CHIRAL DESIGN
Cross-Reference to Related Applications
[0001] This application claims priority to United States Provisional Application Serial
No. 61/928,405, filed January 16, 2014, and 62/063,359, filed October 13, 2014, the entirety of
each of which is incorporated herein by reference.
Background of the Invention
[0002] Oligonucleotides are useful in therapeutic, diagnostic, research and nanomaterials
applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) for
therapeutics can be limited, for example, because of their instability against extra- and
intracellular nucleases and/or their poor cell penetration and distribution. Additionally, in vitro
studies have shown that properties of antisense oligonucleotides such as binding affinity,
sequence specific binding to the complementary RNA (Cosstick and Eckstein, 1985; LaPlanche
et a , 1986; Latimer et a , 1989; Hacia et a , 1994; Mesmaeker et a , 1995), and stability to
nucleases can be affected by the absolute stereochemical configurations of the phosphorus atoms
(Cook, et al. US005599797A). Therefore, there is a need for new and improved oligonucleotides
and oligonucleotide compositions, such as, e.g., new antisense and siRNA oligonucleotides and
oligonucleotide compositions.
Summary of the Invention
[0003] Among other things, the present invention encompasses the recognition that
stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that
differ from one another in the stereochemical structure of individual backbone chiral centers
within the oligonucleotide chain. Moreover, the present invention encompasses the insight that it
is typically unlikely that a stereorandom oligonucleotide preparation will include every possible
stereoisomer of the relevant oligonucleotide. Thus, among other things, the present invention
provides new chemical entities that are particular stereoisomers of oligonucleotides of interest.
That is, the present invention provides substantially pure preparations of single oligonucleotide
compounds, where a particular oligonucleotide compound may be defined by its base sequence,
its length, its pattern of backbone linkages, and its pattern of backbone chiral centers.
[0004] The present invention demonstrates, among other things, that individual
stereoisomers of a particular oligonucleotide can show different stability and/or activity from
each other. Moreover, the present disclosure demonstrates that stability improvements achieved
through inclusion and/or location of particular chiral structures within an oligonucleotide can be
comparable to, or even better than those achieved through use of certain modified backbone
linkages, bases, and/or sugars (e.g., through use of certain types of modified phophates, 2'-
modifications, base modifications, etc.).
[0005] Among other things, the present invention recognizes that properties and activities
of an oligonucleotide can be adjusted by optimizing its pattern of backbone chiral centers. In
some embodiments, the present invention provides compositions of oligonucleotides, wherein
the oligonucleotides have a common pattern of backbone chiral centers which, unexpectedly,
greatly enhances the stability and/or biological activity of the oligonucleotides. In some
embodiments, a pattern of backbone chiral centers provides increased stability. In some
embodiments, a pattern of backbone chiral centers provides surprisingly increased activity. In
some embodiments, a pattern of backbone chiral centers provides increased stability and activity.
In some embodiments, when an oligonucleotide is utilized to cleave a nucleic acid polymer, a
pattern of backbone chiral centers of the oligonucleotide, surprisingly by itself, changes the
cleavage pattern of a target nucleic acid polymer. In some embodiments, a pattern of backbone
chiral centers effectively prevents cleavage at secondary sites. In some embodiments, a pattern
of backbone chiral centers creates new cleavage sites. In some embodiments, a pattern of
backbone chiral centers minimizes the number of cleavage sites. In some embodiments, a
pattern of backbone chiral centers minimizes the number of cleavage sites so that a target nucleic
acid polymer is cleaved at only one site within the sequence of the target nucleic acid polymer
that is complementary to the oligonucleotide. In some embodiments, a pattern of backbone
chiral centers enhances cleavage efficiency at a cleavage site. In some embodiments, a pattern of
backbone chiral centers of the oligonucleotide improves cleavage of a target nucleic acid
polymer. In some embodiments, a pattern of backbone chiral centers increases selectivity. In
some embodiments, a pattern of backbone chiral centers minimizes off-target effect. In some
embodiments, a pattern of backbone chiral centers increase selectivity, e.g., cleavage selectivity
between two target sequences differing only by a single nucleotide polymorphism (SNP).
[0006] All publications and patent documents cited in this application are incorporated
herein by reference in their entirety.
