U.S. patent application number 14/496955 was filed with the patent office on 2015-01-08 for methods and compositions for treating insects.
The applicant listed for this patent is ALNYLAM PHARMACEUTICALS, INC.. Invention is credited to Jason Rhodes, Donna T. WARD.
Application Number | 20150011614 14/496955 |
Document ID | / |
Family ID | 43544870 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150011614 |
Kind Code |
A1 |
WARD; Donna T. ; et
al. |
January 8, 2015 |
METHODS AND COMPOSITIONS FOR TREATING INSECTS
Abstract
Provided herein are methods and compositions for modulating gene
expression in insects by administering a composition comprising an
RNA effector molecule and a delivery agent. Methods are provided
for controlling pest populations by inhibiting insect growth,
development, survival, reproduction and/or viability. Also provided
herein are methods for treating or preventing disease in an insect
caused by a pathogen or by external factors (e.g., pollution,
environment, stress, weather, etc.).
Inventors: |
WARD; Donna T.; (Cambridge,
MA) ; Rhodes; Jason; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALNYLAM PHARMACEUTICALS, INC. |
Cambridge |
MA |
US |
|
|
Family ID: |
43544870 |
Appl. No.: |
14/496955 |
Filed: |
September 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13364477 |
Feb 2, 2012 |
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14496955 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
C12N 15/85 20130101;
C12N 2710/14043 20130101; C12N 2310/14 20130101; A61P 31/04
20180101; C12N 2320/00 20130101; A01N 57/16 20130101; C12N 15/86
20130101; C12N 2320/32 20130101; C12N 15/1131 20130101; A61P 33/00
20180101; A61P 31/10 20180101; A01K 67/0333 20130101; C12N 15/113
20130101; A01N 25/002 20130101; C12N 2320/30 20130101; A61K 48/00
20130101; A61P 31/12 20180101; A61P 31/00 20180101 |
Class at
Publication: |
514/44.A |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/85 20060101 C12N015/85 |
Claims
1. A method for treating or preventing disease in an insect, the
method comprising administering to the insect a composition
comprising an RNA effector molecule or a vector encoding an RNA
effector molecule, and a delivery agent, wherein the RNA effector
molecule modulates gene expression of an insect or an insect
pathogen.
2. The method of claim 1, wherein the disease is caused by an
insect pathogen selected from the group consisting of a virus,
mite, nematode, bacteria, fungus, or parasite.
3. The method of claim 1, wherein the disease is caused by
pollution, exposure to electromagnetic radiation, exposure to
pesticides, environment, or stress.
4. The method of claim 1, wherein the RNA effector molecule
inhibits or activates gene expression.
5. The method of claim 2, wherein modulating gene expression
inhibits pathogen infectivity, virulence, reproduction, viability,
growth, translation, protein production, viral uptake or
transmission.
6. The method of claim 2, wherein modulating gene expression
decreases insect susceptibility to a pathogen.
7. The method of claim 1, wherein said administering comprises
providing a food source for the insect, wherein the food source
comprises the composition.
8. The method of claim 7, wherein the food source comprises a
virus, a bacterium, a fungus, a plant, or a yeast cell expressing
the RNA effector molecule.
9. The method of claim 1, wherein said administering comprises
contacting the insect with a solution comprising the
composition.
10. The method of claim 9, wherein the composition is administered
topically.
11. The method of claim 1, wherein the RNA effector molecule
comprises an oligonucleotide.
12. The method of claim 11, wherein the oligonucleotide is a single
stranded or double stranded oligonucleotide.
13. The method of claim 11, wherein the oligonucleotide comprises
an siRNA, an miRNA, an shRNA, a ribozyme, an antisense RNA, a decoy
oligonucleotide, an antimir, a supermir, or an RNA activator.
14. The method of claim 1, wherein the vector is a viral vector, an
expression vector, or a plasmid.
15. The method of claim 14, wherein the viral vector comprises a
baculoviral vector.
16. The method of claim 1, wherein the insect is a bee, wasp,
butterfly, ant or ladybug.
17. The method of claim 1, wherein the composition is administered
to adult insects.
18. The method of claim 1, wherein the composition is administered
to a breeding or feeding locus.
19. The method of claim 1, wherein the insect is a hive bee or a
forager bee, and the pathogen is selected from the group consisting
of IAPV, Acute Bee Paralysis Virus and Kashmir Bee Paralysis
Virus.
20. A method for modulating gene expression in an insect, the
method comprising: administering to the insect a composition
comprising an RNA effector molecule or a vector encoding an RNA
effector molecule and a delivery agent, wherein the RNA effector
molecule modulates gene expression in the insect.
21. The method of claim 20, wherein modulation of gene expression
inhibits viability, survival, growth, development, and/or
reproduction of the insect.
22. The method of claim 21, wherein the insect is a pest.
23. The method of claim 20, wherein modulation of gene expression
increases insect susceptibility to a pathogen.
24. The method of claim 20, wherein the insect is a hive-dwelling
insect and modulation of gene expression in the insect is delayed
until the insect returns to the hive.
25. The method of claim 22, wherein the composition is specific to
the pest and does not affect other insects.
26. A composition comprising an RNA effector molecule or a vector
encoding an RNA effector molecule, and a delivery agent, wherein
the RNA effector molecule modulates gene expression of an insect or
an insect pathogen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation application under 35
U.S.C. .sctn.120 of U.S. patent application Ser. No. 13/364,477
filed on Feb. 2, 2012, which is a Continuation Application of
International Application No. PCT/US2010/043458 filed on Jul. 28,
2010, which designates the U.S., and which claims benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Application No. 61/230,911
filed on Aug. 3, 2009, the contents of which are incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The field of the invention relates to the treatment of
insects with a composition comprising an RNA effector molecule.
BACKGROUND
[0003] Pests including insects, arachnids, crustaceans, fungi,
bacteria, viruses, nematodes, flatworms, roundworms, pinworms,
hookworms, tapeworms, trypanosomes, schistosomes, botflies, fleas,
ticks, mites, lice and the like are pervasive in the human
environment, and a multitude of means have been utilized for
attempting to control infestations by these pests. Compositions for
controlling infestations by microscopic pests such as bacteria,
fungi, and viruses have been provided in the form of antibiotic
compositions, antiviral compositions, and antifungal compositions.
Compositions for controlling infestations by larger pests such as
nematodes, flatworm, roundworms, pinworms, heartworms, tapeworms,
trypanosomes, schistosomes, and the like have typically been in the
form of chemical compositions which can either be applied to the
surfaces of substrates on which pests are known to infest, or to be
ingested by an infested animal in the form of pellets, powders,
tablets, pastes, or capsules and the like.
SUMMARY OF THE INVENTION
[0004] Described herein are compositions comprising an RNA effector
molecule and methods for administering such compositions to an
insect or group of insects, wherein the RNA effector molecule
modulates gene expression. The compositions are useful for
controlling insect pest populations by inhibiting survival,
viability, reproductions, growth and/or development of a pest
population. Alternatively, the compositions are useful for treating
or preventing a disease, including, but not limited to,
pathogen-borne disease or disease caused by environmental factors
(e.g., pollution, agricultural chemicals), in insects having a
beneficial function by modulating gene expression of the pathogen
or of the insect.
[0005] One aspect described herein relates to a method for
modulating gene expression in an insect, the method comprising:
administering to the insect a composition comprising an RNA
effector molecule or a vector encoding an RNA effector molecule,
and a delivery agent, wherein the RNA effector molecule modulates
gene expression in the insect.
[0006] Another aspect described herein relates to a method for
treating or preventing disease in an insect, the method comprising
administering to the insect a composition comprising an RNA
effector molecule or a vector encoding an RNA effector molecule,
and a delivery agent, wherein the RNA effector molecule modulates
gene expression of an insect or insect pathogen.
[0007] As used herein, an "RNA effector molecule" refers to a
molecule that modulates the expression of a gene. In certain
embodiments, the RNA effector molecule is an oligonucleotide. As
used herein, the oligonucleotide can comprise an RNA interference
agent, an RNA activator, an miRNA, an shRNA, a ribozyme, an
antisense RNA, a decoy oligonucleotide, an antimir, or a
supermir.
[0008] As used herein, the terms "RNA interference agent," "RNAi"
or "iRNA" refer to an oligonucleotide as that term is defined
herein, and which mediates the targeted cleavage of an RNA
transcript via an RNA-induced silencing complex (RISC) pathway.
[0009] The iRNAs included in the compositions featured herein
encompass a dsRNA having an RNA strand (the antisense strand)
having a region that is typically 9-36 nucleotides in length, e.g.,
30 nucleotides or less, generally 19-24 nucleotides in length, that
is substantially complementary to at least part of an mRNA
transcript of an insect pest or an insect pathogen.
[0010] In one embodiment, an iRNA for modulating expression of an
insect or insect pathogen gene includes at least two sequences that
are complementary to each other. The iRNA includes a sense strand
having a first sequence and an antisense strand having a second
sequence. The antisense strand includes a nucleotide sequence that
is substantially complementary to at least part of an mRNA of a
target gene, and the region of complementarity is 30 nucleotides or
less, and at least 15 nucleotides in length. Generally, the iRNA is
19 to 24, e.g., 19 to 21 nucleotides in length. In some embodiments
the iRNA is from about 15 to about 25 nucleotides in length, and in
other embodiments the iRNA is from about 25 to about 30 nucleotides
in length. In another embodiment of this aspect, the
oligonucleotide comprises 9-36 base pairs.
[0011] The iRNA, upon contacting with an insect or insect pathogen,
inhibits the expression of a target gene by at least 10%, at least
20%, at least 25%, at least 30%, at least 35% or at least 40% or
more. In one embodiment, the iRNA is formulated in a stable nucleic
acid lipid particle (SNALP).
[0012] In another embodiment of this aspect, the oligonucleotide is
a single stranded or double stranded oligonucleotide.
[0013] In another embodiment of this aspect, the oligonucleotide is
modified. The oligonucleotide molecules featured herein can include
naturally occurring nucleotides or can include at least one
modified nucleotide, including, but not limited to a 2'-O-methyl
modified nucleotide, a nucleotide having a 5'-phosphorothioate
group, and a terminal nucleotide linked to a cholesteryl
derivative. Alternatively, the modified nucleotide can be chosen
from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide.
[0014] In one aspect, the invention provides a vector for
inhibiting the expression of a insect or insect pathogen gene. In
one embodiment, the vector includes at least one regulatory
sequence operably linked to a nucleotide sequence that encodes at
least one strand of an iRNA featured in the invention.
[0015] In one embodiment of these aspects, the disease is caused by
an insect pathogen selected from the group consisting of a virus,
mite, nematode, bacteria, fungus, or parasite. Alternatively, the
disease can be caused by external factors including, but not
limited to, pollution, exposure to electromagnetic radiation,
exposure to pesticides, environment, or stress.
[0016] In one embodiment of this aspect, the insect is a pest.
Alternatively, the insect comprises a beneficial insect such as
e.g., a bee, wasp, butterfly, ant or ladybug.
[0017] In another embodiment of the aspects described herein, the
RNA effector molecule inhibits or activates gene expression.
[0018] In another embodiment of the aspects described herein, the
modulation of gene expression inhibits viability, survival, growth,
development, and/or reproduction of the insect. In another
embodiment of the aspects described herein modulation of gene
expression increases insect susceptibility to a pathogen.
[0019] In another embodiment of the aspects described herein,
administering comprises providing a food source for the insect,
wherein the food source comprises the composition. A food source
can be provided as a liquid, solid, gel, semi-solid composition,
sugar composition, or lipid composition. Alternatively, the food
source comprises a bacterium, a virus, a fungus, a plant or a yeast
cell expressing the oligonucleotide.
[0020] In another embodiment of the aspects described herein, the
insect is a hive-dwelling insect and modulation of gene expression
in the insect is delayed until the insect returns to the hive.
[0021] In another embodiment of the aspects described herein, the
hive-dwelling insect spreads the composition to other insects in
the hive.
[0022] In another embodiment of the aspects described herein, the
insect is a bee such as e.g., a forager bee, a hive bee, a queen
bee, a drone bee, a worker bee etc. In some embodiments the
pathogen is a bee pathogen selected from the group consisting of
IAPV, Acute Bee Paralysis Virus and Kashmir Bee Paralysis
Virus.
[0023] In another embodiment of the aspects described herein,
administering comprises contacting the insect with a solution
comprising the composition. The composition can be administered
topically, or alternatively the insect, its habitat or a field is
sprayed or soaked with the solution.
[0024] In another embodiment of the aspects described herein, the
delivery agent is a lipid, a liposome, a food source, a solution,
an emulsion, a micelle or other membranous formulation, a lipid
particle, a bacteria, a fungi, a plant, a yeast cell, or a yeast
cell particle.
[0025] In another embodiment, the vector is a viral vector (e.g., a
baculoviral vector), an expression vector or a plasmid.
[0026] In another embodiment of the aspects described herein, the
lipid particle comprises about 15-25% triacylglycerol, about 0.5-2%
phospholipids, about 1-3% glycerol, and at least one lipid-binding
protein.
[0027] In another embodiment of this aspect, the composition is
provided in a spray, solution, gel, bait, a food source, or powder
form. The composition can further comprise an attractant, such as
e.g., an insect pheromone or hormone.
[0028] In another embodiment of the aspects described herein, the
composition is specific to the pest and does not affect other
insects.
[0029] In another embodiment of the aspects described herein, the
composition is administered to adult insects.
[0030] In another embodiment of the aspects described herein, the
composition is administered to a breeding or feeding locus.
[0031] In another embodiment of the aspects described herein, the
composition further comprises an additional agent, including but
not limited to antivirals, antifungals, antibacterials, pesticides,
antihelminthics, nutrients, pollen, sucrose and/or agents that stun
or slow insect movement.
[0032] In another embodiment of the aspects described herein, the
insect pathogen is a virus, mite, nematode, bacteria, fungus, or
parasite.
[0033] In another embodiment of the aspects described herein,
modulating gene expression inhibits pathogen infectivity,
virulence, reproduction, viability, growth, translation, protein
production, viral uptake or transmission of the insect
pathogen.
[0034] In another embodiment of the aspects described herein,
modulating gene expression decreases insect susceptibility to a
pathogen.
[0035] Another aspect described herein relates to a composition
comprising an RNA effector molecule or a vector encoding an RNA
molecule, and a delivery agent, wherein the RNA effector molecule
modulates gene expression of an insect or an insect pathogen. The
composition can further comprise an insect attractant.
[0036] In one embodiment of this aspect, the oligonucleotide
comprises an siRNA, an miRNA, an shRNA, a ribozyme, an antisense
RNA, a decoy oligonucleotide, an antimir, a supermir, or an RNA
activator. The oligonucleotide can be a single stranded or double
stranded oligonucleotide. In some embodiments, the oligonucleotide
comprises 9-36 base pairs. The oligonucleotide can be modified in
any manner as known in the art and/or described herein.
[0037] In another embodiment of this aspect, the delivery agent is
a viral vector, a plasmid, a lipid, a liposome, a food source, an
expression vector, a solution, an emulsion, a micelle or other
membranous formulation, a lipid particle (e.g., INTRALIPID.TM.), a
bacteria, a fungi, a plant, a yeast cell, or a yeast cell
particle.
[0038] In another embodiment of this aspect, the food source is a
bacteria, fungus, plant, or yeast expressing the
oligonucleotide.
[0039] In another embodiment of this aspect, the composition
inhibits viability, survival, growth, development, and/or
reproduction of the insect.
[0040] In another embodiment of this aspect, the composition
inhibits pathogen infectivity, virulence, reproduction, viability,
growth, translation, protein production, viral uptake or
transmission of the insect pathogen.
[0041] The composition can be provided in a spray, solution, gel,
topical formulation, or powder form. In addition, the composition
can further comprise an antibiotic, antiviral, antifungal
pesticides, antihelminthics, nutrients, pollen, sucrose and/or
agents that stun or slow insect movement. In another embodiment of
this aspect, the attractant comprises an insect pheromone or
hormone.
DEFINITIONS
[0042] As used herein the term "administering" encompasses any
method by which an insect can come into contact with an
oligonucleotide as that term is used herein. In one embodiment, the
oligonucleotide is a dsRNA comprising annealed complementary
strands, one of which has a nucleotide sequence which is
complementary to at least part of the nucleotide sequence of an
insect target gene to be modulated. An insect can be exposed to a
composition (e.g., an oligonucleotide and a delivery agent) by
direct uptake (e.g. by feeding), which does not require expression
of the oligonucleotide within the insect. Alternatively, an insect
can come into direct contact with a composition comprising the
oligonucleotide. For example, an insect can come into contact with
a surface or material treated with a composition comprising an
oligonucleotide. An oligonucleotide can be expressed by a
prokaryotic (for instance, but not limited to, a bacterial) or
eukaryotic (for instance, but not limited to, a yeast) host cell or
host organism. (also virally encoded source)
[0043] As used herein the term "additional agent" refers to a small
molecule, chemical, organic, or inorganic molecule that can be used
to treat insects. In one embodiment, the "additional agent" is a
pesticide. As used herein, the term "pesticide" refers to any
substance or mixture of substances intended for preventing,
destroying, repelling, or mitigating any pest. A pesticide can be a
chemical substance or biological agent used against pests including
insects, pathogens, weeds, nematodes, and microbes that compete
with humans for food, destroy property, spread disease, or are a
nuisance. The term "additional agent" further encompasses other
bioactive molecules such as antibiotics, antivirals pesticides,
antifungals, antihelminthics, nutrients, pollen, sucrose and/or
agents that stun or slow insect movement.
[0044] As used herein the term "plant" is used to refer to any of
various photosynthetic, eukaryotic, multicellular organisms of the
kingdom Plantae characteristically producing embryos, containing
chloroplasts, and having cellulose cell walls. A part of a plant,
i.e., a "plant tissue" can be treated according to the methods
described herein to prevent pest infestation on the plant or on
part of the plant. Alternatively, a plant can be treated to improve
the health of a beneficial insect population by modulating gene
expression of an insect pathogen. In addition, a plant can be
engineered to express an oligonucleotide useful with the methods
described herein. Many suitable plant tissues can be treated
according to the present invention and include, but are not limited
to, somatic embryos, pollen, leaves, stems, calli, stolons,
microtubers, and shoots. Thus, the present invention envisions the
treatment of angiosperm and gymnosperm plants such as acacia,
alfalfa, apple, apricot, artichoke, ash tree, asparagus, avocado,
banana, barley, beans, beet, birch, beech, blackberry, blueberry,
broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot,
cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry,
Chinese cabbage, citrus, clemintine, clover, coffee, corn, cotton,
cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus,
fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground
cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,
lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,
maple, melon, millet, mushroom, mustard, nuts, oak, oats, okra,
onion, orange, an ornamental plant or flower or tree, papaya, palm,
parsley, parsnip, pea, peach, peanut, pear, peat, pepper,
persimmon, pigeon pea, pine, pineapple, plantain, plum,
pomegranate, potato, pumpkin, radicchio, radish, rapeseed,
raspberry, rice, rye, sorghum, sallow, soybean, spinach, spruce,
squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato,
sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf
grasses, turnips, a vine, walnut, watercress, watermelon, wheat,
yams, yew, and zucchini.
[0045] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0046] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0047] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of elements that do not materially affect the basic
and novel or functional characteristic(s) of that embodiment of the
invention.
[0048] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0049] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0050] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
DETAILED DESCRIPTION
[0051] The methods and compositions described herein provide for
modulation of gene expression in insects by administering a
composition comprising an RNA effector molecule and a delivery
agent. Methods are provided for controlling pest populations by
inhibiting insect growth, development, survival, reproduction
and/or viability. Also provided herein are methods for treating or
preventing disease in an insect caused by a pathogen or by external
factors (e.g., pollution, environment, stress, weather, etc.).
Insect Pests
[0052] The present invention provides methods and compositions for
controlling pest infestations by administering, or otherwise
exposing, to a pest a composition comprising an oligonucleotide
that post-transcriptionally modulates (e.g., represses, inhibits,
or activates) a requisite biological function in the pest.
[0053] As used herein, the term "pest" refers to insects that cause
damage to plants, other organisms or otherwise causes a nuisance. A
pest can ingest or contact one or more cells, tissues, or products
produced by an organism transformed with an oligonucleotide
composition as described herein, as well as a surface or material
treated with such an oligonucleotide composition.
[0054] As used herein the term "insect" describes any insect,
meaning any organism belonging to the Kingdom Animals, more
specific to the Phylum Arthropoda, and to the Class Insecta or the
Class Arachnida.
[0055] In one embodiment of the invention, the insect can belong to
the following orders: Acari, Araneae, Anoplura, Coleoptera,
Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera,
Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera,
Isoptera, Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata,
Orthoptera, Phasmida, Plecoptera, Protura, Psocoptera,
Siphonaptera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera,
Trichoptera, and Zoraptera.
[0056] As used herein, the terms "pest" or "insect pests" include
but are not limited to the following examples: from the order
Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria
spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia
gemmatalis, Archips spp, Argyrotaenia spp., Autographa spp.,
Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp.,
Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp.,
Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia
binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp.,
Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp.,
Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp.,
Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria
cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis
spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma
spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia
Nubilalis, Pammene spp., Pandemis spp., Panolis flammea,
Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae,
Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp.,
Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp.,
Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta
spp.; from the order Coleoptera, for example, Agriotes spp.,
Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,
Cosmopolites spp., Curculio spp., Dermestes spp., Epilachna spp.,
Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp.,
Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus
spp., Popillia spp., Psylliodes spp., Rhizopertha spp.,
Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp.,
Tribolium spp. and Trogoderma spp.; from the order Orthoptera, for
example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea
maderae, Locusta spp., Periplaneta ssp., and Schistocerca spp.;
from the order Isoptera, for spp; from the order Psocoptera, for
spp.; from the order Anoplura, for example jfaematopinus spp.,
Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera
spp.; from the order Mallophaga, for
example.sub.([Hat])Dalphamalpha/mealpha spp. and Trichodectes spp.;
from the order Thysanoptera, for spp., Hercinothrips spp.,
Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips
aurantii; from the order Heteroptera, for example, Cimex spp.,
Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster
spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp.,
Sahlbergella singularis, Scotinophara spp., Triatoma spp., Miridae
family spp. such as Lygus hesperus and Lygus lineoloris, LygaeidaQ
family spp. such as Blissus leucopterus, and Pentatomidae family
spp.; from the order Homoptera, for example, Aleurothrixus
floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis
spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp.,
Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus
hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,
Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp.,
Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp.,
Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis
spp., Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica,
Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp.,
Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes
vaporariorum, Trioza erytreae and Unaspis citri; from the order
Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp.,
Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp.,
Lasius sppp., Monomorium pharaonis, Neodiprion spp, Solenopsis spp.
and Vespa ssp.; from the order Diptera, for example, Aedes spp.,
Antherigona soccata, Bibio hortulanus, CalHphora erythrocephala,
Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus
spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp.,
Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp.,
Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp.,
Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp.,
Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp.,
Tannia spp. and Tipula spp., from the order Siphonaptera, for
example, Ceratophyllus spp. and Xenopsylla cheopis and from the
order Thysanura, for example Lepisma saccharina.
[0057] In one embodiment the insect is chosen from the group
consisting of: an insect which is a plant pest, such as but not
limited to Nilaparvata spp. (e.g. N. lugens (brown planthopper));
Laodelphax spp. (e.g. L. striatellus (small brown planthopper));
Nephotettix spp. (e.g. N. virescens or N. cincticeps (green
leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp.
(e.g. S. furcifera (white-backed planthopper)); Blissus spp. (e.g.
B. leucopterus leucopterus (chinch bug)); Scotinophora spp. (e.g.
S. vermidulate (rice blackbug)); Acrosternum spp. (e.g. A. Mare
(green stink bug)); Parnara spp. (e.g. P. guttata (rice skipper));
Chilo spp. (e.g. C. suppressalis (rice striped stem borer), C.
auricilius (gold-fiinged stem borer), or C. polychrysus
(dark-headed stem borer)); Chilotraea spp. (e.g. C. polychrysa
(rice stalk borer)); Sesamia spp. (e.g. S. inferens (pink rice
borer)); Tryporyza spp. (e.g. T. innotata (white rice borer), or T.
incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g. C.
medinalis (rice leafroller)); Agromyza spp. (e.g. A. oryzae
(leafminer), or A. parvicornis (corn blot leafminer)); Diatraea
spp. (e.g. D. saccharalis (sugarcane borer), or D. grandiosella
(southwestern corn borer)); Narnaga spp. (e.g. N. aenescens (green
rice caterpillar)); Xanthodes spp. (e.g. X. transversa (green
caterpillar)); Spodoptera spp. (e.g. S. frugiperda (fall armyworm),
S. exigua (beet armyworm), S. littoralis (climbing cutworm) or S.
praefica (western yellowstriped armyworm)); Mythimna spp. (e.g.
Mythmna (Pseudaletia) seperata (armyworm)); Helicoverpa spp. (e.g.
H. zea (corn earworm)); Colaspis spp. (e.g. C. brunnea (grape
colaspis)); Lissorhoptrus spp. (e.g. L. oryzophilus (rice water
weevil)); Echinocnemus spp. (e.g. E. squamos (rice plant weevil));
Diclodispa spp. (e.g. D. armigera (rice hispa)); Oulema spp. (e.g.
O. oryzae (leaf beetle); Sitophilus spp. (e.g. S. oryzae (rice
weevil)); Pachydiplosis spp. (e.g. P. oryzae (rice gall midge));
Hydrellia spp. (e.g. H. griseola (small rice leafminer), or H.
sasakii (rice stem maggot)); Chlorops spp. (e.g. C. oryzae (stem
maggot)); Ostrinia spp. (e.g. O. nubilalis (European corn borer));
Agrotis spp. (e.g. A. ipsilon (black cutworm)); Elasmopalpus spp.
(e.g. E. lignosellus (lesser cornstalk borer)); Melanotus spp.
(wireworms); Cyclocephala spp. (e.g. C. borealis (northern masked
chafer), or C. immaculata (southern masked chafer)); Popillia spp.
(e.g. P. japonica (Japanese beetle)); Chaetocnema spp. (e.g. C.
pulicaria (corn flea beetle)); Sphenophorus spp. (e.g. S. maidis
(maize billbug)); Rhopalosiphum spp. (e.g. R. maidis (corn leaf
aphid)); Anuraphis spp. (e.g. A. maidiradicis (corn root aphid));
Melanoplus spp. (e.g. M. femurrubrum (redlegged grasshopper) M.
differentialis (differential grasshopper) or M. sanguinipes
(migratory grasshopper)); Hylemya spp. (e.g. H. platura (seedcorn
maggot)); Anaphothrips spp. (e.g. A. obscrurus (grass thrips));
Solenopsis spp. (e.g. S. milesta (thief ant)); or spp. (e.g. T.
urticae (twospotted spider mite), T. cinnabarinus (carmine spider
mite); Helicoverpa spp. (e.g. H. zea (cotton bollworm), or H.
armigera (American bollworm)); Pectinophora spp. (e.g. P.
gossypiella (pink bollworm)); Earias spp. (e.g. E. vittella
(spotted bollworm)); Heliothis spp. (e.g. H. virescens (tobacco
budworm)); Anthonomus spp. (e.g. A. grandis (boll weevil));
Pseudatomoscelis spp. (e.g. P. seriatus (cotton fleahopper));
Trialeurodes spp. (e.g. T. abutiloneus (banded-winged whitefly) T.
vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B.
argentifol (silverleaf whitefly)); Aphis spp. (e.g. A. gossypii
(cotton aphid), A. mellifera); Lygus spp. (e.g. L. lineolaris
(tarnished plant bug) or L. hesperus (western tarnished plant
bug)); Euschistus spp. (e.g. E. conspersus (consperse stink bug));
Chlorochroa spp. (e.g. C. sayi (Say stinkbug)); Nezara spp. (e.g.
N. viridula (southern green stinkbug)); Thrips spp. (e.g. T. tabaci
(onion thrips)); Frankliniella spp. (e.g. F. fusca (tobacco
thrips), or F. occidentalis (western flower thrips)); Leptinotarsa
spp. (e.g. L. decemlineata (Colorado potato beetle), L. juncta
(false potato beetle), or L. texana (Texan false potato beetle));
Lema spp. (e.g. L. trilineata (three-lined potato beetle)); Epitrix
spp. (e.g. E. cucumeris (potato flea beetle), E. hirtipennis (flea
beetle), or E. tuberis (tuber flea beetle)); Epicauta spp. (e.g. E.
vittata (striped blister beetle)); Empoasca spp. (e.g. E. fabae
(potato leafhopper)); Myzus spp. (e.g. M. persicae (green peach
aphid)); Paratrioza spp. (e.g. P. cockerelli (psyllid)); Conoderus
spp. (e.g. C. falli (southern potato wireworm), or C. vespertinus
(tobacco wireworm)); Phthorimaea spp. (e.g. P. operculella (potato
tuberworm)); Macrosiphum spp. (e.g. M. euphorbiae (potato aphid));
Thyanta spp. (e.g. T. pallidovirens (redshouldered stinkbug));
Phthorimaea spp. (e.g. P. operculella (potato tuberworm));
Helicoverpa spp. (e.g. H. zea (tomato fruitworm); Keiferia spp.
(e.g. K. lycopersicella (tomato pinworm)); Limonius spp.
(wireworms); Manduca spp. (e.g. M. sexta (tobacco hornworm), or M.
quinquemaculata (tomato hornworm)); Liriomyza spp. (e.g. L.
sativae, L. trifolli or L. huidobrensis (leafminer)); Drosophilla
spp. (e.g. D. melanogaster, D. yakuba, D. pseudoobscura or D.
simulans); Carabus spp. (e.g. C. granulatus); Chironomus spp. (e.g.
C. tentanus); Ctenocephalides spp. (e.g. C. felis (cat flea));
Diaprepes spp. (e.g. D. abbreviatus (root weevil)); Ips spp. (e.g.
Ips. pini (pine engraver)); Tribolium spp. (e.g. T. castaneum (red
floor beetle)); Glossina spp. (e.g. G. morsitans (tsetse fly));
Anopheles spp. (e.g. A. gambiae (malaria mosquito)); Helicoverpa
spp. (e.g. H. armigera (African Bollworm)); Acyrthosiphon spp.
(e.g. A. pisum (pea aphid)); Apis spp. (e.g. A. melifera (honey
bee)); Homalodisca spp. (e.g. H. coagulate (glassy-winged
sharpshooter)); Aedes spp. (e.g. Ae. aegypti (yellow fever
mosquito)); Bombyx spp. (e.g. B. mori (silkworm), B. mandarins);
Locusta spp. (e.g. L. migratoa (migratory locust)); Boophilus spp.
(e.g. B. microplus (cattle tick)); Acanthoscurria spp. (e.g. A.
gomesiana (red-haired chololate bird eater)); Diploptera spp. (e.g.
D. punctata (pacific beetle cockroach)); Heliconius spp. (e.g. H.
erato (red passion flower butterfly) or H. melpomene (postman
butterfly)); CurcuHo spp. (e.g. C. glandium (acorn weevil));
Plutella spp. (e.g. P. xylostella (diamondback moth)); Amblyomma
spp. (e.g. A. variegatum (cattle tick)); Anteraea spp. (e.g. A.
yamamai (silkmoth)); Belgica spp. (e.g. B. antartica), Bemisa spp.
(e.g. B. tabaci), Bicyclus spp., Biphillus spp., Collosobruchus
spp., Choristoneura spp., Cicindela spp., Culex spp., Culicoides
spp., Diaphorina spp., Diaprepes spp., Euclidia spp., Glossina
spp., Gryllus spp., Hydropsyche spp., Julodis spp., Lonomia spp.,
Lutzomyia spp., Lysiphebus spp, Meladema spp, Mycetophagus spp.,
Nasonia spp., Oncometopia spp., Papilio spp., Pediculus spp.,
Plodia spp., Rhynchosciara spp., Sphaenius spp., Toxoptena spp.,
Tchoplusa spp., and Anmigenes spp.
[0058] In another embodiment an insect is chosen for gene
expression modulation that is capable of infesting or injuring
humans and/or animals such as, but not limited to those with
piercing-sucking mouthparts, as found in Hemiptera and some
Hymenoptera and Diptera such as mosquitoes, bees, wasps, lice,
fleas and ants, as well as members of the Arachnidae such as ticks
and mites; order, class or family of Acarina (ticks and mites) e.g.
representatives of the families Argasidae, Dermanyssidae, Ixodidae,
Psoroptidae or Sarcoptidae and representatives of the species
Amblyomma spp., Anocenton spp., Argas spp., Boophilus spp.,
Cheyletiella spp., Chorioptes spp., Demodex spp., Dermacentor spp.,
Denmanyssus spp., Haemophysalis spp., Hyalomma spp., Ixodes spp.,
Lynxacarus spp., Mesostigmata spp., Notoednes spp., Ornithodoros
spp., Ornithonyssus spp., Otobius spp., otodectes spp.,
Pneumonyssus spp., Psoroptes spp., Rhipicephalus spp., Sancoptes
spp., or Trombicula spp.; Anoplura (sucking and biting lice) e.g.
representatives of the species Bovicola spp., Haematopinus spp.,
Linognathus spp., Menopon spp., Pediculus spp., Pemphigus spp.,
Phylloxera spp., or Solenopotes spp.; Diptera (flies) e.g.
representatives of the species Aedes spp., Anopheles spp.,
Calliphora spp., Chrysomyia spp., Chrysops spp., Cochliomyia spp.,
Cw/ex spp., CuUcoides spp., Cuterebra spp., Dermatobia spp.,
Gastrophilus spp., Glossina spp., Haematobia spp., Haematopota
spp., Hippobosca spp., Hypoderma spp., Lucilia spp., Lyperosia
spp., Melophagus spp., Oestrus spp., Phaenicia spp., Phlebotomus
spp., Phormia spp., Sarcophaga spp., Simulium spp., Stomoxys spp.,
Tabanus spp., Tannia spp. or Zzpu/alpha spp.; Mallophaga (biting
lice) e.g. representatives of the species Damalina spp., Felicola
spp., Heterodoxus spp. or Trichodectes spp.; or Siphonaptera
(wingless insects) e.g. representatives of the species
Ceratophyllus spp., Xenopsylla spp; Cimicidae (true bugs) e.g.
representatives of the species Cimex spp., Tritominae spp.,
Rhodinius spp., or Triatoma spp.
[0059] In another embodiment a target insect is treated as
described herein to prevent unwanted damage to substrates or
materials, such as insects that attack foodstuffs, seeds, wood,
paint, plastic, clothing etc.
[0060] Target Genes
[0061] In practicing the present invention, the expression of
target gene derived from any pest that causes damage to another
organism can be modulated using the methods and compositions
described herein. Several criteria can be employed in the selection
of preferred target genes. In one embodiment, the gene is one whose
protein product has a rapid turnover rate, so that inhibition of
gene expression will result in a rapid decrease in protein levels.
In certain embodiments it is advantageous to select a gene for
which a small change (e.g., reduction) in expression level results
in deleterious effects for the recipient pest. If it is desired to
target a broad range of insect species, for example, a gene is
selected that is highly conserved across these species. Conversely,
for the purpose of conferring specificity, in certain embodiments
of the invention, a gene is selected that contains regions that are
poorly conserved between individual insect species, or between
insects and other organisms; in certain embodiments it can be
desirable to select a gene that has no known homologs in other
organisms.
[0062] In one embodiment, a gene is selected that is expressed in
the insect gut. Target genes for use in the present invention can
include, for example, those that share substantial homologies to
the nucleotide sequences of known gut-expressed genes that encode
protein components of the plasma membrane proton V-ATPase. This
protein complex is the sole energizer of epithelial ion transport
and is responsible for alkalinization of the midgut lumen. The
V-ATPase is also expressed in the Malpighian tubule, an outgrowth
of the insect hindgut that functions in fluid balance and
detoxification of foreign compounds in a manner analogous to a
kidney organ of a mammal.
[0063] In another embodiment, a gene is selected that is
essentially involved in the growth, development, and reproduction
of an insect. Exemplary genes include but are not limited to the
structural subunits of ribosomal proteins and a beta-coatamer gene,
CHD3 gene. Ribosomal proteins such as S4 (RpS4) and S9(RpS9) are
structural constituents of the ribosome involved in protein
biosynthesis and which are components of the cytosolic small
ribosomal subunit, the ribosomal proteins such as L9 and L19 are
structural constituent of ribosome involved in protein biosynthesis
which is localized to the ribosome. The beta-coatamer gene in C.
elegans encodes a protein which is a subunit of a multimeric
complex that forms a membrane vesicle coat. Similar sequences have
been found in diverse organisms such as Arabidopsis thaliana,
Drosophila melanogaster, and Saccharomyces cerevisiae. Related
sequences are found in diverse organisms such as Leptinotarsa
decemlineata, Phaedon cochleariae, Epilachna varivetis, Anthonomus
grandis, Tribolium castaneum, Myzus per sicae, Nilaparvata lugens,
Chilo suppressalis, Plutella xylostella and Acheta domesticus.
