverse condition in a patient.
Thus within additional embodiments the invention provides pharmaceutical compositions and methods
featuring the presence or administration of one or more polynucleic acid(s), typically one or more siNAs, combined,
complexed, or conjugated with a polynucleotide delivery-enhancing polypeptide, optionally formulated with a
pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, and the like.
The present invention satisfies additional objects and advantages by providing short interfering nucleic acid
(siNA) molecules that modulate expression of genes associated with a particular disease state or other adverse
condition in a subject. Typically, the siNA will target a gene that is expressed at an elevated level as a causal or
contributing factor associated with the subject disease state or adverse condition. In this context, the siNA will
effectively downregulate expression of the gene to levels that prevent, alleviate, or reduce the severity or recurrence of
one or more associated disease symptoms. Alternatively, for various distinct disease models where expression of the
target gene is not necessarily elevated as a consequence or sequel of disease or other adverse condition, down
regulation of the target gene will nonetheless result in a therapeutic result by lowering gene expression (i.e., to reduce
levels of a selected mRNA and/or protein product of the target gene). Alternatively, siNAs of the invention may be
targeted to lower expression of one gene, which can result in upregulation of a "downstream" gene whose expression
is negatively regulated by a product or activity of the target gene.
Within exemplary embodiments, the compositions and methods of the invention are useful as therapeutic tools
to regulate expression of tumor necrosis factor-a (TNF-a) to treat or prevent symptoms of rheumatoid arthritis (RA).
In this context the invention further provides compounds, compositions, and methods useful for modulating expression
and activity of TNF- a by RNA interference (RNAi) using small nucleic acid molecules. In more detailed
embodiments, the invention provides small nucleic acid molecules, such as short interfering nucleic acid (siNA), short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), and short hairpin RNA (shRNA)
molecules, and related methods, that are effective for modulating expression of TNF-a and/or TNF-a genes to prevent
or alleviate symptoms of RA in mammalian subjects. Within these and related therapeutic compositions and methods,
me use of chemically-modified siNAs will often improve properties of the modified siNAs in comparison to properties
of native siNA molecules, for example by providing increased resistance to nuclease degradation in vivo, and/or
through improved cellular uptake. As can be readily determined according to the disclosure herein, useful siNAs
having multiple chemical modifications will retain their RNAi activity. The siNA molecules of the instant invention
thus provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery,
genetic engineering, and pharmacogenomic applications.
This siNAs of the present invention may be administered in any form, for example transdermally or by local
injection (e.g., local injection at sites of psoriatic plaques to treat psoriasis, or into the joints of patients afflicted with
psoriatic arthritis or RA). In more detailed embodiments, the invention provides formulations and methods to
administer therapeutically effective amounts of siNAs directed against of a mRNA of TNF-a, which effectively downregulate
the TNF- a RNA and thereby reduce or prevent one or more TNF-a-associated inflammatory condition(s).
Comparable methods and compositions are provided that target expression of one or more different genes associated
with a selected disease condition in animal subjects, including any of a large number of genes whose expression is
known to be aberrantly increased as a causal or contributing factor associated with the selected disease condition.
The siNA/polynucleotide delivery-enhancing polypeptide mixtures of the invention can be administered in
conjunction with other standard treatments for a targeted disease condition, for example in conjunction with
therapeutic agents effective against inflammatory diseases, such as RA or psoriasis. Examples of combinatorially
useful and effective agents in this context include non-steroidal antiinflammatory drugs (NSAIDs), methotrexate, gold
compounds, D-penicillamine, the antimalarials, sulfasalazine, glucocorticoids, and other TNF-a neutralizing agents
such as infliximab and entracept.
Negatively charged polynucleotides of the invention (e.g., RNA or DNA) can be administered to a patient by
any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it
is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The
compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral
administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and
the other compositions known in the art.
The present invention also includes pharmaceutically acceptable formulations of the compositions described
herein. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of
hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for
administration, e.g., systemic administration, into a cell or patient, including for example a human. Suitable forms, in
part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not
prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic
acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be
soluble. Other factors are known in the art, and include considerations such as toxicity.
By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood
stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption
include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and
intramuscular. Each of these administration routes exposes the desired negatively charged polymers, e.g., nucleic
acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function
of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant
invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular
endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells,
such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to
target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal
cells, such as cancer cells.
By "pharmaceutically acceptable formulation" is meant, a composition or formulation that allows for the
effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for
their desired activity. Nonlimiting examples of agents suitable for formulation with the nucleic acid molecules of the
instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the
CNS [Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol., 13:16-26 (1999)]; biodegradable polymers, such as poly
(DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et
a/., Cell Transplant, 8: 47-58 (1999)] (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those
made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake
mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23: 941-949, (1999)]. Other non-limiting examples of
delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., J.
Pharm. Sci., 87:1308-1315 (1998); Tyler et al., FEES Lett, 421: 280-284 (1999); Pardridge et al., PNAS USA., 92:
5592-5596 (1995); Boado, Adv. Drug Delivery Rev., 15: 73-107 (1995); Aldrian-Herrada et al., Nucleic Acids Res.,
26: 4910-4916 (1998); and Tyler et al., PNAS USA, 96: 7053-7058 (1999).
The present invention also includes compositions prepared for storage or administration, which include a
pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example,
preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and
esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence of, or treat (alleviate
a symptom to some extent, preferably all of the symptoms) a disease state. The pharmaceutically effective dose
depends on the type of disease, the composition used, the route of administration, the type of mammal being treated,
the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that
those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of
aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose,
methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;
dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products
of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene
oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of
ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate,
or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for
example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for
example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or
more sweetening agents, such as sucrose or saccharin.
Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example
arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can
contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring
agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an
anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water
provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more
preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned
above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase
can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring
gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin,
and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
The emulsions can also contain sweetening and flavoring agents.
The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.
This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and
suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable
solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-
butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic
sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition,
ratty acids such as oleic acid find use in the preparation of injectables.
The siNAs can also be administered in the form of suppositories, e.g., for rectal administration of the drug.
These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary
temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such
materials include cocoa butter and polyethylene glycols.
The siNAs can be modified extensively to enhance stability by modification with nuclease resistant groups, for
example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H. [For a review see Usman and Cedergren, TIBS 17: 34
(1992); Usman et al., Nucleic Acids Symp. Ser. 31: 163 (1994)]. SiNA constructs can be purified by gel
electrophoresis using general methods or can be purified by high pressure liquid chromatography and re-suspended in
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent
their degradation by serum ribonucleases, which can increase their potency. See e.g., Eckstein et al., International
Publication No. WO 92/07065; Perrault et al., Nature 344: 565 (1990); Pieken et al., Science 253, 314 (1991); Usman
and Cedergren, Trends in Biochem. Sci. 17: 334 (1992); Usman et al., International Publication No. WO 93/15187;
and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat.
No. 6,300,074. All of the above references describe various chemical modifications that can be made to the base,
phosphate and/or sugar moieties of the nucleic acid molecules described herein.
There are several examples in the art describing sugar, base and phosphate modifications that can be
introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For
example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with
nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base
modifications. For a review see Usman and Cedergren, , TIBS. 17: 34 (1992); Usman et al., Nucleic Acids Symp. Ser.