Definitions
[0007] Aliphatic: The term "aliphatic" or "aliphatic group", as used herein, means a
straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that
is completely saturated or that contains one or more units of unsaturation, or a monocyclic
hydrocarbon or bicyclic or polycyclic hydrocarbon that is completely saturated or that contains
one or more units of unsaturation, but which is not aromatic (also referred to herein as
"carbocycle" "cycloaliphatic" or "cycloalkyl"), that has a single point of attachment to the rest of
the molecule. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms.
Unless otherwise specified, aliphatic groups contain 1-10 aliphatic carbon atoms. In some
embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments,
aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups
contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3
aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic
carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers
to a monocyclic or bicyclic C3-C10 hydrocarbon that is completely saturated or that contains one
or more units of unsaturation, but which is not aromatic, that has a single point of attachment to
the rest of the molecule. In some embodiments, "cycloaliphatic" (or "carbocycle" or
"cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that
contains one or more units of unsaturation, but which is not aromatic, that has a single point of
attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to,
linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof
such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
[0008] Alkylene: The term "alkylene" refers to a bivalent alkyl group. An "alkylene
chain" is a polymethylene group, i.e., -(CH 2) - , wherein n is a positive integer, preferably from
1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a
polymethylene group in which one or more methylene hydrogen atoms are replaced with a
substituent. Suitable substituents include those described below for a substituted aliphatic group.
[0009] Alkenylene: The term "alkenylene" refers to a bivalent alkenyl group. A
substituted alkenylene chain is a polymethylene group containing at least one double bond in
which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include
those described below for a substituted aliphatic group.
[0010] Animal: As used herein, the term "animal" refers to any member of the animal
kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In
some embodiments, "animal" refers to non-human animals, at any stage of development. In
certain embodiments, the non-human animal is a mammal {e.g. , a rodent, a mouse, a rat, a rabbit,
a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In
some embodiments, an animal may be a transgenic animal, a genetically-engineered animal,
and/or a clone.
[0011] Approximately: As used herein, the terms "approximately" or "about" in
reference to a number are generally taken to include numbers that fall within a range of 5%,
10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise
stated or otherwise evident from the context (except where such number would be less than 0%>
or exceed 100% of a possible value). In some embodiments, use of the term "about" in reference
to dosages means ± 5 mg/kg/day.
[0012] Aryl: The term "aryl" used alone or as part of a larger moiety as in "aralkyl,"
"aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein
each ring in the system contains three to seven ring members. The term "aryl" may be used
interchangeably with the term "aryl ring." In certain embodiments of the present invention,
"aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl,
naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within
the scope of the term "aryl," as it is used herein, is a group in which an aromatic ring is fused to
one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl,
or tetrahydronaphthyl, and the like.
[0013] Characteristic portion: As used herein, the phrase a "characteristic portion" of a
protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of
continuous stretches of amino acids, that together are characteristic of a protein or polypeptide.
Each such continuous stretch generally will contain at least two amino acids. Furthermore, those
of ordinary skill in the art will appreciate that typically at least 5, 10, 15, 20 or more amino acids
are required to be characteristic of a protein. In general, a characteristic portion is one that, in
addition to the sequence identity specified above, shares at least one functional characteristic
with the relevant intact protein.
[0014] Characteristic sequence: A "characteristic sequence" is a sequence that is found
in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of
ordinary skill in the art to define members of the family.
[0015] Characteristic structural element: The term "characteristic structural element"
refers to a distinctive structural element (e.g., core structure, collection of pendant moieties,
sequence element, etc) that is found in all members of a family of polypeptides, small molecules,
or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members
of the family.
[0016] Comparable: The term "comparable" is used herein to describe two (or more) sets
of conditions or circumstances that are sufficiently similar to one another to permit comparison
of results obtained or phenomena observed. In some embodiments, comparable sets of
conditions or circumstances are characterized by a plurality of substantially identical features and
one or a small number of varied features. Those of ordinary skill in the art will appreciate that
sets of conditions are comparable to one another when characterized by a sufficient number and
type of substantially identical features to warrant a reasonable conclusion that differences in
results obtained or phenomena observed under the different sets of conditions or circumstances
are caused by or indicative of the variation in those features that are varied.