Other target genes for use with the methods described herein can
include, for example, those that play important roles in viability,
growth, survival, development, reproduction, and infectivity. These
target genes include, for example, house keeping genes,
transcription factors, and insect specific genes or lethal knockout
mutations in Caenorhabditis or Drosophila. The target genes for use
with the methods described herein can also be those that are from
other organisms, e.g., from a nematode (e.g., Meloidogyne spp. or
Heterodera spp.), other insects or arachnidae (e.g. Leptinotarsa
spp., Phaedon spp., Epilachna spp., Anthonomus spp., Tribolium
spp., Myzus spp., Nilaparvata spp., Chilo spp., Plutella spp., or
Acheta spp.
[0064] In one embodiment, the target gene is a gene which can
induces cell death (e.g., apoptosis). The gene can be directly
responsible for inducing apoptosis or can be a gene that indirectly
induces apoptosis, e.g., by affecting activity of other genes
leading to apoptosis. As used herein, a gene directly induces
apoptosis when expression of the gene leads to cell death. A gene
indirectly induces apoptosis when expression of the gene modulates
the expression of other genes that induce apoptosis.
[0065] Additionally, the nucleotide sequences for use as a target
sequence in the methods described herein can also be derived from
viral, bacterial, insect or fungal genes whose functions have been
established from literature and the nucleotide sequences of which
share substantial similarity with the target genes in the genome of
an insect.
[0066] For many of the insects that are potential targets for
control by the present invention, there may be limited information
regarding the sequences of most genes or the phenotype resulting
from mutation of particular genes. Therefore, genes can be selected
based on available information available concerning corresponding
genes in a model organism, such as Caenorhabditis or Drosophila, or
in some other insect species. Genes can also be selected based on
available sequence information for other species, such as nematode
or fungal species, in which the genes have been characterized. In
some cases it will be possible to obtain the sequence of a
corresponding gene from a target insect by searching databases,
such as GenBank, using either the name of the gene or the gene
sequence. Once the sequence is obtained, PCR can be used to amplify
an appropriately selected segment of the gene in the insect for use
in the present invention.
[0067] In one embodiment, the expression of a target gene is
modulated such that the insect has an increased susceptibility to a
pathogen. Target genes that can be inhibited to increase pathogen
susceptibility include those that confer immunity to the insect
and/or are a part of the insect immune system. Alternatively, one
can select a target gene in the insect to be activated by RNA
activation that is involved in the uptake, reproduction, or
virulence of the pathogen in the host. In such an example, the
expression of a receptor necessary for uptake of a pathogen in the
insect or the expression of a protein involved in viral
reproduction would speed up infection and reproduction of a virus,
resulting in enhanced pathogen susceptibility. One of skill in the
art can determine which gene products of the immune system can be
inhibited, or which gene products involved in a pathogen life cycle
can be activated in the insect host. Exemplary genes involved in
pathogen-borne disease in honeybees can be found in e.g., Navajas,
M et al., BMC Genomics 2008; 9:301. Other exemplary genes can be
found in e.g., Feldhaar, and Gross. Microbes and Infection 2008;
10(9):1082-1088. In addition, the genetic structure of the innate
immune system is well-conserved in insects, which permits one of
skill in the art to determine an appropriate gene target from the
sequence of the Drosophila genome.
[0068] Beneficial Insects
[0069] The methods and compositions described herein can be
formulated for treating or preventing disease in beneficial
insects. As used herein, the term "beneficial insect" is used to
describe an insect that provides benefit to humans, mammals, an
ecosystem and/or the environment by e.g., pollinating crops,
spreading seeds, reducing numbers of pest insects, providing a
useable product (e.g., honey, beeswax, silk, etc.). It is
contemplated that many of the insects in the list of pests can also
be beneficial in particular embodiments. The term "beneficial" and
"pest" are determined by one of skill in the art with reference to
the occurrence of a desired outcome. For example, silkworms can be
beneficial should one desire to produce silk products, however
silkworms can also be a pest for those interested in growing
mulberry leaves (i.e., a preferred source of food for the
silkworm). Thus, for a desired outcome, one of skill in the art can
determine if an insect is beneficial or a pest.
[0070] Exemplary "beneficial insects" include e.g., ladybugs, bees,
wasps, ants and butterflies. As used herein, the term "bee" is
defined as any of several winged, hairy-bodied, usually stinging
insects of the superfamily Apoidea in the order Hymenoptera,
including both solitary and social species and characterized by
sucking and chewing mouthparts for gathering nectar and pollen.
Exemplary bee species include, but are not limited to Apis, Bombus,
Trigona, Osmia and the like. In one embodiment, bees include, but
are not limited to bumblebees (Bombus terrestris) and honeybees
(Apis mellifera).
[0071] Target Pathogens
[0072] As used herein, the term "pathogen" is defined as a nucleic
acid-containing agent capable of proliferation within a beneficial
insect and/or colony, the pathogen causing disease in an insect
and/or colonies (e.g., a virus, a bacteria, a mite, a spore, a
parasite, and a fungus). In one embodiment, the insect pathogen is
a bee pathogen. A bee or bee colony pathogenic agent can be an
intracellular or extra-cellular parasite. According to one
embodiment of the invention, the pathogen is a "bee pathogen",
causing or facilitating a bee or bee colony disease, such as Colony
Collapse Disorder, Sacbrood virus disease, Deformed Wing Disease,
Cloudy Wing Disease, Chronic Paralysis, Nosemosis, American Foul
Brood and the like.
[0073] The importance of honeybees and other pollinating insects to
the global world economy far surpasses their contribution in terms
of honey production. The United States Department of Agriculture
(USDA) estimates that every third bite we consume in our diet is
dependent on a honeybee to pollinate that food. The total
contribution of pollination in terms of added value to fruit crops
exceeds $15 billion per annum, with indirect potential consequence
of $75 billion dollars.
[0074] The health and vigor of honeybee colonies are threatened by
numerous parasites and pathogens, including viruses, bacteria,
protozoa, fungi, and mites, each with characteristic modes of
transmission.
[0075] In general, transmission of viruses can occur via two
pathways: horizontal and vertical transmission. In horizontal
transmission, viruses are transmitted among individuals of the same
generation, while vertical transmission occurs from adults to their
offspring. Transmission can occur through multiple routes in social
organisms (for a detailed review see Chen Y P, et al (2006) Appl
Environ Microbiol. 72(1):606-11). Recently, horizontal transmission
of honeybee viruses has been documented in bee colonies, for
example, transmission of deformed wing virus (DWV) and Kashmir Bee
Virus (KBV) by the parasitic mite Varroa destructor, as well as
some evidence of virus in honeybee eggs and young larvae, life
stages not parasitized by Varroa mites. Vertical transmission of
multiple viruses from mother queens to their offspring in honeybees
has also been recently demonstrated, as well as viruses in feces of
queens, suggesting a role for feeding in virus transmission.
Moreover, honeybee viruses have been detected in tissues of the
gut, suggesting that viruses could be ingested by queens from
contaminated foods and passed into the digestive tract, which then
acts as a major reservoir for viral replication. Indeed, viruses
might penetrate the gut wall and move into the insect hemocoel,
spreading infections to other tissues.
[0076] In honeybees viruses often persist as latent infections.
Thus, group living activities such as trophylaxis and nurse bee
brood feeding, can potentially drive high levels of horizontal
transmission or amplification of existing infections.
[0077] Colony Collapse Disorder (CCD) of honeybees is threatening
to annihilate U.S. and world agriculture. Indeed, in the recent
outbreak of CCD in the U.S in the winter of 2006-2007, an estimated
25% or more of the 2.4 million honeybee hives were lost because of
CCD. An estimated 23% of beekeeping operations in the United States
suffered from CCD over the winter of 2006-2007, affecting an
average of 45% of the beekeepers operations. In the winter of
2007-2008, the CCD action group of the USDA-ARS estimated that a
total of 36% of all hives from commercial operations were destroyed
by CCD.
[0078] CCD is characterized by the rapid loss from a colony of its
adult bee population, with dead adult bees usually found at a
distance from the colony. At the final stages of collapse, a queen
is attended only by a few newly emerged adult bees. Collapsed
colonies often have considerable capped brood and food reserves.
The phenomenon of CCD was first reported in 2006; however,
beekeepers noted unique colony declines consistent with CCD as
early as 2004. Various factors such as mites and infectious agents,
weather patterns, electromagnetic (cellular antennas) radiation,
pesticides, poor nutrition and stress have been postulated as
causes. To date, control of CCD has focused on varroa mite control,
sanitation and removal of affected hives, treating for
opportunistic infections (such as Nosema) and improved nutrition.
No effective preventative measures have been developed to date.
[0079] That CCD is due to the introduction of a previously
unrecognized infectious agent is supported by preliminary evidence
that CCD is transmissible through the reuse of equipment from CCD
colonies and that such transmission can be broken by irradiation of
the equipment before use.
[0080] Recently, Israeli acute paralysis virus of bees (IAPV, SEQ
ID NO: 1), was strongly correlated with CCD. In contrast, IAPV was
not only found in 83% of CCD colonies, but was almost completely
absent from apparently healthy colonies. Moreover, it was recently
shown that when injected or fed to the bees, IAPV causes paralysis
and death in 98% of bees within days, further confirming IAPV as
the infective agent in CCD.
[0081] Israeli acute paralysis virus (IAPV) has been characterized
as a bee-affecting dicistrovirus. Recently, DNA versions of genomic
segments of non-retro RNA viruses have been found in their
respective host genomes, and the reciprocal exchange of genome
sequences between host and virus has been demonstrated (Maori et
al. Virology 2007; 362:342). These authors showed that the bees who
harbored integrated viral sequences were found to be resistant to
subsequent viral infection, and a RNAi mechanism of resistance was
postulated. A metagenomic survey has indicated a close association
between CCD and IAPV (Cox-Foster et al., Science, 2007; 318:283).
It thus follows that prevention of IAPV infection using the methods
and compositions described herein may prevent development of CCD,
significantly improving the state of the beekeeping industry and
world agriculture.
[0082] According to some embodiments of the invention, the virus is
Israel Acute Paralysis Virus and said polypeptide of said virus is
selected from the group consisting of IAPV polymerase polyprotein
and IAPV structural polyprotein. In further embodiments, other IAPV
polypeptides (including viral nucleic acid sequences detected in
honeybee nucleic acid) can be targeted for gene modulation as
described in the International PCT Publication WO2009/060429, filed
Nov. 3, 2008 (Inventor: Paldi, N et al.), which is herein
incorporated by reference in its entirety.
[0083] Administration to Insects
[0084] An insect (e.g., a pest insect or a beneficial insect) can
be exposed to an oligonucleotide in combination with a delivery
agent in any suitable manner that permits administering the
composition to the insect. For example, the insect can be contacted
with the composition in a pure or substantially pure form, for
example a solution containing the oligonucleotide. Preferably, the
composition comprises at a minimum, an oligonucleotide and a
delivery agent. In one embodiment, the insect can be simply
"soaked" or "sprayed" with a solution comprising the
oligonucleotide.
[0085] Alternatively, the oligonucleotide can be linked to a food
component of the insect, such as a food component for a mammalian
pathogenic insect and/or agricultural pest for ease of delivery
and/or in order to increase uptake of the oligonucleotide by the
insect. Ingestion by an insect permits delivery of the insect
control agents and results in modulation of a target gene in the
host. Methods for oral introduction include, for example, directly
mixing an oligonucleotide with the insect's food, spraying the
oligonucleotide in the insect's habitat or field, as well as
engineered approaches in which a species that is used as food is
engineered to express an oligonucleotide, then fed to the insect to
be affected. For example, a bacterium, such as Lactobacillus, can
be transformed with a target sequence and then fed to an insect. In
one embodiment, for example, the oligonucleotide composition can be
incorporated into, or overlaid on the top of, the insect's diet.
For example, the oligonucleotide composition can be sprayed onto a
field of crops which a pest insect attacks.
[0086] The oligonucleotide can also be incorporated in the medium
in which the insect grows, lives, reproduces, feeds, or infests.
For example, an oligonucleotide can be incorporated into a food
container or protective wrapping as a means for inhibiting pest
infestation. Wood, for example, can be treated with a solution
comprising an oligonucleotide to prevent pest infestation.
[0087] In other embodiments, the oligonucleotide is expressed in a
bacterial or fungal cell and the bacterial or fungal cell is taken
up or eaten by the insect species. Bacteria can be engineered to
produce any of the oligonucleotide or oligonucleotide constructs
contemplated herein. These bacteria can be eaten by the insect
species, When taken up, the oligonucleotide can initiate gene
expression modulation and can lead to e.g., degradation of the
target mRNA and weakening, killing of a pest or decreasing pathogen
susceptibility of a beneficial insect.
[0088] In some embodiments, the oligonucleotide composition is
sprayed directly onto a plant e.g., crops, by e.g., backpack
spraying, aerial spraying, crop spraying/dusting etc. In another
embodiment, an oligonucleotide producing bacteria or yeast cells
can be sprayed directly onto the crops.
[0089] Some bacteria have a very close interaction with the host
plant, such as, but not limited to, symbiotic Rhizobium with the
Legminosea (for example Soy). Such recombinant bacteria could be
mixed with the seeds (for instance as a coating) and used as soil
improvers.
[0090] A virus such as a baculovirus which specifically infects
insects can also be used. This ensures safety for mammals,
especially humans, since the virus will not infect the mammal, so
no unwanted gene modulation effect will occur.
[0091] Possible applications include intensive greenhouse cultures,
for instance crops that are less interesting from a GMO point of
view, as well as broader field crops such as soy.
[0092] A composition can be a coating or a powder that can be
applied to a substrate as a means for protecting the substrate from
infestation by an insect and thereby preventing pest-induced damage
to the substrate or material. Thus, in one embodiment, the
composition is in the form of a coating on a suitable surface which
adheres to, and is eventually ingested by an insect which comes
into contact with the coating. Such a composition can be used to
protect any substrate or material that is susceptible to
infestation by or damage caused by a pest, for example foodstuffs
and other perishable materials, and substrates such as wood.
[0093] For example, the composition can be a liquid that is brushed
or sprayed onto or imprinted into the material or substrate to be
treated. Thus, a human user can spray the insect or the substrate
directly with the composition. For example, houses and other wood
products can be destroyed by termites, powder post beetles, and
carpenter ants. By treating wood or house siding with a composition
comprising an oligonucleotide, it can be possible to reduce pest
infestation. Likewise, a tree trunk can be treated with a
composition comprising an oligonucleotide.
[0094] Flour beetles, grain weevils, meal moths, and other pests
feed on stored grain, cereals, pet food, powdered chocolate, and
almost everything else in the kitchen pantry that is not protected.
Accordingly, the present invention provides a means for treating
cereal boxes and other food storage containers and wrapping with a
composition comprising an oligonucleotide composition.
[0095] Larvae of clothes moths eat clothes made from animal
products, such as fur, silk and wool. Thus, it can be desirable to
treat hangers, closet organizers, and garment bags with the
oligonucleotide as described herein. Book lice and silverfish are
pests of libraries because they eat the starchy glue in the
bindings of books. Accordingly, the present invention provides
compositions for treating books from pest infestation and
destruction.
[0096] In one embodiment, the composition is in the form of a bait.
The bait is designed to lure the insect to come into contact with
the composition. In one embodiment, upon coming into contact
therewith, the composition is then internalized by the insect, by
ingestion for example and mediates modulation of gene expression to
thus kill, or otherwise affect the insect. The bait can depend on
the species being targeted. An attractant can also be used. The
attractant can be a pheromone, such as a male or female pheromone.
The attractant acts to lure the insect to the bait, and can be
targeted for a particular insect or can attract a whole range of
insects. The bait can be in any suitable form, such as a solid,
paste, pellet or powdered form.
[0097] The bait can also be carried away by the insect back to the
colony. The bait can then act as a food source for other members of
the colony, thus providing an effective control of a large number
of insects and potentially an entire insect pest colony. This is an
advantage associated with use of the oligonucleotide or bacteria
expressing the oligonucleotide as described herein, because the
delayed action of the gene modulation effects on the pests allows
the bait to be carried back to the colony, thus delivering maximal
impact in terms of exposure to the insects.
[0098] The baits can be provided in a suitable "housing" or "trap".
Such housings and traps are commercially available and existing
traps can be adapted to include the compositions of the invention.
The housing or trap can be box-shaped for example, and can be
provided in pre-formed condition or can be formed of foldable
cardboard for example. Suitable materials for a housing or trap
include plastics and cardboard, particularly corrugated cardboard.
The inside surfaces of the traps can be lined with a sticky
substance in order to restrict movement of the insect once inside
the trap. The housing or trap can contain a suitable trough inside
which can hold the bait in place. A trap is distinguished from a
housing because the insect can not readily leave a trap following
entry, whereas a housing acts as a "feeding station" which provides
the insect with a preferred environment in which they can feed and
feel safe from predators.
[0099] It is clear that numerous products and substrates can be
treated with the inventive compositions for reducing pest
infestation. Of course, the nature of the excipients and the
physical form of the composition can vary depending upon the nature
of the substrate that is desired to be treated. For example, the
composition can be a liquid that is brushed or sprayed onto or
imprinted into the material or substrate to be treated, or a
coating that is applied to the material or substrate to be
treated.
[0100] The compositions described herein can further be delivered
to beneficial insects (e.g., bees) as described above. The
following exemplary methods and compositions provided for bees can
be extended to other beneficial insects by one of skill in the
art.
[0101] Bee Pathogens and Administration to Bees
[0102] As used herein, the term "bee" is defined as any of several
winged, hairy-bodied, usually stinging insects of the superfamily
Apoidea in the order Hymenoptera, including both solitary and
social species and characterized by sucking and chewing mouthparts
for gathering nectar and pollen. Exemplary bee species include, but
are not limited to Apis, Bombus, Trigona, Osmia and the like. In
one embodiment, bees include, but are not limited to bumblebees
(Bombus terrestris) and honeybees (Apis mellifera).
[0103] As used herein, the term "colony" is defined as a population
of dozens to typically several tens of thousand honeybees that
cooperate in nest building, food collection, and brood rearing. A
colony normally has a single queen, the remainder of the bees being
either "workers" (females) or "drones" (males). The social
structure of the colony is maintained by the queen and workers and
depends on an effective system of communication. Division of labor
within the worker caste primarily depends on the age of the bee but
varies with the needs of the colony. Reproduction and colony
strength depend on the queen, the quantity of food stores, and the
size of the worker force. Honeybees can also be subdivided into the
categories of "hive bees", usually for the first part of a workers
lifetime, during which the "hive bee" performs tasks within the
hive, and "forager bee", during the latter part of the bee's
lifetime, during which the "forager" locates and collects pollen
and nectar from outside the hive, and brings the nectar or pollen
into the hive for consumption and storage.
[0104] As used herein, the term "tolerance" is defined as the
ability of a bee or bee colony to resist infestation by and/or
proliferation of a pathogen, including, but not limited to, degree
of infection, severity of symptoms, infectivity to other
individuals (contagion), and the like. Tolerance can be assessed,
for example, by monitoring infectivity, presence of symptoms or
time course of a disease in a population following a challenge with
the pathogen.
[0105] As used herein, the term "pathogen" is defined as a nucleic
acid-containing agent capable of proliferation within the bee
and/or bee colony, the pathogen causing disease in bees or bee
colonies, especially, but not exclusively, a virus, a bacteria and
a fungus. A bee or bee colony pathogenic agent can be an
intracellular or extra-cellular parasite. According to one
embodiment of the invention, the pathogen is a "bee pathogen",
causing or facilitating a bee or bee colony disease, such as Colony
Collapse Disorder, Sacbrood virus disease, Deformed Wing Disease,
Cloudy Wing Disease, Chronic Paralysis, Nosemosis, American Foul
Brood and the like.
[0106] As used herein, the terms "bee disease" or "bee colony
disease" are defined as undesirable changes in the behavior,
physiology, morphology, reproductive fitness, economic value, honey
production, pollination capability, resistance to infection and/or
infestation of a bee, a population of bees and/or a bee colony,
directly or indirectly resulting from contact with a bee or bee
colony pathogenic agent.
[0107] As detailed herein, bee feeding is common practice amongst
bee-keepers, for providing both nutritional and other, for example,
supplemental needs. Bees typically feed on honey and pollen, but
have been known to ingest non-natural feeds as well. Bees can be
fed various foodstuffs including, but not limited to Wheast (a
dairy yeast grown on cottage cheese), soybean flour, yeast (e.g.
brewer's yeast, torula yeast) and yeast products-fed singly or in
combination and soybean flour fed as a dry mix or moist cake inside
the hive or as a dry mix in open feeders outside the hive. Also
useful is sugar, or a sugar syrup. The addition of 10 to 12 percent
pollen to a supplement fed to bees can be used improve
palatability. The addition of 25 to 30 percent pollen can be used
to improve the quality and quantity of essential nutrients that are
required by bees for vital activity.
[0108] Cane or beet sugar, isomerized corn syrup, and type-50 sugar
syrup can be substituted for honey in the natural diet of honey
bees. The last two can be supplied as a liquid to bees. Liquid feed
can be supplied to bees inside the hive by, for example, any of the
following methods: friction-top pail, combs within the brood
chamber, division board feeder, boardman feeder, etc. Dry sugar can
be fed by placing a pound or two on an inverted inner cover. A
supply of water can be provided to the bees. In one embodiment,
pans or trays in which floating supports-such as wood chips, cork,
or plastic sponge-are present are envisaged. Detailed descriptions
of supplemental feeds for bees can be found in, for example, USDA
publication by Standifer, et al 1977, entitled "Supplemental
Feeding of Honey Bee Colonies" (USDA, Agriculture Information
Bulletin No. 413).
[0109] Bees in a hive are potentially susceptible to the pathogenic
diseases described above. Thus, according to some embodiments, the
bees can be honeybees, forager bees, hive bees and the like.
[0110] Methods for reducing the susceptibility of a bee colony or
bee-hive to bee pathogens by feeding oligonucleotides and/or
polynucleotides are envisaged. Thus, in some embodiments, the
present invention can be used to benefit any numbers of bees, from
a few in the hive, to the entire bee population within a hive and
its surrounding area. It will be appreciated, that in addition to
feeding of oligonucleotides and/or polynucleotides for reduction of
the bee pathogen infection and infestation, enforcement of proper
sanitation (for example, refraining from reuse of infested hives)
can augment the effectiveness of treatment and prevention of
infections.
[0111] According to an aspect of some embodiments of the present
invention there is provided a method for increasing the tolerance
of a bee to a disease caused by a pathogen comprising feeding the
bee an effective amount of the oligonucleotide comprising a nucleic
acid sequence down-regulating expression of a gene product of a bee
pathogen or a nucleic acid construct comprising the
oligonucleotide, thereby increasing the tolerance of the bee to the
pathogen.
[0112] According to a further aspect of some embodiments described
herein there is provided a method for increasing the tolerance of a
bee colony to a disease caused by a pathogen comprising feeding
bees of the colony an effective amount of the oligonucleotide
composition comprising a nucleic acid sequence down-regulating
expression of a gene product of a bee pathogen or a nucleic acid
construct comprising the oligonucleotide, thereby increasing the
tolerance of the colony to the pathogen.
[0113] According to some embodiments of the invention the bee is a
honeybee, including e.g., a forager, a drone bee, a hive bee or a
queen bee. In some embodiments a composition comprising an
oligonucleotide is administered to bees to treat and/or prevent
Colony Collapse Disorder, and/or infection by Israel Acute
Paralysis Virus.
[0114] According to some embodiments of the invention the feeding
comprises providing a liquid bee-ingestible composition or a solid
bee-ingestible composition.
[0115] The methods and compositions described herein can also be
used to increase the tolerance of bees to Colony Collapse Disorder
(CCD), the method comprising feeding to the honeybee hive an
effective amount of an oligonucleotide (e.g., double stranded
ribonucleic nucleic acid (RNA), said double stranded RNA being
homologous to a contiguous sequence of at least 21 nucleotides of
Israel Acute Paralysis Virus) and a delivery agent.
[0116] A non-limiting list of exemplary disease-causing pathogens,
and diseases of bees and bee colonies associated with the
pathogenic agents, suitable for treatment according to some
embodiments of the methods and compositions of the present
invention is found in Table I below. The complete genomes of
several known isolates of IAPV and information on possible
phylogenic relationships between strains that can be similarly
targeted with the methods and compositions of the present invention
are provided in Palacios et al. 2008 (published online ahead of
print on 23 Apr. 2008, Journal of Virology)
TABLE-US-00001 TABLE I Bee and Bee Colony Pathogens Parasitic
Organism Genes Parasitic Organism Genes Acute bee paralysis Acute
bee paralysis virus, complete genome. virus Accession NC_002548
Israel acute paralysis Accession: NC_009025, israel acute paralysis
virus virus of bees, complete genome Deformed wing virus Deformed
wing virus, complete genome. Accession NC_004830 Kashmir bee virus
Accession: AY275710, kashmir bee virus, complete genome Black queen
cell virus Black queen cell virus strain poland-6 non- structural
polyprotein and structural polyprotein genes, complete cds.
Accession: EF517521 Chronic paralysis Chronic bee paralysis virus
rna 2, complete virus sequence. Accession: NC_010712 Cloudy wing
virus Cloudy wing virus rna polymerase (pol) gene, partial cds.
Accession AF034543 Paenibacillus larvae Accession: NZ_AARF01000646,
whole genome (American Foul (shotgun) sequenced. Brood)
Melissococcus pluton Accession: EF666055 Melissococcus plutonius
(European Foul superoxide dismutase (soda) gene Brood) Nosema apis,
1) Accession DQ996230, Nosema apis RNA polymerase II largest
subunit 2) Accessions EU545140, EF584425, EF584423, EF584418 all
are 16S ribosomal RNA gene Nosema cerana F091883, EF091884, and
EF091885 are accessions of 5S ribosomal RNA gene, intergenic
spacer, and small subunit ribosomal RNA gene.
[0117] For example, a suitable bee pathogen siRNA can be an
IAPV-specific oligonucleotide corresponding to IAPV sequences as
described in WO2009/060429, which is herein incorporated by
reference in its entirety. Additional suitable bee pathogen siRNAs
can be designed according to sequences from any bee pathogens, for
example, the sequences detailed in Table I, including, but not
limited to Acute Bee Paralysis Virus, Deformed Wing Virus, Kashmir
Bee Virus, Black Queen Cell Virus, Chronic Paralysis Virus, Cloudy
Wing Virus, Paenibacillus larvae, Melissococcus pluton, Nosema
apis, and Nosema cerana (described in WO2009/064029, herein
incorporated by reference in its entirety.
[0118] Multiple bee-pathogen sequences can be designed to include
sequences suitable for producing oligonucleotides effective against
more than one bee pathogen, such as the multiple bee-virus dsRNA
described in detail in WO2009/064029, herein incorporated by
reference in its entirety. Such multiple bee-pathogen dsRNA can be
of the long or short variety, and can include sequences
corresponding to homologous sequences within a class of bee
pathogens (multiple bee-virus sequences, for example), or sequences
corresponding to diverse classes of pathogens (e.g.
viral+bacterial+fungal sequences, etc). Further, multiple sequences
can be designed to include two or more oligonucleotides (e.g.,
dsRNA sequences) of the same bee-pathogen.
[0119] According to yet another embodiment of the present
invention, synthesis of RNA silencing agents suitable for use with
the present invention can be effected according to bee pathogen
target sequences known to integrate into the host genome, target
sequences suspected associated with resistance to a bee pathogen
infection, target sequences representing intergenic regions of the
bee pathogen genome and pathogen-specific sequences shown to be
critical for pathogen growth and/or replication. It will be
appreciated that, in a further embodiment of the present invention,
oligonucleotides targeted to sequences having a conserved homology
between different strains of the bee pathogen, or even between
diverse bee pathogens, once such sequences are identified, can be
effective against more than one strain of the bee pathogen, or even
against different bee pathogens.
[0120] For example, a suitable antisense oligonucleotide targeted
against the IAPV mRNA would be of the sequences as described in
WO2009/064029, herein incorporated by reference in its
entirety.
Transgenic Plants
[0121] In another aspect, the oligonucleotide can be administered
to the insect via contact with a plant expressing the
oligonucleotide. The plant can be engineered to express the
oligonucleotide in all or some tissues via transformation with an
appropriate construct In one embodiment, the insect can be a pest
insect which ingests a part of the plant. Alternatively, the insect
can have a beneficial function, and comes into contact with the
oligonucleotide expressed in the plant or a portion thereof (e.g.,
the oligonucleotide can be expressed in the pollen).
[0122] The term "transgenic plant cell" or "transgenic plant"
refers to a plant cell or a plant that expresses an
oligonucleotide, as that term is used herein. The transgenic plants
are also meant to comprise progeny (decedent, offspring, etc.) of
any generation of such a transgenic plant or a seed of any
generation of all such transgenic plants wherein said progeny or
seed comprises an oligonucleotide, or fragment thereof.
[0123] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single simple recombinant DNA sequence
inserted into one chromosome and is referred to as a transgenic
event. Such transgenic plants can be referred to as being
heterozygous for the inserted exogenous sequence. A transgenic
plant homozygous with respect to a transgene can be obtained by
sexually mating (selfing) an independent segregant transgenic plant
that contains a single exogenous gene sequence to itself, for
example an FO plant, to produce F1 seed. One fourth of the F1 seed
produced will be heterozygous with respect to the transgene.
Germinating F1 seed results in plants that can be tested for
heterozygosity, typically using a SNP assay or a thermal
amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay). Crossing a
heterozygous plant with itself or another heterozygous plant
results in only heterozygous progeny.
[0124] In addition to direct transformation of a plant with a
recombinant DNA construct, transgenic plants can be prepared by
crossing a first plant having a recombinant DNA construct with a
second plant lacking the construct. For example, recombinant DNA
for gene suppression can be introduced into a first plant line that
is amenable to transformation to produce a transgenic plant which
can be crossed with a second plant line to introgress the
recombinant DNA for gene suppression into the second plant
line.
[0125] Transgenic plants, that can be generated for use with the
methods and compositions described herein include, but are not
limited to, alfalfa, aneth, apple, apricot, artichoke, arugula,
asparagus, avocado, banana, barley, beans, beet, blackberry,
blueberry, broccoli, brussel sprouts, cabbage, canola, cantaloupe,
carrot, cassava, cauliflower, celery, cherry, cilantro, citrus,
clementine, coffee, corn, cotton, cucumber, Douglas fir, eggplant,
endive, escarole, eucalyptus, fennel, figs, gourd, grape,
grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon,
lime, Loblolly pine, mango, melon, mushroom, nut, oat, okra, onion,
orange, an ornamental plant, papaya, parsley, pea, peach, peanut,
pear, pepper, persimmon, pine, pineapple, plantain, plum,
pomegranate, poplar, potato, pumpkin, quince, radiata pine,
radicchio, radish, raspberry, rice, rye, sorghum, Southern pine,
soybean, spinach, squash, strawberry, sugarbeet, sugarcane,
sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato,
turf, a vine, watermelon, wheat, yams, and zucchini.
[0126] The skilled artisan will recognize that a wide variety of
transformation techniques exist in the art, and new techniques are
continually becoming available. Any technique that is suitable for
the target host plant can be employed within the scope of the
present invention. For example, the constructs can be introduced in
a variety of forms including, but not limited to as a strand of
DNA, in a plasmid, or in an artificial chromosome. The introduction
of the constructs into the target plant cells can be accomplished
by a variety of techniques, including, but not limited to
Agrobacterium-mediated transformation, electroporation,
microinjection, microprojectile bombardment calcium-phosphate-DNA
co-precipitation or liposome-mediated transformation of a
heterologous nucleic acid. The transformation of the plant is
preferably permanent, i.e. by integration of the introduced
expression constructs into the host plant genome, so that the
introduced constructs are passed onto successive plant
generations.
[0127] Any promoter capable of driving expression in the plant of
interest may be used in the practice of the invention. The promoter
may be native or analogous or foreign or heterologous to the plant
host. The choice of promoters to be included depends upon several
factors, including, but not limited to, efficiency, selectability,
inducibility, desired expression level, and cell- or
tissue-preferential expression. It is a routine matter for one of
skill in the art to modulate the expression of a sequence by
appropriately selecting and positioning promoters and other
regulatory regions relative to that sequence.
[0128] Promoters active in photosynthetic tissue in order to drive
transcription in green tissues such as leaves and stems are of
particular interest for the present invention. Most suitable are
promoters that drive expression only or predominantly in such
tissues. The promoter may confer expression constitutively
throughout the plant, or differentially with respect to the green
tissues, or differentially with respect to the developmental stage
of the green tissue in which expression occurs, or in response to
external stimuli.
[0129] Examples of such promoters include the
ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the
RbcS promoter from eastern larch (Larix laricina), the pine cab6
promoter (Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778),
the Cab-1 gene promoter from wheat (Fejes et al. (1990) Plant Mol.
Biol. 15:921-932), the CAB-1 promoter from spinach (Lubberstedt et
al. (1994) Plant Physiol. 104:997-1006), the cab1 promoter from
rice (Luan et al. (1992) Plant Cell 4:971-981), the pyruvate
orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al.
(1993) Proc Natl Acad Sci USA 90:9586-9590), the tobacco Lhcbl*2
promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), the
Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit
et al. (1995) Planta 196:564-570), and thylakoid membrane protein
promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab,
rbcS. Other promoters that drive transcription in stems, leafs and
green tissue are described in U.S. Patent Publication No.
2007/0006346. The TrpA promoter is a pith preferred promoter and
has been described in U.S. Pat. No. 6,018,104.
[0130] A maize gene encoding phosphoenol carboxylase (PEPC) has
been described by Hudspeth & Grula (Plant Molec Biol 12:
579-589 (1989)). Using standard molecular biological techniques the
promoter for this gene can be used to drive the expression of any
gene in a green tissue-specific manner in transgenic plants.
[0131] In some other embodiments of the present invention,
inducible promoters may be desired. Inducible promoters drive
transcription in response to external stimuli such as chemical
agents or environmental stimuli. For example, inducible promoters
can confer transcription in response to hormones such as giberellic
acid or ethylene, or in response to light or drought.
Oligonucleotides
[0132] In the context of this invention, the term "oligonucleotide"
refers to a polymer or oligomer of nucleotide or nucleoside
monomers consisting of naturally occurring bases, sugars and
intersugar (backbone) linkages. The term "oligonucleotide" also
includes polymers or oligomers comprising non-naturally occurring
monomers, or portions thereof, which function similarly. Such
modified or substituted oligonucleotides are often preferred over
native forms because of properties such as, for example, enhanced
cellular uptake and increased stability in the presence of
nucleases.
[0133] The oligonucleotide as used herein can be single-stranded or
double-stranded. A single-stranded oligonucleotide can have
double-stranded regions and a double-stranded oligonucleotide can
have single-stranded regions. Exemplary oligonucleotides include,
but are not limited to structural genes, genes including control
and termination regions, self-replicating systems such as viral or
plasmid DNA, single-stranded and double-stranded siRNAs and other
RNA interference reagents (RNAi agents or iRNA agents), shRNA,
antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics,
supermirs, aptamers, antimirs, antagomirs, triplex-forming
oligonucleotides, RNA activators, and decoy oligonucleotides.
[0134] Double-stranded and single-stranded oligonucleotides that
are effective in inducing RNA interference are also referred to as
siRNA, RNAi agent, or iRNA agent, herein. These RNA interference
inducing oligonucleotides associate with a cytoplasmic
multi-protein complex known as RNAi-induced silencing complex
(RISC). In many embodiments, single-stranded and double-stranded
RNAi agents are sufficiently long that they can be cleaved by an
endogenous molecule, e.g. by Dicer, to produce smaller
oligonucleotides that can enter the RISC machinery and participate
in RISC mediated cleavage of a target sequence, e.g. a target
mRNA.
[0135] Oligonucleotides of the present invention can be of various
lengths. In particular embodiments, oligonucleotides can range from
about 10 to 100 nucleotides in length. In various related
embodiments, oligonucleotides, single-stranded, double-stranded,
and triple-stranded, can range in length from about 10 to about 50
nucleotides, from about 20 to about 50 nucleotides, from about 15
to about 30 nucleotides, from about 20 to about 30 nucleotides in
length. In certain embodiments, oligonucleotide is from about 9 to
about 39 nucleotides in length. In some other embodiments,
oligonucleotide is at least 30 nucleotides in length.
[0136] The oligonucleotides of the invention can comprise any
oligonucleotide modification described herein and below. In certain
instances, it can be desirable to modify one or both strands of a
double-stranded oligonucleotide. In some cases, the two strands
will include different modifications. In other instances, multiple
different modifications can be included on each of the strands. The
various modifications on a given strand can differ from each other,
and can also differ from the various modifications on other
strands. For example, one strand can have a modification, e.g., a
modification described herein, and a different strand can have a
different modification, e.g., a different modification described
herein. In other cases, one strand can have two or more different
modifications, and the another strand can include a modification
that differs from the at least two modifications on the first
strand.