31:163 (1994); Burgin et al., Biochemistry, 35: 14090 (1996). Sugar modification of nucleic acid molecules have been
extensively described in the art. See Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al.
Nature,344, 565-568 (1990); Pieken et al. Science, 253: 314-317 (1991); Usman and Cedergren, Trends in Biochem.
Sci., 17: 334-339 (1992); Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No.
5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No.
WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,
International PCT Publication No. WO 98/13526; Thompson et al., Karpeisky et al., Tetrahedron Lett., 39: 1131
(1998); Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences, 48: 39-55 (1998); Verma and Eckstein, Annu. Rev.
Biochem., 67: 99-134 (1998); and Burlina et al., Bioorg. Med. Chem., 5: 1999-2010 (1997). Such publications
describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate
modifications and the like into nucleic acid molecules without modulating catalysis. In view of such teachings, similar
modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so
long as the ability of siNA to promote RNAi in cells is not significantly inhibited.
While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate,
phosphorodithioate, and/or 5'-methylphosphonate linkages improves stability, excessive modifications can cause some
toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide
linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in
increased efficacy and higher specificity of these molecules.
In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications
comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino,
amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,
thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker
and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417
(1995), and Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in
Antisense Research, ACS, 24-39 (1994).
Methods for the delivery of nucleic acid molecules are described in Akhtar et al., Trends Cell Bio., 2: 139
(1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer et al., Mol.
Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol., 137: 165-192 (1999); and Lee et al.,
ACS Symp. Ser., 752: 184-192 (2000). Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO
94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized
for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a
TOriety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by
iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see
for example Gonzalez et al., Bioconjugate Chem., 10: 1068-1074 (1999); Wang et al., International PCT publication
Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)ac- id (PLGA) and PLCA microspheres (see for
example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable
nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT
Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct
injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether
subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by
needle-free technologies such as those described in Conry et al., Clin. Cancer Res., 5: 2330-2337 (1999) and Barry et
al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as
pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some
extent, preferably all of the symptoms) of a disease state in a subject.
In this specification and the appended claims, the singular forms of "a", "an" and "the" include plural
reference unless the context clearly dictates otherwise.
Production and Characterization of Compositions Comprising a siRNA
Complexed With a Polynucleotide Delivery-Enhancing Polypeptide
To form complexes between candidate siRNAs and polynucleotide delivery-enhancing polypeptides of the
invention, an adequate amount of siRNA is combined with a pre-determined amount of polynucleotide deliveryenhancing
polypeptide, for example in Opti-MEM® cell medium (Invitrogen), in defined ratios and incubated at room
temperature for about 10-30 min. Subsequently a selected volume, e.g., about 50 ul, of this mixture is brought into
contact with target cells and the cells are incubated for a predetermined incubation period, which in the present
example was about 2 hr. The siRNA/peptide mixture can optionally include cell culture medium or other additives
such as fetal bovine serum. For H3, H4 and H2b, a series of experiments was performed to complex these
polynucleotide delivery-enhancing polypeptides with siRNA in different ratios. Generally this was initiated with a
1:0.01 to 1:50 of siRNA/histone ratio. To each well in a 96-well microtiter plate, 40 pm siRNA was added. Each well
contained beta-gal cells at 50% confluency. Exemplary optimized ratios for transfection efficiency are shown in Table
Transfections were performed with either regular siRNA or siRNA complexed with one of the aboveidentified
histone proteins on 9L/beta-gal cells. The siRNA was designed to specifically knock down betagalactosidase
mRNA, and activities are expressed as percentage of beta-gal activities from control (control cells were
transfected using lipofectamine without the polynucleotide delivery-enhancing polypeptide).
Assays for detecting and/or quantifying the efficiency of siRNA delivery are carried out using conventional
methods, for example beta-galactosidase assay or flow cytometry methods.
For beta-galactosidase assays, 9L/LacZ cells, a cell line constitutively expressing beta-galactosidase, were
used. 9L/LacZ cells are rat gliosarcoma fibroblast cells that constitutively express LacZ and were obtained from ATCC
(#CRL-2200). 9L/LacZ cells were grown in Dulbecco's Modified Essential Medium (DMEM) media with a
supplement of 1 mM sodium pyruvate, nonessential amino acids, and 20% fetal bovine serum. Cells were cultured at
37°C and 5% CO2 supplemented with an antibiotic mixture containing 100 units/ml penicillin, 100 n.g/ml streptomycin
and 0.25 mg/ml Fungizone (Invitrogen). The siRNA duplex designed against beta-gal mRNA was chemically
synthesized and used with delivery reagents to evaluate knock-down efficiency.
siRNA Synthesis and Preparation
Synthesis of oligonucleotides was carried out using the standard 2-cyanoethyl phosphoramidite method on
long chain alkylamine controlled pore glass derivatized with 5'-O-Dimethyltrityl-2'-O-/-butyldimethylsilyl-3'-6-
succinyl ribonucleoside of choice or 5'-O-Dimethyltrityl-2'-deoxy-3'-O-succinyl thymidine support where applicable.
All oligonucleotides were synthesized at either the 0.2 or 1-umol scale using an ABI 3400 DNA/RNA synthesizer,
cleaved from the solid support using concentrated NH4OH, and deprotected using a 3:1 mixture of NHjOH : EtOH at
55 °C. The deprotection of 2'-TBDMS protecting groups was achieved by incubating the base-deprotected RNA with a
ablution (600 |LiL per rnol) of N-methylpyrrolidone/triethylamine/triethylamine tris(hydrofluoride) (6:3:4 by volume)
at 65 °C for 2.5 hours. The corresponding building blocks, 5'-dimethoxytrityl-A-(tac)-2'-O-(/-butyldimethylsilyl)-3'-
[(2-cyanoethyl)-(A,Ar-diisopropyl)]-phosphoramidites of A, U, C and G (Proligo, Boulder CO) as well the modified
phosphoramidites, 5'DMTr-5-methyl-U-TOM-CE-Phosphoramidite, 5'-DMTr-2'-OMe-Ac-C-CE Phosphoramidite, 5'-
DMTr-2'-OMe-G-CE Phosphoramidite, 5'-DMTr-2'-OMe-U-CE Phosphoramidite, 5'-DMTr-2'-OMe-A-CE
Phosphoramidite (Glen Research) were purchased directly from suppliers. Triethylamine-trihydrofluride, Nmethylpyrrolidinone
and concentrated ammonium hydroxide was purchased from Aldrich. All HPLC analysis and
purifications were performed on a Waters 2690 with Xterra™ columns. All other reagents were purchased from Glen
Research Inc. Oligonucleotides were purified to greater than 97% purity as determined by RP-HPLC. siRNAs for
mouse injection were purchased from Qiagen, which were HPLC purified after annealing with acceptable endotoxin
level for in vivo injection.
On the first day of the procedure, saturated 9L/LacZ cultures are taken from T75 flasks, and the cells are
detached and diluted into 10ml of complete medium (DMEM, IxPS, IxNa Pyruvate, Ix NEAA). The cells are further
diluted to 1:15, and lOOul of this preparation are aliquoted into wells of 96 well plates, which will generally yield
about 50% cell confluence by the next day for the transfection. Edges of the wells are left empty and filled with 250 ul
water, and the plates are placed un-stacked in the incubator overnight at 37°C (5% CO2 incubator).