[0017] Dosing regimen: As used herein, a "dosing regimen" or "therapeutic regimen"
refers to a set of unit doses (typically more than one) that are administered individually to a
subject, typically separated by periods of time. In some embodiments, a given therapeutic agent
has a recommended dosing regimen, which may involve one or more doses. In some
embodiments, a dosing regimen comprises a plurality of doses each of which are separated from
one another by a time period of the same length; in some embodiments, a dosing regime
comprises a plurality of doses and at least two different time periods separating individual doses.
In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In
some embodiments, different doses within a dosing regimen are of different amounts. In some
embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or
more additional doses in a second dose amount different from the first dose amount. In some
embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or
more additional doses in a second dose amount same as the first dose amount.
[0018] Equivalent agents: Those of ordinary skill in the art, reading the present
disclosure, will appreciate that the scope of useful agents in the context of the present invention
is not limited to those specifically mentioned or exemplified herein. In particular, those skilled
in the art will recognize that active agents typically have a structure that consists of a core and
attached pendant moieties, and furthermore will appreciate that simple variations of such core
and/or pendant moieties may not significantly alter activity of the agent. For example, in some
embodiments, substitution of one or more pendant moieties with groups of comparable threedimensional
structure and/or chemical reactivity characteristics may generate a substituted
compound or portion equivalent to a parent reference compound or portion. In some
embodiments, addition or removal of one or more pendant moieties may generate a substituted
compound equivalent to a parent reference compound. In some embodiments, alteration of core
structure, for example by addition or removal of a small number of bonds (typically not more
than 5, 4, 3, 2, or 1 bonds, and often only a single bond) may generate a substituted compound
equivalent to a parent reference compound. In many embodiments, equivalent compounds may
be prepared by methods illustrated in general reaction schemes as, for example, described below,
or by modifications thereof, using readily available starting materials, reagents and conventional
or provided synthesis procedures. In these reactions, it is also possible to make use of variants,
which are in themselves known, but are not mentioned here.
[0019] Equivalent Dosage: The term "equivalent dosage" is used herein to compare
dosages of different pharmaceutically active agents that effect the same biological result.
Dosages of two different agents are considered to be "equivalent" to one another in accordance
with the present invention if they achieve a comparable level or extent of the biological result. In
some embodiments, equivalent dosages of different pharmaceutical agents for use in accordance
with the present invention are determined using in vitro and/or in vivo assays as described herein.
In some embodiments, one or more lysosomal activating agents for use in accordance with the
present invention is utilized at a dose equivalent to a dose of a reference lysosomal activating
agent; in some such embodiments, the reference lysosomal activating agent for such purpose is
selected from the group consisting of small molecule allosteric activators (e.g.,
pyrazolpyrimidines), imminosugars (e.g., isofagomine), antioxidants (e.g., n-acetyl-cysteine),
and regulators of cellular trafficking (e.g., Rabla polypeptide).
[0020] Heteroaliphatic: The term "heteroaliphatic" refers to an aliphatic group wherein
one or more units selected from C, CH, CH2, or CH3 are independently replaced by a heteroatom.
In some embodiments, a heteroaliphatic group is heteroalkyl. In some embodiments, a
heteroaliphatic group is heteroalkenyl.
[0021] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a
larger moiety, e.g., "heteroaralkyl," or "heteroaralkoxy," refer to groups having 5 to 10 ring
atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 p electrons shared in a cyclic array;
and having, in addition to carbon atoms, from one to five heteroatoms. The term "heteroatom"
refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and
any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl,
furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,
purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroar-," as used herein,
also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or
heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,
indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,
quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl,
phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-
3(4H)-one. A heteroaryl group may be mono- or bicyclic. The term "heteroaryl" may be used
interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of
which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an
alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently
are optionally substituted.
[0022] Heteroatom: The term "heteroatom" means one or more of oxygen, sulfur,
nitrogen, phosphorus, boron, selenium, or silicon (including, any oxidized form of nitrogen,
boron, selenium, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a
substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH
(as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).
[0023] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl,"
"heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3-
to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either
saturated or partially unsaturated, and having, in addition to carbon atoms, one or more,
preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of
a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated
or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen,
the nitrogen may be N (as in 3,4-dihydro-2 H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in
N-substituted pyrrolidinyl).
[0024] A heterocyclic ring can be attached to its pendant group at any heteroatom or
carbon atom that results in a stable structure and any of the ring atoms can be optionally
substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include,
without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl,
dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The
terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic
moiety," and "heterocyclic radical," are used interchangeably herein, and also include groups in
which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as
indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or
point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic.