Double-Stranded Oligonucleotides
[0137] The skilled person is well aware that double-stranded
oligonucleotides comprising a duplex structure of between 20 and
23, but specifically 21, base pairs have been characterized as
particularly effective in inducing RNA interference (Elbashir et
al., EMBO 2001, 20:6877-6888). However, others have found that
shorter or longer double-stranded oligonucleotides can be effective
as well.
[0138] The double-stranded oligonucleotides comprise two
oligonucleotide strands that are sufficiently complementary to
hybridize to form a duplex structure. Generally, the duplex
structure is between 15 and 30, more generally between 18 and 25,
yet more generally between 19 and 24, and most generally between 19
and 21 base pairs in length. In certain embodiments, longer
double-stranded oligonucleotides of between 25 and 30 base pairs in
length are preferred. In certain embodiments, shorter
double-stranded oligonucleotides of between 10 and 15 base pairs in
length are preferred. In another embodiment, the double-stranded
oligonucleotide is at least 21 nucleotides long.
[0139] In one embodiment, the double-stranded oligonucleotide
comprises a sense strand and an antisense strand, wherein the
antisense RNA strand has a region of complementarity which is
complementary to at least a part of a target sequence, and the
duplex region is 14-30 nucleotides in length. Similarly, the region
of complementarity to the target sequence is between 14 and 30,
more generally between 18 and 25, yet more generally between 19 and
24, and most generally between 19 and 21 nucleotides in length.
[0140] By "target sequence" or "target gene" is meant any nucleic
acid sequence whose expression or activity is to be modulated. The
target nucleic acid can be DNA or RNA, such as endogenous DNA or
RNA, viral DNA or viral RNA, or other RNA encoded by a gene, virus,
bacteria, fungus, insect or plant.
[0141] Suitable oligonucleotides can be designed according to the
target sequence. Various methods and tools are available to one of
skill in the art to design siRNAs and antisense oligonucleotides
that can target a given target sequence. Exemplary insect and
insect pathogen sequences to be target include, but are not
limited, Acute bee paralysis virus (accession: NC 002548, Israel
acute paralysis virus, (accession: NC.sub.--009025), Deformed wing
virus (accession: NC.sub.--004830, Kashmir bee virus (accession:
AY275710), Black queen cell virus (accession: EF517521), Chronic
paralysis virus (accession: NC.sub.--010712), Cloudy wing virus
(accession: AF034543), Paenibacillus larvae (accession:
NZ_AARF01000646), Melissococcus pluton (European Foul Brood,
accession: EF666055), Ascophaera apis (Chalkbrood), Nosema apis
(accession: DQ996230, EU545140, EF584425, EF584423 and EF584418),
Nosema cerana (accession: EF091883, EF091884, and EF091885),
Spodoptera frugiperda ascovirus 1a (accession: NC008361), Triatoma
virus (accession: NC003783 and AF178440), HZ-1 insect virus late
gene (accession: L8840), Autographa californica nuclear
polyhedrosis virus helicase gene (M57687), Spodoptera frugiperda
ascovirus 1a (accession: AM398843), Nudaureila capensis omega virus
capid protein (accession: S43937), and Trichoplusia ni granulovirus
(accession: AF079223).
[0142] 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, /. 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, 10 out of 10 being 50%,
60%, 70%, 80%, 90%, and 100% complementary). "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.
[0143] In many embodiments, the double-stranded oligonucleotide is
sufficiently large that it can be cleaved by an endogenous
molecule, e.g., by Dicer, to produce smaller double-stranded
oligonucleotides, e.g., RNAi agents. In one embodiment, the
double-stranded oligonucleotide modulates the expression of a
target gene via RISC mediated cleavage of the target sequence.
[0144] In certain embodiments, the double-stranded region of a
double-stranded oligonucleotide is equal to or at least, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27,
28, 29, or 30 nucleotide pairs in length.
[0145] In certain embodiments, the antisense strand of a
double-stranded oligonucleotide is equal to or at least 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides in length.
[0146] In certain embodiments, the sense strand of a
double-stranded oligonucleotide is equal to or at least 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28,
29, or 30 nucleotides in length.
[0147] In certain embodiments, one strand has at least one stretch
of 1-5 single-stranded nucleotides in the double-stranded region.
In certain other embodiments, both strands have at least one
stretch of 1-5 single-stranded nucleotides in the double stranded
region. When both strands have a stretch of 1-5 single-stranded
nucleotides in the double stranded region, such single-stranded
nucleotides can be opposite to each other or they can be located
such that the second strand has no single-stranded nucleotides
opposite to the single-stranded oligonucleotides of the first
strand and vice versa.
[0148] In certain embodiments, each strand of the double-stranded
oligonucleotide has a ZXY structure, such as is described in PCT
Application No. PCT/US2004/07070 filed on Mar. 8, 2004, contents of
which are hereby incorporated in their entireties.
Hairpins and Dumbbells
[0149] The present invention also includes double-stranded
oligonucleotide wherein the two strands are linked together. The
two strands be linked to each other at both ends, or at one end
only. The two strands can be linked together by an oligonucleotide
linker including, but not limited to, (N).sub.n; wherein N is
independently a modified or unmodified nucleotide and n is 3-23.
Some of the nucleotides in the linker can be involved in base-pair
interactions with other nucleotides in the loop. The two strands
can also be linked together by a non-nucleosidic linker, e.g. a
linker described herein. It will be appreciated by one of skill in
the art that any oligonucleotide chemical modifications or
variations describe herein can be used in the oligonucleotide
linker.
[0150] Hairpin and dumbbell type RNAi agents will have a duplex
region equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 nucleotide pairs. The duplex region can be equal to or
less than 200, 100, or 50, in length. In certain embodiments,
ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19
to 21 nucleotides pairs in length.
[0151] The hairpin RNAi agents can have a single strand overhang or
terminal unpaired region, in some embodiments at the 3', and in
certain embodiments on the antisense side of the hairpin. In
certain embodiments, the overhangs are 1-4, more generally 2-3
nucleotides in length.
[0152] The hairpin oligonucleotides are also referred to as "shRNA"
herein.
Single-Stranded Oligonucleotides
[0153] The single-stranded oligonucleotides of the present
invention also comprise nucleotide sequence that is substantially
complementary to a "sense" nucleic acid encoding a gene expression
product, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an RNA sequence,
e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. The single-stranded
oligonucleotides of the invention include antisense
oligonucleotides, single-stranded RNAi agents, antimirs and triplex
forming oligonucleotides. The region of complementarity can be less
than 30 nucleotides in length, and at least 15 nucleotides in
length. Generally, the single stranded oligonucleotides are 10 to
25 nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, or 24 nucleotides in length). In one embodiment the
strand is 25-30 nucleotides. In one embodiment, the single-stranded
oligonucleotide is 15-29 nucleotides in length. Single strands
having less than 100% complementarity to the target mRNA, RNA or
DNA are also embraced by the present invention. In certain
embodiments, the single-stranded oligonucleotide has a ZXY
structure, such as is described in PCT Application No.
PCT/US2004/07070 filed on Mar. 8, 2004.
[0154] The single-stranded oligonucleotide can hybridize to a
complementary RNA, e.g., mRNA, pre-mRNA, and prevent access of the
translation machinery to the target RNA transcript, thereby
preventing protein synthesis. The single-stranded oligonucleotide
can also hybridize to a complementary RNA and the RNA target can be
subsequently cleaved by an enzyme such as RNase H and thus
preventing translation of target RNA. In other embodiments, the
single-stranded oligonucleotide modulates the expression of a
target gene via RISC mediated cleavage of the target sequence.
[0155] A "single-stranded RNAi agent" as used herein, is an RNAi
agent which is made up of a single molecule. A single-stranded RNAi
agent can include a duplexed region, formed by intra-strand
pairing, e.g., it can be, or include, a hairpin or pan-handle
structure. Single-stranded RNAi agents can be antisense with regard
to the target molecule. A single-stranded RNAi agent can be
sufficiently long that it can enter the RISC and participate in
RISC mediated cleavage of a target mRNA.
[0156] A single-strand RNAi agent is at least 14, and in other
embodiments at least 15, at least 20, at least 25, at least 29, at
least 35, at least 40, or at least 50 nucleotides in length. In
certain embodiments, it is less than 200, 100, or 60 nucleotides in
length. In certain embodiments single-stranded RNAi agents are 5'
phosphorylated or include a phosphoryl analog at the 5' prime
terminus.
[0157] In certain embodiments, single-stranded RNAi agents and/or
at least one strand of the double-stranded RNAi agent, includes at
least one of the following motifs: [0158] (a) 5'-phosphorothioate
or 5'-phosphorodithioate; [0159] (b) a cationic modification of
nucleotides 1 and 2 on the 5' terminal, wherein the cationic
modification is at C5 position of pyrimidines and C2, C6, C8,
exocyclic N2 or exocyclic N6 of purines; [0160] (c) at least one
G-clamp nucleotide in the first two terminal nucleotides at the 5'
end and the other nucleotide having a cationic modification,
wherein the cationic modification is at C5 position of pyrimidines
or C2, C6, C8, exocyclic N2 or exocyclic N6 position of purines;
[0161] (d) at least one 2'-F modified nucleotide comprising a
nucleobase base modification; [0162] (e) at least one
gem-2'-O-methyl/2'-F modified nucleotide comprising a nucleobase
modification, preferably the methyl substituent is in the up
configuration, e.g. in the arabinose configuration; [0163] (f) a
5'-PuPu-3' dinucleotide at the 3' terminal wherein both nucleotides
comprise a modified MOE at 2'-position as described in U.S.
Provisional Application No. 61/226,017 filed Jul. 16, 2009; [0164]
(g) a 5'-PuPu-3' dinucleotide at the 5' terminal wherein both
nucleotides comprise a modified MOE at 2'-position as described in
U.S. Provisional Application No. 61/226,017 filed Jul. 16, 2009;
[0165] (h) nucleotide at the 5' terminal having a modified MOE at
2'-position as described in U.S. Provisional Application No.
61/226,017 filed Jul. 16, 2009; [0166] (i) nucleotide at the 5'
terminal having a 3'-F modification; [0167] (j) 5' terminal
nucleotide comprising a 4'-substituent; [0168] (k) 5' terminal
nucleotide comprising a replacement of 04' with N(alkyl), S or
CH.sub.2; [0169] (l) 3' terminal nucleotide comprising a
4'-substituent; and [0170] (m) combinations thereof
MicroRNAs
[0171] MicroRNAs (miRNAs or mirs) are a highly conserved class of
small RNA molecules that are transcribed from DNA in the genomes of
plants and animals, but are not translated into protein.
Pre-microRNAs are processed into miRNAs. Processed microRNAs are
single stranded .about.17-25 nucleotide (nt) RNA molecules that
become incorporated into the RNA-induced silencing complex (RISC)
and have been identified as key regulators of development, cell
proliferation, apoptosis and differentiation. They are believed to
play a role in regulation of gene expression by binding to the
3'-untranslated region of specific mRNAs. RISC mediates
down-regulation of gene expression through translational
inhibition, transcript cleavage, or both. RISC is also implicated
in transcriptional silencing in the nucleus of a wide range of
eukaryotes.
[0172] MicroRNAs have also been implicated in modulation of
pathogens in hosts. For example, see Jopling, C. L., et al.,
Science (2005) vol. 309, pp 1577-1581. Without wishing to be bound
by theory, administration of a microRNA, microRNA mimic, and/or
anti microRNA oligonucleotide, leads to modulation of pathogen
viability, growth, development, and/or replication.
[0173] In certain embodiments, the oligonucleotide is a microRNA,
microRNA mimic, and/or anti microRNA, wherein microRNA is a host
microRNA.
[0174] The number of miRNA sequences identified to date is large
and growing, illustrative examples of which can be found, for
example, in: "miRBase: microRNA sequences, targets and gene
nomenclature" Griffiths-Jones S, Grocock R J, van Dongen S, Bateman
A, Enright A J. NAR, 2006, 34, Database Issue, D140-D144; "The
microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database
Issue, D109-D111; and also on the worldwide web at
microrna.dot.sanger.dot.ac.dot.uk/sequences/.
Ribozymes
[0175] Ribozymes are oligonucleotides having specific catalytic
domains that possess endonuclease activity (Kim and Cech, Proc Natl
Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons,
Cell. 1987 Apr. 24; 49(2):211-20). At least six basic varieties of
naturally-occurring enzymatic RNAs are known presently. In general,
enzymatic nucleic acids act by first binding to a target RNA. Such
binding occurs through the target binding portion of an enzymatic
nucleic acid which is held in close proximity to an enzymatic
portion of the molecule that acts to cleave the target RNA. Thus,
the enzymatic nucleic acid first recognizes and then binds a target
RNA through complementary base-pairing, and once bound to the
correct site, acts enzymatically to cut the target RNA. Strategic
cleavage of such a target RNA will destroy its ability to direct
synthesis of an encoded protein. After an enzymatic nucleic acid
has bound and cleaved its RNA target, it is released from that RNA
to search for another target and can repeatedly bind and cleave new
targets.
[0176] Methods of producing a ribozyme targeted to any target
sequence are known in the art. Ribozymes can be designed as
described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat.
Appl. Publ. No. WO 94/02595, each specifically incorporated herein
by reference, and synthesized to be tested in vitro and in vivo, as
described therein.
Aptamers
[0177] Aptamers are nucleic acid or peptide molecules that bind to
a particular molecule of interest with high affinity and
specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and
Szostak, Nature 346:818 (1990)). DNA or RNA aptamers have been
successfully produced which bind many different entities from large
proteins to small organic molecules. See Eaton, Curr. Opin. Chem.
Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol.
9:324-9(1999), and Hermann and Patel, Science 287:820-5 (2000).
Aptamers can be RNA or DNA based. Generally, aptamers are
engineered through repeated rounds of in vitro selection or
equivalently, SELEX (systematic evolution of ligands by exponential
enrichment) to bind to various molecular targets such as small
molecules, proteins, nucleic acids, and even cells, tissues and
organisms. The aptamer can be prepared by any known method,
including synthetic, recombinant, and purification methods, and can
be used alone or in combination with other aptamers specific for
the same target. Further, as described more fully herein, the term
"aptamer" specifically includes "secondary aptamers" containing a
consensus sequence derived from comparing two or more known
aptamers to a given target.
Decoy Oligonucleotides
[0178] Because transcription factors recognize their relatively
short binding sequences, even in the absence of surrounding genomic
DNA, short oligonucleotides bearing the consensus binding sequence
of a specific transcription factor can be used as tools for
manipulating gene expression in living cells. This strategy
involves the intracellular delivery of such "decoy
oligonucleotides", which are then recognized and bound by the
target factor. Occupation of the transcription factor's DNA-binding
site by the decoy renders the transcription factor incapable of
subsequently binding to the promoter regions of target genes.
Decoys can be used as therapeutic agents, either to inhibit the
expression of genes that are activated by a transcription factor,
or to upregulate genes that are suppressed by the binding of a
transcription factor. Examples of the utilization of decoy
oligonucleotides can be found in Mann et al., J. Clin. Invest.,
2000, 106: 1071-1075, which is expressly incorporated by reference
herein, in its entirety.
miRNA Mimics
[0179] miRNA mimics represent a class of molecules that can be used
to imitate the gene modulating activity of one or more miRNAs.
Thus, the term "microRNA mimic" refers to synthetic non-coding RNAs
(i.e. the miRNA is not obtained by purification from a source of
the endogenous miRNA) that are capable of entering the RNAi pathway
and regulating gene expression. miRNA mimics can be designed as
mature molecules (e.g. single stranded) or mimic precursors (e.g.,
pri- or pre-miRNAs).
[0180] In one design, miRNA mimics are double stranded molecules
(e.g., with a duplex region of between about 16 and about 31
nucleotides in length) and contain one or more sequences that have
identity with the mature strand of a given miRNA. Double-stranded
miRNA mimics have designs similar to as described above for
double-stranded oligonucleotides.
[0181] In one embodiment, a miRNA mimic comprises a duplex region
of between 16 and 31 nucleotides and one or more of the following
chemical modification patterns: the sense strand contains
2'-O-methyl modifications of nucleotides 1 and 2 (counting from the
5' end of the sense oligonucleotide), and all of the Cs and Us; the
antisense strand modifications can comprise 2' F modification of
all of the Cs and Us, phosphorylation of the 5' end of the
oligonucleotide, and stabilized internucleotide linkages associated
with a 2 nucleotide 3' overhang.
Supermirs
[0182] A supermir refers to an oligonucleotide, e.g., single
stranded, double stranded or partially double stranded, which has a
nucleotide sequence that is substantially identical to an miRNA and
that is antisense with respect to its target. This term includes
oligonucleotides which comprise at least one
non-naturally-occurring portion which functions similarly. In a
preferred embodiment, the supermir does not include a sense strand,
and in another preferred embodiment, the supermir does not
self-hybridize to a significant extent. An supermir featured in the
invention can have secondary structure, but it is substantially
single-stranded under physiological conditions. A supermir that is
substantially single-stranded is single-stranded to the extent that
less than about 50% (e.g., less than about 40%, 30%, 20%, 10%, or
5%) of the supermir is duplexed with itself. The supermir can
include a hairpin segment, e.g., sequence, preferably at the 3' end
can self hybridize and form a duplex region, e.g., a duplex region
of at least 1, 2, 3, or 4 and preferably less than 8, 7, 6, or 5
nucleotides, e.g., 5 nucleotides. The duplexed region can be
connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or
6 dTs, e.g., modified dTs. In another embodiment the supermir is
duplexed with a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10
nucleotides in length, e.g., at one or both of the 3' and 5' end or
at one end and in the non-terminal or middle of the supermir.
Antimirs or miRNA Inhibitors
[0183] The terms "antimir" "microRNA inhibitor" or "miR inhibitor"
are synonymous and refer to oligonucleotides or modified
oligonucleotides that interfere with the activity of specific
miRNAs Inhibitors can adopt a variety of configurations including
single stranded, double stranded (RNA/RNA or RNA/DNA duplexes), and
hairpin designs, in general, microRNA inhibitors comprise one or
more sequences or portions of sequences that are complementary or
partially complementary with the mature strand (or strands) of the
miRNA to be targeted, in addition, the miRNA inhibitor can also
comprise additional sequences located 5' and 3' to the sequence
that is the reverse complement of the mature miRNA. The additional
sequences can be the reverse complements of the sequences that are
adjacent to the mature miRNA in the pri-miRNA from which the mature
miRNA is derived, or the additional sequences can be arbitrary
sequences (having a mixture of A, G, C, U, or dT). In some
embodiments, one or both of the additional sequences are arbitrary
sequences capable of forming hairpins. Thus, in some embodiments,
the sequence that is the reverse complement of the miRNA is flanked
on the 5' side and on the 3' side by hairpin structures. MicroRNA
inhibitors, when double stranded, can include mismatches between
nucleotides on opposite strands. Furthermore, microRNA inhibitors
can be linked to conjugate moieties in order to facilitate uptake
of the inhibitor into a cell.
[0184] MicroRNA inhibitors, including hairpin miRNA inhibitors, are
described in detail in Vermeulen et al., "Double-Stranded Regions
Are Essential Design Components Of Potent Inhibitors of RISC
Function," RNA 13: 723-730 (2007) and in WO2007/095387 and WO
2008/036825 each of which is incorporated herein by reference in
its entirety. A person of ordinary skill in the art can select a
sequence from the database for a desired miRNA and design an
inhibitor useful for the methods disclosed herein.
Antagomirs
[0185] Antagomirs are RNA-like oligonucleotides that harbor various
modifications for RNAse protection and pharmacologic properties,
such as enhanced tissue and cellular uptake. They differ from
normal RNA by, for example, complete 2'-O-methylation of sugar,
phosphorothioate backbone and, for example, a cholesterol-moiety at
3'-end. In a preferred embodiment, antagomir comprises a
2'-O-methylmodification at all nucleotides, a cholesterol moiety at
3'-end, two phsophorothioate backbone linkages at the first two
positions at the 5'-end and four phosphorothioate linkages at the
3'-end of the molecule. Antagomirs can be used to efficiently
silence endogenous miRNAs by forming duplexes comprising the
antagomir and endogenous miRNA, thereby preventing miRNA-induced
gene silencing. An example of antagomir-mediated miRNA silencing is
the silencing of miR-122, described in Krutzfeldt et al, Nature,
2005, 438: 685-689, which is expressly incorporated by reference
herein in its entirety.
RNA Activators
[0186] Recent studies have found that dsRNA can also activate gene
expression, a mechanism that has been termed "small RNA-induced
gene activation" or RNAa. See for example Li, L. C. et al. Proc
Natl Acad Sci USA. (2006), 103(46):17337-42 and Li L. C. (2008).
"Small RNA-Mediated Gene Activation". RNA and the Regulation of
Gene Expression: A Hidden Layer of Complexity. Caister Academic
Press. ISBN 978-1-904455-25-7. It has been shown that dsRNAs
targeting gene promoters induce potent transcriptional activation
of associated genes. Endogenous miRNA that cause RNAa have also
been found in humans. Check E. Nature (2007). 448 (7156):
855-858.
[0187] Another surprising observation is that gene activation by
RNAa is long-lasting. Induction of gene expression has been seen to
last for over ten days. The prolonged effect of RNAa could be
attributed to epigenetic changes at dsRNA target sites.
[0188] In certain embodiments, the oligonucleotide is an RNA
activator, wherein the oligonucleotide increases the expression of
a gene. In one embodiment, increased gene expression inhibits
viability, growth development, and/or reproduction of a pest insect
or an insect pathogen.
Triplex Forming Oligonucleotides
[0189] Studies have shown that triplex forming oligonucleotides
(TFO) can be designed which can recognize and bind to
polypurine/polypyrimidine regions in double-stranded helical DNA in
a sequence-specific manner. These recognition rules are outline by
Maher III, L. J., et al., Science (1989) vol. 245, pp 725-730;
Moser, H. E., et al., Science (1987) vol. 238, pp 645-630; Beal, P.
A., et al., Science (1992) vol. 251, pp 1360-1363; Conney, M., et
al., Science (1988) vol. 241, pp 456-459 and Hogan, M. E., et al.,
EP Publication 375408. Modification of the oligonucleotides, such
as the introduction of intercalators and backbone substitutions,
and optimization of binding conditions (pH and cation
concentration) have aided in overcoming inherent obstacles to TFO
activity such as charge repulsion and instability, and it was
recently shown that synthetic oligonucleotides can be targeted to
specific sequences (for a recent review see Seidman and Glazer, J
Clin Invest 2003; 1 12:487-94). in general, the triplex-forming
oligonucleotide has the sequence correspondence:
TABLE-US-00002 oligo 3'-A G G T duplex 5'-A G C T duplex 3'-T C G
A
[0190] However, it has been shown that the A-AT and G-GC triplets
have the greatest triple helical stability (Reither and Jeltsch,
BMC Biochem, 2002, Se.rho.tl2, Epub). The same authors have
demonstrated that TFOs designed according to the A-AT and G-GC rule
do not form non-specific triplexes, indicating that the triplex
formation is indeed sequence specific.
[0191] Thus for any given sequence a triplex forming sequence can
be devised. Triplex-forming oligonucleotides preferably are at
least 15, more preferably 25, still more preferably 30 or more
nucleotides in length, up to 50 or 100 nucleotides.
[0192] Formation of the triple helical structure with the target
DNA induces steric and functional changes, blocking transcription
initiation and elongation, allowing the introduction of desired
sequence changes in the endogenous DNA and resulting in the
specific down-regulation of gene expression. Examples of such
suppression of gene expression in cells treated with TFOs include
knockout of episomal supFG1 and endogenous HPRT genes in mammalian
cells (Vasquez et al., Nucl Acids Res. 1999; 27: 1176-81, and Puri,
et al, J Biol Chem, 2001; 276:28991-98), and the sequence- and
target specific downregulation of expression of the Ets2
transcription factor, important in prostate cancer etiology
(Carbone, et al, Nucl Acid Res. 2003; 31:833-43), and the
pro-inflammatory ICAM-I gene (Besch et al, J Biol Chem, 2002;
277:32473-79). In addition, Vuyisich. and Beal have recently shown
that sequence specific TFOs can bind to dsRNA, inhibiting activity
of dsRA-dependent enzymes such as RNA-dependent kinases (Vuyisich
and Beal, Nuc. Acids Res 2000; 28:2369-74).
[0193] Additionally, TFOs designed according to the abovementioned
principles can induce directed mutagenesis capable of effecting DNA
repair, thus providing both down-regulation and up-regulation of
expression of endogenous genes (Seidman and Glazer, J Clin Invest
2003; 112:487-94). Detailed description of the design, synthesis
and administration of effective TFOs can be found in U.S. Patent
Application Nos. 2003 017068 and 2003 0096980 to Froehler et al,
and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat.
No. 5,721,138 to Lawn, contents of which are herein incorporated in
their entireties.
Oligonucleotide Modifications
[0194] Unmodified oligonucleotides can be less than optimal in some
applications, e.g., unmodified oligonucleotides can be prone to
degradation by e.g., cellular nucleases. However, chemical
modifications to one or more of the subunits of oligonucleotide can
confer improved properties, e.g., can render oligonucleotides more
stable to nucleases. Typical oligonucleotide modifications can
include one or more of: (i) alteration, e.g., replacement, of one
or both of the non-linking phosphate oxygens and/or of one or more
of the linking phosphate oxygens in the phosphodiester backbone
linkage; (ii) alteration, e.g., replacement, of a constituent of
the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;
(iii) wholesale replacement of the phosphate moiety with
"dephospho" linkers; (iv) modification or replacement of a
naturally occurring base with a non-natural base; (v) replacement
or modification of the ribose-phosphate backbone, e.g. peptide
nucleic acid (PNA); (vi) modification of the 3' end or 5' end of
the oligonucleotide, e.g., removal, modification or replacement of
a terminal phosphate group or conjugation of a moiety, e.g.,
conjugation of a ligand, to either the 3' or 5' end of
oligonucleotide; and (vii) modification of the sugar, e.g., six
membered rings.
[0195] The terms replacement, modification, alteration, and the
like, as used in this context, do not imply any process limitation,
e.g., modification does not mean that one must start with a
reference or naturally occurring ribonucleic acid and modify it to
produce a modified ribonucleic acid bur rather modified simply
indicates a difference from a naturally occurring molecule. As
described below, modifications, e.g., those described herein, can
be provided as asymmetrical modifications.
[0196] A modification described herein can be the sole
modification, or the sole type of modification included on multiple
nucleotides, or a modification can be combined with one or more
other modifications described herein. The modifications described
herein can also be combined onto an oligonucleotide, e.g. different
nucleotides of an oligonucleotide have different modifications
described herein.
[0197] In certain embodiments, the oligonucleotide is a modified
oligonucleotide in that the oligonucleotide comprises at least one
modification, e.g., sugar modification, non-phosphodiester backbone
linkage and/or nucleobase modification.
The Phosphate Group
[0198] The phosphate group can be modified by replacing one of the
oxygens with a different substituent. One result of this
modification to RNA phosphate backbones can be increased resistance
of the oligonucleotide to nucleolytic breakdown. Examples of
modified phosphate groups include phosphorothioate,
phosphoroselenates, borano phosphates, borano phosphate esters,
hydrogen phosphonates, phosphoroamidates, alkyl or aryl
phosphonates and phosphotriesters. In certain embodiments, one of
the non-bridging phosphate oxygen atoms in the phosphate backbone
moiety can be replaced by any of the following: S, Se, BR.sub.3 (R
is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an aryl group,
etc.), H, NR.sub.2 (R is hydrogen, optionally substituted alkyl,
aryl), or OR (R is optionally substituted alkyl or aryl). The
phosphorous atom in an unmodified phosphate group is achiral.
However, replacement of one of the non-bridging oxygens with one of
the above atoms or groups of atoms renders the phosphorous atom
chiral; in other words a phosphorous atom in a phosphate group
modified in this way is a stereogenic center. The stereogenic
phosphorous atom can possess either the "R" configuration (herein
Rp) or the "S" configuration (herein Sp).
[0199] Phosphorodithioates have both non-bridging oxygens replaced
by sulfur. The phosphorus center in the phosphorodithioates is
achiral which precludes the formation of oligonucleotides
diastereomers. Thus, while not wishing to be bound by theory,
modifications to both non-bridging oxygens, which eliminate the
chiral center, e.g. phosphorodithioate formation, can be desirable
in that they cannot produce diastereomer mixtures. Thus, the
non-bridging oxygens can be independently any one of O, S, Se, B,
C, H, N, or OR (R is alkyl or aryl).
[0200] The phosphate linker can also be modified by replacement of
bridging oxygen, (i.e. oxygen that links the phosphate to the
nucleoside), with nitrogen (bridged phosphoroamidates), sulfur
(bridged phosphorothioates) and carbon (bridged
methylenephosphonates). The replacement can occur at the either one
of the linking oxygens or at both linking oxygens. When the
bridging oxygen is the 3'-oxygen of a nucleoside, replacement with
carbon is preferred. When the bridging oxygen is the 5'-oxygen of a
nucleoside, replacement with nitrogen is preferred.
[0201] Modified phosphate linkages where at least one of the oxygen
linked to the phosphate has been replaced or the phosphate group
has been replaced by a non-phosphorous group, are also referred to
as "non-phosphodiester backbone linkage" or "non-phosphodiester
linker."
Replacement of the Phosphate Group
[0202] The phosphate group can be replaced by non-phosphorus
containing connectors, e.g. dephospho linkers. Dephospho linkers
are also referred to as non-phosphodiester linkers herein. While
not wishing to be bound by theory, it is believed that since the
charged phosphodiester group is the reaction center in nucleolytic
degradation, its replacement with neutral structural mimics should
impart enhanced nuclease stability. Again, while not wishing to be
bound by theory, it can be desirable, in some embodiment, to
introduce alterations in which the charged phosphate group is
replaced by a neutral moiety.
[0203] Examples of moieties which can replace the phosphate group
include, but are not limited to, amides (for example amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5') and amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5')), hydroxylamino, siloxane
(dialkylsiloxxane), carboxamide, carbonate, carboxymethyl,
carbamate, carboxylate ester, thioether, ethylene oxide linker,
sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal
(3'-S--CH.sub.2--O-5'), formacetal (3'-O--CH.sub.2--O-5'), oxime,
methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI,
3'-CH.sub.2--N(CH.sub.3)--O-5'), methylenehydrazo,
methylenedimethylhydrazo, methyleneoxymethylimino, ethers
(C3'-O--C5'), thioethers (C3'-S--C5'), thioacetamido
(C3'-N(H)--C(.dbd.O)--CH.sub.2--S--C5', C3'-O--P(O)--O--SS--C5',
C3'-CH.sub.2--NH--NH--C5', 3'-NHP(O)(OCH.sub.3)--O-5' and
3'-NHP(O)(OCH.sub.3)--O-5' and nonionic linkages containing mixed
N, O, S and CH.sub.2 component parts. See for example, Carbohydrate
Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook
Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65).
Preferred embodiments include methylenemethylimino (MMI),
methylenecarbonylamino, amides, carbamate and ethylene oxide
linker.
[0204] One skilled in the art is well aware that in certain
instances replacement of a non-bridging oxygen can lead to enhanced
cleavage of the backbone linkage by the neighboring 2'-OH, thus in
many instances, a modification of a non-bridging oxygen can
necessitate modification of 2'-OH, e.g., a modification that does
not participate in cleavage of the neighboring backbone linkage,
e.g. a "2'-deoxy" modification, e.g., arabinose sugar, 2'-O-alkyl,
2'-F, LNA and ENA.
[0205] Preferred non-phosphodiester backbone linkages include
phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more enantiomeric
excess of Sp isomer, phosphorothioates with an at least 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more
enantiomeric excess of Rp isomer, phosphorodithioates,
phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters
(e.g., methyl-phosphonate), selenophosates, phosphoramidates (e.g.,
N-alkylphosphoramidate), and boranophosphonates.
Replacement of Ribophosphate Backbone
[0206] Oligonucleotide-mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates. While not
wishing to be bound by theory, it is believed that the absence of a
repetitively charged backbone diminishes binding to proteins that
recognize polyanions (e.g. nucleases). Again, while not wishing to
be bound by theory, it can be desirable in some embodiment, to
introduce alterations in which the bases are tethered by a neutral
surrogate backbone. Examples include the morpholino, cyclobutyl,
pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA
(aegPNA) and backbone-extended pyrrolidine PNA (bepPNA) nucleoside
surrogates. A preferred surrogate is a PNA surrogate.
Sugar Modifications
[0207] An oligonucleotide can include modification of all or some
of the sugar groups of the nucleic acid. E.g., the 2' hydroxyl
group (OH) can be modified or replaced with a number of different
"oxy" or "deoxy" substituents. While not being bound by theory,
enhanced stability is expected since the hydroxyl can no longer be
deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can
catalyze degradation by intramolecular nucleophilic attack on the
linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0208] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR, n=1-50; "locked"
nucleic acids (LNA) in which the oxygen at the 2' position is
connected by (CH.sub.2).sub.n, wherein n=1-4, to the 4' carbon of
the same ribose sugar, preferably n is 1 (LNA) or 2 (ENA); O-AMINE
or O--(CH.sub.2).sub.nAMINE (n=1-10, AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, ethylene diamine or polyamino); and
O--CH.sub.2CH.sub.2(NCH.sub.2CH.sub.2NMe.sub.2).sub.2.
[0209] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the single-strand
overhangs); halo (e.g., fluoro); amino (e.g. NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino); --NHC(O)R (R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl;
alkenyl and alkynyl, which can be optionally substituted with e.g.,
an amino functionality.
[0210] Other suitable 2'-modifications, e.g., modified MOE, are
described in U.S. Provisional Application No. 61/226,017 filed Jul.
16, 2009, contents of which are herein incorporated by
reference.
[0211] A modification at the 2' position can be present in the
arabinose configuration The term "arabinose configuration" refers
to the placement of a substituent on the C2' of ribose in the same
configuration as the 2'-OH is in the arabinose.
[0212] The sugar group can comprise two different modifications at
the same carbon in the sugar, e.g., gem modification. The sugar
group can also contain one or more carbons that possess the
opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, an oligonucleotide can
include nucleotides containing e.g., arabinose, as the sugar. The
monomer can have an alpha linkage at the 1' position on the sugar,
e.g., alpha-nucleosides. The monomer can also have the opposite
configuration at the 4'-position, e.g., C5' and H4' or substituents
replacing them are interchanged with each other. When the C5' and
H4' or substituents replacing them are interchanged with each
other, the sugar is said to be modified at the 4' position.
[0213] Oligonucleotides can also include "abasic" sugars, which
lack a nucleobase at C-1'. These abasic sugars can also be further
containing modifications at one or more of the constituent sugar
atoms. Oligonucleotides can also contain one or more sugars that
are the L isomer, e.g. L-nucleosides. Modification to the sugar
group can also include replacement of the 4'-O with a sulfur,
optionally substituted nitrogen or CH.sub.2 group. In certain
embodiments, linkage between C1' and nucleobase is in the a
configuration.
[0214] Modifications can also include acyclic nucleotides, wherein
at least one of ribose carbons (C1', C2', C3', C4' or C5') are
independently or in combination absent from the nucleotide, e.g.,
acyclic nucleotide. In certain embodiments, acyclic nucleotide
is
##STR00001##
wherein R.sub.1 and R.sub.2 independently are H, halogen, OR.sub.3,
or alkyl; and R.sub.3 is H, alkyl, cycloalkyl, aryl, aralkyl,
heteroaryl or sugar).
[0215] Preferred sugar modifications are 2'-H, 2'-O-Me
(2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F,
2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl,
2'-O--CH.sub.2-(4'-C) (LNA), 2'-O--CH.sub.2CH.sub.2-(4'-C) (ENA),
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) and gem 2'-OMe/2'F
with 2'-O-Me in the arabinose configuration.
[0216] It is to be understood that when a particular nucleotide is
linked through its 2'-position to the next nucleotide, the sugar
modifications described herein can be placed at the 3'-position of
the sugar for that particular nucleotide, e.g., the nucleotide that
is linked through its 2'-position. A modification at the 3'
position can be present in the xylose configuration The term
"xylose configuration" refers to the placement of a substituent on
the C3' of ribose in the same configuration as the 3'-OH is in the
xylose sugar.