On the second day, the transfection complex is prepared in Opti-MEM, 50ul each well. The medium is
removed from the plates, and the wells are washed once with 200ul PBS or Opti-MEM. The plates are blotted and
dried completely with tissue by inversion. The transfection mixture is then added (50ul/well) into each well, and
250ul water is added to the wells on the edge to prevent them from drying. The cells are then incubated for at least 3
hours at 37°C (5% CO2 incubator). The transfection mixture is removed and replaced with lOOul of complete medium
(DMEM, IxPS, IxNa Pyruvate, Ix NEAA). The cells are cultured for a defined length of time, and then harvested for
the enzyme assay.
The siRNA sequence used to silence the beta-galactosidase mRNA was the following:
C.U.A.C.A.C.A.A.A.U.C.A.G.C.G.A.U.U.U.dT.dT (Sense) (SEQ ID NO: 32)
A.A.A.U.C.G.C.U.G.A.U.U.U.G.U.G.U.A.G.dT.dT (Antisense) (SEQ ID NO: 33)
The data for the present example is shown in Table 2. The transfection efficiency inversely correlates
with the amount of beta-galactosidase activity measured from the cell lysate. Upon transfection, a measured
decrease in beta-galactosidase activity indicates a successful transfection. Thus, in the absence of transfection, the
measured beta-galactosidase activity is 100% and the transfection efficiency is 0%. As beta-galactosidase
activity decreases, the transfection efficiency increases. For example, in Table 2, Histone H2B plus siRNA
results in a transfection efficiency of 62.03% indicating that the measured beta-galactosidase activity decreased to
37.97%. The same approach for determining transfection efficiency was used for the data presented in Table 3.
To evaluate the effects of adding a cationic lipid to a siRNA/polynucleotide delivery-enhancing polypeptide
mixture, complex or conjugate, the above procedures were followed except the lipofectamine (Invitrogen) was added
to siRNA/polynucleotide delivery formulation in constant concentrations, following manufacturer's instructions (0 2
To produce the composition comprised of GKINLKALAALAKKIL (SEQ ID NO: 28), siRNA and
LIPOFECTIN® (Invitrogen), the siRNA and peptide were mixed together first in Opti-MEM cell culture medium at
room temperature, after which LIPOFECTIN® was added at room temperature to the mixture to form the
siRNA/peptide/cationic lipid composition.
To produce the composition comprised of RVIRVWFQNKRCKDKK (SEQ ID NO: 29), siRNA and
LIPOFECTIN*, the peptide and the LIPOFECTIN® were mixed together first in Opti-MEM cell culture medium, into
this mixture was added the siRNA to form the siRNA/peptide/LIPOFECTIN® composition
To produce the siRNA/peptide/cationic lipid composition using
GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30) or
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31) it does not matter in which order the components are added
together to produce the siRNA/peptide/cationic lipid composition.
To produce the siRNA/mellitin/LIPOFECTIN®, the siRNA and mellitin were first mixed together in Opti-
MEM cell culture medium and then the LIPOFECTIN® was added to the mixture.
To produce the siRNA/histone HI /LIPOFECTIN® composition, the histone HI and LIPOFECTIN® were first
added together in Opti-MEM cell culture medium thoroughly mixed and then the siRNA was added, thoroughly and
mixed with the histone LIPOFECTIN® mixture to form the siRNA/histone HI/ LIPOFECTIN® composition.
Based on the foregoing results, it is apparent that exemplary polynucleotide delivery-enhancing polypeptides
of the invention can substantially enhance cellular uptake of siRNAs, while the addition of an optional cationic lipid to
certain siRNA/polypeptide mixtures of the invention may substantially improve siRNA delivery efficiency.
Production and Characterization of Compositions Comprising a siRNA
Conjugated With a TAT-HA Polynucleotide Delivery-Enhancing Polvpeptide
The present example describes the synthesis and uptake activity of specific peptides covalently conjugated to
one strand of a siRNA duplex. These conjugates efficiently deliver siRNA into the cytoplasm.
Both peptides and RNAs are prepared using standard solid phase synthesis methods.
To form conjugates, the peptide and RNA molecules must be functionalized with specific moieties to allow for
covalent attachment to each other. For the peptide, the N-terminus is functionalized with 3-maleimidopropionic acid.
For the RNA molecule the 5' end of the sense strand or 3' end of the antisense strand is functionalized with a l-Odimethoxytrityl-
hexyl-disulfide linker using standard procedures.
Structure of the peptide-siRNA conjugate (SEQ ID NOS 34 and 35)
Cells were plated the day before so that they were ~50-80% confluent at time of transfection. For complexes,
siRNA and peptide were diluted in Opti-MEM® media (Invitrogen), then mixed and allowed to complex 5-10 minutes
before adding to cells washed with PBS. Final concentration of siRNA was SOOnM at each peptide concentration (2-
SOM). The conjugate, also diluted in Opti-MEM® media, was added to cells at final concentrations ranging from
62.5nM to SOOnM. At SOOnM concentration, we also combined with 20% FBS just before adding to washed cells.
Cells were transfected for 3 hours at 37°C, 5%CO2. Cells were washed with PBS, treated with trypsin and then
analyzed by flow cytometry. siRNA uptake was measured by intensity of Cy5 fluorescence and cellular viability
assessed by addition of propidium iodide.
As shown in Figure 1, the peptide/siRNA conjugates achieve a greater percent uptake in mouse tail fibroblast
cells than peptide/siRNA complexes. Further, the peptide/siRNA conjugates achieved a higher mean fluorescence
intensity (MFI; Figure 2) than the peptide/siRNA complex. Thus, these data indicate that in certain embodiments it
will be desirable to conjugate the polynucleotide delivery-enhancing polypeptide to the siRNA molecule.
Screening of siRN A/Delivery Peptide Complexes for siRNA
Uptake in 9L/LacZ Cells
The present example provides additional evidence that a broad and diverse assemblage of rationally-designed
polynucleotide delivery-enhancing polypeptides of the invention enhance siRNA uptake when complexed with
Uptake was measured using 9L/lacZ cells transfected as described in Example 2. Cells were washed with
PBS, treated with trypsin and then analyzed by flow cytometry. siRNA uptake was measured by intensity of FAM
fluorescence and cellular viability assessed by addition of propidium iodide. Table 4 below summarizes the percent
cell uptake data in 9L/LacZ cells for the various rationally-designed polynucleotide delivery-enhancing polypeptides.
Included in this table, is the concentration of the peptide and siRNA used.
SiRN A/Deli very is Enhanced by Polynucleotide Delivery-Enhancing
Polypeptides In Vitro
The present example illustrates the enhancement of siRNA uptake by polynucleotide delivery-enhancing
polypeptides of the invention in LacZ cells, murine primary fibroblasts and human monocytes. The materials and
methods used for the experiments performed in 9L/LacZ cells and mouse fibroblast cells are generally the same as
described above, except that for the murine experiments, 9L/LacZ cells were replaced with mouse tail fibroblasts. The
results for transfections performed with mouse tail fibroblast (MTF) cells are summarized in Table 5, which shows the
concentration of the peptide and siRNA used along with the label conjugated to the siRNA molecule. The results for
transfections performed with both MTF and 9L/LacZ cells are summarized in Table 6. The data presented in Table 6
compares transfection efficiencies for some peptide/siRNA complexes in different cell types.