The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the
alkyl and heterocyclyl portions independently are optionally substituted.
[0025] Intraperitoneal: The phrases "intraperitoneal administration" and "administered
intraperitonealy" as used herein have their art-understood meaning referring to administration of
a compound or composition into the peritoneum of a subject.
[0026] In vitro: As used herein, the term "in vitro" refers to events that occur in an
artificial environment, e.g. , in a test tube or reaction vessel, in cell culture, etc. , rather than within
an organism {e.g., animal, plant, and/or microbe).
[0027] In vivo: As used herein, the term " vivo" refers to events that occur within an
organism {e.g., animal, plant, and/or microbe).
[0028] Lower alkyl: The term "lower alkyl" refers to a Ci_4 straight or branched alkyl
group. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and
tert-butyl.
[0029] Lower haloalkyl: The term "lower haloalkyl" refers to a Ci_4 straight or branched
alkyl group that is substituted with one or more halogen atoms.
[0030] Optionally substituted: As described herein, compounds of the invention may
contain "optionally substituted" moieties. In general, the term "substituted," whether preceded
by the term "optionally" or not, means that one or more hydrogens of the designated moiety are
replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted"
group may have a suitable substituent at each substitutable position of the group, and when more
than one position in any given structure may be substituted with more than one substituent
selected from a specified group, the substituent may be either the same or different at every
position. Combinations of substituents envisioned by this invention are preferably those that
result in the formation of stable or chemically feasible compounds. The term "stable," as used
herein, refers to compounds that are not substantially altered when subjected to conditions to
allow for their production, detection, and, in certain embodiments, their recovery, purification,
and use for one or more of the purposes disclosed herein.
[0031] Suitable monovalent substituents on a substitutable carbon atom of an "optionally
substituted" group are independently halogen; -(CH 2)o 4R°; -(CH 2)0 4OR°; -O(CH 2)0_4R°,
-0-(CH 2)o 4C(0)OR°; -(CH 2)0 4CH(OR°)2; -(CH 2)0 4SR°; -(CH 2)0 4Ph, which may be
substituted with R°; -(CH 2)0 4O(CH2)0 iPh which may be substituted with R°; -CH=CHPh,
which may be substituted with R°; -(CH 2)0 4O(CH2)0 i-pyridyl which may be substituted with
R°; -N0 2; -CN; - N3; -(CH 2)0 4N(R°)2; -(CH 2)0 4N(R°)C(0)R°; -N(R°)C(S)R°;
- (CH2)o 4N(R°)C(0)NR° 2; -N(R°)C(S)NR°2; -(CH 2)0 4N(R°)C(0)OR°;
-N(R°)N(R°)C(0)R°; -N(R°)N(R°)C(0)NR° 2; -N(R°)N(R°)C(0)OR°; -(CH 2)0 4C(0)R°;
-C(S)R°; - (CH2)o 4C(0)OR°; -(CH 2)0 4C(0)SR°; -(CH2)0 4C(0)OSiR° 3; -(CH 2)0 4OC(0)R°;
-OC(0)(CH 2)o 4SR, -SC(S)SR°; -(CH 2)0 4SC(0)R°; -(CH 2)0 4C(0)NR° 2; -C(S)NR° 2;
-C(S)SR°; -SC(S)SR°, -(CH2)0 4OC(0)NR° 2; -C(0)N(OR°)R°; -C(0)C(0)R°;
-C(0)CH 2C(0)R°; -C(NOR°)R°; -(CH2)0 4SSR°; -(CH 2)0 4S(0) 2R°; -(CH 2)0 4S(0) 2OR°;
- (CH2)o 4OS(0) 2R°; -S(0) 2NR°2; -(CH 2)0 4S(0)R°; -N(R°)S(0) 2NR°2; -N(R°)S(0) 2R°;
-N(OR°)R°; -C(NH)NR° 2; -P(0) 2R°; -P(0)R° 2; -OP(0)R° 2; -OP(0)(OR°) 2; -SiR° 3; -(C
straight or branched alkylene)0-N(R°) 2; or - (Ci 4 straight or branched alkylene)C(0)0-N(R°) 2,
wherein each R° may be substituted as defined below and is independently hydrogen,
Ci 6 aliphatic, -CH 2Ph, -O(CH 2)0 iPh, -CH 2-(5-6 membered heteroaryl ring), or a 5-6
membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two
independent occurrences of R°, taken together with their intervening atom(s), form a 3-12
membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted
as defined below.