[0217] The hydrogen attached to C4' and/or C1' can be replaced by a
straight- or branched-optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, wherein
backbone of the alkyl, alkenyl and alkynyl can contain one or more
of O, S, S(O), SO.sub.2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'),
CH(Z'), phosphorous containing linkage, optionally substituted
aryl, optionally substituted heteroaryl, optionally substituted
heterocyclic or optionally substituted cycloalkyl, where R' is
hydrogen, acyl or optionally substituted aliphatic, Z' is selected
from the group consisting of OR.sub.11, COR.sub.11,
CO.sub.2R.sub.11,
##STR00002##
NR.sub.21R.sub.31, CONR.sub.21R.sub.31, CON(H)NR.sub.21R.sub.31,
ONR.sub.21R.sub.31, CON(H)N.dbd.CR.sub.41R.sub.51,
N(R.sub.21)C(.dbd.NR.sub.31)NR.sub.21R.sub.31,
N(R.sub.21)C(O)NR.sub.21R.sub.31, N(R.sub.21)C(S)NR.sub.21R.sub.31,
OC(O)NR.sub.21R.sub.31, SC(O)NR.sub.21R.sub.31,
N(R.sub.21)C(S)OR.sub.11, N(R.sub.21)C(O)OR.sub.11,
N(R.sub.21)C(O)SR.sub.11, N(R.sub.21)N.dbd.CR.sub.41R.sub.51,
ON.dbd.CR.sub.41R.sub.51, SO.sub.2R.sub.11, SOR.sub.11, SR.sub.11
and substituted or unsubstituted heterocyclic; R.sub.21 and
R.sub.31 for each occurrence are independently hydrogen, acyl,
unsubstituted or substituted aliphatic, aryl, heteroaryl,
heterocyclic, OR.sub.11, COR.sub.11, CO.sub.2R.sub.11, or
NR.sub.11R.sub.11'; or R.sub.21 and R.sub.31, taken together with
the atoms to which. they are attached, form a heterocyclic ring;
R.sub.41 and R.sub.51 for each occurrence are independently
hydrogen, acyl, unsubstituted or substituted aliphatic, aryl,
heteroaryl, heterocyclic, OR.sub.11, COR.sub.11, or
CO.sub.2R.sub.11, or NR.sub.11R.sub.11'; and R.sub.11 and R.sub.11'
are independently hydrogen, aliphatic, substituted aliphatic, aryl,
heteroaryl, or heterocyclic. In one embodiment, the hydrogen
attached to the C4' of the 5' terminal nucleotide is replaced.
[0218] In certain embodiments, C4' and C5' together form an
optionally substituted heterocyclic, preferably comprising at least
one --PX(Y)-, wherein X is H, OH, OM, SH, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted
alkylthio, optionally substituted alkylamino or optionally
substituted dialkylamino, where M is independently for each
occurrence an alki metal or transition metal with an overall charge
of +1; and Y is O, S, or NR', where R' is hydrogen, optionally
substituted aliphatic. Preferably this modification is at the 5
terminal of the oligonucleotide.
Terminal Modifications
[0219] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. For example, the 3' and/or 5' ends of an oligonucleotide
can be conjugated to other functional molecular entities such as
labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA,
fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on
sulfur, silicon, boron or ester). The functional molecular entities
can be attached to the sugar through a phosphate group and/or a
linker. The terminal atom of the linker can connect to or replace
the linking atom of the phosphate group or the C-3' or C-5' O, N, S
or C group of the sugar. Alternatively, the linker can connect to
or replace the terminal atom of a nucleotide surrogate (e.g.,
PNAs).
[0220] When a linker/phosphate-functional molecular
entity-linker/phosphate array is interposed between two strands of
a dsRNA, this array can substitute for a hairpin RNA loop in a
hairpin-type RNA agent.
[0221] Terminal modifications useful for modulating activity
include modification of the 5' end with phosphate or phosphate
analogs. For example, in certain embodiments antisense strands of
dsRNAs, are 5' phosphorylated or include a phosphoryl analog at the
5' terminus. 5'-phosphate modifications include those which are
compatible with RISC mediated gene silencing. In certain
embodiments, the 5'-end of the oligonucleotide comprises the
modification
##STR00003##
wherein W, X and Y are each independently selected from the group
consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR.sub.3
(R is hydrogen, alkyl, aryl), BH.sub.3.sup.-, C (i.e. an alkyl
group, an aryl group, etc. . . . ), H, NR.sub.2 (R is hydrogen,
alkyl, aryl), or OR (R is hydrogen, alkyl or aryl); A and Z are
each independently for each occurrence absent, O, S, CH.sub.2, NR
(R is hydrogen, alkyl, aryl), or optionally substituted alkylene,
wherein backbone of the alkylene can comprise one or more of O, S,
SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the
end; and n is 0-2. It is understood that A is replacing the oxygen
linked to 5' carbon of sugar. When n is 0, W and Y together with
the P to which they are attached can form an optionally substituted
5-8 membered heterocyclic, wherein W an Y are each independently O,
S, NR' or alkylene. Preferably the heterocyclic is substituted with
an aryl or heteroaryl. In certain embodiments, one or both hydrogen
on C5' of the 5'-terminal nucleotides are replaced with a halogen,
e.g., F.
[0222] Exemplary 5'-modifications include, but are not limited to,
5'-monophosphate ((HO).sub.2(O)P--O-5); 5'-diphosphate
((HO).sub.2(O)P--O--P(HO)(O)--O-5); 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5);
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5);
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); 5'-alpha-thiotriphosphate;
5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate;
5'-phosphoramidates ((HO).sub.2(O)P--NH-5',
(HO)(NH.sub.2)(O)P--O-5'). Other 5'-modification include
5'-alkylphosphonates (R(OH)(O)P--O-5', R=alkyl, e.g., methyl,
ethyl, isopropyl, propyl, etc. . . . ), 5'-alkyletherphosphonates
(R(OH)(O)P--O-5', R=alkylether, e.g., methoxymethyl (CH.sub.2OMe),
ethoxymethyl, etc. . . . ). Other exemplary 5'-modifications
include where Z is optionally substituted alkyl at least once,
e.g.,
((HO).sub.2(X)P--O[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
((HO)2(X)P--O[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
((HO)2(X)P-[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5'; dialkyl
terminal phosphates and phosphate mimics:
HO[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
H.sub.2N[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
H[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
Me.sub.2N[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
HO[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
H.sub.2N[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
H[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
Me.sub.2N[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5', wherein a and
b are each independently 1-10. Other embodiments, include
replacement of oxygen and/or sulfur with BH.sub.3, BH.sub.3.sup.-
and/or Se.
[0223] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorescein or an Alexa dye, e.g.,
Alexa 488. Terminal modifications can also be useful for enhancing
uptake, useful modifications for this include targeting ligands.
Terminal modifications can also be useful for cross-linking an
oligonucleotide to another moiety; modifications useful for this
include mitomycin C, psoralen, and derivatives thereof.
Nucleobases
[0224] Adenine, cytosine, guanine, thymine and uracil are the most
common bases (or nucleobases) found in nucleic acids. These bases
can be modified or replaced to provide oligonucleotides having
improved properties. For example, nuclease resistant
oligonucleotides can be prepared with these bases or with synthetic
and natural nucleobases (e.g., inosine, xanthine, hypoxanthine,
nubularine, isoguanisine, or tubercidine) and any one of the above
modifications. Alternatively, substituted or modified analogs of
any of the above bases and "universal bases" can be employed. When
a natural base is replaced by a non-natural and/or universal base,
the nucleotide is said to comprise a modified nucleobase and/or a
nucleobase modification herein. Modified nucleobase and/or
nucleobase modifications also include natural, non-natural and
universal bases, which comprise conjugated moieties, e.g. a ligand
described herein. Preferred conjugate moieties for conjugation with
nucleobases include cationic amino groups which can be conjugated
to the nucleobase via an appropriate alkyl, alkenyl or a linker
with an amide linkage. Examples of non-natural bases include, but
are not limited to, 2-(halo)adenine, 2-(alkyl)adenine,
2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,
2-(aminopropyl)adenine,
2-(methylthio)-N.sup.6-(isopentenyl)adenine, 6-(alkyl)adenine,
6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine,
8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine,
8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine,
8-(thiol)adenine, N.sup.6-(isopentyl)adenine,
N.sup.6-(methyl)adenine, N.sup.6,N.sup.6-(dimethyl)adenine,
2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine,
6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine,
7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine,
8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine,
8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine,
N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine,
3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine,
5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine,
5-(propynyl)cytosine, 5-(propynyl)cytosine,
5-(trifluoromethyl)cytosine, 6-(azo)cytosine,
N.sup.4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil,
2-(thio)uracil, 5-(methyl)-2-(thio)uracil,
5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil,
5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil,
5-(methyl)-2,4-(dithio)uracil,
5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil,
5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil,
5-(aminoallyl)uracil, 5-(aminoalkyl)uracil,
5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil,
5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil,
5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil,
uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil,
5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil,
5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil,
dihydrouracil, N.sup.3-(methyl)uracil, 5-uracil (i.e.,
pseudouracil),
2-(thio)pseudouracil, 4-(thio)pseudouracil,
2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil,
5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil,
5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil,
5-(methyl)-4-(thio)pseudouracil,
5-(alkyl)-2,4-(dithio)pseudouracil,
5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil,
1-substituted 2(thio)-pseudouracil, 1-substituted
4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil,
1-(aminocarbonylethylenyl)-pseudouracil,
1-(aminocarbonylethylenyl)-2(thio)-pseudouracil,
1-(aminocarbonylethylenyl)-4-(thio)pseudouracil,
1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine,
hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl,
2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl,
nitrobenzimidazolyl, nitroindazolyl, aminoindolyl,
pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl,
5-(methyl)isocarbostyrilyl,
3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,
6-(methyl)-7-(aza)indolyl, imidizopyridinyl,
9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,
7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,
2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,
phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl,
stilbenyl, tetracenyl, pentacenyl, difluorotolyl,
4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,
6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,
6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine,
5-substituted pyrimidines, N.sup.2-substituted purines,
N.sup.6-substituted purines, O.sup.6-substituted purines,
substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl,
6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,
2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated
derivatives thereof.
[0225] As used herein, a universal nucleobase is any modified,
unmodified, naturally occurring or non-naturally occurring
nucleobase that can base pair with all of the four naturally
occurring nucleobases without substantially affecting the melting
behavior, recognition by intracellular enzymes or activity of the
oligonucleotide duplex. Some exemplary universal nucleobases
include, but are not limited to, 2,4-difluorotoluene,
nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine,
4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl
isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl
isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl,
imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl,
isocarbostyrilyl, 7-propynyl isocarbostyrilyl,
propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl,
4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl,
phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and
structural derivatives thereof (see for example, Loakes, 2001,
Nucleic Acids Research, 29, 2437-2447).
[0226] Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, hereby incorporated by reference, those disclosed in
International Application No. PCT/US09/038425, filed Mar. 26, 2009,
hereby incorporated by reference, those disclosed in the Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those
disclosed by English et al., Angewandte Chemie, International
Edition, 1991, 30, 613.
General References
[0227] The oligonucleotides used in accordance with this invention
can be synthesized with solid phase synthesis, see for example
"Oligonucleotide synthesis, a practical approach", Ed. M. J. Gait,
IRL Press, 1984; "Oligonucleotides and Analogues, A Practical
Approach", Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,
Modern machine-aided methods of oligodeoxyribonucleotide synthesis,
Chapter 2, Oligoribonucleotide synthesis, Chapter 3,
2'-O-Methyloligoribonucleotides: synthesis and applications,
Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis
of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of
oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7,
Oligodeoxynucleotides containing modified bases. Other particularly
useful synthetic procedures, reagents, blocking groups and reaction
conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,
486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,
2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993,
49, 6123-6194, or references referred to therein. Modification
described in WO 00/44895, WO01/75164, or WO02/44321 can be used
herein. The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
Phosphate Group References
[0228] The preparation of phosphinate oligonucleotides is described
in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate
oligonucleotides is described in U.S. Pat. No. 4,469,863. The
preparation of phosphoramidite oligonucleotides is described in
U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation
of phosphotriester oligonucleotides is described in U.S. Pat. No.
5,023,243. The preparation of boranophosphate oligonucleotide is
described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The
preparation of 3'-Deoxy-3'-amino phosphoramidate oligonucleotides
is described in U.S. Pat. No. 5,476,925.
3'-Deoxy-3'-methylenephosphonate oligonucleotides is described in
An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of
sulfur bridged nucleotides is described in Sproat et al.
Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
[0229] Modifications to the 2' modifications can be found in Verma,
S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references
therein. Specific modifications to the ribose can be found in the
following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem.,
1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79,
1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310).
Replacement of the Phosphate Group References
[0230] Methylenemethylimino linked oligonucleosides, also
identified herein as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
herein as MDH linked oligonucleosides, and methylenecarbonylamino
linked oligonucleosides, also identified herein as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified herein as amide-4 linked
oligonucleosides as well as mixed backbone compounds having, as for
instance, alternating MMI and PO or PS linkages can be prepared as
is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and
in published PCT applications PCT/US92/04294 and PCT/US92/04305
(published as WO 92/20822 WO and 92/20823, respectively).
Formacetal and thioformacetal linked oligonucleosides can be
prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564.
Ethylene oxide linked oligonucleosides can be prepared as is
described in U.S. Pat. No. 5,223,618. Siloxane replacements are
described in Cormier, J. F. et al. Nucleic Acids Res. 1988, 16,
4583. Carbonate replacements are described in Tittensor, J. R. J.
Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described
in Edge, M. D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991.
Carbamate replacements are described in Stirchak, E. P. Nucleic
Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
[0231] Cyclobutyl sugar surrogate compounds can be prepared as is
described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate
can be prepared as is described in U.S. Pat. No. 5,519,134.
Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos. 5,142,047 and 5,235,033, and other related patent
disclosures. Peptide Nucleic Acids (PNAs) are known per se and can
be prepared in accordance with any of the various procedures
referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties
and Potential Applications, Bioorganic & Medicinal Chemistry,
1996, 4, 5-23. They can also be prepared in accordance with U.S.
Pat. No. 5,539,0a83.
Terminal Modification References
[0232] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
Nucleobases References
[0233] N-2 substituted purine nucleoside amidites can be prepared
as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine
nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can
be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl
pyrimidine nucleoside amidites can be prepared as is described in
U.S. Pat. No. 5,484,908. Additional references are disclosed in the
above section on base modifications.
Placement of Modifications within an Oligonucleotide
[0234] As oligonucleotides are polymers of subunits or monomers,
many of the modifications described herein can occur at a position
which is repeated within an oligonucleotide, e.g., a modification
of a nucleobase, a sugar, a phosphate moiety, or the non-bridging
oxygen of a phosphate moiety. It is not necessary for all positions
in a given oligonucleotide to be uniformly modified, and in fact
more than one of the aforementioned modifications can be
incorporated in a single oligonucleotide or even at a single
nucleoside within an oligonucleotide.
[0235] In some cases the modification will occur at all of the
subject positions in the oligonucleotide but in many, and in fact
in most cases it will not. By way of example, a modification can
only occur at a 3' or 5' terminal position, can only occur in the
internal region, can only occur in 3', 5' or both terminal regions,
e.g. at a position on a terminal nucleotide or in the last 2, 3, 4,
5, 6, 7, 8, 9, or 10 nucleotides of an oligonucleotide. A
modification can occur in a double strand region, a single strand
region, or in both. A modification can occur only in the double
strand region of an oligonucleotide or can only occur in a single
strand region of an oligonucleotide. In certain embodiments, a
modification described herein does not occur in the region
corresponding to the target cleavage site region. For example, a
phosphorothioate modification at a non-bridging oxygen position can
only occur at one or both termini, can only occur in a terminal
regions, e.g., at a position on a terminal nucleotide or in the
last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a strand, or can
occur in double strand and single strand regions, particularly at
termini.
[0236] Some modifications can preferably be included on an
oligonucleotide at a particular location, e.g., at an internal
position of a strand, or on the 5' or 3' end of an oligonucleotide.
A preferred location of a modification on an oligonucleotide, can
confer preferred properties on the oligonucleotide. For example,
preferred locations of particular modifications can confer optimum
gene silencing properties, or increased resistance to endonuclease
or exonuclease activity.
[0237] In certain embodiments, the oligonucleotide comprises at
least one of 5'-5', 3'-3', 3'-2', 2'-5', 2'-3' or 2'-2' backbone
linkage. In certain embodiments, the last nucleotide on the
terminal end is linked via a 5'-5', 3'-3', 3'-2', 2'-5', 2'-3' or
2'-2' backbone linkage to the rest of the oligonucleotide. In some
preferred embodiments, the last nucleotide on the terminal end is
linked via a 5'-5', 3'-3', 3'-2', 2'-3' or 2'-2' backbone linkage
to the rest of the oligonucleotide.
5'-Pyrimidine-Purine-3' and 5'-Pyrimidine-Pyrimidine-3'
Dinucleotide Motif
[0238] An oligonucleotide can comprise at least 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10, 5'-pyrimidine-purine-3' (5'-PyPu-3') and/or
5'-pyrimidine-pyrimidine-3' (5'-PyPy-3') dinucleotide sequence
motif, wherein the 5'-most pyrimidine ribose sugar is modified at
the 2'-position. Preferred 2'-modifications include, but are not
limited to, 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE
(2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl]
(2'-O-NMA),
2'-O--CH.sub.2CH.sub.2N(CH.sub.2CH.sub.2NMe.sub.2).sub.2,
2'-S-methyl, 2'-O--CH.sub.2-(4'-C) (LNA) and
2'-O--CH.sub.2CH.sub.2-(4'-C) (ENA). Double-stranded
oligonucleotides including these modifications are particularly
stabilized against endonuclease activity. In one embodiment, the 3'
most nucleotide in the dinucleotide motif also comprises a ribose
sugar which is modified at the 2'-position. When both nucleotides
of the dinucleotide motif comprise ribose sugar with
2'-modification, the modification can be the same or different on
the two nucleotides. In another embodiment, the 5' most pyrimidine
in all occurrences of the dinucleotide motif in the oligonucleotide
comprises a ribose sugar which is modified at the 2'-position. In
yet another embodiment, both nucleotides in all occurrences of the
dinucleotide motif comprise a ribose sugar comprising a
2'-modification. In yet another embodiment, the 5'-most pyrimidine
in the dinucleotide motif is uridine. In yet still another
embodiment, the 5'-most pyrimidine in the dinucleotide motif is
cytidine.
[0239] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein the backbone linkage between
the two nucleotides is not a phosphodiester. In certain
embodiments, the backbone linkage is a non-phosphodiester linkage
described herein. Preferred non-phosphodiester backbone linkages
include, but are not limited to, phosphorothioate,
phosphorodithioate, N-alkyl phosphoramidate, alkyl phosphonate
(e.g., methyl phosphonate) and borano phosphonate. In one
embodiment, the backbone linkage between the two nucleotides in all
occurrences of the dinucleotide motif is a non-phosphodiester
linkage. In another embodiment, the 5'-most pyrimidine in the
dinucleotide motif is uridine. In yet another embodiment, the
5'-most pyrimidine in the dinucleotide motif is cytidine.
[0240] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein at least one of the
nucleotides comprises a nucleobase modification, e.g. a modified
nucleobase or a nucleobase with one or more conjugated moieties. In
one embodiment, the 5' most pyrimidine in the dinucleotide sequence
motif comprises the nucleobase modification. In another embodiment,
the 3' most nucleotide in the dinucleotide motif also comprises the
nucleobase modification. In yet another embodiment, both
nucleotides in the dinucleotide motif comprise a nucleobase
modification. In certain embodiments, at least one nucleotides in
all occurrences of the dinucleotide motif comprises a nucleobase
modification. In still another embodiment, the 5'-most pyrimidine
in the dinucleotide motif is uridine. In yet still another
embodiment, the 5'-most pyrimidine in the dinucleotide motif is
cytidine.
[0241] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein the 5'-most pyrimidine ribose
sugar is modified at the 2'-position and the oligonucleotide
further comprises at least one of a non-phosphodiester backbone
linkage, a nucleobase modification or a 2' modification. In one
embodiment, the 5'-most pyrimidines in all occurrences of the
dinucleotide motif comprise a ribose sugar modified at the
2'-position, and the oligonucleotide further comprises at least one
of a non-phosphodiester backbone linkage, a nucleobase modification
or a 2' modification. In further embodiments, the
non-phosphodiester backbone linkage, the nucleobase modification
and/or the 2'-modification is comprised within the dinucleotide
motif, e.g. the internucleotide linkage between the two nucleotides
of the dinucleotide motif is a non-phosphodiester backbone linkage,
one or both nucleotides comprise a nucleobase modification and/or
the 3'-nucleotide of the motif comprises a 2'-modification. In one
embodiment, the 5'-most pyrimidine in the dinucleotide motif is
uridine. In another embodiment, the 5'-most pyrimidine in the
dinucleotide motif is cytidine.
[0242] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, Or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif, wherein the ribose sugar of the
5'-most pyrimidine is replaced by a non ribose moiety, e.g., a six
membered ring. In one embodiment, the 5'-most pyrimidines all
occurrences of the dinucleotide motif comprise a non ribose sugar,
e.g. a six membered ring. In one embodiment, the 5'-most pyrimidine
in the dinucleotide motif is uridine. In another embodiment, the
5'-most pyrimidine in the dinucleotide motif is cytidine.
[0243] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein the C.sup.5 position of the
5'-most pyrimidine is conjugated with a ligand, e.g. a cationic
group, e.g. a cationic amino group. In one embodiment, the 5'-most
pyrimidines all occurrences of dinucleotide motif are conjugated
with a ligand, at the C.sup.5 position, wherein each ligand is
selected independently of other ligands. In another embodiment, the
5'-most pyrimidine in the dinucleotide motif is uridine. In yet
another embodiment, the 5'-most pyrimidine in the dinucleotide
motif is cytidine.
[0244] In some embodiments, the oligonucleotide comprises at least
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' dinucleotide wherein
the N.sup.2,N.sup.6, and/or C.sup.8 position of the purine is
conjugated with a ligand, e.g. a cationic group, e.g. a cationic
amino group. In one embodiment, the 3'-most purines in all
occurrences of the dinucleotide motif are conjugated with a ligand
at the N.sup.2,N.sup.6, and/or C.sup.8 positions, wherein each
ligand is selected independently of other ligands. In another
embodiment, the 5'-most pyrimidine in the dinucleotide motif is
uridine. In yet another embodiment, the 5'-most pyrimidine in the
dinucleotide motif is cytidine.
[0245] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein both nucleotides comprise
nucleobase modifications, e.g., the C.sup.5 position of the
pyrimidine and the N.sup.2,N.sup.6, and/or C.sup.8 position of the
purine is conjugated with a ligand, e.g. a cationic group, wherein
each ligand is selected independently. In one embodiment, both
nucleotides in all occurrences of the dinucleotide motif are
conjugated with a ligand, wherein each ligand is selected
independently of other ligands. In another embodiment, the 5'-most
pyrimidine in the dinucleotide motif is uridine. In yet another
embodiment, the 5'-most pyrimidine in the dinucleotide motif is
cytidine.
[0246] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein at least one of the
nucleotides comprises a nucleobase modification and neither
nucleotide comprises a modification at the 2' position of the
ribose sugar. In another embodiment, at least one nucleotide in all
occurrences of the dinucleotide motif comprise a nucleobase
modification and neither nucleotide comprises a modification at the
2' position of the ribose sugar. In yet another embodiment, both
nucleotides in the dinucleotide motif comprise a nucleobase
modification and neither nucleotide comprises a modification at the
2' position of the ribose sugar.
[0247] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein the backbone linkage between
the two nucleotides is not a phosphodiester and neither nucleotide
comprises a modification at the 2' position of the ribose sugar. In
certain embodiments, the backbone linkage is a non-phosphodiester
linkage described herein. In one embodiment, the backbone linkage
between the two nucleotides in all occurrences of the dinucleotide
motif is a non-phosphodiester linkage and neither nucleotide
comprises a modification at the 2' position of the ribose
sugar.
[0248] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3' dinucleotide motif wherein the 5'-most pyrimidine
comprises a modification at the 2'-position, backbone linkage
between the two nucleotides is a non-phosphodiester linkage and at
least one of the nucleotides comprises a nucleobase modification.
In one embodiment, the 5' most pyrimidine in the dinucleotide motif
comprises the nucleobase modification. In another embodiment, the
3' most nucleotide in the dinucleotide motif comprises the
nucleobase modification. In yet another embodiment, both the
nucleotides in the dinucleotide motif comprise the nucleobase
modification. In yet still another embodiment, the 5' most
pyrimidine in all occurrences of the dinucleotide motif comprises a
2'-modified ribose sugar, backbone linkage between the two
nucleotides is a non-phosphodiester linkage and at least one of the
nucleotides comprises a nucleobase modification.
[0249] In certain embodiments, the oligonucleotide comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 5'-PyPu-3' and/or
5'-PyPy-3'dinucleotide motif wherein the 3' most nucleotide
comprises a modification at the 2'-position, backbone linkage
between the two nucleotides is a non-phosphodiester linkage and at
least one of the nucleotides comprises a nucleobase modification.
In one embodiment, the 5'-most nucleotide in the dinucleotide motif
comprises the nucleobase modification. In another embodiment, the
3'-most nucleotide in the dinucleotide motif comprises the
nucleobase modification. In yet another embodiment, both
nucleotides in the dinucleotide comprise the nucleobase
modification. In yet still another embodiment, the 3'most
nucleotide in all occurrences of the dinucleotide motif comprises a
2'-modified ribose sugar, backbone linkage between the two
nucleotides is a non-phosphodiester linkage and at least one of the
nucleotides comprises a nucleobase modification.
[0250] In one embodiment, oligonucleotide comprises a motif
selected from the group consisting of: 2'-modified uridines in all
occurrences of the sequence motif 5'-uridine-adenosine-3'
(5'-UA-3'), 2'-modified uridines in all occurrences of the sequence
motif 5'-uridine-guanosine-3' (5'-UG-3'), 2'-modified cytidines in
all occurrences of the sequence motif 5'-cytidine-adenosine-3'
(5'-CA-3'), 2'-modified cytidines in all occurrences of the
sequence motif 5'-cytidine-Guanosine-3' (5'-CA-3'), 2'-modified
5'-most uridines in all occurrences of the sequence motif
5'-uridine-uridine-3' (5'-UU-3'), 2'-modified 5'-most cytidines in
all occurrences of the sequence motif 5'-cytidine-cytidine-3'
(5'-CC-3'), 2'-modified cytidines in all occurrences of the
sequence motif 5'-cytidine-uridine-3' (5'-CU-3'), 2'-modified
uridines in all occurrences of the sequence motif
5'-uridine-cytidine-3' (5'-UC-3'), and combinations thereof; and
wherein the oligonucleotide further comprises at least one
non-phosphodiester backbone linkage, nucleobase modification and/or
sugar modification, e.g. a 2' sugar modification. Preferably, the
non-phosphodiester backbone linkage, nucleobase modification and/or
sugar modification is within the dinucleotide motif.
[0251] In certain embodiments, the oligonucleotide comprises a
5'-purine-purine-3' (5'-PuPu-3') dinucleotide motif at the 5'
and/or 3' terminal end, wherein both nucleotide sugars are
modified, e.g., 2'-modified. In one embodiment, at least one of the
purines is modified at the 2, 6, 7, 8, N.sup.2 exocyclic, and/or
N.sup.6 exocyclic positions, or combinations thereof. In another
embodiment, the backbone linkage between the purines is a
non-phosphodiester linkage.
[0252] In certain embodiments, the 5' terminal nucleotide of the
oligonucleotide comprises sugar modification, e.g., a 2'
modification, a 4' modification or an 04' modification, e.g.,
replacement of 04' with S, substituted N or CH.sub.2. In one
embodiment, the 5' terminal nucleotide further comprises a modified
nucleobase or nucleobase modification.
Overhangs
[0253] Double-stranded oligonucleotides having at least one
single-stranded nucleotide overhang have unexpectedly superior
inhibitory properties than their blunt-ended counterparts. As used
herein, the term "overhang" refers to a double-stranded structure
where at least one end of one strand is longer than the
corresponding end of the other strand forming the double-stranded
structure. Generally, the single-stranded overhang is located at
the 3'-terminal end of the antisense strand or, alternatively, at
the 3'-terminal end of the sense strand. The double-stranded
oligonucleotide can also have a blunt end, generally located at the
5'-end of the antisense strand. Generally, the antisense strand of
the double-stranded oligonucleotide has a single-stranded overhang
at the 3'-end, and the 5'-end is blunt.
[0254] In one embodiment, at least one end of the double-stranded
region has a single-stranded nucleotide overhang of 1 to 4,
generally 1 or 2 nucleotides. In certain other embodiment, both
ends of the double-stranded region have a single-stranded
nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.
[0255] In some embodiments it is particularly preferred, e.g., to
enhance stability, to include particular nucleobases in the
single-stranded overhangs, or to include modified nucleotides or
nucleotide surrogates, in single-strand overhangs. For example, it
can be desirable to include purine nucleotides in overhangs. In
some embodiments all or some of the bases in the single strand
overhang will be modified, e.g., with a modification described
herein. Modifications in the single-stranded overhangs can include,
e.g., the use of modifications at the 2' OH group of the ribose
sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,
instead of ribonucleotides, and modifications in the phosphate
group, e.g., phosphothioate modifications. Overhangs need not be
homologous with the target sequence. In certain embodiments, the
single strand overhangs are asymmetrically modified with a
modification described herein, e.g. a first single stand overhang
comprises a modification that is not present in a second single
strand overhang.
[0256] In certain embodiments, the unpaired nucleotide adjacent to
the terminal nucleotide base pair on the end of the double-stranded
region is a purine. In one embodiment, the single-stranded overhang
has the sequence 5'-GCNN-3', wherein N is independently for each
occurrence, A, G, C, U, dT, dU or absent. In certain embodiment,
the single-stranded overhang has the sequence 5'-NN-3', wherein N
is a modified or unmodified nucleotide described herein. In one
preferred embodiment, the single-stranded overhang has the sequence
5'-dTdT-3' (dT=deoxythymidine). In another preferred embodiment,
the single-stranded overhang has the sequence 5'-dTdT-3' (dT=deoxy
thymidine) and the internucleotide linkage between the dTs is a
non-phosphodiester backbone linkage.
[0257] In one embodiment, the antisense strand of the
double-stranded oligonucleotide has 1-10 single-stranded nucleotide
overhangs each at the 3' end and the 5' end over the sense strand.
In another embodiment, the sense strand of the double-stranded
oligonucleotide has 1-10 single-stranded nucleotide overhangs each
at the 3' end and the 5' end over the antisense strand.
Mismatches
[0258] The antisense strand of the double-stranded oligonucleotide
can contain one or more mismatches to the target sequence. In a
preferred embodiment, the antisense strand contains no more than 3
mismatches. If the antisense strand contains mismatches to a target
sequence, it is preferable that the area of mismatch not be located
in the center of the region of complementarity between the
antisense strand and the target sequence. If the antisense strand
contains mismatches to the target sequence, it is preferable that
the mismatch be restricted to 5 nucleotides from either end, for
example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of
the region of complementarity between the antisense strand and the
target sequence. The methods known in the art can be used to
determine whether a double-stranded oligonucleotide containing a
mismatch to a target sequence is effective in inhibiting the
expression of the target gene.
[0259] In certain embodiments, the sense-strand comprises a
mismatch to the antisense strand. In one embodiment, the sense
strand comprises no more than 1, 2, 3, 4 or 5 mismatches to the
antisense strand. In preferred embodiments, the sense strand
comprises no more than 3 mismatches to the antisense strand.
[0260] In some embodiments, the sense-strand comprises a mismatch
to the antisense strand and the mismatch is within the 5
nucleotides from the 3'-end of the sense strand, for example 5, 4,
3, 2, or 1 nucleotides from the end of the region of
complementarity between the sense and the antisense strands.
[0261] In certain embodiments, the sense-strand comprises a
mismatch to the antisense strand and the mismatch is located in the
target cleavage site region. In certain embodiments, the sense
strand comprises a nucleobase modification, e.g. an optionally
substituted natural or non-natural nucleobase, a universal
nucleobase, in the target cleavage site region.
[0262] The "target cleavage site" herein means the backbone linkage
in the target gene, e.g. target mRNA, or the sense strand that is
cleaved by the RISC mechanism by utilizing the RNAi agent. And the
"target cleavage site region" comprises at least one or at least
two nucleotides on 3', 5' or both sides of the cleavage site.
Preferably, the target cleavage site region comprises two
nucleotides on both sides of the cleavage site. For the sense
strand, the target cleavage site is the backbone linkage in the
sense strand that would get cleaved if the sense strand itself was
the target to be cleaved by the RNAi mechanism. The target cleavage
site can be determined using methods known in the art, for example
the 5'-RACE assay as detailed in Soutschek et al., Nature (2004)
432, 173-178. Without wishing to be bound by theory, the cleavage
site region for a conical double stranded RNAi agent comprising two
21-nucleotides long strands (wherein the strands form a double
stranded region of 19 consecutive nucleotide base pairs having
2-nucleotide single stranded overhangs at the 3'-ends), the
cleavage site region corresponds to positions 9-12 from the 5'-end
of the sense strand.
[0263] Consideration of the efficacy of RNAi agents with mismatches
in inhibiting expression of the target gene is important,
especially if the particular region of complementarity in the
target gene is known to have polymorphic sequence variation within
the population.
Multi-Targeting
[0264] Sequences that are different from each other at 1, 2, 3, 4
or 5 positions can be targeted by a single RNAi agent, e.g.,
double-stranded or single-stranded RNAi agent. As used in this
context, the phrase "different from each other" refers to the
target sequences having different nucleotides at that position. In
these cases the RNAi agent strand that is complementary to the
target sequences comprises universal nucleobases at positions
complementary to where the target sequences are different from each
other. For example, the antisense strand of the double-stranded
RNAi agent comprises universal nucleobases at positions
complementary to where the sequences to be targeted do not match
each other.
[0265] These multi targeting RNAi agents can be used to alter the
expression of different transcripts/alleles of a single gene,
different isoforms of a single gene, different splice variants of a
single gene, different transcripts of more than one gene, wild-type
and mutated form of a gene or homolog of a gene in different
species.
[0266] The double-stranded RNAi agents described herein can also
target more than one RNA region by having each strand targeting a
sequence or part thereof independently. For example, a
double-stranded RNAi agent can include a first and second sequence
that are sufficiently complementary to each other to hybridize. The
first sequence can be complementary to a first target sequence and
the second sequence can be complementary to a second target
sequence. The first and second sequences of the RNAi agent can be
on different RNA strands, and the mismatch between the first and
second sequences can be less than 50%, 40%, 30%, 20%, 10%, 5%, or
1%. The first and second sequences of the RNAi agent can be on the
same RNA strand, and in a related embodiment more than 50%, 60%,
70%, 80%, 90%, 95%, or 1% of the RNAi agent can be in bimolecular
form. The first and second sequences of the RNAi agent can be fully
complementary to each other.
[0267] The first target sequence can be a first target gene and the
second target sequence can be a second target gene, or the first
and second target sequences can be different regions of a single
target gene. The first and second sequences can differ by at least
1 nucleotide.
[0268] The first and second target sequences can be transcripts
encoded by first and second sequence variants, e.g., first and
second alleles, of a gene. The sequence variants can be mutations,
or polymorphisms, for example. The first target sequence can
include a nucleotide substitution, insertion, or deletion relative
to the second target sequence, or the second target sequence can be
a mutant or variant of the first target sequence. The first and
second target sequences can comprise viral or human genes. The
first and second target sequences can also be on variant
transcripts of an oncogene or include different mutations of a
tumor suppressor gene transcript. In addition, the first and second
target sequences can correspond to hot-spots for genetic
variation.
Terminal End Thermal Stability
[0269] The double stranded oligonucleotides can be optimized for
RNA interference by increasing the propensity of the duplex to
disassociate or melt (decreasing the free energy of duplex
association), in the region of the 5' end of the antisense strand
This can be accomplished, e.g., by the inclusion of modifications
or modified nucleosides which increase the propensity of the duplex
to disassociate or melt in the region of the 5' end of the
antisense strand. It can also be accomplished by inclusion of
modifications or modified nucleosides or attachment of a ligand
that increases the propensity of the duplex to disassociate of melt
in the region of the 5'end of the antisense strand. While not
wishing to be bound by theory, the effect can be due to promoting
the effect of an enzyme such as helicase, for example, promoting
the effect of the enzyme in the proximity of the 5' end of the
antisense strand.
[0270] Modifications which increase the tendency of the 5' end of
the antisense strand in the duplex to dissociate can be used alone
or in combination with other modifications described herein, e.g.,
with modifications which decrease the tendency of the 3' end of the
antisense in the duplex to dissociate. Likewise, modifications
which decrease the tendency of the 3' end of the antisense in the
duplex to dissociate can be used alone or in combination with other
modifications described herein, e.g., with modifications which
increase the tendency of the 5' end of the antisense in the duplex
to dissociate.
[0271] Nucleic acid base pairs can be ranked on the basis of their
propensity to promote dissociation or melting (e.g., on the free
energy of association or dissociation of a particular pairing, the
simplest approach is to examine the pairs on an individual pair
basis, though next neighbor or similar analysis can also be used).
In terms of promoting dissociation: A:U is preferred over G:C; G:U
is preferred over G:C; I:C is preferred over G:C (I=inosine);
mismatches, e.g., non-canonical or other than canonical pairings
are preferred over canonical (A:T, A:U, G:C) pairings; pairings
which include a universal base are preferred over canonical
pairings.