(SEQ ID NO: 35)
(SEQ ID NO: 59)
(SEQ ID NO: 90)
(SEQ ID NO: 58)
(SEQ ID NO: 38)
NH2-RQIKIWFQNRRMKWKK-amide (SEQ ID NO: 53)
NH2-RVIRWFQNKRCKDKK amide (SEQ ID NO: 67)
(SEQ ID NO: 92)
(SEQ ID NO: 76)
(SEQ ID NO: 39)
GRKKRRQRRRPPQC (SEQ ID NO: 36)
KLALKLALKALKAALKLA-amide (SEQ ID NO: 13)
(SEQ ID NO: 94)
(SEQ ID NO: 89)
NH2-AAVALLPAVLLALLAPSGASGLDKRDYV-amide (SEQ ID NO: 47)
NH2-RQIKIWFQNRRMKWKK-amide (SEQ ID NO: 53)
(SEQ ID NO: 56)
(SEQ ID NO: 59)
NH2-RVIRWFQNKRCKDKK-amide (SEQ ID NO: 67)
NH2-GRKKRRQRRRPPQC-amide (SEQ ID NO: 36)
NH2-KETWWETWWTEWSQPKKKRKV-amide (SEQ ID NO: 95)
(SEQ ID NO: 50)
NH2-C(YGRKKRRQRRRG)2-amide (SEQ ID NO: 42)
(SEQ ID NO: 35)
NH2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 58)
NH2-AAVALLPAVLLALLAPRRRRRR-amide (SEQ ID NO: 45)
ID NO: 91)
(SEQ ID NO: 96)
To further characterize the ability of polynucleotide delivery-enhancing polypeptides to transfect cells in
culture, human monocytes were incubated with FITC labeled siRNA complexed with various concentrations of PN73,
PN250, PN182, PN58 and PN158. Human monocytes were used in addition to LacZ and mouse fibroblast cells
because they are the targeted cell type in the treatment of rheumatoid arthritis.
Monocytes were isolated from fresh human blood samples from healthy donors, to a purity greater than 95%
by FACS analysis.
Monocytes were transfected using procedures of Example 2 using Cy5- or FAM-conjugated siRNA and
peptide, and siRNA uptake was measured by the intensity of intracellular Cy5 or FAM fluorescence. Cell viability was
determined using propidium iodide (uptake) or AnnexinV-PE (staining).
Figure 3 illustrates the ability of several different polynucleotide delivery-enhancing polypeptides to enhance
siRNA uptake in human monocytes in culture. Transfection by lipofectamine was used as a comparator. Cell viability
was also assessed for each peptide. The results showed that the polynucleotide delivery-enhancing polypeptide, PN73
is an ideal candidate for the treatment of rheumatoid arthritis. The data show the surprising and unexpected discovery
that the PN73 peptide enhances uptake of siRNA by human monocytes with high efficiency and low toxicity indicating
that suggests it can be used for the treatment of rheumatoid arthritis in vivo.
siRNA/Deliverv is Enhanced by peptide-siRNA Conjugates
The present example provides results from screens to evaluate activity of siRNA/polynucleotide deliveryenhancing
polypeptide conjugates for inducing or enhancing siRNA uptake in 9L/LacZ culture cell lines and primary
fibroblast from mouse tail. The results for transfections performed with 9L/LacZ cells are summarized in Table 7.
The results for transfections performed with MTF are summarized in Table 8.
of siRNA uptake mediated by rationally-designed polynucleotide delivery-enhancing polypeptides
conjugated to siRNAs in LacZ cells
Efficiency of siRNA uptake mediated by rationally-designed polynucleotide delivery-enhancing polypeptides
conjugated to siRNAs in murine tail fibroblast cells
(SEQ ID NO: 102)
(SEQ ID NO: 103)
(SEQ ID NO: 37)
Maleimide-Dmt-r-FKQqQqQqQqQq-amide (SEQ ID NO: 74)
Maleimide-RQIKIWFQNRRMKWKK-amide (SEQ ID NO:
(SEQ ID NO: 104)
Maleimido-RVIRWFQNKRSKDKK-amide (SEQ ID NO: 92)
Maleimide-WRFKQqQqQqQqQq-amide (SEQ ID NO: 76)
(SEQ ID NO: 105)
NH2-WRFKC-amide (SEQ ID NO: 106)
Maleimide-GRKKRRQRRRPPQ-amide (SEQ ID NO: 43)
Maleimido-KLALKLALKALKAALKLA-amide (SEQ ID
(SEQ ID NO: 108)
The foregoing data evince that a diverse assemblage of siRNA/peptide conjugates of the invention mediate
delivery of siRNAs into different cell types at high efficiency.
9 siRNA Gene Expression Knock Down is Enhanced by
Polvnucleotide Delivery-Enhancing Polvpeptides Complexed to siRNA
The instant example demonstrates effective knockdown of target gene expression by siRNA/polynucleotide
delivery-enhancing polypeptide complexes of the invention. In the current studies, the ability of peptide/siRNA
complexes to modulate expression of a human tumor necrosis factor-a (hTNF-a) gene, implicated as mediating the
occurrence or progression of RA when overexpressed in human and other mammalian subjects, was tested.
Human monocytes were isolated and transfected as described in Example 4. For mRNA measurement, branch
DNA technology from Genospectra (CA) was used according to manufacturer's specification. Human monocytes
(CD 14+) treated with LPS induce TNF-a-specific mRNA within approximately 2 hrs, followed by peak levels of TNFa
protein 2 hrs later. siRNAs were screened for knockdown activity by transfecting monocytes with siRNA candidate
sequences using Lipofectamine 2000, treating infected cells with LPS, and measuring TNF-a mRNA levels
approximately 16 hrs later. Various siRNA sequences were screened (Table 9) for their ability to knockdown TNF-a
mRNA and protein levels in activated human primary monocytes.
To quantitate mRNA level in the cells, both house keeping gene (cypB) and target gene (TNF-a) mRNA were
measured, and the reading for TNF- a was normalized with cypB to obtain relative luminescence unit. To quantify
protein level, the TNF-a ELISA from BD Bioscience was used according to manufacturer's specification.
Table 10, 11 and 12 illustrate the effectiveness of specific oligos complexed to a polynucleotide deliveryenhancing
polypeptides of the invention to target and knock down TNF-a gene expression levels in human monocytes.
Activities for a representative set of siRNA sequences ranged from 80% mRNA knockdown activity to no
detectable activity. In general, TNF-a protein levels were reduced more than mRNA levels, e.g., a 50% knockdown in
TNF-a mRNA (TNF-a-1) resulted in a 75% reduction in TNF-a protein level. Dose response curves for selected
exhibited knockdown levels from 30% to 60 % were obtained. Calculated IC50values were in the 10
olar to 200 pMolar range. While the siRNA sequences evaluated were distributed throughout the TNF-a transcript,
the most potent siRNAs identified were located in two areas: the middle of the coding region and the 3'-UTR.
The foregoing results (Tables 10, 11 and 12) evince that effective levels of TNF-a gene expression knock
down can be achieved in mammalian cells using the novel siRNA/polynucleotide delivery-enhancing polypeptide
compositions of the invention.
siRNA Gene Expression Knock Down is Enhanced by
Polynucleotide Delivery-Enhancing Polypeptides Conjugated with siRNA
The present example demonstrates knockdown of target gene expression by peptide-siRNA conjugates of the
invention. The materials and methods for these studies are the same as those described above . The results of this
example are illustrated in Table 13.
The results show that a diverse assemblage of polynucleotide delivery-enhancing polypeptides of the invention
conjugated with siRNAs function to enhance siRNA-mediated knockdown of TNF- gene expression in mammalian
Time Course of siRNA Gene Expression Knock Down
The instant example presents studies relating to the time course of siRNA-mediated gene expression
Knockdown. To test the duration of the siRNA effect, the siRNA transfection procedures as noted above were
employed, except that fibroblasts derived from eGFP expressing mice were used. The transfection reagent used here
was lipofectamine. The cells were replated on the 18th day due to overgrowth. The second transfection was performed
on the 19th day post first transfection. On the 19th day the eGFP levels were measured after the transfection. Scramble
or nonsense siRNA (Qiagen) was used as a control, along with a GFPI siRNA (GFPI) and a hairpin siRNA (D#21).
The knockdown activities were calibrated with scramble siRNA (Qiagen control)
Time Course of EGFP Gene Expression Knock Down by Lipofectamine Mediated Transfection
The foregoing studies (Table 14) demonstrate that siRNA knockdown activity became apparent around day 3,
and was sustained through day 9, whereafter target gene expression returned to baseline levels around day 17. After
the second transfection on day 18, another reduction of eGFP expression occurred indicating that the reagent can be
repeatedly administered to cells to yield repeated or enduring gene expression knockdown.
Multiple Dosing Protocol to Extend siRNA Knockdown Effect in Mammalian Cells
The instant example demonstrates that multiple dosing schedules will effectively extend gene expression
knockdown effects in mammalian cells mediated by siRNA/polynucleotide delivery-enhancing polypeptide
compositions of the invention. The materials and methods employed for these studies are the same as described above,
with the exception that repeated transfections were conducted at the times indicated. The scramble siRNA (Qiagen)
was utilized for side by side controls. Table 15 summarizes the data for multiple transfections with a peptide/siRNA
complex. The percent knockdown activity of the TNF-a gene represents the percent of total gene expression.
The foregoing results demonstrate that when multiple transfections are performed timely (in this case between
about the 5th-7th day post first transfection), TNF-a gene expression knockdown effects in mammalian cells can be
maintained or re-induced.
Optimizing Rational Design of Polvnucleotide Delivery-Enhancing Polvpeptides
The instant example provides an exemplary design and data for optimizing polynucleotide delivery-enhancing
polypeptides of the invention. The subject rational design manipulations were conducted for a histone H2B-derived
polynucleotide delivery-enhancing polypeptide.
In order to better understand the function-structural activity relationships of this and other polynucleotide
delivery-enhancing polypeptides, primary structural studies were performed by characterizing C- and N-terminal
function, and activity of conjugates between PN73 and other chemical moieties.
The amino acid sequence for the human histone 2B (H2B) protein is shown below.
PN73, PN360 and PN361 are peptide fragments of H2B and the portion of the H2B protein that they represent are
identified below in parentheses following the peptide name. The amino acids sequence for PN360 and PN361 listed
below are aligned with the corresponding amino acid sequence found in PN73. The PN73 peptide fragment is
underlined in the H2B amino acid sequence and represents H2B amino acids 13 through 48. It may also be represented
by H2B amino acids 12 through 48. PN360 shares the N-terminus with PN73 but lacks PN73's C-terminus while
PN361 shares the C-terminus with PN73 but lacks PN73's N-terminus. The PN73 conjugate is PN73 covalently linked
to a single siRNA strand (e.g., sense strand). PN404 is a version of PN73 in which all of lysines are replaced with
arginines and PN509 is a pegylated PN73 (PEG molecular weight Ik Dalton ) derivative that is pegylated at the Nterminus.
H2B (histone 2B) amino acid sequence
LLPGELAKHAVSEGTKAVTKYTSSK (SEQ ID NO: 164)
ID NO: 59)
PN360 (13-35; N-terminus of PN73)
NH2-KGSKKAVTKAQKKDGKKRKRSRK-amide (SEQ ID NO: 57)
PN361 (24-48; C-terminus of PN73)
NH2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 58)
PN73 (13-48)-siRNA (sense strand) conjugates
siRNA-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO 59)
PN404 (PN73 where all lysines are replaced with arginines)
ID NO: 91)
PN509 (pegylated PN73)
PEG- RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 59).
Figure 5 provides the results of uptake efficacy and viability studies in mouse tail fibroblast cells for the
foregoing PN73 rationally-designed derivative polynucleotide delivery-enhancing polypeptides. The activity changes
of modified PN73 in mouse tail fibroblast cells are illustrated. Unlike PN404, PN509 increases uptake without
increasing toxicity. While deleting part of the N-terminus of PN73 reduces activity, removal of C-terminal residues
abolishes the activity. Both PN73 and PN509 show higher activity in primary cells than Lipofectamine (Invitrogen,
Acetylated Polynucleotide Delivery-Enhancing Polypeptide Increases siRNA Stability
The purpose of the instant example was to determine if modification of the exemplary polynucleotide deliveryenhancing
polypeptide PN73 would provide increased stability to the peptide and consequently enhance its transfection
activity. The stability of unmodified, N-terminus pegylated and N-terminus acetylated forms of PN73 in plasma was
compared. The C-terminus of the PN73 is amidated. Size exclusion chromatography coupled with an ultraviolet
detector were used to characterize the stability of the unmodified and modified forms of PN73 before and after
incubation in plasma.
In the absence of plasma, the unmodified, pegylated and acetylated forms of PN73 showed distinct yet
overlapping UV traces at approximately 9 minutes. However, after 4 hours of exposure to plasma, UV traces specific
to unmodified PN73 and pegylated PN73 were no longer present indicating significant degradation. In contrast, the
distinct UV trace for acetylated PN73 remained indicating that this modification significantly increased stability of the
PN73 in plasmid compared to the unmodified and pegylated PN73 forms.
These data show the surprisingly and unexpected discovery that PN73 stability in plasma can be enhanced by
N-terminus acetylation of the PN73 peptide. The primary structure of the acetylated PN73 peptide is as follows:
Ac-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59)
Polynucleotide Delivery-Enhancing Polvpeptide Does Not Elicit an Interferon Response
The purpose of the instant example was to compare the interferon response of cells transfected with either
lipofectamine plus siRNA or the exemplary polynucleotide delivery-enhancing polypeptide, PN73 peptide plus siRNA.
Interferon responsiveness was assayed by ELISA (protein) and bDNA (mRNA levels).
Traditionally, siRNA molecules are delivered into cells by a liposomal mediated transfection. However, this
typically results in a poor efficiency of delivery, an inflammatory response in vivo and an upregulation of interferon
gene expression which results in an inhibition of cell growth. Consequently, there is a limited reduction in targeted
gene expression levels thus making siRNA an ineffective method of treatment and tool for studying gene expression.
Delivery of siRNA by PN73 overcomes this problem.