[0272] It is preferred that pairings which decrease the propensity
to form a duplex are used at 1 or more of the positions in the
duplex at the 5' end of the antisense strand. The terminal pair
(the most 5' pair in terms of the antisense strand), and the
subsequent 4 base pairing positions (going in the 3' direction in
terms of the antisense strand) in the duplex are preferred for
placement of modifications to decrease the propensity to form a
duplex. More preferred are placements in the terminal most pair and
the subsequent 3, 2, or 1 base pairings. It is preferred that at
least 1, and more preferably 2, 3, 4, or 5 of the base pairs from
the 5'-end of antisense strand in the duplex be chosen
independently from the group of: A:U, G:U, I:C, mismatched pairs,
e.g., non-canonical or other than canonical pairings or pairings
which include a universal base. In a preferred embodiment at least
one, at least 2, or at least 3 base-pairs include a universal
base.
[0273] Modifications or changes which promote dissociation are
preferably made in the sense strand, though in some embodiments,
such modifications/changes will be made in the antisense
strand.
[0274] Nucleic acid base pairs can also be ranked on the basis of
their propensity to promote stability and inhibit dissociation or
melting (e.g., on the free energy of association or dissociation of
a particular pairing, the simplest approach is to examine the pairs
on an individual pair basis, though next neighbor or similar
analysis can also be used). In terms of promoting duplex stability:
G:C is preferred over A:U, Watson-Crick matches (A:T, A:U, G:C) are
preferred over non-canonical or other than canonical pairings,
analogs that increase stability are preferred over Watson-Crick
matches (A:T, A:U, G:C), e.g. 2-amino-A:U is preferred over A:U,
2-thio U or 5 Me-thio-U:A, are preferred over U:A, G-clamp (an
analog of C having 4 hydrogen bonds):G is preferred over C:G,
guanadinium-G-clamp:G is preferred over C:G, pseudo uridine:A, is
preferred over U:A, sugar modifications, e.g., 2' modifications,
e.g., 2'F, ENA, or LNA, which enhance binding are preferred over
non-modified moieties and can be present on one or both strands to
enhance stability of the duplex.
[0275] It is preferred that pairings which increase the propensity
to form a duplex are used at 1 or more of the positions in the
duplex at the 3' end of the antisense strand. The terminal pair
(the most 3' pair in terms of the antisense strand), and the
subsequent 4 base pairing positions (going in the 5' direction in
terms of the antisense strand) in the duplex are preferred for
placement of modifications to increase the propensity to form a
duplex. More preferred are placements in the terminal most pair and
the subsequent 3, 2, or 1 base pairings. It is preferred that at
least 1, and more preferably 2, 3, 4, or 5 of the pairs of the
recited regions be chosen independently from the group of: G:C, a
pair having an analog that increases stability over Watson-Crick
matches (A:T, A:U, G:C), 2-amino-A:U, 2-thio U or 5 Me-thio-U:A,
G-clamp (an analog of C having 4 hydrogen bonds):G,
guanadinium-G-clamp:G, pseudo uridine:A, a base pair in which one
or both subunits has a sugar modification, e.g., a 2' modification,
e.g., 2'F, ENA, or LNA, which enhance binding. In some embodiments,
at least one, at least, at least 2, or at least 3, of the base
pairs promote duplex stability.
[0276] In a preferred embodiment, at least one, at least 2, or at
least 3, of the base pairs are a pair in which one or both subunits
has a sugar modification, e.g., a 2' modification, e.g.,
2'-O-methyl, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl),
2'-F, 2'-O--CH.sub.2-(4'-C) (LNA) and 2'-O--CH.sub.2CH.sub.2-(4'-C)
(ENA), which enhance binding.
[0277] G-clamps and guanidinium G-clamps are discussed in the
following references: Holmes and Gait, "The Synthesis of
2'-O-Methyl G-Clamp Containing Oligonucleotides and Their
Inhibition of the HIV-1 Tat-TAR Interaction," Nucleosides,
Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al.,
"Steric inhibition of human immunodeficiency virus type-1
Tat-dependent trans-activation in vitro and in cells by
oligonucleotides containing 2'-O-methyl G-clamp ribonucleoside
analogues," Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et
al., "Structural basis for recognition of guanosine by a synthetic
tricyclic cytosine analogue: Guanidinium G-clamp," Helvetica
Chimica Acta, 86:966-978, 2003; Rajeev, et al., "High-Affinity
Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine
Analogues," Organic Letters, 4:4395-4398, 2002; Ausin, et al.,
"Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers," Organic
Letters, 4:4073-4075, 2002; Maier et al., "Nuclease resistance of
oligonucleotides containing the tricyclic cytosine analogues
phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and
origins of their nuclease resistance properties," Biochemistry,
41:1323-7, 2002; Flanagan, et al., "A cytosine analog that confers
enhanced potency to antisense oligonucleotides," Proceedings Of The
National Academy Of Sciences Of The United States Of America,
96:3513-8, 1999.
[0278] As is discussed above, an oligonucleotide can be modified to
both decrease the stability of the antisense 5'end of the duplex
and increase the stability of the antisense 3' end of the duplex.
This can be effected by combining one or more of the stability
decreasing modifications in the antisense 5' end of the duplex with
one or more of the stability increasing modifications in the
antisense 3' end of the duplex.
Nuclease Stability
[0279] In vivo applications of oligonucleotides is limited due to
presence of nucleases in the serum and/or blood. Thus in certain
instances it is preferable to modify the 3', 5' or both ends of an
oligonucleotide to make the oligonucleotide resistant against
exonucleases, e.g., 3' to 5' exonucleases.
[0280] In certain embodiments, a double-stranded oligonucleotide
comprises, on at least one end of the duplex region, a G-C base
pair at the terminal position of the duplex region (e.g., the last
base pair of the duplex) or the four consecutive base from the
duplex region end comprise at least two G-C base pairs. In one
embodiment, both ends of duplex region comprise a terminal G-C base
pair and/or the first four consecutive base pairs from the terminal
end comprise at least two G-C base pairs.
Off-Target Effects
[0281] In the RNA interference pathway, both strands of the
double-stranded RNAi agent have the potential to enter the RISC
complex and reduce the gene expression of corresponding
complementary sequences. Without wishing to be bound by theory, one
way an unwanted off-target effect happens ins when the sense strand
enters the RISC complex and reduces the gene expression of a
complementary sequence which is not the desired target of the RNAi
agent.
[0282] A number of strategies can be applied to reduce the
off-target effects due to sense strand mediated RNA interference.
The sense strand can be chemically modified so that it can no
longer act in the RISC mediated cleavage of a target sequence.
Without wishing to be bound by theory, such modifications minimize
off-target RNAi effects due to sense strand.
[0283] In one embodiment, the sense strand does not have a free
terminal 5' --OH group. In another embodiment, the sense strand
does not have a 5'-phosphate group. In certain embodiments, the
5'-OH of sense strand is modified so that it can not be
phosphorylated, e.g. with a cap moiety. In certain embodiments, the
cap moiety comprises L-sugar nucleotide, an alpha nucleotide, a
hydroxy protecting group, an alkyl, a cycloalkyl or a heterocycle.
In certain embodiments, the linkage between the 5' end of sense
strand and a conjugate is a non-phosphodiester backbone linkage. In
a preferred embodiment, the linkage between the 5' end of sense
strand and a conjugate does not have a phosphate group.
[0284] In certain embodiments, the sense strand comprises at least
one modified nucleotide in the target cleavage site region.
Preferably, the modification include modification at 2' position of
ribose sugar or more preferably a nucleobase modification.
[0285] In certain embodiments, ends of double-stranded
oligonucleotide can be modified so that the end corresponding to
5'end of sense strand has a higher thermal stability as compared to
the end corresponding 3' end of sense strand, as described above in
the Terminal end thermal stability section above. Without wishing
to be bound by theory, this allows preferential incorporation of
the antisense strand into the RISC complex and reduces off-target
effects of sense strand.
[0286] Specificity of the oligonucleotides of the invention can
also be increased by selecting target sequences that are different
at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more position from
other sequences. Other sequences can be related genes, similar
genes in closely related species, and variations and combinations
thereof.
Asymmetric Modifications
[0287] Modifications described herein can be used to asymmetrically
modified a double-stranded oligonucleotide. An asymmetrically
modified double-stranded oligonucleotide is one in which one strand
has a modification which is not present on the other strand. As
such, an asymmetrical modification is a modification found on one
strand but not on the other strand. Any modification, e.g., any
modification described herein, can be present as an asymmetrical
modification. An asymmetrical modification can confer any of the
desired properties associated with a modification, e.g., those
properties discussed herein. For example, an asymmetrical
modification can confer resistance to degradation, an alteration in
half life; target the oligonucleotide to a particular target, e.g.,
to a particular tissue; modulate, e.g., increase or decrease, the
affinity of a strand for its complement or target sequence; or
hinder or promote modification of a terminal moiety, e.g.,
modification by a kinase or other enzymes involved in the RISC
mechanism pathway. The designation of a modification as having one
property does not mean that it has no other property, e.g., a
modification referred to as one which promotes stabilization might
also enhance targeting. Asymmetrical modifications can include
those in which only one strand is modified as well as those in
which both are modified.
[0288] When the two strands of double-stranded oligonucleotide are
linked together, e.g. a hairpin or a dumbbell, the two strands of
the double stranded region can also be asymmetrically modified. For
example, first strand of the double-stranded region comprises at
least one asymmetric modification that is not present in the second
strand of the double stranded region or vice versa.
[0289] While not wishing to be bound by theory or any particular
mechanistic model, it is believed that asymmetrical modification
allows a double-stranded RNAi agent to be optimized in view of the
different or "asymmetrical" functions of the sense and antisense
strands. For example, both strands can be modified to increase
nuclease resistance, however, since some changes can inhibit RISC
activity, these changes can be chosen for the sense stand. In
addition, since some modifications, e.g., a ligand, can add large
bulky groups that, e.g., can interfere with the cleavage activity
of the RISC complex, such modifications are preferably placed on
the sense strand. Thus, ligands, especially bulky ones (e.g.
cholesterol), are preferentially added to the sense strand. The
ligand can be present at either (or both) the 5' or 3' end of the
sense strand of a RNAi agent.
[0290] Each strand can include one or more asymmetrical
modifications. By way of example: one strand can include a first
asymmetrical modification which confers a first property on the
oligonucleotide and the other strand can have a second asymmetrical
modification which confers a second property on the
oligonucleotide. For example, one strand, e.g., the sense strand
can have a modification which targets the oligonucleotide to a
tissue, and the other strand, e.g., the antisense strand, has a
modification which promotes hybridization with the target gene
sequence.
[0291] In some embodiments both strands can be modified to optimize
the same property, e.g., to increase resistance to nucleolytic
degradation, but different modifications are chosen for the sense
and the antisense strands, because the modifications affect other
properties as well.
[0292] Multiple asymmetric modifications can be introduced into
either or both of the sense and antisense strand. A strand can have
at least 1, 2, 3, 4, 5, 6, 7, 8, or more modifications and all or
substantially all of the monomers, e.g., nucleotides of a strand
can be asymmetrically modified.
[0293] In certain embodiments, the asymmetric modifications are
chosen so that only one of the two strands of double-stranded RNAi
agent is effective in inducing RNAi. Inhibiting the induction of
RNAi by one strand can reduce the off target effects due to
cleavage of a target sequence by that strand.
[0294] In preferred embodiments asymmetrical modifications which
result in one or more of the following are used: modifications of
the 5' end of the sense strand which inhibit kinase activation of
the sense strand, including, e.g., attachments of ligands or the
use modifications which protect against 5' exonucleolytic
degradation; or modifications of either strand, but preferably the
sense strand, which enhance binding between 5'-end of the sense and
3'-end of the antisense strand and thereby promote a "tight"
structure at this end of the molecule.
[0295] The end region of the RNAi agent defined by the 3' end of
the sense strand and the 5'end of the antisense strand is also
important for function. This region can include the terminal 2, 3,
or 4 paired nucleotides and any 3' overhang. Preferred embodiments
include asymmetrical modifications of either strand, but preferably
the sense strand, which decrease binding between 3'-end of the
sense and 5'-end of the antisense strand and thereby promote an
"open" structure at this end of the molecule. Such modifications
include placing conjugates which target the molecule or
modifications which promote nuclease resistance on the sense strand
in this region. Modification of the antisense strand which inhibit
kinase activation are avoided in preferred embodiments.
[0296] Particularly preferred asymmetric modification are
modifications of 2'-OH of ribose sugar and modification of backbone
phosphodiester linkage. Other preferred asymmetric modifications
include conjugation of ligands. Each strand can be conjugated with
ligands that are different between the two strands.
[0297] In certain embodiments, one strand has an asymmetrical
2'-modification, e.g., a 2'-O-alkyl modification, and the other
strand has an asymmetrical modification of the backbone
phosphodiester linkage. In certain embodiments, one strand has an
asymmetrical 2'-modification, e.g., a 2'-O-alkyl modification, and
the other strand also has an asymmetrical 2'-modification that is
different from the first strand's asymmetrical 2'-modification,
e.g., 2'-fluoro modification.
[0298] In certain embodiments, one strand is asymmetrically
modified with 2'-O-alkyl, e.g. 2'-OMe modification and the second
strand is asymmetrically modified with 2'-fluoro modification.
[0299] In certain embodiments, one strand is asymmetrically
modified with 2'-O-alkyl, e.g. 2'-OMe modification and the second
strand is asymmetrically modified with backbone phosphodiester
linkage modification, e.g. a phosphorothioate modification.
[0300] In certain embodiments, one strand is asymmetrically
modified with 2'-fluoro modification and the second strand is
asymmetrically modified with backbone phosphodiester linkage
modification, e.g. a phosphorothioate modification.
[0301] It is preferable to have RNAi agents wherein there are
multiple 2'-O-alkyl, e.g., 2'-OMe modifications on the sense strand
and multiple 2'-fluoro and/or multiple modified backbone
phosphodiester linkages on the antisense strand.
[0302] Modifications, e.g., those described herein, which modulate,
e.g., increase or decrease, the affinity of a strand for its
compliment or target, can be provided as asymmetrical
modifications.
Chimeric Oligonucleotides
[0303] The present invention also includes oligonucleotides which
are chimeric oligonucleotides. "Chimeric" oligonucleotides or
"chimeras," in the context of this invention, are oligonucleotide
which contain two or more chemically distinct regions, each made up
of at least one monomer unit, i.e., a modified or unmodified
nucleotide in the case of an oligonucleotide. Chimeric
oligonucleotides can be described as having a particular motif. In
certain embodiments, the motifs include, but are not limited to, an
alternating motif, a gapped motif, a hemimer motif, a uniformly
fully modified motif and a positionally modified motif. As used
herein, the phrase "chemically distinct region" refers to an
oligonucleotide region which is different from other regions by
having a modification that is not present elsewhere in the
oligonucleotide or by not having a modification that is present
elsewhere in the oligonucleotide. An oligonucleotide can comprise
two or more chemically distinct regions. As used herein, a region
that comprises no modifications is also considered chemically
distinct.
[0304] A chemically distinct region can be repeated within an
oligonucleotide. Thus, a pattern of chemically distinct regions in
an oligonucleotide can be realized such that a first chemically
distinct region is followed by one or more second chemically
distinct regions. This sequence of chemically distinct regions can
be repeated one or more times. Preferably, the sequence is repeated
more than one time. Both strands of a double-stranded
oligonucleotides can comprise these sequences. Each chemically
distinct region can actually comprise as little as a single
nucleotide. In certain embodiments, each chemically distinct region
independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17 or 18 nucleotides.
[0305] In certain embodiments, alternating nucleotides comprise the
same modification, e.g. all the odd number nucleotides in a strand
have the same modification and/or all the even number nucleotides
in a strand have the similar modification to the first strand. In
certain other embodiments, all the odd number nucleotides in an
oligonucleotide have the same modification and all the even
numbered nucleotides have a modification that is not present in the
odd number nucleotides and vice versa.
[0306] When both strands of a double-stranded oligonucleotide
comprise the alternating modification patterns, nucleotides of one
strand can be complementary in position to nucleotides of the
second strand which are similarly modified. In an alternative
embodiment, there is a phase shift between the patterns of
modifications of the first strand, respectively, relative to the
pattern of similar modifications of the second strand. Preferably,
the shift is such that the similarly modified nucleotides of the
first strand and second strand are not in complementary position to
each other.
[0307] In certain embodiments, the first strand has an alternating
modification pattern wherein alternating nucleotides comprise a
2'-modification, e.g., 2'-O-Methyl modification. In certain
embodiments, the first strand comprises an alternating 2'-O-Methyl
modification and the second strand comprises an alternating
2'-fluoro modification. In other embodiments, both strands of a
double-stranded oligonucleotide comprise alternating 2'-O-methyl
modifications.
[0308] When both strands of a double-stranded oligonucleotide
comprise alternating 2'-O-methyl modifications, such 2'-modified
nucleotides can be in complementary position in the duplex region.
Alternatively, such 2'-modified nucleotides can not be in
complementary positions in the duplex region.
[0309] In certain embodiments, the oligonucleotide comprises two
chemically distinct regions, wherein each region is 1-10
nucleotides in length.
[0310] In other embodiments, the oligonucleotide comprises three
chemically distinct regions. The middle region is about 5-15
nucleotide in length and each flanking or wing region is 1-5
nucleotides in length. All three regions can have different
modifications or the wing regions can be similarly modified to each
other.
[0311] As used herein the term "alternating motif" refers to an
oligonucleotide comprising at least two different chemically
distinct regions that that alternate for essentially the entire
sequence of the oligonucleotide. In an alternating motif length of
each region is independent of the length of other regions.
[0312] As used herein, the term "uniformly fully modified motif"
refers to an oligonucleotide wherein all nucleotides in the
oligonucleotide have at least one modification that is the
same.
[0313] As used herein, the term "hemimer motif" refers to an
oligonucleotide having two chemically distinct regions, wherein one
region is at the 5' end of the oligonucleotide and the other region
is at the 3 end of the oligonucleotide. In one embodiment, length
of each chemically distinct region is independently 1 nucleotide to
1 nucleotide less than the length of the oligonucleotide.
[0314] As used herein the term "gapped motif" refers to an
oligonucleotide having three chemically distinct regions. In one
embodiment, the gapped motif is a symmetric gapped motif, wherein
the two outer chemically distinct regions (wing regions) are
identically modified. In another embodiment, the gapped motif is an
asymmetric gaped motif in that the three regions are chemically
distinct from each other.
[0315] As used herein the term "positionally modified motif" refers
to an oligonucleotide having three or more chemically distinct
regions. Positionally modified oligonucleotides are distinguished
from gapped motifs, hemimer motifs, blockmer motifs and alternating
motifs because the pattern of regional substitution defined by any
positional motif does not fit into the definition provided herein
for one of these other motifs. The term positionally modified
oligomeric compound includes many different specific substitution
patterns.
[0316] It is to be understood that when an oligonucleotide
comprises two or more different modifications, modification pattern
for each modification is independent of the pattern for the other
modification. In certain embodiments, the modification pattern of
two or more modifications do not overlap, partially overlap or
fully overlap with each other.
[0317] In certain embodiments, oligonucleotide comprises two or
more chemically distinct regions and has a structure as described
in International Application No. PCT/US09/038433, filed Mar. 26,
2009, contents of which are herein incorporated in their
entirety.
Ligands
[0318] A wide variety of entities, e.g., ligands, can be coupled to
the oligonucleotides described herein. Ligands can include
naturally occurring molecules, or recombinant or synthetic
molecules. Exemplary ligands include, but are not limited to,
polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,
styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K,
PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG].sub.2, polyvinyl
alcohol (PVA), polyurethane, poly(2-ethylacryllic acid),
N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine,
cationic groups, spermine, spermidine, polyamine,
pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer
polyamine, arginine, amidine, protamine, cationic lipid, cationic
porphyrin, quaternary salt of a polyamine, thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, mucin,
glycosylated polyaminoacids, transferrin, bisphosphonate,
polyglutamate, polyaspartate, an aptamer, asialofetuin, hyaluronan,
procollagen, insulin, transferrin, albumin, sugar-albumin
conjugates, intercalating agents (e.g., acridines), cross-linkers
(e.g. psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin,
Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases (e.g., EDTA),
lipophilic molecules (e.g., steroids, bile acids, cholesterol,
cholic acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl
group, hexadecylglycerol, borneol, menthol, 1,3-propanediol,
heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha helical
peptide, amphipathic peptide, RGD peptide, cell permeation peptide,
endosomolytic/fusogenic peptide), alkylating agents, phosphate,
amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled
markers, enzymes, haptens (e.g. biotin), transport/absorption
facilitators (e.g., naproxen, aspirin, vitamin E, folic acid),
synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine,
imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes
of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies,
hormones and hormone receptors, lectins, carbohydrates, multivalent
carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K,
vitamin B, e.g., folic acid, B12, riboflavin, biotin and
pyridoxal), vitamin cofactors, lipopolysaccharide, an activator of
p38 MAP kinase, an activator of NF-.kappa.B, taxon, vincristine,
vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin
A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis
factor alpha (TNFalpha), interleukin-1 beta, gamma interferon,
natural or recombinant low density lipoprotein (LDL), natural or
recombinant high-density lipoprotein (HDL), and a cell-permeation
agent, preferably a helical cell-permeation agent.
[0319] Peptide and peptidomimetic ligands include those having
naturally occurring or modified peptides, e.g., D or L peptides;
.alpha., .beta., or .gamma. peptides; N-methyl peptides;
azapeptides; peptides having one or more amide, i.e., peptide,
linkages replaced with one or more urea, thiourea, carbamate, or
sulfonyl urea linkages; or cyclic peptides. A peptidomimetic (also
referred to herein as an oligopeptidomimetic) is a molecule capable
of folding into a defined three-dimensional structure similar to a
natural peptide. The peptide or peptidomimetic ligand can be about
5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40,
45, or 50 amino acids long.
[0320] Exemplary amphipathic peptides include, but are not limited
to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like
peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides,
hagfish intestinal antimicrobial peptides (HFIAPs), magainines,
brevinins-2, dermaseptins, melittins, pleurocidin, H.sub.2A
peptides, Xenopus peptides, esculentinis-1, and caerins.
[0321] As used herein, the term "endosomolytic ligand" refers to
molecules having endosomolytic properties. Endosomolytic ligands
promote the lysis of and/or transport of the composition of the
invention, or its components, from the cellular compartments such
as the endosome, lysosome, endoplasmic reticulum (ER), golgi
apparatus, microtubule, peroxisome, or other vesicular bodies
within the cell, to the cytoplasm of the cell. Some exemplary
endosomolytic ligands include, but are not limited to, imidazoles,
poly or oligoimidazoles, linear or branched polyethyleneimines
(PEIs), linear and branched polyamines, e.g. spermine, cationic
linear and branched polyamines, polycarboxylates, polycations,
masked oligo or poly cations or anions, acetals, polyacetals,
ketals/polyketals, orthoesters, linear or branched polymers with
masked or unmasked cationic or anionic charges, dendrimers with
masked or unmasked cationic or anionic charges, polyanionic
peptides, polyanionic peptidomimetics, pH-sensitive peptides,
natural and synthetic fusogenic lipids, natural and synthetic
cationic lipids.
[0322] Exemplary endosomolytic/fusogenic peptide include, but are
not limited to, AALEALAEALEALAEALEALAEAAAAGGC (GALA),
AALAEALAEALAEALAEALAEALAAAAGGC (EALA), ALEALAEALEALAEA,
GLFEAIEGFIENGWEGMIWDYG (INF-7), GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2),
GLF EAI EGFI ENGW EGMI DGWYGC GLF EAI EGFI ENGW EGMI DGWYGC
(diINF-7), GLF EAI EGFI ENGW EGMI DGGC GLF EAI EGFI ENGW EGMI DGGC
(diINF-3), GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF),
GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3), GLF EAI EGFI ENGW
EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine),
GLFEALLELLESLWELLLEA (JTS-1), GLFKALLKLLKSLWKLLLKA (ppTG1),
GLFRALLRLLRSLWRLLLRA (ppTG20), WEAKLAKALAKALAKHLAKALAKALKACEA
(KALA), GLFFEAIAEFIEGGWEGLIEGC (HA), GIGAVLKVLTTGLPALISWIKRKRQQ
(Melittin), and histidine rich peptides H.sub.5WYG and
CHK.sub.6HC.
[0323] Without wishing to be bound by theory, fusogenic lipids fuse
with and consequently destabilize a membrane. Fusogenic lipids
usually have small head groups and unsaturated acyl chains.
Exemplary fusogenic lipids include, but are not limited to,
1,2-dileoyl-sn-3-phosphoethanolamine (DOPE),
phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine
(POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol
(Di-Lin),
N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanam-
ine (DLin-k-DMA) and
N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethan-
amine (XTC).
[0324] Synthetic polymers with endosomolytic activity amenable to
the present invention are described in United States Patent
Application Publications Nos. 2009/0048410; 2009/0023890;
2008/0287630; 2008/0287628; 2008/0281044; 2008/0281041;
2008/0269450; 2007/0105804; 20070036865; and 2004/0198687, contents
of which are hereby incorporated by reference in their
entirety.
[0325] Exemplary cell permeation peptides include, but are not
limited to, RQIKIWFQNRRMKWKK (penetratin), GRKKRRQRRRPPQC (Tat
fragment 48-60), GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based
peptide), LLIILRRRIRKQAHAHSK (PVEC), GWTLNSAGYLLKINLKALAALAKKIL
(transportan), KLALKLALKALKAALKLA (amphiphilic model peptide),
RRRRRRRRR (Arg9), KFFKFFKFFK (Bacterial cell wall permeating
peptide), LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37),
SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1),
ACYCRIPACIAGERRYGTCIYQGRLWAFCC (.alpha.-defensin),
DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (.beta.-defensin),
RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39),
ILPWKWPWWPWRR-NH2 (indolicidin), AAVALLPAVLLALLAP (RFGF),
AALLPVLLAAP (RFGF analogue) and RKCRIVVIRVCR (bactenecin).
[0326] Exemplary cationic groups include, but are not limited to,
protonated amino groups, derived from e.g., O-AMINE
(AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene
diamine, polyamino); aminoalkoxy, e.g., O(CH.sub.2).sub.nAMINE,
(e.g., AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino); amino (e.g. NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid); and
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino).
[0327] As used herein the term "targeting ligand" refers to any
molecule that provides an enhanced affinity for a selected target,
e.g., a cell, cell type, tissue, organ, region of the body, or a
compartment, e.g., a cellular, tissue or organ compartment. Some
exemplary targeting ligands include, but are not limited to,
antibodies, antigens, folates, receptor ligands, carbohydrates,
aptamers, integrin receptor ligands, chemokine receptor ligands,
transferrin, biotin, serotonin receptor ligands, PSMA, endothelin,
GCPII, somatostatin, LDL and HDL ligands.
[0328] Carbohydrate based targeting ligands include, but are not
limited to, D-galactose, multivalent galactose,
N-acetyl-D-galactose (GalNAc), multivalent GalNAc, e.g. GalNAC2 and
GalNAc3; D-mannose, multivalent mannose, multivalent lactose,
N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent fucose,
glycosylated polyaminoacids and lectins. The term multivalent
indicates that more than one monosaccharide unit is present. Such
monosaccharide subunits can be linked to each other through
glycosidic linkages or linked to a scaffold molecule.
[0329] A number of folate and folate analogs amenable to the
present invention as ligands are described in U.S. Pat. Nos.
2,816,110; 51410,104; 5,552,545; 6,335,434 and 7,128,893, contents
which are herein incorporated in their entireties by reference.
[0330] As used herein, the terms "PK modulating ligand" and "PK
modulator" refers to molecules which can modulate the
pharmacokinetics of the composition of the invention. Some
exemplary PK modulator include, but are not limited to, lipophilic
molecules, bile acids, sterols, phospholipid analogues, peptides,
protein binding agents, vitamins, fatty acids, phenoxazine,
aspirin, naproxen, ibuprofen, PEGs, and biotin. Oligonucleotides
that comprise a number of phosphorothioate linkages are also known
to bind to serum protein, thus short oligonucleotides, e.g.
oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,
comprising plurality of phosphorothioate linkages in the backbone
are also amenable to the present invention as ligands (e.g. as PK
modulating ligands). In addition, aptamers that bind serum
components (e.g. serum proteins) are also amenable to the present
invention as PK modulating ligands.
[0331] When two or more ligands are present, the ligands can all
have same properties, all have different properties or some ligands
have the same properties while others have different properties.
For example, a ligand can have targeting properties, have
endosomolytic activity or have PK modulating properties. In a
preferred embodiment, all the ligands have different
properties.
[0332] In some embodiments, ligand on one strand of double-stranded
oligonucleotide has affinity for a ligand on the second strand. In
certain other embodiments, a ligand is covalently linked to both
strands of a double-stranded oligonucleotide. As used herein, when
a ligand is linked to more than oligonucleotide strand, point of
attachment for an oligonucleotide can be an atom of the ligand self
or an atom on a carrier molecule to which the ligand itself is
attached.
[0333] Ligands can be coupled to the oligonucleotides at various
places, for example, 3'-end, 5'-end, and/or at an internal
position. When two or more ligands are present, the ligand can be
on opposite ends of an oligonucleotide. In preferred embodiments,
the ligand is attached to the oligonucleotides via an intervening
tether/linker. The ligand or tethered ligand can be present on a
monomer when said monomer is incorporated into the growing strand.
In some embodiments, the ligand can be incorporated via coupling to
a "precursor" monomer after said "precursor" monomer has been
incorporated into the growing strand. For example, a monomer
having, e.g., an amino-terminated tether (i.e., having no
associated ligand), e.g., monomer-linker-NH.sub.2 can be
incorporated into a growing oligonucleotide strand. In a subsequent
operation, i.e., after incorporation of the precursor monomer into
the strand, a ligand having an electrophilic group, e.g., a
pentafluorophenyl ester or aldehyde group, can subsequently be
attached to the precursor monomer by coupling the electrophilic
group of the ligand with the terminal nucleophilic group of the
precursor monomer's tether.
[0334] In another example, a monomer having a chemical group
suitable for taking part in Click Chemistry reaction can be
incorporated e.g., an azide or alkyne terminated tether/linker. In
a subsequent operation, i.e., after incorporation of the precursor
monomer into the strand, a ligand having complementary chemical
group, e.g. an alkyne or azide can be attached to the precursor
monomer by coupling the alkyne and the azide together.
[0335] For double-stranded oligonucleotides, ligands can be
attached to one or both strands. In some embodiments, a
double-stranded RNAi agent comprises a ligand conjugated to the
sense strand. In other embodiments, a double-stranded RNAi agent
comprises a ligand conjugated to the antisense strand.
[0336] In some embodiments, ligand can be conjugated to
nucleobases, sugar moieties, or internucleosidic linkages of
nucleic acid molecules. Conjugation to purine nucleobases or
derivatives thereof can occur at any position including, endocyclic
and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or
8-positions of a purine nucleobase are attached to a conjugate
moiety. Conjugation to pyrimidine nucleobases or derivatives
thereof can also occur at any position. In some embodiments, the
2-, 5-, and 6-positions of a pyrimidine nucleobase can be
substituted with a conjugate moiety. When a ligand is conjugated to
a nucleobase, the preferred position is one that does not interfere
with hybridization, i.e., does not interfere with the hydrogen
bonding interactions needed for base pairing.
[0337] Conjugation to sugar moieties of nucleosides can occur at
any carbon atom. Example carbon atoms of a sugar moiety that can be
attached to a conjugate moiety include the 2', 3', and 5' carbon
atoms. The 1' position can also be attached to a conjugate moiety,
such as in an abasic residue. Internucleosidic linkages can also
bear conjugate moieties. For phosphorus-containing linkages (e.g.,
phosphodiester, phosphorothioate, phosphorodithiotate,
phosphoroamidate, and the like), the conjugate moiety can be
attached directly to the phosphorus atom or to an O, N, or S atom
bound to the phosphorus atom. For amine- or amide-containing
internucleosidic linkages (e.g., PNA), the conjugate moiety can be
attached to the nitrogen atom of the amine or amide or to an
adjacent carbon atom.
[0338] There are numerous methods for preparing conjugates of
oligomeric compounds. Generally, an oligomeric compound is attached
to a conjugate moiety by contacting a reactive group (e.g., OH, SH,
amine, carboxyl, aldehyde, and the like) on the oligomeric compound
with a reactive group on the conjugate moiety. In some embodiments,
one reactive group is electrophilic and the other is
nucleophilic.
[0339] For example, an electrophilic group can be a
carbonyl-containing functionality and a nucleophilic group can be
an amine or thiol. Methods for conjugation of nucleic acids and
related oligomeric compounds with and without linking groups are
well described in the literature such as, for example, in Manoharan
in Antisense Research and Applications, Crooke and LeBleu, eds.,
CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is
incorporated herein by reference in its entirety.
[0340] Representative United States patents that teach the
preparation of oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578, 717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578, 718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904, 582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082, 830;
5,112,963; 5,149,782; 5,214,136; 5,245,022; 5,254, 469; 5,258,506;
5,262,536; 5,272,250; 5,292,873; 5,317, 098; 5,371,241, 5,391,723;
5,416,203, 5,451,463; 5,510, 475; 5,512,667; 5,514,785; 5,565,552;
5,567,810; 5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;
5,599, 923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153, 737;
6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395, 437; 6,444,806;
6,486,308; 6,525,031; 6,528,631; 6,559, 279; contents which are
herein incorporated in their entireties by reference.
Ligand Carriers
[0341] In some embodiments, the ligands, e.g. endosomolytic
ligands, targeting ligands or other ligands, are linked to a
monomer which is then incorporated into the growing oligonucleotide
strand during chemical synthesis. Such monomers are also referred
to as carrier monomers herein. The carrier monomer is a cyclic
group or acyclic group; preferably, the cyclic group is selected
from the group consisting of pyrrolidinyl, pyrazolinyl,
pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,
piperazinyl, [1,3]-dioxolane, oxazolidinyl, isoxazolidinyl,
morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,
pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic
group is selected from serinol backbone or diethanolamine backbone.
In certain embodiments, the cyclic carrier monomer is based on
pyrrolidinyl such as 4-hydroxyproline or a derivative thereof.
Linkers
[0342] In certain embodiments, the covalent linkages between the
oligonucleotide and other components, e.g. a ligand or a ligand
carrying monomer can be mediated by a linker. This linker can be
cleavable or non-cleavable, depending on the application. In
certain embodiments, a cleavable linker can be used to release the
nucleic acid after transport to the desired target. The intended
nature of the conjugation or coupling interaction, or the desired
biological effect, will determine the choice of linker group.
[0343] As used herein, the term "linker" means an organic moiety
that connects two parts of a compound. Linkers typically comprise a
direct bond or an atom such as oxygen or sulfur, a unit such as
NR.sup.1, C(O), C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of
atoms, such as substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,
heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,
heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,
heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,
alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,
alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
alkylheteroarylalkenyl, alkylheteroarylalkynyl,
alkenylheteroarylalkyl, alkenylheteroarylalkenyl,
alkenylheteroarylalkynyl, alkynylheteroarylalkyl,
alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,
alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or
more methylenes can be interrupted or terminated by O, S, S(O),
SO.sub.2, N(R.sup.1).sub.2, C(O), cleavable linking group,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocyclic; where
R.sup.1 is hydrogen, acyl, aliphatic or substituted aliphatic.
[0344] In one embodiment, the linker is
--[(P-Q-R).sub.q--X--(P'-Q'-R').sub.q'].sub.q''-T-, wherein:
[0345] P, R, T, P' and R' are each independently for each
occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2,
CH.sub.2NH, CH.sub.2O; NHCH(R.sup.a)C(O),
--C(O)--CH(R.sup.a)--NH--, C(O)-- (optionally substituted
alkyl)-NH--, CH.dbd.N--O,
##STR00004##
cyclyl, heterocycyclyl, aryl or heteroaryl; R.sub.50 and R.sub.51
are independently alkyl, substituted alkyl, or R.sub.50 and
R.sub.51 taken together to form a cyclic ring;
[0346] Q and Q' are each independently for each occurrence absent,
--(CH.sub.2).sub.n--, --C(R.sup.100)(R.sup.200)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.100)(R.sup.200)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--;
[0347] X is absent or a cleavable linking group;
[0348] R.sup.a is H or an amino acid side chain;
[0349] R.sup.100 and R.sup.200 are each independently for each
occurrence H, CH.sub.3, OH, SH or N(R.sup.X).sub.2;
[0350] R.sup.X is independently for each occurrence H, methyl,
ethyl, propyl, isopropyl, butyl or benzyl;
[0351] q, q' and q'' are each independently for each occurrence
0-20 and wherein the repeating unit can be the same or
different;
[0352] n is independently for each occurrence 1-20; and
[0353] m is independently for each occurrence 0-50.