Interferon responsiveness of lipofectamine was compared to PN73 peptide in transfection of several different
siRNAs. siRNAs were complexed with either lipofectamine or PN73 at concentrations of 1 nM, 10 nM, 100 nM or
200 nM. Interleukin ip (IL-1P) served as a molecular marker to determine interferon responsiveness and Qneg was
used as a negative control. Results showed that lipofectamine complexed with the 100 nM or 200 nM TNF-a9 siRNA
caused a significant increase in IL-lp mRNA levels. Furthermore, all other siRNAs tested caused a mild increase in
IL-lp mRNA levels. In contrast, the same siRNAs complexed with the PN73 peptide did not cause an increase in IL-
1P mRNA levels.
To further characterize the difference in interferon responsiveness observed between cells transfected with
either lipofectamine and PN73 transfection, an ELISA assay was performed to determine the protein expression levels
of the following molecular markers: Interleukin ip (IL-lp), Interferon-ct (INF-ct), Interleukin-6 (IL-6), Interleukin-8
(IL-8), Interleukin-12 (IL-12), MEP-la, Interferon-y (IFN-y), and Tumor Necrosis Factor-a (TNF-a). Table 16
summarizes the relative protein expression levels of cells transfected with lipofectamine complexed with siRNA or
PN73 complexed with siRNA.
The results showed both siRNA LC20 and LCI7 had no interferon response regardless of what transfection
rfttgent was used. However, transfection of EFN-1 or TNF-a9 with lipofectamine caused an increase in IL-1J3, IL-6,
and MlP-la, protein expression levels. In contrast, transfection of all tested siRNAs with PN73 caused no observable
induction in protein expression in any of the interferon response markers tested.
These data from the ELISA assay show the surprisingly and unexpected discovery that PN73 mediated
transfection of siRNAs does not elicit an interferon response.
siRNA Conjugated with a Polvnucleotide Delivery-Enhancing Polypeptide Provides Greater Knockdown
Activity than siRNA Complexed with Polynucleotide Delivery-Enhancing Polypeptide.
The purpose of the instant example was to compare the knockdown activities in human monocytes of the
siRNAs LCI3 and LC20 either conjugated or complexed with the exemplary polynucleotide delivery-enhancing
polypeptide PN73. Isolation and transfection of human monocytes as well as the methods used to measure knockdown
activity were discussed earlier. Qneg represents a random siRNA sequence and functions as the negative control in
these experiments. The observed Qneg knockdown activity is normalized to 100% (100% gene expression levels) and
the activity of LC20 and LCI3 is presented as a relative percentage of the negative control. LC20 and LCI3 are
siRNAs targeted against the human TNF-a gene. The knockdown activity for the siRNAs LC20 and LCI3 without
PN73, in a complex with PN73 or conjugated with PN73 were tested over a concentration range of 0 nM to 2.5 nM.
PN73 was kept at a 1:1 ratio in both the complex and conjugate experiments.
In the absence of PN73, LCI3 and L20 showed little knockdown activity (Figure 6-C). Both LCI3 and LC20
caused an approximate 15% and 30% decrease in TNF-a gene expression relative to the Qneg control when
complexed with PN73 (Figure 6-B). However, knockdown activity for TNF-a was reduced to below 60% when the
siRNA was conjugated to PN73 (Figure 6-A). This is significant increase the siRNA knockdown activity compared to
the PN73/siRNA complex. Thus, these data show the surprisingly and unexpected discovery that siRNA knockdown
activity is significantly enhanced when the siRNA is conjugated to the exemplary polynucleotide delivery-enhancing
Deletion Analysis of the Exemplary Polynucleotide Delivery-Enhancing Polypeptide
The purpose of the present example was to determine the minimum length of the
PN73 peptide that is critical for its ability to enhance the delivery of small and macromolecules into cells. A shown in
Table 17,10 truncated forms of PN73 were created by sequentially deleting 3 residues at a time from the N-terminus
of the peptide. Below is an explanation of the primary structure of PN73 and the truncated forms that will be
examined for transfection activity. All peptides tested in this Example were tagged with a C-terminus FITC
(fluorescein-5-isothiocyanate) label (i.e., -GK[EPSILON]G-amide).
PN643 or PN73 (13-48) KGSKJCAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 59)
PN661 (16-48) KXAVTKJ\QKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 165)
PN685 (19-48) VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 166)
PN660 (22-48) AQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 167)
PN735 (25-48) KDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 168)
PN655 (28-48) KKRKRSRKESYSVYVYKVLKQ (SEQ ID NO: 169)
PN654 (31 -48) KRSRKESYSVYVYKVLKQ (SEQ ED NO: 170)
PN708 (34-48) RKESYSVYVYKVLKQ (SEQ ID NO: 171)
PN653 (37-48) SYSVYVYKVLKQ (SEQ ID NO: 172)
PN652 (40-48) VYVYKVLKQ (SEQ ID NO: 173)
PN651 (43-48) YKVLKQ (SEQ ID NO: 174)
In Vivo siRNA-Mediated Gene Expression Knock Down Activity
A. eGFP mice
eGFP transgenic mice, 20-25g body weight, were purchased from Jackson Laboratory. For siRNA injections,
was used at a dose of 5mg/kg at a 1:5 ratio of siRNA/PN73. All treatments were by tail vein injections once
daily for three days. The tissues were clollected on day 4. The experimental protocols were performed at R&R
Rabbitary (Standwood, WA) and approved by their Institutional Animal Care and Use Committee (IACUC).
The tissues from animal were placed in PBS immediately and stored on ice. For isolation cells from tissues,
the sample tissues were placed between 2 frosted glass microscope slides with frosted side facing tissue. Smear
together to mash tissue several times. Then rinse off the smashed tissue from slides into the well of 12-well plate with
PBS. One ml of mashed tissue was transferred to 12-well plate containing 1 ml of 2X collagenase (type I for muscle
and II for liver tissue); the final concentration is 100 units/ml. Incubation of tissues in collagenase solution for 3 hours
at 37°C; In order to remove the small pieces and connective tissue, filter the digested tissue mixture through cell
strainer into 6 well plate. Transfer 500 ul cells into labeled FACS tubes. Fluorescence intensity of the isolated cells
was determined by flow cytometry.
A double stranded siRNA duplex specific against eGFP were chemically synthesized. siRNA was dissolved
either in PBS buffer or saline or glucose buffer.
The three groups of animals (specific eGFP siRNA alone, peptide/eGFP siRNA complex and control
siRNA/peptide complex) were iv injected in varied doses and durations as indicated in Table 1. The peripheral blood
of treated mice was drawn through orbital bleeding and stored on ice in the presence of anticoagulant. The PBMC
were isolated by Ficoll based centrifugation method. And the knockdown activities were assessed by determining the
eGFP fluorescent intensity in the cells by flow cytometry
Reduction of eGFP protein in muscle cells in EGFP transgenic animal
Transgenic mice were treated with siRNA in eGFP transgenic mouse complexed with peptide PN73 (1:5 ratio)
(5 mg/kg for three days). For determination of the knockdown activities, cells were isolated from muscle tissue
following treatment with collagenase as mentioned in methods section. The fluorescence intensity of isolated cells
were analyzed by flow cytomethry.
In comparison with control siRNA and GFP siRNA alone, GFP siRNA/PN509 (pegylated PN73) complex
showed effective knockdown of EGFP in vivo after consecutive three day injections.
Reduction of eGFP protein and niRNA in liver cells in EGFP transgenic animal
Transgenic mice were treated with siRNA in eGFP transgenic mouse complexed with peptide PN73 (1:5 ratio)
(5 mg/kg for three days). For determination of the knockdown activities, cells were isolated from liver tissue following
treatment with collagenase as mentioned in methods section. The fluorescence intensity of isolated cells were
analyzed by flow cytomethry.
In comparison with control siRNA/PN602 and GFP siRNA alone, GFP siRNA/PN509 (pegylated PN73)
complex showed effective knockdown of EGFP in vivo after consecutive three day injections. Knockdown of eGFP
mRNA are here summarized in Table 18.. Knockdown of protein expression is comparable.
The delivery peptide can efficiently delivery siRNA and knockdown eGFP in PBMC in vivo (up to 50%). In
comparison with negative control, naked siRNA also showed some activity in knocking down of eGFP, but it is much
B. Taconic Mice
For human TNF-a animal disease models, two transgenic models were used. The tg!97 mice were acquired
from Pasteur Hellenic Institute (Athens, Greece), and the B6.SJL-Tg(TNF) N21 mice were purchased from Taconic,
Inc. (Germantown, NY). The genotypes of both human TNF-a transgenic mouse models were performed by suppliers.
For tg!97 mice, 6-week old mice were divided into three treatment groups for infliximab, siRNA/PN73 complex and
saline. Infliximab, a TNF-a neutralizing antibody based drug, was purchased from a local drug store (as Remicade)
and used at a dose of 10 mg/kg, once per week. siRNA was used at a dose of 2mg/kg at a 1:5 ratio of siRNA/PN73.
All treatments were by tail vein injection twice a week. The experimental protocols were performed at SkeleTech Inc.
(Bothell, WA) and approved by their Institutional Animal Care and Use Committee (IACUC). A similar dose regimen
was used for B6.SJL-Tg(TNF) N21 mice. Clinical scores were based on a previously described scoring system (2, 3)
as follows: 0 (normal), 1 (edema or distortion of paw or ankle joints), 2 (distortion of paw and ankle joints), or 3
(ankylosis of wrist or ankle joints). The sum of all four paws was scored twice a week with a maximum score of 12
For determination of TNF-a level, For the analysis of experiments involving transgenic mice, plasma samples
were separated from whole blood collected by orbital bleeding. Plasma levels of hTNF-a were determined by ELISA
at a 1:2 dilution according to the manufacturer's instructions (R&D systems, Minneapolis, MN).
bDNA assays (Quantigene assay from Genospectra, Fremont, CA) were performed following the
Reduction of human TNF-a plasma protein level and RA scores in transgenic mice after treatment with
siRNA/PN73 complex or infliximab.
Human TNF-a transgenic mice were treated starting on 5 weeks of age with infliximab (10|xg/kg, ip injection)
and siRNA (LC20) against TNF-a complexed with peptide PN73 (1:5 ratio) (2 mg/kg, iv injection, twice weekly). For
Hellenic transgenic mice, RA scores were determined (blinded to treatment) using the following criteria: 0, normal; 1,
edema or distortion of paw or ankle joints; 2 distortion of paw and ankle joints; 3, ankylosis of wrist or ankle joints.
The LC20/PN73 treated group show dramatic suppression of clinic progressions of joint arthritis during three
week treatment period.
Reduction of human TNF-a plasma protein level after treatment with siRNA/PN73 complex or
For Taconic mice, TNF-a levels in plasma were determined by ELISA (R&D systems, Minneapolis, MN) and
treatment schedules were same as Hellenic transgenic hTNF-a transgenic mice.
C. Helenic Mice
The present example provides in vivo data demonstrating the efficacy of siRNA/polynucleotide deliveryenhancing
polypeptide compositions of the invention to mediate systemic delivery and therapeutic gene knockdown by
siRNA, effective to modulate target gene expression and modify phenotype of cells in a therapeutic manner.
Human NF-a expressing mice were purchase from the Hellenic Pasture Institute, Greece) at 5weeks old. Mice
were administered through intra venous (IV) with 300ul saline twice a week (4 mice), with the RA drug Ramicade
(5mg/kg) once a week (2 mice), or with LC20 siRNA (2mg/kg) mixed with PN73 at 1:5 molar ratio twice a week (2
mice). During the injection periods, plasma samples were collected for ELISA testing (R&D Systems,
Cat#SSTAOOC), and paw scores were taken twice a week as an accepted index of RA disease progression and
therapeutic efficacy (Table 19).
The foregoing results demonstrate effective reduction of TNF-a protein levels in peptide/siRNA-treated mice
ill the circulating blood as compared to levels in Ramicade or saline (control) treated mice.
Additional evidence of in vivo efficacy of the siRNA/polynucleotide delivery-enhancing polypeptide
compositions and methods of the invention were obtained from the above murine subjects using paw scores, an
accepted phenotypic index for RA disease status and treatment efficacy. Due to the difference in the starting point
(some animals present with scores at earlier points), the scores have been adjusted to 0 for all animals in the
experiments. Each paw is given a score between 0 and 3, with the highest score of 12, according to the following
1: edema or distortion of paw or ankle joints
2: distortion of paw and ankle joints
3: ankylosis of wrist or ankle joints.
The results of these paw score evaluations are presented graphically in Figure 4. The data demonstrate that the
polynucleotide delivery-enhancing polypeptide PN73 can deliver therapeutic amounts of siRNAs (e.g. LC20, TNF-a2,
and TNF-a9 (UAGCCCAUGUUGUAGCAAA (SEQ ID NO. 175))) when injected into animals as shown by a
delayed RA progression at week 8. The PN73/siRNA treated mice faired better on the paw scoring index at week 8
compared to the Ramicade-treated mice. When paw score evaluations were carried out to 11 weeks post-treatment,
PN73/LC20 complex achieved comparable paw score evaluations to the Ramicade-treated mice. At a ratio of 1:5 for
the PN73 peptide/LC20 siRNA, 2 mg/kg LC20 achieved the greatest relative observed delay in RA progression
compared to the lower doses of LC20 tested. Table 20 below summarizes the relative effectiveness of several siRNAs
for 5 different groups evaluated after treatment with PN73 and siRNAs.
LC20, YC12 and LC17 Overall low paw score.
YC12 and LC17 not as
effective as LC20
TNF-a2 and TNF- LC20 and TNF-a9 are more
effective than Ramicade by
week 8; LC20 is equally
effective as Ramicade by
'siRNAs were tested in the presence of absence ofPN73; Ramicade is a positive treatment control; PBS is a negative
The foregoing results demonstrate that siRNA and polynucleotide delivery-enhancing polypeptide
compositions of the invention provide promising new therapeutic tools for regulating gene expression and treating and
managing disease. siRNAs of the invention, for example siRNAs targeting human TNF-a-specific mRNAs for
degradation, offer higher specificity, lower immunogenicity and greater disease modification than current small
molecule, soluble receptor, or antibody therapies for RA. More than 50 candidate siRNA sequences were screened
that targeted hTNF-a and yielded single administration knockdowns of 30-85%. Over 20 in silica designed peptide
complex and/or covalent molecules were compared for fluorescent RNA uptake by monocytes and a number were
found to have significantly better uptake than Lipofectamine or cholesterol-conjugated siRNA and with 10 pM IC50
values. The peptide-siRNA formulations efficiently knockdown TNF-a mRNA and protein levels in activated human
monocytes in vitro.