[0354] In one embodiment, the linker comprises at least one
cleavable linking group.
[0355] In certain embodiments, the linker is a branched linker. The
branchpoint of the branched linker may be at least trivalent, but
can be a tetravalent, pentavalent or hexavalent atom, or a group
presenting such multiple valencies. In certain embodiments, the
branchpoint is, --N, --N(Q)-C, --O--C, --S--C, --SS--C,
--C(O)N(Q)-C, --OC(O)N(Q)-C, --N(Q)C(O)--C, or --N(Q)C(O)O--C;
wherein Q is independently for each occurrence H or optionally
substituted alkyl. In one embodiment, the branchpoint is glycerol
or derivative thereof.
[0356] In some embodiments, the carrier monomer can be based on the
pyrroline ring system as shown in formula (I)
##STR00005##
[0357] wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH,
SO, SO.sub.2, or SO.sub.2NH;
[0358] R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16,
R.sup.17, and R.sup.18 are each independently for each occurrence
H, --CH.sub.2OR.sup.a, or OR.sup.b,
[0359] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, or a solid support;
[0360] R.sup.30 is independently for each occurrence
-linker-R.sup.L;
[0361] R.sup.L is hydrogen or a ligand, e.g. an endosomolytic
agent, a targeting ligand or other ligand described herein; and
[0362] provided that R.sup.L is a ligand at least once.
[0363] For the pyrroline-based monomers, R.sup.11 is
--CH.sub.2OR.sup.a and R1.sup.3 is OR.sup.b; or R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.9 is OR.sup.b; or R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.11 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.15 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b. In certain
embodiments, CH.sub.2OR.sup.a and OR.sup.b can be geminally
substituted. For the 4-hydroxyproline-based monomers, R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b. The pyrroline- and
4-hydroxyproline-based compounds can therefore contain linkages
(e.g., carbon-carbon bonds) wherein bond rotation is restricted
about that particular linkage, e.g. restriction resulting from the
presence of a ring. Thus, CH.sub.2OR.sup.a and OR.sup.b can be cis
or trans with respect to one another in any of the pairings
delineated above Accordingly, all cis/trans isomers are expressly
included. The compounds can also contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of the compounds are expressly included
(e.g., the centers bearing CH.sub.2OR.sup.a and OR.sup.b can both
have the R configuration; or both have the S configuration; or one
center can have the R configuration and the other center can have
the S configuration and vice versa).
[0364] In one preferred embodiment, R.sup.11 is CH.sub.2OR.sup.a
and R.sup.15 is OR.sup.b.
[0365] In one embodiment, R.sup.b is a solid support.
[0366] In another embodiment, carrier of formula (I) is a
phosphoramidite, i.e., one of R.sup.a or R.sup.b is
--P(O-alkyl)N(alkyl).sub.2, e.g.,
--P(OCH.sub.2CH.sub.2CN)N(i-propyl).sub.2. In one embodiment,
R.sup.b is --P(O-alkyl)N(alkyl).sub.2.
[0367] In some embodiments, the carrier can be based on the ribose
ring system as shown in formula (II).
##STR00006##
[0368] wherein:
[0369] X is O, S, NR.sup.N or CR.sup.P.sub.2;
[0370] B is independently for each occurrence hydrogen, optionally
substituted natural or non-natural nucleobase, optionally
substituted natural nucleobase conjugated with -linker-R.sup.L or
optionally substituted non-natural nucleobase conjugated with
-linker-R.sup.L;
[0371] R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are each
independently for each occurrence H, OR.sup.6, F, N(R.sup.N).sub.2,
or -J-linker-R.sup.L;
[0372] J is absent, O, S, NR.sup.N, OC(O)NH, NHC(O)O, C(O)NH,
NHC(O), NHSO, NHSO.sub.2, NHSO.sub.2NH, OC(O), C(O)O, OC(O)O,
NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R.sup.P).sub.2)O, or
OP(N(R.sup.P).sub.2);
[0373] R.sup.6 is independently for each occurrence hydrogen,
hydroxyl protecting group, optionally substituted alkyl, optionally
substituted aryl, optionally substituted cycloalkyl, optionally
substituted aralkyl, optionally substituted alkenyl, optionally
substituted heteroaryl, polyethyleneglycol (PEG), a phosphate, a
diphosphate, a triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite or a solid support;
[0374] R.sup.N is independently for each occurrence H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted heteroaryl or an amino protecting group;
[0375] R.sup.P is independently for each occurrence H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted cycloalkyl or optionally substituted heteroaryl;
[0376] R.sup.L is hydrogen or a ligand, e.g. an endosomolytic
agent, a targeting ligand or other ligand described herein; and
[0377] provided that R.sup.L is present at least once and further
provided that R.sup.L is a ligand at least once.
[0378] In some embodiments, the carrier monomer is based on an
acyclic group and is termed an "acyclic carrier". Preferred acyclic
carriers can have the structure shown in formula (III) or formula
(IV) below.
[0379] In some embodiments, the acyclic carrier has the structure
shown in formula (III).
##STR00007##
[0380] wherein:
[0381] W is absent, O, S and N(R.sup.N), where R.sup.N is
independently for each occurrence H, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted cycloalkyl,
optionally substituted aralkyl, optionally substituted heteroaryl
or an amino protecting group;
[0382] E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO,
SO.sub.2, or SO.sub.2NH;
[0383] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite or a solid support;
[0384] R.sup.30 is independently for each occurrence
-linker-R.sup.L;
[0385] R.sup.L is hydrogen or a ligand, e.g. an endosomolytic
agent, a targeting ligand or other ligand described herein; and
[0386] r, s and t are each independently for each occurrence 0, 1,
2 or 3;
[0387] provided that R.sup.L is a ligand at least once.
[0388] When r and s are different, then the tertiary carbon can be
either the R or S configuration. In preferred embodiments, x and y
are one and z is zero (e.g. carrier is based on serinol). The
acyclic carriers can optionally be substituted, e.g. with hydroxy,
alkoxy, perhaloalky.
[0389] In some embodiments, the acyclic carrier has the structure
shown in formula (IV)
##STR00008##
[0390] wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH,
SO, SO.sub.2, or SO.sub.2NH;
[0391] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite or a solid support;
[0392] R.sup.30 is independently for each occurrence
-linker-R.sup.L;
[0393] R.sup.L is hydrogen or a ligand; and
[0394] r and s are each independently for each occurrence 0, 1, 2
or 3;
[0395] provided that R.sup.L is a ligand at least once.
[0396] Other ligands and ligand conjugated monomers amenable to the
invention are described in U.S. patent application Ser. No.
10/916,185, filed Aug. 10, 2004; Ser. No. 10/946,873, filed Sep.
21, 2004; Ser. No. 10/985,426, filed Nov. 9, 2004; Ser. No.
10/833,934, filed Aug. 3, 2007; Ser. No. 11/115,989 filed Apr. 27,
2005, Ser. No. 11/119,533, filed Apr. 29, 2005; Ser. No.
11/197,753, filed Aug. 4, 2005; Ser. No. 11/944,227, filed Nov. 21,
2007; Ser. No. 12/328,528, filed Dec. 4, 2008; and Ser. No.
12/328,537, filed Dec. 4, 2008, contents which are herein
incorporated in their entireties by reference for all purposes.
Ligands and ligand conjugated monomers amenable to the invention
are also described in International Application Nos.
PCT/US04/001461, filed Jan. 21, 2004; PCT/US04/010586, filed Apr.
5, 2004; PCT/US04/011255, filed Apr. 9, 2005; PCT/US05/014472,
filed Apr. 27, 2005; PCT/US05/015305, filed Apr. 29, 2005;
PCT/US05/027722, filed Aug. 4, 2005; PCT/US08/061289, filed Apr.
23, 2008; PCT/US08/071576, filed Jul. 30, 2008; PCT/US08/085574,
filed Dec. 4, 2008 and PCT/US09/40274, filed Apr. 10, 2009,
contents which are herein incorporated in their entireties by
reference for all purposes.
Cleavable Linking Groups
[0397] A cleavable linking group is one which is sufficiently
stable outside the cell, but which upon entry into a target cell is
cleaved to release the two parts the linker is holding together. In
a preferred embodiment, the cleavable linking group is cleaved at
least 10 times or more, preferably at least 100 times faster in the
target cell or under a first reference condition (which can, e.g.,
be selected to mimic or represent intracellular conditions) than in
the blood or serum of a subject, or under a second reference
condition (which can, e.g., be selected to mimic or represent
conditions found in the blood or serum).
[0398] Cleavable linking groups are susceptible to cleavage agents,
e.g., pH, redox potential or the presence of degradative molecules.
Generally, cleavage agents are more prevalent or found at higher
levels or activities inside cells than in serum or blood. Examples
of such degradative agents include: redox agents which are selected
for particular substrates or which have no substrate specificity,
including, e.g., oxidative or reductive enzymes or reductive agents
such as mercaptans, present in cells, that can degrade a redox
cleavable linking group by reduction; esterases; endosomes or
agents that can create an acidic environment, e.g., those that
result in a pH of five or lower; enzymes that can hydrolyze or
degrade an acid cleavable linking group by acting as a general
acid, peptidases (which can be substrate specific), and
phosphatases.
[0399] A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The pH of human serum is 7.4, while the average
intracellular pH is slightly lower, ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and
lysosomes have an even more acidic pH at around 5.0. Some linkers
will have a cleavable linking group that is cleaved at a preferred
pH, thereby releasing the cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
[0400] A linker can include a cleavable linking group that is
cleavable by a particular enzyme. The type of cleavable linking
group incorporated into a linker can depend on the cell to be
targeted. For example, liver targeting ligands can be linked to the
cationic lipids through a linker that includes an ester group.
Liver cells are rich in esterases, and therefore the linker will be
cleaved more efficiently in liver cells than in cell types that are
not esterase-rich. Other cell-types rich in esterases include cells
of the lung, renal cortex, and testis. Linkers that contain peptide
bonds can be used when targeting cell types rich in peptidases,
such as liver cells and synoviocytes.
[0401] In general, the suitability of a candidate cleavable linking
group can be evaluated by testing the ability of a degradative
agent (or condition) to cleave the candidate linking group. It will
also be desirable to also test the candidate cleavable linking
group for the ability to resist cleavage in the blood or when in
contact with other non-target tissue. Thus one can determine the
relative susceptibility to cleavage between a first and a second
condition, where the first is selected to be indicative of cleavage
in a target cell and the second is selected to be indicative of
cleavage in other tissues or biological fluids, e.g., blood or
serum. The evaluations can be carried out in cell free systems, in
cells, in cell culture, in organ or tissue culture, or in whole
animals. It can be useful to make initial evaluations in cell-free
or culture conditions and to confirm by further evaluations in
whole animals. In preferred embodiments, useful candidate compounds
are cleaved at least 2, 4, 10 or 100 times faster in the cell (or
under in vitro conditions selected to mimic intracellular
conditions) as compared to blood or serum (or under in vitro
conditions selected to mimic extracellular conditions).
Redox Cleavable Linking Groups
[0402] One class of cleavable linking groups are redox cleavable
linking groups that are cleaved upon reduction or oxidation. An
example of reductively cleavable linking group is a disulphide
linking group (--S--S--). To determine if a candidate cleavable
linking group is a suitable "reductively cleavable linking group,"
or for example is suitable for use with a particular iRNA moiety
and particular targeting agent one can look to methods described
herein. For example, a candidate can be evaluated by incubation
with dithiothreitol (DTT), or other reducing agent using reagents
know in the art, which mimic the rate of cleavage which would be
observed in a cell, e.g., a target cell. The candidates can also be
evaluated under conditions which are selected to mimic blood or
serum conditions. In a preferred embodiment, candidate compounds
are cleaved by at most 10% in the blood. In preferred embodiments,
useful candidate compounds are degraded at least 2, 4, 10 or 100
times faster in the cell (or under in vitro conditions selected to
mimic intracellular conditions) as compared to blood (or under in
vitro conditions selected to mimic extracellular conditions). One
preferred embodiment is --C(R).sub.2--S--S--, wherein R is H or
C.sub.1-C.sub.6 alkyl and at least one R is C.sub.1-C.sub.6 alkyl
such as CH.sub.3 or CH.sub.2CH.sub.3.
Phosphate-Based Cleavable Linking Groups
[0403] Phosphate-based cleavable linking groups are cleaved by
agents that degrade or hydrolyze the phosphate group. An example of
an agent that cleaves phosphate groups in cells are enzymes such as
phosphatases in cells. Examples of phosphate-based linking groups
are --O--P(O)(OR)--O--, --O--P(S)(OR)--O--, --O--P(S)(SR)--O--,
--S--P(O)(OR)--O--, --O--P(O)(OR)--S--, --S--P(O)(ORk)-S--,
--O--P(S)(ORk)-S--, --S--P(S)(OR)--O--, --O--P(O)(R)--O--,
--O--P(S)(R)--O--, --S--P(O)(R)--O--, --S--P(S)(R)--O--,
--S--P(O)(R)--S--, --O--P(S)(R)--S--. Preferred embodiments are
--O--P(O)(OH)--O--, --O--P(S)(OH)--O--, --O--P(S)(SH)--O--,
--S--P(O)(OH)--O--, --O--P(O)(OH)--S--, --S--P(O)(OH)--S--,
--O--P(S)(OH)--S--, --S--P(S)(OH)--O--, --O--P(O)(H)--O--,
--O--P(S)(H)--O--, --S--P(O)(H)--O--, --S--P(S)(H)--O--,
--S--P(O)(H)--S--, --O--P(S)(H)--S--, wherein R is optionally
substituted linear or branched C.sub.1-C.sub.10 alkyl A preferred
embodiment is --O--P(O)(OH)--O--.
Acid Cleavable Linking Groups
[0404] Acid cleavable linking groups are linking groups that are
cleaved under acidic conditions. In preferred embodiments acid
cleavable linking groups are cleaved in an acidic environment with
a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower),
or by agents such as enzymes that can act as a general acid. In a
cell, specific low pH organelles, such as endosomes and lysosomes
can provide a cleaving environment for acid cleavable linking
groups. Examples of acid cleavable linking groups include but are
not limited to hydrazones, esters, and esters of amino acids. Acid
cleavable groups can have the general formula --C.dbd.NN-- or
--OC(O)--.
Ester-Based Cleavable Linking Groups
[0405] Ester-based cleavable linking groups are cleaved by enzymes
such as esterases and amidases in cells. Examples of ester-based
cleavable linking groups include but are not limited to esters of
alkylene, alkenylene and alkynylene groups. Ester cleavable linking
groups have the general formula --C(O)O--.
Peptide-Based Cleavable Linking Groups
[0406] Peptide-based cleavable linking groups are cleaved by
enzymes such as peptidases and proteases in cells. A peptide based
cleavable linking group comprises two or more amino acids.
Peptide-based cleavable linking groups have the general formula
--NHCHR.sup.AC(O)NHCHR.sup.BC(O)--, where R.sup.A and R.sup.B are
the R groups of the two adjacent amino acids. In certain
embodiments, the peptide-based cleavage linkage comprises the amino
acid sequence that is the substrate for a peptidase or a protease
found in cells.
Oligonucleotide Production
[0407] The oligonucleotide compounds of the invention can be
prepared using solution-phase or solid-phase organic synthesis, or
enzymatically by methods known in the art. Organic synthesis offers
the advantage that the oligonucleotide strands comprising
non-natural or modified nucleotides can be easily prepared. Any
other means for such synthesis known in the art can additionally or
alternatively be employed. It is also known to use similar
techniques to prepare other oligonucleotides, such as the
phosphorothioates, phosphorodithioates and alkylated derivatives.
The double-stranded oligonucleotide compounds of the invention can
be prepared using a two-step procedure. First, the individual
strands of the double-stranded molecule are prepared separately.
Then, the component strands are annealed.
[0408] Regardless of the method of synthesis, the oligonucleotide
can be prepared in a solution (e.g., an aqueous and/or organic
solution) that is appropriate for formulation. For example, the
oligonucleotide preparation can be precipitated and redissolved in
pure double-distilled water, and lyophilized. The dried
oligonucleotide can then be resuspended in a solution appropriate
for the intended formulation process.
[0409] Teachings regarding the synthesis of particular modified
oligonucleotides can be found in the following U.S. patents or
pending patent applications: U.S. Pat. Nos. 5,138,045 and
5,218,105, drawn to polyamine conjugated oligonucleotides; U.S.
Pat. No. 5,212,295, drawn to monomers for the preparation of
oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.
5,378,825 and 5,541,307, drawn to oligonucleotides having modified
backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified
oligonucleotides and the preparation thereof through reductive
coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases
based on the 3-deazapurine ring system and methods of synthesis
thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases
based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to
processes for preparing oligonucleotides having chiral phosphorus
linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids;
U.S. Pat. No. 5,554,746, drawn to oligonucleotides having
.beta.-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods
and materials for the synthesis of oligonucleotides; U.S. Pat. No.
5,578,718, drawn to nucleosides having alkylthio groups, wherein
such groups can be used as linkers to other moieties attached at
any of a variety of positions of the nucleoside; U.S. Pat. Nos.
5,587,361 and 5,599,797, drawn to oligonucleotides having
phosphorothioate linkages of high chiral purity; U.S. Pat. No.
5,506,351, drawn to processes for the preparation of 2'-O-alkyl
guanosine and related compounds, including 2,6-diaminopurine
compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides
having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to
oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168,
and U.S. Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl
nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn
to backbone-modified oligonucleotide analogs; and U.S. Pat. Nos.
6,262,241, and 5,459,255, drawn to, inter alia, methods of
synthesizing 2'-fluoro-oligonucleotides.
Gene Expression Modulation
[0410] As used herein the term "modulate gene expression" means
that expression of the gene, or level of RNA molecule or equivalent
RNA molecules encoding one or more proteins or protein subunits is
up regulated of down regulated, such that expression, level, or
activity is greater than or less than that observed in the absence
of the modulator. For example, the term "modulate" can mean
"inhibit," but the use of the word "modulate" is not limited to
this definition.
[0411] As used herein, gene expression modulation happens when the
expression of the gene, or level of RNA molecule or equivalent RNA
molecules encoding one or more proteins or protein subunits is
different by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more from that
observed in the absence of modulator, e.g., oligonucleotide. The %
and(/or) fold difference can be calculated relative to the control
or the non-control.
[0412] As used herein, the term "inhibit", "down-regulate", or
"reduce", means that the expression of the gene, or level of RNA
molecules or equivalent RNA molecules encoding one or more proteins
or protein subunits, or activity of one or more proteins or protein
subunits, is reduced below that observed in the absence of
modulator. The gene expression is down-regulated when expression of
the gene, or level of RNA molecules or equivalent RNA molecules
encoding one or more proteins or protein subunits, or activity of
one or more proteins or protein subunits, is reduced at least 10%
lower relative to a corresponding non-modulated control, and
preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
98%, 99% or most preferably, 100% (i.e., no gene expression).
[0413] As used herein, the term "increase" or "up-regulate", means
that the expression of the gene, or level of RNA molecules or
equivalent RNA molecules encoding one or more proteins or protein
subunits, or activity of one or more proteins or protein subunits,
is increased above that observed in the absence of modulator. The
gene expression is up-regulated when expression of the gene, or
level of RNA molecules or equivalent RNA molecules encoding one or
more proteins or protein subunits, or activity of one or more
proteins or protein subunits, is increased at least 10% relative to
a corresponding non-modulated control, and preferably at least 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% 100%, 2-fold,
5-fold, 10-fold, 100-fold or 1000-fold higher than in the absence
of a modulator.
[0414] By "gene" or "target gene" is meant, a nucleic acid that
encodes an RNA, for example, nucleic acid sequences including, but
not limited to, structural genes encoding a polypeptide. The target
gene can be a gene derived from a cell, an endogenous gene, a
transgene, or exogenous genes such as genes of a pathogen, for
example a virus, which is present in the cell after infection
thereof. The cell containing the target gene can be derived from or
contained in any organism, for example a plant, animal, protozoan,
virus, bacterium, or fungus.
Formulations/Delivery Agents
[0415] For ease of exposition the formulations, compositions,
delivery agents and methods in this section are discussed largely
with regard to RNAi agents. It may be understood, however, that
these formulations, compositions and methods can be practiced with
other oligonucleotides described herein, e.g., antisense,
antagomir, aptamer, microRNA, antimir and ribozyme, and such
practice is within the invention.
[0416] A formulated RNAi composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., less
than 80, 50, 30, 20, or 10% water). In another example, the RNAi is
in an aqueous phase, e.g., in a solution that includes water.
[0417] The aqueous phase or the crystalline compositions can, e.g.,
be incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase) or a particle (e.g., a micro
particle as can be appropriate for a crystalline composition).
Generally, the RNAi composition is formulated in a manner that is
compatible with the intended method of administration.
[0418] In particular embodiments, the composition is prepared by at
least one of the following methods: spray drying, lyophilization,
vacuum drying, evaporation, fluid bed drying, or a combination of
these techniques; or sonication with a lipid, freeze-drying,
condensation and other self-assembly.
[0419] An RNAi preparation can be formulated in combination with
another agent, e.g., another therapeutic agent or an agent that
stabilizes the RNAi agent, e.g., a protein that complex with RNAi
agent to form an iRNP. Still other agents include chelators, e.g.,
EDTA (e.g., to remove divalent cations such as Mg.sup.2+), salts,
RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as
RNAsin) and so forth.
[0420] In one embodiment, the RNAi preparation includes another
RNAi agent, e.g., a second RNAi that can mediated RNAi with respect
to a second gene, or with respect to the same gene. Still other
preparation can include at least 3, 5, ten, twenty, fifty, or a
hundred or more different RNAi species. Such RNAi agents can
mediate RNAi with respect to a similar number of different
genes.
[0421] In one embodiment, the RNAi preparation includes at least a
second therapeutic agent (e.g., an agent other than RNA or DNA).
For example, an RNAi composition for the treatment of a viral
disease, e.g., HIV, might include a known antiviral agent (e.g., a
protease inhibitor or reverse transcriptase inhibitor). In another
example, an RNAi agent composition for the treatment of a cancer
might further comprise a chemotherapeutic agent.
[0422] Exemplary formulations and/or delivery agents are discussed
below:
Liposomes
[0423] The oligonucleotides of the invention, e.g. antisense,
antagomir, aptamer, ribozyme and RNAi agent can be formulated in
liposomes. As used herein, a liposome is a structure having
lipid-containing membranes enclosing an aqueous interior. Liposomes
can have one or more lipid membranes. Liposomes can be
characterized by membrane type and by size. Small unilamellar
vesicles (SUVs) have a single membrane and typically range between
0.02 and 0.05 .mu.m in diameter; large unilamellar vesicles (LUVS)
are typically larger than 0.05 .mu.m. Oligolamellar large vesicles
and multilamellar vesicles have multiple, usually concentric,
membrane layers and are typically larger than 0.1 .mu.m. Liposomes
with several non-concentric membranes, i.e., several smaller
vesicles contained within a larger vesicle, are termed
multivesicular vesicles.
[0424] Liposomes can further include one or more additional lipids
and/or other components such as cholesterol. Other lipids can be
included in the liposome compositions for a variety of purposes,
such as to prevent lipid oxidation, to stabilize the bilayer, to
reduce aggregation during formation or to attach ligands onto the
liposome surface. Any of a number of lipids can be present,
including amphipathic, neutral, cationic, and anionic lipids. Such
lipids can be used alone or in combination.
[0425] Additional components that can be present in a liposomes
include bilayer stabilizing components such as polyamide oligomers
(see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins,
detergents, lipid-derivatives, such as PEG conjugated to
phosphatidylethanolamine, PEG conjugated to phosphatidic acid, PEG
conjugated to ceramides (see, U.S. Pat. No. 5,885,613), PEG
conjugated dialkylamines and PEG conjugated
1,2-diacyloxypropan-3-amines.
[0426] Liposome can include components selected to reduce
aggregation of lipid particles during formation, which can result
from steric stabilization of particles which prevents
charge-induced aggregation during formation. Suitable components
that reduce aggregation include, but are not limited to,
polyethylene glycol (PEG)-modified lipids, monosialoganglioside
Gm1, and polyamide oligomers ("PAO") such as (described in U.S.
Pat. No. 6,320,017). Exemplary suitable PEG-modified lipids
include, but are not limited to, PEG-modified
phosphatidylethanolamine and phosphatidic acid, PEG-ceramide
conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified
dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines.
Particularly preferred are PEG-modified diacylglycerols and
dialkylglycerols. Other compounds with uncharged, hydrophilic,
steric-barrier moieties, which prevent aggregation during
formation, like PEG, Gm1, or ATTA, can also be coupled to lipids to
reduce aggregation during formation. ATTA-lipids are described,
e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid conjugates are
described, e.g., in U.S. Pat. Nos. 5,820,873, 5,534,499 and
5,885,613. Typically, the concentration of the lipid component
selected to reduce aggregation is about 1 to 15% (by mole percent
of lipids). It should be noted that aggregation preventing
compounds do not necessarily require lipid conjugation to function
properly. Free PEG or free ATTA in solution can be sufficient to
prevent aggregation. If the liposomes are stable after formulation,
the PEG or ATTA can be dialyzed away before administration to a
subject.
[0427] Neutral lipids, when present in the liposome composition,
can be any of a number of lipid species which exist either in an
uncharged or neutral zwitterionic form at physiological pH. Such
lipids include, for example diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin,
dihydrosphingomyelin, cephalin, and cerebrosides. The selection of
neutral lipids for use in liposomes described herein is generally
guided by consideration of, e.g., liposome size and stability of
the liposomes in the bloodstream. Preferably, the neutral lipid
component is a lipid having two acyl groups, (i.e.,
diacylphosphatidylcholine and diacylphosphatidylethanolamine).
Lipids having a variety of acyl chain groups of varying chain
length and degree of saturation are available or can be isolated or
synthesized by well-known techniques. In one group of embodiments,
lipids containing saturated fatty acids with carbon chain lengths
in the range of C.sub.14 to C.sub.22 are preferred. In another
group of embodiments, lipids with mono or diunsaturated fatty acids
with carbon chain lengths in the range of C.sub.14 to C.sub.22 are
used. Additionally, lipids having mixtures of saturated and
unsaturated fatty acid chains can be used. Preferably, the neutral
lipids used in the present invention are DOPE, DSPC, POPC, DMPC,
DPPC or any related phosphatidylcholine. The neutral lipids useful
in the present invention can also be composed of sphingomyelin,
dihydrosphingomyeline, or phospholipids with other head groups,
such as serine and inositol.
[0428] The sterol component of the lipid mixture, when present, can
be any of those sterols conventionally used in the field of
liposome, lipid vesicle or lipid particle preparation. A preferred
sterol is cholesterol.
[0429] Cationic lipids, when present in the liposome composition,
can be any of a number of lipid species which carry a net positive
charge at about physiological pH. Such lipids include, but are not
limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride ("DOTMA");
N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt
("DOTAP.Cl");
3.beta.-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,
N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"),
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"), 5-carboxyspermylglycine diocaoleyamide ("DOGS"),
and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide
("DPPES"). Additionally, a number of commercial preparations of
cationic lipids can be used, such as, e.g., LIPOFECTIN (including
DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE
(comprising DOSPA and DOPE, available from GIBCO/BRL). Other
cationic lipids suitable for lipid particle formation are described
in WO98/39359, WO96/37194. Other cationic lipids suitable for
liposome formation are described in US Provisional applications No.
61/018,616 (filed Jan. 2, 2008), No. 61/039,748 (filed Mar. 26,
2008), No. 61/047,087 (filed Apr. 22, 2008) and No. 61/051,528
(filed May 21-2008), all of which are incorporated by reference in
their entireties for all purposes.
[0430] Anionic lipids, when present in the liposome composition,
can be any of a number of lipid species which carry a net negative
charge at about physiological pH. Such lipids include, but are not
limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,
N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and
other anionic modifying groups joined to neutral lipids.
[0431] "Amphipathic lipids" refer to any suitable material, wherein
the hydrophobic portion of the lipid material orients into a
hydrophobic phase, while the hydrophilic portion orients toward the
aqueous phase. Such compounds include, but are not limited to,
phospholipids, aminolipids, and sphingolipids. Representative
phospholipids include sphingomyelin, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, palmitoyloleoyl phosphatdylcholine,
lysophosphatidylcholine, lysophosphatidylethanolamine,
dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.
Other phosphorus-lacking compounds, such as sphingolipids,
glycosphingolipid families, diacylglycerols, and
.beta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols.
[0432] Also suitable for inclusion in the liposome compositions of
the present invention are programmable fusion lipids. Liposomes
containing programmable fusion lipids have little tendency to fuse
with cell membranes and deliver their payload until a given signal
event occurs. This allows the liposome to distribute more evenly
after injection into an organism or disease site before it starts
fusing with cells. The signal event can be, for example, a change
in pH, temperature, ionic environment, or time. In the latter case,
a fusion delaying or "cloaking" component, such as an ATTA-lipid
conjugate or a PEG-lipid conjugate, can simply exchange out of the
liposome membrane over time. By the time the liposome is suitably
distributed in the body, it has lost sufficient cloaking agent so
as to be fusogenic. With other signal events, it is desirable to
choose a signal that is associated with the disease site or target
cell, such as increased temperature at a site of inflammation.
[0433] A liposome can also include a targeting moiety, e.g., a
targeting moiety that is specific to a cell type or tissue.
Targeting of liposomes with a surface coating of hydrophilic
polymer chains, such as polyethylene glycol (PEG) chains, for
targeting has been proposed (Allen, et al., Biochimica et
Biophysica Acta 1237: 99-108 (1995); DeFrees, et al., Journal of
the American Chemistry Society 118: 6101-6104 (1996); Blume, et
al., Biochimica et Biophysica Acta 1149: 180-184 (1993); Klibanov,
et al., Journal of Liposome Research 2: 321-334 (1992); U.S. Pat.
No. 5,013,556; Zalipsky, Bioconjugate Chemistry 4: 296-299 (1993);
Zalipsky, FEBS Letters 353: 71-74 (1994); Zalipsky, in Stealth
Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press, Boca Raton
Fla. (1995). Other targeting moieties, such as ligands, cell
surface receptors, glycoproteins, vitamins (e.g., riboflavin),
aptamers and monoclonal antibodies, can also be used. The targeting
moieties can include the entire protein or fragments thereof.
Targeting mechanisms generally require that the targeting agents be
positioned on the surface of the liposome in such a manner that the
targeting moiety is available for interaction with the target, for
example, a cell surface receptor.
[0434] In one approach, a targeting moiety, such as receptor
binding ligand, for targeting the liposome is linked to the lipids
forming the liposome. In another approach, the targeting moiety is
attached to the distal ends of the PEG chains forming the
hydrophilic polymer coating (Klibanov, et al., Journal of Liposome
Research 2: 321-334 (1992); Kirpotin et al., FEBS Letters 388:
115-118 (1996)). A variety of different targeting agents and
methods are known and available in the art, including those
described, e.g., in Sapra, P. and Allen, T M, Prog. Lipid Res.
42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res. 12:1-3,
(2002). Other lipids conjugated with targeting moieties are
described in US provisional application No. 61/127,751 (filed May
14, 2008) and PCT application #PCT/US2007/080331 (filed Oct. 3,
2007), all of which are incorporated by reference in their
entireties for all purposes.
[0435] A liposome composition of the invention can be prepared by a
variety of methods that are known in the art. See e.g., U.S. Pat.
No. 4,235,871, No. 4,897,355 and No. 5,171,678; published PCT
applications WO 96/14057 and WO 96/37194; Felgner, P. L. et al.,
Proc. Natl. Acad. Sci., USA (1987) 8:7413-7417, Bangham, et al. M.
Mol. Biol. (1965) 23:238, Olson, et al. Biochim. Biophys. Acta
(1979) 557:9, Szoka, et al. Proc. Natl. Acad. Sci. (1978) 75: 4194,
Mayhew, et al. Biochim. Biophys. Acta (1984) 775:169, Kim, et al.
Biochim. Biophys. Acta (1983) 728:339, and Fukunaga, et al.
Endocrinol. (1984) 115:757.
[0436] For example, a liposome composition of the invention can be
prepared by first dissolving the lipid components of a liposome in
a detergent so that micelles are formed with the lipid component.
The detergent can have a high critical micelle concentration and
can be nonionic. Exemplary detergents include, but are not limited
to, cholate, CHAPS, octylglucoside, deoxycholate and lauroyl
sarcosine. The RNAi agent preparation e.g., an emulsion, is then
added to the micelles that include the lipid components. After
condensation, the detergent is removed, e.g., by dialysis, to yield
a liposome containing the RNAi agent. If necessary a carrier
compound that assists in condensation can be added during the
condensation reaction, e.g., by controlled addition. For example,
the carrier compound can be a polymer other than a nucleic acid
(e.g., spermine or spermidine). To favor condensation, pH of the
mixture can also be adjusted.
[0437] In another example, liposomes of the present invention can
be prepared by diffusing a lipid derivatized with a hydrophilic
polymer into preformed liposome, such as by exposing preformed
liposomes to micelles composed of lipid-grafted polymers, at lipid
concentrations corresponding to the final mole percent of
derivatized lipid which is desired in the liposome. Liposomes
containing a hydrophilic polymer can also be formed by
homogenization, lipid-field hydration, or extrusion techniques, as
are known in the art.
[0438] In another exemplary formulation procedure, the RNAi agent
is first dispersed by sonication in a lysophosphatidylcholine or
other low CMC surfactant (including polymer grafted lipids). The
resulting micellar suspension of RNAi agent is then used to
rehydrate a dried lipid sample that contains a suitable mole
percent of polymer-grafted lipid, or cholesterol. The lipid and
active agent suspension is then formed into liposomes using
extrusion techniques as are known in the art, and the resulting
liposomes separated from the unencapsulated solution by standard
column separation.
[0439] In one aspect of the present invention, the liposomes are
prepared to have substantially homogeneous sizes in a selected size
range. One effective sizing method involves extruding an aqueous
suspension of the liposomes through a series of polycarbonate
membranes having a selected uniform pore size; the pore size of the
membrane will correspond roughly with the largest sizes of
liposomes produced by extrusion through that membrane. See e.g.,
U.S. Pat. No. 4,737,323.
[0440] Other suitable formulations for RNAi agents are described in
PCT application #PCT/US2007/080331 (filed Oct. 3, 2007) and U.S.
Provisional applications No. 61/018,616 (filed Jan. 2, 2008), No.
61/039,748 (filed Mar. 26, 2008), No. 61/047,087 (filed Apr. 22,
2008) and No. 61/051,528 (filed May 21-2008), #61/113,179 (filed
Nov. 10, 2008) all of which are incorporated by reference in their
entireties for all purposes.
Micelles and Other Membranous Formulations
[0441] Recently, the pharmaceutical industry introduced
microemulsification technology to improve bioavailability of some
lipophilic (water insoluble) pharmaceutical agents. Examples
include Trimetrine (Dordunoo, S. K., et al., Drug Development and
Industrial Pharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen,
P. C., et al., J Pharm Sci 80(7), 712-714, 1991). Among other
things, microemulsification provides enhanced bioavailability by
preferentially directing absorption to the lymphatic system instead
of the circulatory system, which thereby bypasses the liver, and
prevents destruction of the compounds in the hepatobiliary
circulation.
[0442] In one aspect of invention, the formulations contain
micelles formed from a compound of the present invention and at
least one amphiphilic carrier, in which the micelles have an
average diameter of less than about 100 nm. More preferred
embodiments provide micelles having an average diameter less than
about 50 nm, and even more preferred embodiments provide micelles
having an average diameter less than about 30 nm, or even less than
about 20 nm.
[0443] As defined herein, "micelles" are a particular type of
molecular assembly in which amphipathic molecules are arranged in a
spherical structure such that all hydrophobic portions on the
molecules are directed inward, leaving the hydrophilic portions in
contact with the surrounding aqueous phase. The converse
arrangement exists if the environment is hydrophobic.
[0444] While all suitable amphiphilic carriers are contemplated,
the presently preferred carriers are generally those that have
Generally-Recognized-as-Safe (GRAS) status, and that can both
solubilize the compound of the present invention and microemulsify
it at a later stage when the solution comes into a contact with a
complex water phase (such as one found in human gastro-intestinal
tract). Usually, amphiphilic ingredients that satisfy these
requirements have HLB (hydrophilic to lipophilic balance) values of
2-20, and their structures contain straight chain aliphatic
radicals in the range of C-6 to C-20. Examples are
polyethylene-glycolized fatty glycerides and polyethylene
glycols.
[0445] Exemplary amphiphilic carriers include, but are not limited
to, lecithin, hyaluronic acid, pharmaceutically acceptable salts of
hyaluronic acid, glycolic acid, lactic acid, chamomile extract,
cucumber extract, oleic acid, linoleic acid, linolenic acid,
monoolein, monooleates, monolaurates, borage oil, evening of
primrose oil, menthol, trihydroxy oxo cholanyl glycine and
pharmaceutically acceptable salts thereof, glycerin, polyglycerin,
lysine, polylysine, triolein, polyoxyethylene ethers and analogues
thereof, polidocanol alkyl ethers and analogues thereof,
chenodeoxycholate, deoxycholate, and mixtures thereof.
[0446] Particularly preferred amphiphilic carriers are saturated
and monounsaturated polyethyleneglycolyzed fatty acid glycerides,
such as those obtained from fully or partially hydrogenated various
vegetable oils. Such oils can advantageously consist of tri-. di-
and mono-fatty acid glycerides and di- and mono-polyethyleneglycol
esters of the corresponding fatty acids, with a particularly
preferred fatty acid composition including capric acid 4-10, capric
acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid
4-14 and stearic acid 5-15%. Another useful class of amphiphilic
carriers includes partially esterified sorbitan and/or sorbitol,
with saturated or mono-unsaturated fatty acids (SPAN-series) or
corresponding ethoxylated analogs (TWEEN-series).
[0447] Commercially available amphiphilic carriers are particularly
contemplated, including Gelucire-series, Labrafil, Labrasol, or
Lauroglycol (all manufactured and distributed by Gattefosse
Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate,
PEG-mono-laurate and di-laurate, Lecithin, Polysorbate 80, etc
(produced and distributed by a number of companies in USA and
worldwide).
[0448] Mixed micelle formulation suitable for delivery through
transdermal membranes can be prepared by mixing an aqueous solution
of the RNAi composition, an alkali metal C.sub.8 to C.sub.22 alkyl
sulphate, and an amphiphilic carrier. The amphiphilic carrier can
be added at the same time or after addition of the alkali metal
alkyl sulphate. Mixed micelles will form with substantially any
kind of mixing of the ingredients but vigorous mixing in order to
provide smaller size micelles.
[0449] In one method a first micelle composition is prepared which
contains the RNAi composition and at least the alkali metal alkyl
sulphate. The first micelle composition is then mixed with at least
three amphiphilic carriers to form a mixed micelle composition. In
another method, the micelle composition is prepared by mixing the
RNAi composition, the alkali metal alkyl sulphate and at least one
of the amphiphilic carriers, followed by addition of the remaining
micelle amphiphilic carriers, with vigorous mixing.
[0450] Phenol and/or m-cresol can be added to the mixed micelle
composition to stabilize the formulation and protect against
bacterial growth. Alternatively, phenol and/or m-cresol can be
added with the amphiphilic carriers. An isotonic agent such as
glycerin can also be added after formation of the mixed micelle
composition.
[0451] For delivery of the micelle formulation as a spray, the
formulation can be put into an aerosol dispenser and the dispenser
is charged with a propellant, such as hydrogen-containing
chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl
ether, diethyl ether and HFA 134a (1,1,1,2 tetrafluoroethane).
Emulsions
[0452] The oligonucleotides of the present invention can be
prepared and formulated as emulsions. Emulsions are typically
heterogeneous systems of one liquid dispersed in another in the
form of droplets (Idson, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.
301). Emulsions are often biphasic systems comprising two
immiscible liquid phases intimately mixed and dispersed with each
other. In general, emulsions can be of either the water-in-oil
(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is
finely divided into and dispersed as minute droplets into a bulk
oily phase, the resulting composition is called a water-in-oil
(w/o) emulsion. Alternatively, when an oily phase is finely divided
into and dispersed as minute droplets into a bulk aqueous phase,
the resulting composition is called an oil-in-water (o/w) emulsion.
Emulsions can contain additional components in addition to the
dispersed phases, and the active drug which can be present as a
solution in either the aqueous phase, oily phase or itself as a
separate phase. Pharmaceutical excipients such as emulsifiers,
stabilizers, dyes, and anti-oxidants can also be present in
emulsions as needed. Pharmaceutical emulsions can also be multiple
emulsions that are comprised of more than two phases such as, for
example, in the case of oil-in-water-in-oil (o/w/o) and
water-in-oil-in-water (w/o/w) emulsions. Such complex formulations
often provide certain advantages that simple binary emulsions do
not. Multiple emulsions in which individual oil droplets of an o/w
emulsion enclose small water droplets constitute a w/o/w emulsion.
Likewise a system of oil droplets enclosed in globules of water
stabilized in an oily continuous phase provides an o/w/o
emulsion.
[0453] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
can be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that can be incorporated into either
phase of the emulsion. Emulsifiers can broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0454] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants can be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0455] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0456] A large variety of non-emulsifying materials is also
included in emulsion formulations and contributes to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0457] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0458] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that can
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used can be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0459] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of ease of
formulation, as well as efficacy from an absorption and
bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base
laxatives, oil-soluble vitamins and high fat nutritive preparations
are among the materials that have commonly been administered orally
as o/w emulsions.
[0460] In one embodiment of the present invention, the compositions
are formulated as microemulsions. A microemulsion can be defined as
a system of water, oil and amphiphile which is a single optically
isotropic and thermodynamically stable liquid solution (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Typically microemulsions are systems that are prepared by first
dispersing an oil in an aqueous surfactant solution and then adding
a sufficient amount of a fourth component, generally an
intermediate chain-length alcohol to form a transparent system.
Therefore, microemulsions have also been described as
thermodynamically stable, isotropically clear dispersions of two
immiscible liquids that are stabilized by interfacial films of
surface-active molecules (Leung and Shah, in: Controlled Release of
Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0461] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0462] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions can, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase can typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase can include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and triglycerides, polyoxyethylated
glyceryl fatty acid esters, fatty alcohols, polyglycolized
glycerides, saturated polyglycolized C8-C10 glycerides, vegetable
oils and silicone oil.
[0463] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form
spontaneously when their components are brought together at ambient
temperature. This can be particularly advantageous when formulating
thermolabile drugs, peptides or dsRNAs. Microemulsions have also
been effective in the transdermal delivery of active components in
both cosmetic and pharmaceutical applications. It is expected that
the microemulsion compositions and formulations of the present
invention will facilitate the increased absorption of
oligonucleotides as well as improve the local cellular uptake of
oligonucleotides.
[0464] Microemulsions of the present invention can also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
oligonucleotides of the present invention. Penetration enhancers
used in the microemulsions of the present invention can be
classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
Lipid Particles
[0465] It has been shown that lipid-conjugated oligonucleotides,
e.g., cholesterol-conjugated oligonucleotides, bind to HDL and LDL
lipoprotein particles which mediate cellular uptake upon binding to
their respective receptors. Thus in one aspect the invention
provides formulated lipid particles (FLiPs) comprising (a) an
oligonucleotide of the invention, where said oligonucleotide has
been conjugated to a lipophile and (b) at least one lipid
component, for example an emulsion, liposome, isolated lipoprotein,
reconstituted lipoprotein or phospholipid, to which the conjugated
oligonucleotide has been aggregated, admixed or associated. The
stoichiometry of oligonucleotide to the lipid component can be 1:1.
Alternatively the stoichiometry can be 1:many, many:1 or many:many,
where many is two or more.
[0466] The FLiP can comprise triacylglycerol, phospholipids,
glycerol and one or several lipid-binding proteins aggregated,
admixed or associated via a lipophilic linker molecule with a
single- or double-stranded oligonucleotide. Surprisingly, it has
been found that due to said one or several lipid-binding proteins
in combination with the above mentioned lipids, the FLiPs show
affinity to heart, lung and/or muscle tissue. These FLiPs can
therefore serve as carrier for oligonucleotides to these
tissues.
[0467] One or more complementary surface active agents can be added
to the reconstituted lipoproteins, for example as complements to
the characteristics of amphiphilic agent or to improve its lipid
particle stabilizing capacity or enable an improved solubilization
of the protein. Such complementary agents can be pharmaceutically
acceptable non-ionic surfactants which preferably are alkylene
oxide derivatives of an organic compound which contains one or more
hydroxylic groups. For example ethoxylated and/or propoxylated
alcohol or ester compounds or mixtures thereof are commonly
available and are well known as such complements to those skilled
in the art. Other pharmacologically acceptable components can also
be added to the FLiPs when desired, such as antioxidants (e.g.,
alpha-tocopherol) and solubilization adjuvants (e.g.,
benzylalcohol).
[0468] One suitable lipid component for FLiP is Intralipid.
Intralipid.RTM. is a brand name for the first safe fat emulsion for
human use. Intralipid.RTM. 20% (a 20% intravenous fat emulsion) is
a sterile, non-pyrogenic fat emulsion prepared for intravenous
administration as a source of calories and essential fatty acids.
It is made up of 20% soybean oil, 1.2% egg yolk phospholipids,
2.25% glycerin, and water for injection. It is further within the
present invention that other suitable oils, such as safflower oil,
can serve to produce the lipid component of the FLiP. Suitable
lipid particle formulations are also described in U.S. patent
application Ser. No. 12/412,206, filed Mar. 26, 2009, contents of
which are herein incorporated in their entirety.
[0469] In one embodiment of the invention is a FLiP comprising a
lipid particle comprising 15-25% triacylglycerol, about 0.5-2%
phospholipids and 1-3% glycerol, and one or several lipid-binding
proteins. In another embodiment, a FLiP includes a liposome having
about 15-25% triacylglycerol, about 1-2% phospholipids, about 2-3%
glycerol, and one or several lipid-binding proteins. In yet another
embodiment of the invention the lipid particle comprises about 20%
triacylglycerol, about 1.2% phospholipids and about 2.25% glycerol,
which corresponds to the total composition of Intralipid, and one
or several lipid-binding proteins.
[0470] In one embodiment, the FLiP has a particle size of about
20-50 nm or about 30-50 nm, e.g., about 35 nm or about 40 nm.
[0471] In another embodiment, the FLiP has a particle size of at
least about 100 nm. FLiPs can alternatively be between about
100-150 nm, e.g., about 110 nm, about 120 nm, about 130 nm, or
about 140 nm, whether characterized as liposome- or
emulsion-based.
[0472] In another embodiment, multiple FLiPs are aggregated
together. In this embodiment, it is envisioned that multiple FLiPs
are delivered, and hence the size can be larger than 100 nm.
[0473] Another suitable lipid component for FLiPs is lipoproteins,
for example isolated lipoproteins or more preferably reconstituted
lipoproteins. Lipoproteins are particles that contain both proteins
and lipids. The lipids or their derivatives can be covalently or
non-covalently bound to the proteins. Exemplary lipoproteins
include chylomicrons, VLDL (Very Low Density Lipoproteins), IDL
(Intermediate Density Lipoproteins), LDL (Low Density Lipoproteins)
and HDL (High Density Lipoproteins).
[0474] Methods of producing reconstituted lipoproteins are known in
the art, for example see A. Jones, Experimental Lung Res. 6,
255-270 (1984), U.S. Pat. No. 4,643,988 and No. 5128318, PCT
publication WO87/02062, Canadian patent #2,138,925. Other methods
of producing reconstituted lipoproteins, especially for
apolipoproteins A-I, A-II, A-IV, apoC and apoE have been described
in A. Jonas, Methods in Enzymology 128, 553-582 (1986) and G.
Franceschini et al. J. Biol. Chem., 260(30), 16321-25 (1985).
[0475] In the final FLiP, the oligonucleotide component is
aggregated, associated or admixed with the lipid components via a
lipophilic moiety. This aggregation, association or admixture can
be at the surface of the final FLiP formulation. Alternatively,
some integration of any of a portion or all of the lipophilic
moiety can occur, extending into the lipid particle. Any lipophilic
linker molecule that is able to bind oligonucleotides to lipids can
be chosen. Examples include pyrrolidine and hydroxyprolinol.
[0476] In addition to the components described above for the
various formulations, these formulations can also include a
targeting moiety, e.g., a targeting moiety that is specific to a
cell type or tissue. Such targeting moieties can be conjugated with
the formulated oligonucleotide and/or conjugated with a component
of the formulation. Formulations can further comprise one or more
of release modifiers, and penetration enhancers.
[0477] The most frequently used lipid for reconstitution is
phosphatidyl choline, extracted either from eggs or soybeans. Other
phospholipids are also used, also lipids such as triglycerides or
cholesterol. For reconstitution, the lipids are first dissolved in
an organic solvent, which is subsequently evaporated under
nitrogen. In this method the lipid is bound in a thin film to a
glass wall. Afterwards the apolipoproteins and a detergent,
normally sodium cholate, are added and mixed. The added sodium
cholate causes a dispersion of the lipid. After a suitable
incubation period, the mixture is dialyzed against large quantities
of buffer for a longer period of time; the sodium cholate is
thereby removed for the most part, and at the same time lipids and
apolipoproteins spontaneously form themselves into lipoproteins or
so-called reconstituted lipoproteins. As alternatives to dialysis,
hydrophobic adsorbents are available which can adsorb detergents
(Bio-Beads SM-2, Bio Rad; Amberlite XAD-2, Rohm & Haas) (E. A.
Bonomo, J. B. Swaney, J. Lipid Res., 29, 380-384 (1988)), or the
detergent can be removed by means of gel chromatography (Sephadex
G-25, Pharmacia). Lipoproteins can also be produced without
detergents, for example through incubation of an aqueous suspension
of a suitable lipid with apolipoproteins, the addition of lipid
which was dissolved in an organic solvent, to apolipoproteins, with
or without additional heating of this mixture, or through treatment
of an apoA-I-lipid-mixture with ultrasound. With these methods,
starting, for example, with apoA-I and phosphatidyl choline,
disk-shaped particles can be obtained which correspond to
lipoproteins in their nascent state. Normally, following the
incubation, unbound apolipoproteins and free lipid are separated by
means of centrifugation or gel chromatography in order to isolate
the homogeneous, reconstituted lipoproteins particles.
[0478] Phospholipids used for reconstituted lipoproteins can be of
natural origin, such as egg yolk or soybean phospholipids, or
synthetic or semisynthetic origin. The phospholipids can be
partially purified or fractionated to comprise pure fractions or
mixtures of phosphatidyl cholines, phosphatidyl ethanolamines,
phosphatidyl inositols, phosphatidic acids, phosphatidyl serines,
sphingomyelin or phosphatidyl glycerols. According to specific
embodiments of the present invention it is preferred to select
phospholipids with defined fatty acid radicals, such as dimyristoyl
phosphatidyl choline (DMPC), dioleoylphosphatidylethanolamine
(DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg
phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylethanolamine
(POPE), dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and
combinations thereof, and the like phosphatidyl cholines with
defined acyl groups selected from naturally occurring fatty acids,
generally having 8 to 22 carbon atoms. According to a specific
embodiment of the present invention phosphatidyl cholines having
only saturated fatty acid residues between 14 and 18 carbon atoms
are preferred, and of those dipalmitoyl phosphatidyl choline is
especially preferred.
[0479] Other phospholipids suitable for reconstitution with
lipoproteins include, e.g., phosphatidylcholine,
phosphatidylglycerol, lecithin, b, g-dipalmitoyl-a-lecithin,
sphingomyelin, phosphatidylserine, phosphatidic acid,
N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium
chloride, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylinositol, cephalin,
cardiolipin, cerebrosides, dicetylphosphate,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,
palmitoyl-oleoyl-phosphatidylcholine,
di-stearoyl-phosphatidylcholine,
stearoyl-palmitoyl-phosphatidylcholine,
di-palmitoyl-phosphatidylethanolamine,
di-stearoyl-phosphatidylethanolamine,
di-myrstoyl-phosphatidylserine, di-oleyl-phosphatidylcholine, and
the like. Non-phosphorus containing lipids can also be used in the
liposomes of the compositions of the present invention. These
include, e.g., stearylamine, docecylamine, acetyl palmitate, fatty
acid amides, and the like.
[0480] Besides the phospholipids, the lipoprotein can comprise, in
various amounts at least one nonpolar component which can be
selected among pharmaceutical acceptable oils (triglycerides)
exemplified by the commonly employed vegetabilic oils such as
soybean oil, safflower oil, olive oil, sesame oil, borage oil,
castor oil and cottonseed oil or oils from other sources like
mineral oils or marine oils including hydrogenated and/or
fractionated triglycerides from such sources. Also medium chain
triglycerides (MCT-oils, e.g. Miglyol.RTM.), and various synthetic
or semisynthetic mono-, di- or triglycerides, such as the defined
nonpolar lipids disclosed in WO 92/05571 can be used in the present
invention as well as acetylated monoglycerides, or alkyl esters of
fatty acids, such isopropyl myristate, ethyl oleate (see EP 0 353
267) or fatty acid alcohols, such as oleyl alcohol, cetyl alcohol
or various nonpolar derivatives of cholesterol, such as cholesterol
esters.
[0481] One or more complementary surface active agents can be added
to the reconstituted lipoproteins, for example as complements to
the characteristics of amphiphilic agent or to improve its lipid
particle stabilizing capacity or enable an improved solubilization
of the protein. Such complementary agents can be pharmaceutically
acceptable non-ionic surfactants which preferably are alkylene
oxide derivatives of an organic compound which contains one or more
hydroxylic groups. For example, ethoxylated and/or propoxylated
alcohol or ester compounds or mixtures thereof are commonly
available and are well known as such complements to those skilled
in the art. Examples of such compounds are esters of sorbitol and
fatty acids, such as sorbitan monopalmitate or sorbitan
monopalmitate, oily sucrose esters, polyoxyethylene sorbitane fatty
acid esters, polyoxyethylene sorbitol fatty acid esters,
polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers,
polyoxyethylene sterol ethers, polyoxyethylene-polypropoxy alkyl
ethers, block polymers and cethyl ether, as well as polyoxyethylene
castor oil or hydrogenated castor oil derivatives and polyglycerine
fatty acid esters. Suitable non-ionic surfactants, include, but are
not limited to various grades of Pluronic.RTM., Poloxamer.RTM.,
Span.RTM., Tween.RTM., Polysorbate.RTM., Tyloxapol.RTM.,
Emulphor.RTM. or Cremophor.RTM. and the like. The complementary
surface active agents can also be of an ionic nature, such as bile
duct agents, cholic acid or deoxycholic their salts and derivatives
or free fatty acids, such as oleic acid, linoleic acid and others.
Other ionic surface active agents are found among cationic lipids
like C10-C24: alkylamines or alkanolamine and cationic cholesterol
esters.
[0482] The process for making the lipid particles comprises the
steps of:
a) mixing a lipid components with one or several lipophile (e.g.
cholesterol) conjugated oligonucleotides that can be chemically
modified; b) fractionating this mixture; c) selecting the fraction
with particles of 30-50 nm, preferably of about 40 nm in size.
Release Moders
[0483] The release characteristics of a formulation of the present
invention depend on the encapsulating material, the concentration
of encapsulated drug, and the presence of release modifiers. For
example, release can be manipulated to be pH dependent, for
example, using a pH sensitive coating that releases only at a low
pH, as in the stomach, or a higher pH, as in the intestine. An
enteric coating can be used to prevent release from occurring until
after passage through the stomach. Multiple coatings or mixtures of
cyanamide encapsulated in different materials can be used to obtain
an initial release in the stomach, followed by later release in the
intestine. Release can also be manipulated by inclusion of salts or
pore forming agents, which can increase water uptake or release of
drug by diffusion from the capsule. Excipients which modify the
solubility of the drug can also be used to control the release
rate. Agents which enhance degradation of the matrix or release
from the matrix can also be incorporated. They can be added to the
drug, added as a separate phase (i.e., as particulates), or can be
co-dissolved in the polymer phase depending on the compound. In all
cases the amount should be between 0.1 and thirty percent (w/w
polymer). Types of degradation enhancers include inorganic salts
such as ammonium sulfate and ammonium chloride, organic acids such
as citric acid, benzoic acid, and ascorbic acid, inorganic bases
such as sodium carbonate, potassium carbonate, calcium carbonate,
zinc carbonate, and zinc hydroxide, and organic bases such as
protamine sulfate, spermine, choline, ethanolamine, diethanolamine,
and triethanolamine and surfactants such as Tween.RTM. and
Pluronic.RTM.. Pore forming agents which add microstructure to the
matrices (i.e., water soluble compounds such as inorganic salts and
sugars) are added as particulates. The range should be between one
and thirty percent (w/w polymer).
[0484] Uptake can also be manipulated by altering residence time of
the particles in the gut. This can be achieved, for example, by
coating the particle with, or selecting as the encapsulating
material, a mucosal adhesive polymer. Examples include most
polymers with free carboxyl groups, such as chitosan, celluloses,
and especially polyacrylates (as used herein, polyacrylates refers
to polymers including acrylate groups and modified acrylate groups
such as cyanoacrylates and methacrylates).
Polymers
[0485] Hydrophilic polymers suitable for use in the formulations of
the present invention are those which are readily water-soluble,
can be covalently attached to a vesicle-forming lipid, and which
are tolerated in vivo without toxic effects (i.e., are
biocompatible). Suitable polymers include polyethylene glycol
(PEG), polylactic (also termed polylactide), polyglycolic acid
(also termed polyglycolide), a polylactic-polyglycolic acid
copolymer, and polyvinyl alcohol. Preferred polymers are those
having a molecular weight of from about 100 or 120 daltons up to
about 5,000 or 10,000 daltons, and more preferably from about 300
daltons to about 5,000 daltons. In a particularly preferred
embodiment, the polymer is polyethyleneglycol having a molecular
weight of from about 100 to about 5,000 daltons, and more
preferably having a molecular weight of from about 300 to about
5,000 daltons. In a particularly preferred embodiment, the polymer
is polyethyleneglycol of 750 daltons (PEG(750)). Polymers can also
be defined by the number of monomers therein; a preferred
embodiment of the present invention utilizes polymers of at least
about three monomers, such PEG polymers consisting of three
monomers (approximately 150 daltons).
[0486] Other hydrophilic polymers which can be suitable for use in
the present invention include polyvinylpyrrolidone,
polymethoxazoline, polyethyloxazoline, polyhydroxypropyl
methacrylamide, polymethacrylamide, polydimethylacrylamide, and
derivatized celluloses such as hydroxymethylcellulose or
hydroxyethylcellulose.
[0487] In one embodiment, a formulation of the present invention
comprises a biocompatible polymer selected from the group
consisting of polyamides, polycarbonates, polyalkylenes, polymers
of acrylic and methacrylic esters, polyvinyl polymers,
polyglycolides, polysiloxanes, polyurethanes and co-polymers
thereof, celluloses, polypropylene, polyethylenes, polystyrene,
polymers of lactic acid and glycolic acid, polyanhydrides,
poly(ortho)esters, poly(butic acid), poly(valeric acid),
poly(lactide-co-caprolactone), polysaccharides, proteins,
polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or
copolymers thereof.
Surfactants
[0488] The above discussed formulation can also include one or more
surfactants. Surfactants find wide application in formulations such
as emulsions (including microemulsions) and liposomes. The use of
surfactants in drug products, formulations and in emulsions has
been reviewed (Rieger, in "Pharmaceutical Dosage Forms," Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285). Surfactants can be
classified into different classes based on the nature of the
hydrophilic group: nonionic, anionic, cationic and amphoteric
(Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 285).
[0489] Nonionic surfactants include, but are not limited to,
nonionic esters such as ethylene glycol esters, propylene glycol
esters, glyceryl esters, polyglyceryl esters, sorbitan esters,
sucrose esters, and ethoxylated esters. Nonionic alkanolamides and
ethers such as fatty alcohol ethoxylates, propoxylated alcohols,
and ethoxylated/propoxylated block polymers are also included in
this class. The polyoxyethylene surfactants are the most popular
members of the nonionic surfactant class.
[0490] Anionic surfactants include, but are not limited to,
carboxylates such as soaps, acyl lactylates, acyl amides of amino
acids, esters of sulfuric acid such as alkyl sulfates and
ethoxylated alkyl sulfates, sulfonates such as alkyl benzene
sulfonates, acyl isethionates, acyl taurates and sulfosuccinates,
and phosphates. The most important members of the anionic
surfactant class are the alkyl sulfates and the soaps.
[0491] Cationic surfactants include, but are not limited to,
quaternary ammonium salts and ethoxylated amines. The quaternary
ammonium salts are the most used members of this class.
[0492] Amphoteric surfactants include, but are not limited to,
acrylic acid derivatives, substituted alkylamides, N-alkylbetaines
and phosphatides.
[0493] A surfactant can also be selected from any suitable
aliphatic, cycloaliphatic or aromatic surfactant, including but not
limited to biocompatible lysophosphatidylcholines (LPCs) of varying
chain lengths (for example, from about C14 to about C20).
Polymer-derivatized lipids such as PEG-lipids can also be utilized
for micelle formation as they will act to inhibit micelle/membrane
fusion, and as the addition of a polymer to surfactant molecules
decreases the CMC of the surfactant and aids in micelle formation.
Preferred are surfactants with CMCs in the micromolar range; higher
CMC surfactants can be utilized to prepare micelles entrapped
within liposomes of the present invention, however, micelle
surfactant monomers could affect liposome bilayer stability and
would be a factor in designing a liposome of a desired
stability.
Penetration Enhancers
[0494] In one embodiment, the formulations of the present invention
employ various penetration enhancers to affect the efficient
delivery of RNAi agents to the skin of animals. Most drugs are
present in solution in both ionized and nonionized forms. However,
usually only lipid soluble or lipophilic drugs readily cross cell
membranes. It has been discovered that even non-lipophilic drugs
can cross cell membranes if the membrane to be crossed is treated
with a penetration enhancer. In addition to aiding the diffusion of
non-lipophilic drugs across cell membranes, penetration enhancers
also enhance the permeability of lipophilic drugs.
[0495] Some exemplary formulations for oligonucleotides are
described in International Application Nos. PCT/US07/079203, filed
Sep. 21, 2007; PCT/US07/080331, filed Oct. 3, 2007; U.S. patent
application Ser. No. 12/123,922, filed May 28, 2008; U.S. Patent
Application Publication Nos. 20060240093 and 20070135372 and U.S.
Provisional Application Nos. 61/018,616, filed Jan. 2, 2008;
61/039,748, filed Mar. 26; 2008; 61/045,228, filed Apr. 15, 2008;
61/047,087, filed Apr. 22, 2008; and 61/051,528, filed May 21,
2008, contents of which are herein incorporated by reference in
their entireties for all purposes.
[0496] In some embodiments, the oligonucleotide is formulated as
yeast cell particles. Without wishing to be bound by theory, yeast
cell particles comprise an extracted yeast cell wall comprising
beta-glucan and payload trapping molecule. Methods of preparing
yeast cell particle for drug delivery are described in U.S. Patent
Publication No. 2008/0044438 and 2005/0281781, contents of which
are herein incorporated in their entireties. In certain
embodiments, the yeast cell particle comprises a recombinant
vector, e.g., a plasmid, that encodes for the oligonucleotide of
the invention.
Agricultural Formulations and Applications
[0497] Methods of agricultural formulation are well known to one
skilled in the art and are also found in Knowles, D A (1998)
Chemistry and technology of agricultural formulations. Kluwer
Academic, London, which is hereby incorporated by reference in its
entirety. One skilled in the art will, of course, recognize that
the formulation and mode of application can affect the activity of
the active ingredient in a given application. Thus, for
agricultural and/or horticultural use the active ingredient, e.g.,
oligonucleotide, can be formulated as a granular of relatively
large particle size (for example, 8/16 or 4/8 US Mesh), as
water-soluble or water-dispersible granules, as powdery dusts, as
wettable powders, as emulsifiable concentrates, as aqueous
emulsions, as solutions, as suspension concentrate, as capsule
suspensions, as soluble (liquid) concentrates, as soluble powders,
or as any of other known types of agriculturally-useful
formulations, depending on the desired mode of application. It is
to be understood that the amounts specified in this specification
are intended to be approximate only, as if the word "about" were
placed in front of the amounts specified.
[0498] These formulations can be applied either as water-diluted
sprays, or dusts, or granules in the areas of interest. These
formulations can contain as little as 0.1%, 0.2% or 0.5% to as much
as 95% or more by weight of active ingredient.
[0499] Dusts are free flowing admixtures of the active ingredient
with finely divided solids such as talc, natural clays, kieselguhr,
flours such as walnut shell and cottonseed flours, and other
organic and inorganic solids which act as dispersants and carriers
for the toxicant; these finely divided solids have an average
particle size of less than about 50 microns. A typical dust
formulation useful herein is one containing 90 parts, 80 parts, 70
parts, 60 parts, 50 parts, 40 parts, 30 parts, 20 parts, preferably
10 parts, or less of the active ingredient, e.g. oligonucleotide.
In one embodiment, the dust formulation comprises 1 part or less of
the active ingredient and 99 parts or more of talc. As used herein,
the terms "active ingredient" and "active agent" refer to a
compound that modulate gene expression activity of an insect or a
pathogen of insect.
[0500] Wettable powders, useful as formulations, are in the form of
finely divided particles that disperse readily in water or other
dispersant. The wettable powder is ultimately applied either as a
dry dust or as an emulsion in water or other liquid. Typical
carriers for wettable powders include Fuller's earth, kaolin clays,
silicas, and other highly absorbent, readily wet inorganic
diluents. Wettable powders normally are prepared to contain about
5-80% of active ingredient, depending on the absorbency of the
carrier, and usually also contain a small amount of a wetting,
dispersing or emulsifying agent to facilitate dispersion. For
example, a useful wettable powder formulation contains 80.0 parts
of the active ingredient, 17.9 parts of Palmetto clay, and 1.0 part
of sodium lignosulfonate and 0.3 part of sulfonated aliphatic
polyester as wetting agents. Additional wetting agent and/or oil
will frequently be added to a tank mix for to facilitate dispersion
on the foliage of the plant.
[0501] Other useful formulations are emulsifiable concentrates
(ECs) which are homogeneous liquid compositions dispersible in
water or other dispersant, and can consist entirely of the active
ingredient, and a liquid or solid emulsifying agent, or can also
contain a liquid carrier, such as xylene, heavy aromatic naphthas,
isophorone, or other non-volatile organic solvents. For
insecticidal application these concentrates are dispersed in water
or other liquid carrier and normally applied as a spray to the area
to be treated. The percentage by weight of the essential active
ingredient can vary according to the manner in which the
composition is to be applied, but in general comprises 0.5 to 95%
of active ingredient by weight of the insecticidal composition.
[0502] Flowable formulations are similar to ECs, except that the
active ingredient is suspended in a liquid carrier, generally
water. Flowables, like ECs, can include a small amount of a
surfactant, and will typically contain active ingredients in the
range of 0.5 to 95%, frequently from 10 to 50%, by weight of the
composition. For application, flowables can be diluted in water or
other liquid vehicle, and are normally applied as a spray to the
area to be treated.
[0503] Typical wetting, dispersing or emulsifying agents used in
agricultural and/or horticultural formulations include, but are not
limited to, the alkyl and alkylaryl sulfonates and sulfates and
their sodium salts; alkylaryl polyether alcohols; sulfated higher
alcohols; polyethylene oxides; sulfonated animal and vegetable
oils; sulfonated petroleum oils; fatty acid esters of polyhydric
alcohols and the ethylene oxide addition products of such esters;
and the addition product of long-chain mercaptans and ethylene
oxide. Many other types of useful surface-active agents are
available in commerce. Surface-active agents, when used, normally
comprise 1 to 15% by weight of the composition.
[0504] Other useful formulations include suspensions of the active
ingredient in a relatively non-volatile solvent such as water, corn
oil, kerosene, propylene glycol, or other suitable solvents.
[0505] Still other useful formulations for agricultural
applications include simple solutions of the active ingredient in a
solvent in which it is completely soluble at the desired
concentration, such as acetone, alkylated naphthalenes, xylene, or
other organic solvents. Granular formulations, wherein the active
ingredient is carried on relative coarse particles, are of
particular utility for aerial distribution or for penetration of
cover crop canopy. Pressurized sprays, typically aerosols wherein
the active ingredient is dispersed in finely divided form as a
result of vaporization of a low-boiling dispersant solvent carrier
can also be used. Water-soluble or water-dispersible granules are
free flowing, non-dusty, and readily water-soluble or
water-miscible. In use by the farmer on the field, the granular
formulations, emulsifiable concentrates, flowable concentrates,
aqueous emulsions, solutions, etc., can be diluted with water to
give a concentration of active ingredient in the range of say 0.1%
or 0.2% to 1.5% or 2%.
[0506] By far the most frequently used are water-miscible
formulations for mixing with water then applying as sprays. Water
miscible, older formulations include: emulsifiable concentrate,
wettable powder, soluble (liquid) concentrate, and soluble powder.
Newer, non-powdery formulations with reduced or no hazardous
solvents and improved stability include: suspension concentrate,
capsule suspensions, water dispersible granules. Such formulations
are preferably solutions and suspension, e. g., aqueous suspension
and solutions, ethanolic suspension and solutions,
aqueous/ethanolic suspension and solutions, saline solutions, and
colloidal suspensions.
[0507] Alternatively, a sprayable wax emulsion formulation can be
used. The formulation contains the active ingredient, in an amount
from about 0.01% to 75% by weight. The aqueous wax emulsions are
broadly described in U.S. Pat. No. 6,001,346, which is hereby
incorporated by reference in is entirety. Formulations of the
methods described herein can have a viscosity appropriate for use
in aerial or backpack spray applications.
[0508] The biodegradable wax carrier comprises at least about 10%
by weight of the formulation. The biodegradable wax carrier is
selected from the group consisting of paraffin, beeswax, vegetable
based waxes such as soywax (soybean based), and hydrocarbon based
waxes such as Gulf Wax Household Paraffin Wax; paraffin wax, avg.
m.p. 53C (hexacosane), high molecular weight hydrocarbons).
carnauba wax, lanolin, shellac wax, bayberry wax, sugar cane wax,
microcrystalline, ozocerite, ceresin, montan, candelilla wax, and
combinations thereof.
[0509] Formulations can contain an emulsifier in an amount from
about 1% to about 10% by weight. Suitable emulsifiers include
lecithin and modified lecithins, mono- and diglycerides, sorbitan
monopalmitate, sorbitan monooleate, sorbitan monolaurate,
polyoxyethylene-sorbitan monooleate, fatty acids, lipids, etc. The
emulsifiers provide or improve emulsification properties of the
composition. The emulsifier can be selected from many products
which are well known in the art, including, but not limited to,
sorbitan monolaurate (anhydrosorbitol stearate, molecular formula
C.sub.24H.sub.46O.sub.6), ARLACEL 60, ARMOTAN MS, CRILL 3, CRILL
K3, DREWSORB 60, DURTAN 60, EMSORB 2505, GLYCOMUL S, HODAG SMS,
IONET S 60, LIPOSORB S, LIPOSORB S-20, MONTANE 60, MS 33, MS33F,
NEWCOL 60, NIKKOL SS 30, NISSAN NONION SP 60, NONION SP 60, NONION
SP 60R, RIKEMAL S 250, sorbitan c, sorbitan stearate, SORBON 60,
SORGEN 50, SPAN 55, AND SPAN 60; other sorbitan fatty acid ester
that can be used include sorbitan monopalmitate, sorbitan
monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan
sesquioleate, sorbitan trioleate, sorbitan monooleate, sorbitan
trioleate. In certain embodiments, SPAN 60 is preferred.
[0510] In certain embodiments, formulations can includes a
phagostimulant, such as corn oil, molasses, glycerol, or corn
syrup, proteinaceous material (protein or hydrolyzed protein),
sugars like sucrose, or food-based ingredients such as
trimethylamine, putrescine, bacterial or yeast volatiles or
metabolites, ammonium acetate, ammonium carbonate or other
ammonia-emitting compounds. Acetic acid vapor can be provided by
compounds that produce volatilized acetic acid, for example,
aqueous acetic acid, glacial acetic acid, glacial (concentrated)
acetic acid, or ammonium producing compounds such as but not
restricted to ammonium hydroxide, ammonium carbonate, ammonium
bicarbonate, ammonium acetate, etc. Ammonium acetate is most
preferred for providing acetic acid and ammonia vapors.
[0511] The active ingredient can be formulated and/or applied with
one or more second compounds. Various combinations active
ingredients can be used to obtain greater advantage. Without
wishing to be bound by theory, such combinations provide certain
advantages, such as, without limitation, exhibiting synergistic
effects, reducing rates of application thereby minimizing any
impact to the environment and to worker safety, controlling a
broader spectrum of insects and non-insect pests, and improving
tolerance by non-pest species, such as mammals, and fish. Other
second compounds include, without limitation, attractant,
insecticides, pesticides, plant growth regulators, fertilizers,
soil conditioners, or other agricultural and horticultural
chemicals. The formulation can include such second compounds in an
amount from about 0.002% to about 25%.
[0512] Attractants include, but are not limited to, visual
attractants (e.g., food coloring), pheromones, light, mimicking
flowers or plants etc.