One exemplary candidate delivery peptide/siRNA formulation was evaluated in two transgenic mouse models
or rheumatoid arthritis (RA) constitutively expressing human TNF-a. Animals treated with 2 mg/kg siRNA by FV
injection or infliximab twice weekly beginning at age 6 weeks showed RA score stabilization (paw and joint
inflammation) beginning at age 7 weeks, compared to controls where these disease conditions persisted through week
10. At age 9 weeks, siRNA treated animals showed comparable reductions in RA scores, but significantly lower
plasma TNF-a protein levels than infliximab treated animals.
Based on the disclosure herein, the use of siRNA to inhibit the expression of target genes, for example
cytokines such as TNF-a, that play important roles in pathological states, such as inflammation, provides effective
treatments to alleviate or prevent symptoms of disease, as exemplified by RA, in mammalian subjects. Exemplary
peptide/siRNA compositions employed within the methods and compositions of the invention provide advantages
relating to their ability to reduce or eliminate target gene expression, e.g., TNF-a expression, rather than by
complexing with the product of the target gene, e.g., TNF-a, as in the case of antibodies or soluble receptors.
Improving systemic delivery of nucleic acids according to the teachings of the invention provides yet
additional advantages for development of siRNAs as drugs. Specific challenges in this context include delivery
through tissue barriers to a target cell or tissue, maintaining the stability of the siRNA, and intracellular delivery
getting siRNAs across cell membranes into cells in sufficient quantities to be effective. The present disclosure
demonstrates for the first time an effective in vivo delivery system comprising novel peptide/siRNA compositions
targeting specific gene expression, such as expression of human TNF-a, which attenuate disease activity in transgenic
animal models predictive of target diseases, as exemplified by studies using murine models of RA. In related studies,
the compositions and methods of the invention effectively inhibit TNF-a expression in activated monocytes derived
from patients with RA. These results indicate that the RNAi pathway effectively mediates alteration of cellular
phenotype and disease progression through intracellular effects on TNF-pathways, and avoids toxicity effects due to
circulating antibody/TNF-a complexes with residual immunoreactivity that characterize current antibody therapies for
RA. Notably, all of the tests herein were implemented with associated toxicity effects minimized, such that the
dosages of siNAs and polynucleotide delivery-enhancing polypeptides shown in these examples always correlated with
cell viability levels of at least 80-90% or greater.
D. Mouse LPS Response
Normal [mouse type] mice were treated with various concentrations of LPS by intraperitoneal or intravenous
injections. LPS responsiveness was determined by measuring levels of TNF-a in blood that was sampled various
times following the LPS injection. A linear range of TNF-a induction was found between 10 ng and 100 ng for LPS
injection by IP administration and up to 25 ng by IV injection. The average time for maximal TNF-a induction is 70
minutes following LPS injection. On the basis of these results, the following LPS doses were selected for further
experimentation: 25 ng for IP administration; 10 ng for IV administration.
The effects of siRNA on LPS induction of TNF-a were tested by injection, six mice per treatment schedule,
with a 2 mg/kg siRNA dose of (1) LC13/PN73; (2) LC13 alone; (3) Qneg/PN73; (4) PN73; (5) buffer alone. The LPS
induction was performed 24 hr following the last siRNA injection, 0.2 ug LPS and blood was collected at 90 minutes
following LPS injection. Results (below) indicate that LCI3 (samples 1 and 2) lowered amounts of circulating TNF-a
resulting from LPS induction compared to negative controls (samples 3-5).
A second experimental approach was utilized using a 2 mg/kg siRNA dose of the following compositions (1)
LC13/PN73; (2) Inm-2/PN73; (3) Inm-4/PN73; (4) Qneg/PN73; and (5) PBS (Buffer control). Three studies of 30
animals each dosing by the following schedules: 4 consecutive days; 8 consecutive days; and 11 consecutive days.
LPS induction was performed at 24 hr post siRNA injection, 25 ng LPS (IP). Blood was drawn 70 minutes following
LPS injection. The results showed that Inm-4 showed greatest KD activity in 4 consecutive day experiment (n=l test)
compared to LCI3, Inm-2, and Qneg. Results from measurements and 8 and 11 days gave variable results owing to
technical problems partly arising from repeated tail injections.
Although the foregoing invention has been described in detail by way of example for purposes of clarity of
understanding, it will be apparent to the artisan that certain changes and modifications may be practiced within the
scope of the appended claims which are presented by way of illustration not limitation. In this context, various
publications and other references have been cited within the foregoing disclosure for economy of description. Each of
these references is incorporated herein by reference in its entirety for all purposes. It is noted, however, that the
various publications discussed herein are incorporated solely for their disclosure prior to the filing date of the present
application, and the inventors reserve the right to antedate such disclosure by virtue of prior invention.
1. A double stranded ribonucleic acid (dsRNA), comprising a first strand having a ribonucleic acid sequence that is complementary to a tumor necrosis factor-α (TNF-α) mRNA having the nucleotide sequence of SEQ ID NO: 132, useful for inhibiting the production of tumor necrosis factor-α (TNF-α).
2. The double stranded ribonucleic acid (dsRNA) as claimed in claim 1, wherein the first strand has a ribonucleic acid sequence that is complementary to a nucleotide sequence selected from the group consisting of: AAUCGGCCCGACUAUCUCGACUU (SEQ ID NO: 117); AAUGGCGUGGAGCUGAGAGAU (SEQ ID NO: 118); AACCUCCUCUCUGCCAUCAAG (SEQ ID NO: 119); AACUGAAAGCAUGAUCCGGGA (SEQ ID NO: 120); AAUCUCGACUUUGCCGAGUCU (SEQ ID NO: 121); AAGGGUGACCGACUCAGCGCU (SEQ ID NO: 122); AAUCAGCCGCAUCGCCGUCUC (SEQ ID NO: 123); AACCCAUGUGCUCCUCACCCA (SEQ ID NO: 124); AAGCUCCAGUGGCUGAACCGC (SEQ ID NO: 125); AAGUCAGAUCAUCUUCUCGAA (SEQ ID NO: 126); AAGGGACCUCUCUCUAAUCAG (SEQ ID NO: 127); CCUCAGCCUCUUCUCCUUCCUGA (SEQ ID NO: 128); AAUCCUCAGCCUCUUCUCCUU (SEQ ID NO: 129); AACCAAUGCCCUCCUGGCCAA (SEQ ID NO: 130); CUGAUUAAGUUGUCUAAACAA (SEQ ID NO: 131); CCGACUCAGCGCUGAGAUCAA (SEQ ID NO: 132); CUUGUGAUUAUUUAUUAUUUA (SEQ ID NO: 133); AAGCCUGUAGCCCAUGUUGUA (SEQ ID NO: 134); UAGGGUCGGAACCCAAGCUUA (SEQ ID NO: 135); and GCCUGUACCUCAUCUACUC (SEQ ID NO: 147).
3. A double stranded ribonucleic acid (dsRNA) substantially as herein described with reference to foregoing description, examples, tables and drawings.
|Indian Patent Application Number||1892/DELNP/2007|
|PG Journal Number||09/2012|
|Date of Filing||12-Mar-2007|
|Name of Patentee||MARINA BIOTECH|
|Applicant Address||3830 MONTE VILLA PARKWAY, BOTHELL, WA 98021-7266, USA|
|PCT International Classification Number||A61K 48/00|
|PCT International Application Number||PCT/US2005/035259|
|PCT International Filing date||2005-09-27|