[0513] Insecticides include, but are not limited to,
organophosphate insecticides, such as chlorpyrifos, diazinon,
dimethoate, malathion, parathion-methyl, naled, and terbufos;
nicotinic insecticides such as imidacloprid and thiacloprid;
pyrethroid insecticides, such as fenvalerate, delta-methrin,
fenpropathrin, cyfluthrin, flucythrinate, alpha-cypermethrin,
biphenthrin, resolved cyhalothrin, etofenprox, esfenvalerate,
tralomethrin, tefluthrin, cycloprothrin, betacyfluthrin, and
acrinathrin; carbamate insecticides, such as aldecarb, carbaryl,
carbofuran, and methomyl; organochlorine insecticides, such as
endosulfan, endrin, heptachlor, and lindane; benzoylurea
insecticides, such as diflubenuron, triflumuron, teflubenzuron,
chlorfluazuron, flucycloxuron, hexaflumuron, flufenoxuron, dimlin,
novaluron, and lufenuron; diacylhydrazines such as methoxyfenozide;
phenylpyrazoles such as fipronil or ethiprole, chlorfenapyr,
diafenthiuron, indoxacarb, metaflumazone, emamectin benzoate,
abamectin, pyridalyl, flubendiamide, rynaxypyr; and other
insecticides, such as amitraz, clofentezine, fenpyroximate,
hexythiazox, spinosad, and imidacloprid.
[0514] Pesiticides include, but are not limited to, benzimidazine
fungicides, such as benomyl, carbendazim, thia-bendazine, and
thiophanate-methyl; 1,2,4-triazine fungicides, such as
epoxyconazine, cyproconazine, flusilazine, flutriafol,
propiconazine, tebuconazine, triadimefon, and tri-adimenol;
substituted anilide fungicides, such as metalaxyl, oxadixyl,
procymidone, and vinclozolin; organophosphorus fungicides, such as
fosetyl, iprobenfos, pyrazophos, edifen-phos, and tolclofos-methyl;
morpholine fungicides, such as fenpropimorph, tridemorph, and
dodemorph; other systemic fungicides, such as fenarimol, imazalil,
prochloraz, tricycla-zine, and triforine; dithiocarbamate
fungicides, such as mancozeb, maneb, propineb, zineb, and ziram;
non-systemic fungicides, such as chlorothalonil, dichlorofluanid,
dithianon, and iprodione, captan, dinocap, dodine, fluazinam,
gluazatine, PCNB, pencycuron, quintozene, tricylamide, and
validamycin; inorganic fungicides, such as copper and sulphur
products, and other fungicides; nematicides such as carbofuran,
carbosulfan, turbufos, aldecarb, ethoprop, fenamphos, oxamyl,
isazofos, cadusafos, and other nematicides.
[0515] A variety of additives can be incorporated into the
formulation. These additives typically change and/or enhance the
physical characteristics of the carrier material and are,
therefore, suitable for designing compositions having specific
requirements as to the release rate and amount of the active
ingredient, protection of the wax composition from weather
conditions, etc. These additives are, among others, plasticizers,
volatility suppressants, antioxidants, lipids, various ultraviolet
blockers and absorbers, or antimicrobials, typically added in
amounts from about 0.001% to about 10%, more typically between
1-6%, by weight.
[0516] Plasticizers, such as glycerin or soy oil affect physical
properties of the composition and can extend its resistance to
environmental destruction.
[0517] Antioxidants, such as vitamin E, BHA (butylated
hydroxyanisole), BHT (butylated hydroxytoluene), and other
antioxidants which protect the bioactive agent from degradation,
can be added in amounts from about 0.1% to about 3%, by weight.
[0518] Ultraviolet blockers, such as beta-carotene, lignin or
p-aminobenzoic acid protect the bioactive agents from light
degradation can be added in amounts from about 1% to about 3%, by
weight.
[0519] Antimicrobials, such as potassium sorbate, nitrates,
nitrites, and propylene oxide, protect the bioactive agents from
microbial destruction can be added in amounts from 0.1% to about 2%
by weight.
[0520] Adjuvants can also be added to the formulation. An adjuvant
is broadly defined as any substance added to the spray tank,
separate from the pesticide formulation, that will improve the
performance of the pesticide. These includes but are not limited to
wetter-spreaders, stickers, penetrants, compatibility agents,
buffers, and so on.
[0521] Other compounds and materials can be added provided they do
not substantially interfere with the activity of active ingredient.
Whether or not an additive substantially interferes with the active
ingredient's activity can be determined by standard test formats,
involving direct comparisons of efficacy of the composition of the
active ingredient without an added compound and the composition of
the active ingredient with an added compound.
[0522] Frequently used carriers or auxiliaries include magnesium
carbonate, titanium dioxide, lactose, mannitol and other sugars,
talc, milk protein, gelatin, starch, vitamins, cellulose and its
derivatives, animal and vegetable oils, polyethylene glycols and
solvents, such as sterile water, alcohols, glycerol and polyhydric
alcohols.
[0523] Preservatives include antimicrobial, anti-oxidants,
chelating agents and inert gases. Other pharmaceutically acceptable
carriers include aqueous solutions, non-toxic excipients, including
salts, preservatives, buffers and the like, as described, for
instance, in Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing Co. (1990). The pH and exact concentration of the
various components of the pharmaceutical composition are adjusted
according to routine skills in the art. See Goodman and Gilman's
The Pharmacological Basis for Therapeutics, 10th ed., McGraw-Hill
Professional (2001).
[0524] In one embodiment, the active agent can be applied to a
breeding locus of insects. As used herein, the term "breeding
locus" refers to an area where the insects breed, i.e. mate and/or
lay eggs.
[0525] In another embodiment, the active agent is applied to a
feeding locus of insects. As used here in, the term "feeding locus"
refers to an area where an insect feeds. In many instances, a
breeding locus and a feeding locus will be the same area.
[0526] In one embodiment, the active ingredient is preferably
applied topically on plants on which an insect feeds.
[0527] In yet another embodiment, the active agent is applied to
both a breeding and a feeding locus of insects.
[0528] In one embodiment, the active agent is applied as a spray to
locus of insects, e.g., breeding locus, feeding locus.
[0529] In one embodiment, the active agent is applied to insect
traps. For example, the trap can be coated with the active agent or
trap can be loaded with insect food comprising an active agent.
Recombinant Vectors
[0530] In another aspect, oligonucleotides useful for the methods
and/or compositions of the invention can be expressed from
transcription units inserted into DNA or RNA vectors. For example,
see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A.,
et al., International PCT Publication No. WO 00/22113, Conrad,
International PCT Publication No. WO 00/22114, and Conrad, U.S.
Pat. No. 6,054,299. The vector can be either prokaryotic or
eukaryotic, and typically is a virus or a plasmid. Expression can
be transient (on the order of hours to weeks) or sustained (weeks
to months or longer), depending upon the specific construct used
and the target tissue or cell type. These transgenes can be
introduced as a linear construct, a circular plasmid, or a viral
vector, which can be an integrating or non-integrating vector. The
transgene can also be constructed to permit it to be inherited as
an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad.
Sci. USA (1995) 92:1292).
[0531] One type of recombinant vector comprises a polynucleotide
encoding a double-stranded oligonucleotide cooperatively linked to
an expression vector. Alternatively, the two strands of the
double-stranded oligonucleotide are encoded by separate open
reading frames. In one embodiment, a dsRNA is expressed as an
inverted repeat joined by a linker polynucleotide sequence that the
dsRNA has a stem and loop structure. The phrase operatively linked
refers to insertion of a polynucleotide molecule into an expression
vector in a manner such that the molecule is able to be expressed
when transformed into a host cell. As used herein, an expression
vector is a DNA or RNA vector that is capable of transforming a
host cell and of effecting expression of a specified polynucleotide
molecule(s). Preferably, the expression vector is also capable of
replicating within the host cell. Expression vectors can be either
prokaryotic or eukaryotic, and are typically viruses or plasmids.
Expression vectors of the present invention include any vectors
that function (i.e., direct gene expression) in recombinant cells
of the present invention, including in bacterial, fungal,
endoparasite, arthropod, other animal, and plant cells. Preferred
expression vectors of the present invention can direct gene
expression in insect cells.
[0532] In particular, expression vectors of the present invention
contain regulatory sequences such as transcription control
sequences, origins of replication, and other regulatory sequences
that are compatible with the recombinant cell and that control the
expression of the polynucleotide encoding a dsRNA or a strand
thereof. In particular, recombinant molecules of the present
invention include transcription control sequences. Transcription
control sequences are sequences which control the initiation,
elongation, and termination of transcription. Particularly
important transcription control sequences are those which control
transcription initiation, such as promoter, enhancer, operator and
repressor sequences. Suitable transcription control sequences
include any transcription control sequence that can function in at
least one of the recombinant cells of the present invention. A
variety of such transcription control sequences are known to those
skilled in the art. Preferred transcription control sequences
include those which function in arthropod cells. Additional
suitable transcription control sequences include tissue-specific
promoters and enhancers. Some exemplary compositions and methods
for preparing expression vectors are described in U.S. patent
application Ser. Nos. 10/522,962 and 10/531,349 and International
Patent Application No. PCT/US2005/029976, contents of which are
herein incorporated in their entireties.
[0533] In certain embodiments, the expression vector is a
insect-infecting virus. The virus can optionally be disarmed. As
used herein, the term "disarmed" means that pathogenicity of the
virus is reduced and/or abolished in comparison to the wildtype
virus. One of skill in the art knows of methods for producing
viruses with lower and/or abolished pathogenicity. Without wishing
to be bound by theory, a disarmed virus can allow the infection to
spread to other insects in the hive and/or locus before the first
infected virus dies.
[0534] A particularly preferred expression vector is a baculovirus.
By "baculovirus" it is meant any virus of the family Baculoviridae,
such as a nuclear polyhedrosis virus (NPV). Baculoviruses are a
large group of evolutionarily related viruses, which infect only
arthropods; indeed, some baculoviruses only infect insects that are
pests of commercially important agricultural and forestry crops,
while others are known that specifically infect other insect pests.
Because baculoviruses infect only arthropods, they pose little or
no risk to humans, plants, or the environment.
[0535] Of the suitable viruses, in addition to the Baculoviridae
are the entomopox viruses (EPV), such as Melolontha melonotha EPV,
Amsacta moorei EPV, Locusta migratoria EPV, Melanoplus sanguinipes
EPV, Schistocerca gregaria EPV, Aedes aogypti EPV, and Chironomus
luridus EPV. Other suitable viruses are granulosis viruses (GV).
Suitable RNA viruses include togaviruses, flavivi-ruses,
picornaviruses, cytoplasmic polyhedrosis viruses (CPV), and the
like. The subfamily of double stranded DNA viruses Eubaculovirinae
includes two genera, NPVs and GVs, which are particularly useful
for biological control because they produce occlusion bodies in
their life cycle. Examples of GVs include Cydia pomonella GV
(coddling moth GV), Pieris brassicae GV, Trichoplusia ni GV,
Artogeia rapae GV, and Plodia interpunctella GV (Indian meal
moth).
[0536] Suitable baculoviruses for practicing this invention can be
occluded or non-occluded. The nuclear polyhedrosis viruses ("NPV")
are one baculovirus subgroup, which are "occluded." That is, a
characteristic feature of the NPV group is that many virions are
embedded in a crystalline protein matrix referred to as an
"occlusion body." Examples of NPVs include Lymantria dispar NPV
(gypsy moth NPV), Autographa californica MNPV, Anagrapha falcifera
NPV (celery looper NPV), Spodoptera litturalis NPV, Spodoptera
frugiperda NPV, Heliothis armigera NPV, Mamestra brassicae NPV,
Choristoneura fumiferana NPV, Trichoplusia ni NPV, Helicoverpa zea
NPV, and Rachiplusia ou NPV. For field use occluded viruses often
are preferable due to their greater stability since the viral
polyhedrin coat provides protection for the enclosed infectious
nucleocapsids.
[0537] Among illustrative, useful baculoviruses in practicing this
invention are those isolated from Anagrapha falcifera, Anticarsia
gemmatalis, Buzura suppressuria, Cydia pomonella, Helicoverpa zea,
Heliothis armigera, Manestia brassicae, Plutella xylostella,
Spodoptera exigua, Spodoptera littoralis, and Spodoptera litura. A
particularly useful "NPV" baculovirus for practicing this invention
is AcNPV, which is a nuclear polyhedrosis virus from Autographa
californica. Autographa californica is of particular interest
because various major pest species within the genera Spodoptera,
Trichoplusia, and Heliothis are susceptible to this virus.
[0538] The tropism of viral vectors can be modified by pseudotyping
the vectors with envelope proteins or other surface antigens from
other viruses, or by substituting different viral capsid proteins,
as appropriate. For example, lentiviral vectors can be pseudotyped
with surface proteins from vesicular stomatitis virus (VSV),
rabies, Ebola, Mokola, and the like. AAV vectors can be made to
target different cells by engineering the vectors to express
different capsid protein serotypes; see, e.g., Rabinowitz J E et
al. (2002), J Virol 76:791-801, the entire disclosure of which is
herein incorporated by reference.
[0539] The present invention may be as defined in any one of the
following numbered paragraphs.
1. A method for treating or preventing disease in an insect, the
method comprising administering to the insect a composition
comprising an RNA effector molecule or a vector encoding an RNA
effector molecule, and a delivery agent, wherein the RNA effector
molecule modulates gene expression of an insect or an insect
pathogen. 2. The method of paragraph 1, wherein the disease is
caused by an insect pathogen selected from the group consisting of
a virus, mite, nematode, bacteria, fungus, or parasite. 3. The
method of paragraph 1, wherein the disease is caused by pollution,
exposure to electromagnetic radiation, exposure to pesticides,
environment, or stress. 4. The method of paragraph 1, wherein the
RNA effector molecule inhibits or activates gene expression. 5. The
method of paragraph 2, wherein modulating gene expression inhibits
pathogen infectivity, virulence, reproduction, viability, growth,
translation, protein production, viral uptake or transmission. 6.
The method of paragraph 2, wherein modulating gene expression
decreases insect susceptibility to a pathogen. 7. The method of
paragraph 1, wherein the administering comprises providing a food
source for the insect, wherein the food source comprises the
composition. 8. The method of paragraph 7, wherein the food source
is provided as a liquid, solid, gel, semi-solid composition, sugar
composition, or lipid composition. 9. The method of paragraph 7,
wherein the food source comprises a virus, a bacterium, a fungus, a
plant, or a yeast cell expressing the RNA effector molecule. 10.
The method of paragraph 1, wherein the administering comprises
contacting the insect with a solution comprising the composition.
11 The method of paragraph 10, wherein the composition is
administered topically. 12. The method of paragraph 10, wherein the
insect is sprayed or soaked with the solution. 13. The method of
paragraph 1, wherein the RNA effector molecule comprises an
oligonucleotide. 14. The method of paragraph 13, wherein the
oligonucleotide is a single stranded or double stranded
oligonucleotide. 15. The method of paragraph 13, wherein the
oligonucleotide is modified. 16. The method of paragraph 15,
wherein the modification is selected from the group consisting of:
2'-O-methyl modified nucleotide, a nucleotide having a
5'-phosphorothioate group, a terminal nucleotide linked to a
cholesteryl derivative, a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide. 17. The method of paragraph
15, wherein the oligonucleotide comprises an siRNA, an miRNA, an
shRNA, a ribozyme, an antisense RNA, a decoy oligonucleotide, an
antimir, a supermir, or an RNA activator. 18. The method of
paragraph 1, wherein the vector is a viral vector, an expression
vector, or a plasmid. 19. The method of paragraph 1, wherein the
delivery agent is a lipid, a liposome, a food source, a solution,
an emulsion, a micelle or other membranous formulation, a lipid
particle, a bacteria, a fungus, a plant, a yeast cell, or a yeast
cell particle. 20. The method of paragraph 18, wherein the viral
vector comprises a baculoviral vector. 21. The method of paragraph
19, wherein the lipid particle comprises about 15-25%
triacylglycerol, about 0.5-2% phospholipids, about 1-3% glycerol,
and at least one lipid-binding protein. 22. The method of paragraph
1, wherein the composition is provided in a spray, solution, gel,
bait, a food source, or powder form. 23. The method of paragraph 1,
wherein the composition further comprises an attractant. 24. The
method of paragraph 23, wherein the attractant comprises an insect
pheromone or hormone. 25. The method of paragraph 1, wherein the
composition is administered in combination with an antibiotic,
antiviral or anthelmintic agent. 26. The method of paragraph 1,
wherein the insect is a bee, wasp, butterfly, ant or ladybug. 27.
The method of paragraph 15, wherein the oligonucleotide comprises
9-36 base pairs. 28. The method of paragraph 1, wherein the
composition is administered to adult insects. 29. The method of
paragraph 1, wherein the composition is administered to a breeding
or feeding locus. 30. The method of paragraph 1, wherein the
composition further comprises an additional agent. 31. The method
of paragraph 1, wherein the composition further comprises sucrose.
32. The method of any of the preceding paragraphs, wherein the
insect is a hive bee or a forager bee, and the pathogen is selected
from the group consisting of IAPV, Acute Bee Paralysis Virus and
Kashmir Bee Paralysis Virus. 33. A method for modulating gene
expression in an insect, the method comprising: administering to
the insect a composition comprising an RNA effector molecule or a
vector encoding an RNA effector molecule and a delivery agent,
wherein the RNA effector molecule modulates gene expression in the
insect. 34. The method of paragraph 33, wherein the insect is a
pest. 35. The method of paragraph 33, wherein the RNA effector
molecule inhibits or activates gene expression. 36. The method of
paragraph 33, wherein modulation of gene expression inhibits
viability, survival, growth, development, and/or reproduction of
the insect. 37. The method of paragraph 33, wherein modulation of
gene expression increases insect susceptibility to a pathogen. 38.
The method of paragraph 33, wherein the administering comprises
providing a food source for the insect, wherein the food source
comprises the composition. 39. The method of paragraph 38, wherein
the food source is provided as a liquid, solid, gel, semi-solid
composition, sugar composition, or lipid composition. 40. The
method of paragraph 38, wherein the food source comprises a virus,
a bacterium, a fungus, a plant or a yeast cell expressing the
oligonucleotide. 41. The method of paragraph 33, wherein the insect
is a hive-dwelling insect and modulation of gene expression in the
insect is delayed until the insect returns to the hive. 42. The
method of paragraph 41, wherein the hive-dwelling insect spreads
the composition to other insects in the hive. 43. The method of
paragraph 33, wherein the administering comprises contacting the
insect with a solution comprising the composition. 44. The method
of paragraph 43, wherein the composition is administered topically.
45. The method of paragraph 43, wherein the insect is sprayed or
soaked with the solution. 46. The method of paragraph 33, wherein
the RNA effector molecule comprises an oligonucleotide. 47. The
method of paragraph 46, wherein the oligonucleotide is a single
stranded or double stranded oligonucleotide. 48. The method of
paragraph 46, wherein the oligonucleotide is modified. 49. The
method of paragraph 48, wherein the modification is selected from
the group consisting of: 2'-O-methyl modified nucleotide, a
nucleotide having a 5'-phosphorothioate group, a terminal
nucleotide linked to a cholesteryl derivative, a 2'-deoxy-2'-fluoro
modified nucleotide, a 2'-deoxy-modified nucleotide, a locked
nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide,
2'-alkyl-modified nucleotide, morpholino nucleotide, a
phosphoramidate, and a non-natural base comprising nucleotide. 50.
The method of paragraph 46, wherein the oligonucleotide comprises
an siRNA, an miRNA, an shRNA, a ribozyme, an antisense RNA, a decoy
oligonucleotide, an antimir, a supermir, or an RNA activator. 51.
The method of paragraph 33, wherein the vector is a viral vector,
an expression vector, or a plasmid. 52. The method of paragraph 33,
wherein the delivery agent is a lipid, a liposome, a food source, a
solution, an emulsion, a micelle or other membranous formulation, a
lipid particle, a bacteria, a fungus, a plant, a yeast cell, or a
yeast cell particle. 53. The method of paragraph 52, wherein the
viral vector comprises a baculoviral vector. 54. The method of
paragraph 52, wherein the lipid particle comprises about 15-25%
triacylglycerol, about 0.5-2% phospholipids, about 1-3% glycerol,
and at least one lipid-binding protein. 55. The method of paragraph
33, wherein the composition is provided in a spray, solution, gel,
bait, a food source, or powder form. 56. The method of paragraph
33, wherein the composition further comprises an attractant. 57.
The method of paragraph 56, wherein the attractant comprises an
insect pheromone or hormone. 58. The method of paragraph 34,
wherein the composition is specific to the pest and does not affect
other insects. 59. The method of paragraph 46, wherein the
oligonucleotide comprises 9-36 base pairs. 60. The method of
paragraph 33, wherein the composition is administered to adult
insects. 61. The method of paragraph 33, wherein the composition is
administered to a breeding or feeding locus. 62. The method of
paragraph 33, wherein the composition further comprises an
additional agent. 63. The method of paragraph 33, wherein the
composition further comprises sucrose. 64. A composition comprising
an RNA effector molecule or a vector encoding an RNA effector
molecule, and a delivery agent, wherein the RNA effector molecule
modulates gene expression of an insect or an insect pathogen. 65.
The composition of paragraph 64, wherein the RNA effector molecule
comprises an oligonucleotide. 66. The composition of paragraph 65,
wherein the oligonucleotide comprises an siRNA, an miRNA, an shRNA,
a ribozyme, an antisense RNA, a decoy oligonucleotide, an antimir,
a supermir, or an RNA activator. 67. The composition of paragraph
65, wherein the oligonucleotide is a single stranded or double
stranded oligonucleotide. 68. The method of paragraph 64, wherein
the vector is a viral vector, an expression vector, or a plasmid.
69. The method of paragraph 64, wherein the delivery agent is a
lipid, a liposome, a food source, a solution, an emulsion, a
micelle or other membranous formulation, a lipid particle, a
bacteria, a fungus, a plant, a yeast cell, or a yeast cell
particle. 70. The composition of paragraph 64, wherein the lipid
particle comprises about 15-25% triacylglycerol, about 0.5-2%
phospholipids, about 1-3% glycerol, and at least one lipid-binding
protein. 71. The composition of paragraph 64, wherein the
composition is provided as a food source for the insect. 72. The
composition of paragraph 71, wherein the food source is provided as
a liquid, solid, gel, semi-solid composition, sugar composition, or
lipid composition. 73. The composition of paragraph 71, wherein the
food source is a virus, a bacterium, a fungus, a plant, or a yeast
cell expressing the oligonucleotide. 74. The composition of
paragraph 64, wherein the composition inhibits viability, survival,
growth, development, and/or reproduction of the insect. 75. The
composition of paragraph 64, wherein the composition inhibits
pathogen infectivity, virulence, reproduction, viability, growth,
translation, protein production, viral uptake or transmission of
the insect pathogen. 76. The composition of paragraph 64, wherein
the composition is provided in a spray, solution, gel, topical
formulation, or powder form. 77. The composition of paragraph 65,
wherein the oligonucleotide comprises 9-36 base pairs. 78. The
composition of paragraph 64, wherein the composition further
comprises an antibiotic, antiviral or anthelmintic agent. 79. The
composition of paragraph 64, further comprising an insect
attractant. 80. The composition of paragraph 79, wherein the
attractant comprises an insect pheromone or hormone. 81. The
composition of paragraph 65, wherein the oligonucleotide is
modified. 82. The composition of paragraph 81, wherein the
modification is selected from the group consisting of: 2'-O-methyl
modified nucleotide, a nucleotide having a 5'-phosphorothioate
group, a terminal nucleotide linked to a cholesteryl derivative, a
2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified
nucleotide, a locked nucleotide, an abasic nucleotide,
2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide,
morpholino nucleotide, a phosphoramidate, and a non-natural base
comprising nucleotide. 83. The composition of paragraph 64, further
comprising an additional agent. 84. The composition of paragraph
64, further comprising sucrose.
[0540] It is understood that the foregoing detailed description and
the following examples are illustrative only and are not to be
taken as limitations upon the scope of the invention. Various
changes and modifications to the disclosed embodiments, which will
be apparent to those of skill in the art, may be made without
departing from the spirit and scope of the present invention.
Further, all patents, patent applications, and publications
identified in the specification and examples are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents are
based on the information available to the applicants and do not
constitute any admission as to the correctness of the dates or
contents of these documents.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 36 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000
<210> SEQ ID NO 2 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 2 Ala Ala Leu Glu Ala Leu
Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala 1 5 10 15 Leu Glu Ala Leu
Ala Glu Ala Ala Ala Ala Gly Gly Cys 20 25 <210> SEQ ID NO 3
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 3 Ala Ala Leu Ala Glu Ala Leu Ala
Glu Ala Leu Ala Glu Ala Leu Ala 1 5 10 15 Glu Ala Leu Ala Glu Ala
Leu Ala Ala Ala Ala Gly Gly Cys 20 25 30 <210> SEQ ID NO 4
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 4 Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala
Leu Ala Glu Ala 1 5 10 15 <210> SEQ ID NO 5 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 5 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp
Glu Gly 1 5 10 15 Met Ile Trp Asp Tyr Gly 20 <210> SEQ ID NO
6 <211> LENGTH: 23 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 6 Gly Leu Phe Gly Ala Ile Ala Gly Phe
Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly
20 <210> SEQ ID NO 7 <211> LENGTH: 48 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 7 Gly Leu Phe Glu Ala
Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp
Gly Trp Tyr Gly Cys Gly Leu Phe Glu Ala Ile Glu Gly 20 25 30 Phe
Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Cys 35 40
45 <210> SEQ ID NO 8 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 8 Gly Leu Phe Glu Ala
Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp
Gly Gly Cys Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile 20 25 30 Glu
Asn Gly Trp Glu Gly Met Ile Asp Gly Gly Cys 35 40 <210> SEQ
ID NO 9 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 9 Gly Leu Phe Gly Ala Leu Ala Glu
Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 His Leu Ala Glu Ala Leu
Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25 30 Gly Ser Cys 35
<210> SEQ ID NO 10 <211> LENGTH: 34 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 10 Gly Leu Phe Glu Ala
Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Leu Ala Glu
Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly 20 25 30 Ser
Cys <210> SEQ ID NO 11 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (17)..(17) <223>
OTHER INFORMATION: Norleucine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (38)..(38) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 11 Gly Leu Phe
Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Xaa
Ile Asp Gly Lys Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu 20 25
30 Asn Gly Trp Glu Gly Xaa Ile Asp Gly 35 40 <210> SEQ ID NO
12 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 12 Gly Leu Phe Glu Ala Leu Leu Glu
Leu Leu Glu Ser Leu Trp Glu Leu 1 5 10 15 Leu Leu Glu Ala 20
<210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 13 Gly Leu Phe Lys Ala Leu
Leu Lys Leu Leu Lys Ser Leu Trp Lys Leu 1 5 10 15 Leu Leu Lys Ala
20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 14 Gly Leu Phe
Arg Ala Leu Leu Arg Leu Leu Arg Ser Leu Trp Arg Leu 1 5 10 15 Leu
Leu Arg Ala 20 <210> SEQ ID NO 15 <211> LENGTH: 30
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 15
Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5
10 15 Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25
30 <210> SEQ ID NO 16 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 16 Gly Leu Phe
Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu 1 5 10 15 Gly
Leu Ile Glu Gly Cys 20 <210> SEQ ID NO 17 <211> LENGTH:
26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 17 Gly
Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10
15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 <210> SEQ ID
NO 18 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 18 His His His His His Trp Tyr Gly 1
5 <210> SEQ ID NO 19 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 19 Cys His Lys Lys Lys Lys
Lys Lys His Cys 1 5 10 <210> SEQ ID NO 20 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Penetratin peptide <400> SEQUENCE: 20 Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> SEQ
ID NO 21 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Human immunodeficiency virus type 1 <400> SEQUENCE:
21 Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly
1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25
<210> SEQ ID NO 22 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Signal sequence based
peptide <400> SEQUENCE: 22 Gly Ala Leu Phe Leu Gly Trp Leu
Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val 20 25 <210> SEQ ID NO 23 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown: PVEC peptide <400> SEQUENCE: 23 Leu Leu Ile Ile Leu
Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys
<210> SEQ ID NO 24 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Transportan peptide
<400> SEQUENCE: 24 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Lys Ile Asn Leu Lys 1 5 10 15 Ala Leu Ala Ala Leu Ala Lys Lys
Ile Leu 20 25 <210> SEQ ID NO 25 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Amphiphilic model peptide <400> SEQUENCE: 25 Lys Leu Ala Leu
Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala
<210> SEQ ID NO 26 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 26 Arg Arg Arg Arg Arg Arg
Arg Arg Arg 1 5 <210> SEQ ID NO 27 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Bacterial cell wall permeating peptide <400> SEQUENCE: 27 Lys
Phe Phe Lys Phe Phe Lys Phe Phe Lys 1 5 10 <210> SEQ ID NO 28
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: LL-37 polypeptide <400> SEQUENCE: 28
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5
10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu
Val 20 25 30 Pro Arg Thr Glu Ser 35 <210> SEQ ID NO 29
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: Cecropin P1 polypeptide <400>
SEQUENCE: 29 Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser
Ala Lys Lys 1 5 10 15 Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln
Gly Gly Pro Arg 20 25 30 <210> SEQ ID NO 30 <211>
LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown: Alpha-defensin polypeptide <400> SEQUENCE: 30 Ala
Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10
15 Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30
<210> SEQ ID NO 31 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Beta-defensin
polypeptide <400> SEQUENCE: 31 Asp His Tyr Asn Cys Val Ser
Ser Gly Gly Gln Cys Leu Tyr Ser Ala 1 5 10 15 Cys Pro Ile Phe Thr
Lys Ile Gln Gly Thr Cys Tyr Arg Gly Lys Ala 20 25 30 Lys Cys Cys
Lys 35 <210> SEQ ID NO 32 <211> LENGTH: 42 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown: PR-39
polypeptide <400> SEQUENCE: 32 Arg Arg Arg Pro Arg Pro Pro
Tyr Leu Pro Arg Pro Arg Pro Pro Pro 1 5 10 15 Phe Phe Pro Pro Arg
Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro 20 25 30 Arg Phe Pro
Pro Arg Phe Pro Gly Lys Arg 35 40 <210> SEQ ID NO 33
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: Indolicidin peptide <400> SEQUENCE:
33 Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10
<210> SEQ ID NO 34 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: RFGF peptide <400>
SEQUENCE: 34 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu
Leu Ala Pro 1 5 10 15 <210> SEQ ID NO 35 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
RFGF analogue peptide <400> SEQUENCE: 35 Ala Ala Leu Leu Pro
Val Leu Leu Ala Ala Pro 1 5 10 <210> SEQ ID NO 36 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown: Bactenecin peptide <400> SEQUENCE: 36 Arg Lys Cys
Arg Ile Val Val Ile Arg Val Cys Arg 1 5 10
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 36 <210>
SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ ID NO 2
<211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 2 Ala Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu
Ala Leu Ala Glu Ala 1 5 10 15 Leu Glu Ala Leu Ala Glu Ala Ala Ala
Ala Gly Gly Cys 20 25 <210> SEQ ID NO 3 <211> LENGTH:
30 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 3
Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala 1 5
10 15 Glu Ala Leu Ala Glu Ala Leu Ala Ala Ala Ala Gly Gly Cys 20 25
30 <210> SEQ ID NO 4 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 4 Ala Leu Glu Ala Leu Ala
Glu Ala Leu Glu Ala Leu Ala Glu Ala 1 5 10 15 <210> SEQ ID NO
5 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 5 Gly Leu Phe Glu Ala Ile Glu Gly Phe
Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Trp Asp Tyr Gly 20
<210> SEQ ID NO 6 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 6 Gly Leu Phe Gly Ala Ile
Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly
Trp Tyr Gly 20 <210> SEQ ID NO 7 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 7
Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5
10 15 Met Ile Asp Gly Trp Tyr Gly Cys Gly Leu Phe Glu Ala Ile Glu
Gly 20 25 30 Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp
Tyr Gly Cys 35 40 45 <210> SEQ ID NO 8 <211> LENGTH: 44
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 8
Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5
10 15 Met Ile Asp Gly Gly Cys Gly Leu Phe Glu Ala Ile Glu Gly Phe
Ile 20 25 30 Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Gly Cys 35 40
<210> SEQ ID NO 9 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 9 Gly Leu Phe Gly Ala
Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 His Leu Ala
Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25 30 Gly
Ser Cys 35 <210> SEQ ID NO 10 <211> LENGTH: 34
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 10
Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5
10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly
Gly 20 25 30 Ser Cys <210> SEQ ID NO 11 <211> LENGTH:
41 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (17)..(17)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (38)..(38)
<223> OTHER INFORMATION: Norleucine <400> SEQUENCE: 11
Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5
10 15 Xaa Ile Asp Gly Lys Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile
Glu 20 25 30 Asn Gly Trp Glu Gly Xaa Ile Asp Gly 35 40 <210>
SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 12 Gly Leu Phe Glu Ala Leu
Leu Glu Leu Leu Glu Ser Leu Trp Glu Leu 1 5 10 15 Leu Leu Glu Ala
20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 13 Gly Leu Phe
Lys Ala Leu Leu Lys Leu Leu Lys Ser Leu Trp Lys Leu 1 5 10 15 Leu
Leu Lys Ala 20 <210> SEQ ID NO 14 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 14 Gly
Leu Phe Arg Ala Leu Leu Arg Leu Leu Arg Ser Leu Trp Arg Leu 1 5 10
15 Leu Leu Arg Ala 20 <210> SEQ ID NO 15 <211> LENGTH:
30 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 15
Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5
10 15 Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25
30 <210> SEQ ID NO 16 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 16 Gly Leu Phe
Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu 1 5 10 15 Gly
Leu Ile Glu Gly Cys 20 <210> SEQ ID NO 17 <211> LENGTH:
26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 17 Gly
Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10
15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 <210> SEQ ID
NO 18 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 18 His His His His His Trp Tyr Gly 1
5 <210> SEQ ID NO 19 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 19 Cys His Lys Lys Lys Lys
Lys Lys His Cys 1 5 10 <210> SEQ ID NO 20 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Penetratin peptide <400> SEQUENCE: 20 Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> SEQ
ID NO 21 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Human immunodeficiency virus type 1 <400> SEQUENCE:
21 Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly
1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25
<210> SEQ ID NO 22 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Signal sequence based
peptide <400> SEQUENCE: 22 Gly Ala Leu Phe Leu Gly Trp Leu
Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val 20 25 <210> SEQ ID NO 23 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown: PVEC peptide <400> SEQUENCE: 23 Leu Leu Ile Ile Leu
Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys
<210> SEQ ID NO 24 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Transportan peptide
<400> SEQUENCE: 24 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Lys Ile Asn Leu Lys 1 5 10 15 Ala Leu Ala Ala Leu Ala Lys Lys
Ile Leu 20 25 <210> SEQ ID NO 25 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Amphiphilic model peptide <400> SEQUENCE: 25 Lys Leu Ala Leu
Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala
<210> SEQ ID NO 26 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 26 Arg Arg Arg Arg Arg Arg
Arg Arg Arg 1 5 <210> SEQ ID NO 27 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Bacterial cell wall permeating peptide <400> SEQUENCE: 27 Lys
Phe Phe Lys Phe Phe Lys Phe Phe Lys 1 5 10 <210> SEQ ID NO 28
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: LL-37 polypeptide <400> SEQUENCE: 28
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5
10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu
Val 20 25 30 Pro Arg Thr Glu Ser 35 <210> SEQ ID NO 29
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: Cecropin P1 polypeptide
<400> SEQUENCE: 29 Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu
Glu Asn Ser Ala Lys Lys 1 5 10 15 Arg Ile Ser Glu Gly Ile Ala Ile
Ala Ile Gln Gly Gly Pro Arg 20 25 30 <210> SEQ ID NO 30
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: Alpha-defensin polypeptide <400>
SEQUENCE: 30 Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu
Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala
Phe Cys Cys 20 25 30 <210> SEQ ID NO 31 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Beta-defensin polypeptide <400> SEQUENCE: 31 Asp His Tyr Asn
Cys Val Ser Ser Gly Gly Gln Cys Leu Tyr Ser Ala 1 5 10 15 Cys Pro
Ile Phe Thr Lys Ile Gln Gly Thr Cys Tyr Arg Gly Lys Ala 20 25 30
Lys Cys Cys Lys 35 <210> SEQ ID NO 32 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
PR-39 polypeptide <400> SEQUENCE: 32 Arg Arg Arg Pro Arg Pro
Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro 1 5 10 15 Phe Phe Pro Pro
Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro 20 25 30 Arg Phe
Pro Pro Arg Phe Pro Gly Lys Arg 35 40 <210> SEQ ID NO 33
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: Indolicidin peptide <400> SEQUENCE:
33 Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10
<210> SEQ ID NO 34 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: RFGF peptide <400>
SEQUENCE: 34 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu
Leu Ala Pro 1 5 10 15 <210> SEQ ID NO 35 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
RFGF analogue peptide <400> SEQUENCE: 35 Ala Ala Leu Leu Pro
Val Leu Leu Ala Ala Pro 1 5 10 <210> SEQ ID NO 36 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown: Bactenecin peptide <400> SEQUENCE: 36 Arg Lys Cys
Arg Ile Val Val Ile Arg Val Cys Arg 1 5 10
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