[0032] Suitable monovalent substituents on R° (or the ring formed by taking two
independent occurrences of R° together with their intervening atoms), are independently
halogen, -(CH 2)0 2R*, -(haloR*), -(CH 2)0 2OH, -(CH 2)0 2OR*, -(CH 2)0_
2CH(OR*) 2; -O(haloR'), - C , - N3, -(CH 2)0 2C(0)R*, -(CH 2)0_2C(O)OH, -(CH 2)0 2C(0)OR*,
- (CH2)o 2SR*, -(CH2V2SH, -(CH2V2NH2, - (CH2)o 2NHR*, -(CH 2)0 2NR*2, -N0 2, - SiR
-OSiR* 3, -C(0)SR* - (Ci 4 straight or branched alkylene)C(0)OR*, or -SSR* wherein each R
is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and
is independently selected from Ci 4 aliphatic, -CH 2Ph, -O(CH 2)0 iPh, or a 5-6 membered
saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R°
include =0 and =S.
[0033] Suitable divalent substituents on a saturated carbon atom of an "optionally
substituted" group include the following: =0, =S, = R*
2, =NNHC(0)R *, =NNHC(0)OR *,
= HS(0 )2R*, =NR*, =NOR *, -0(C(R *
2))2 30 - or -S(C(R *
2))2_3S-, wherein each independent
occurrence of R is selected from hydrogen, Ci_ aliphatic which may be substituted as defined
below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-
4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent
substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group
include: -0(CR 2)2 30-, wherein each independent occurrence of R is selected from hydrogen,
Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered
saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0034] Suitable substituents on the aliphatic group of R include halogen,
-R*, -(haloR*), -OH, -OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, - H2, -NHR*, -NR* 2,
or -N0 2, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with
one or more halogens, and is independently Ci 4 aliphatic, -CH 2Ph, -O(CH 2)0 iPh, or a 5-6
membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0035] Suitable substituents on a substitutable nitrogen of an "optionally substituted"
group include - R†, -NR †
2, -C(0)R †, -C(0)OR †, -C(0)C(0)R †, -C(0)CH 2C(0)R †,
-S(0) 2R†, -S(0) 2NR†
2, -C(S)NR †
2, -C(NH)NR †
2, or -N(R †)S(0) 2R†; wherein each R† is
independently hydrogen, Ci_6 aliphatic which may be substituted as defined below, unsubstituted
-OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the
definition above, two independent occurrences of R^, taken together with their intervening
atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl mono- or
bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
[0036] Suitable substituents on the aliphatic group of R are independently halogen,
-R*, -(haloR*), -OH, -OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, - H2, -NHR*, -NR* 2,
or -N0 2, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with
one or more halogens, and is independently Ci_4 aliphatic, -CH 2Ph, -O(CH 2)0 iPh, or a 5-6
membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0037] Oral: The phrases "oral administration" and "administered orally" as used herein
have their art-understood meaning referring to administration by mouth of a compound or
composition.
[0038] Parenteral: The phrases "parenteral administration" and "administered
parenterally" as used herein have their art-understood meaning referring to modes of
administration other than enteral and topical administration, usually by injection, and include,
without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.
[0039] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a
ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is
intended to encompass rings having multiple sites of unsaturation, but is not intended to include
aryl or heteroaryl moieties, as herein defined.
[0040] Pharmaceutical composition: As used herein, the term "pharmaceutical
composition" refers to an active agent, formulated together with one or more pharmaceutically
acceptable carriers. In some embodiments, active agent is present in unit dose amount
appropriate for administration in a therapeutic regimen that shows a statistically significant
probability of achieving a predetermined therapeutic effect when administered to a relevant
population. In some embodiments, pharmaceutical compositions may be specially formulated for
administration in solid or liquid form, including those adapted for the following: oral
administration, for example, drenches (aqueous or non-aqueous solutions or suspensions),
tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders,
granules, pastes for application to the tongue; parenteral administration, for example, by
subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution
or suspension, or sustained-release formulation; topical application, for example, as a cream,
ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity;
intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly;
transdermally; or nasally, pulmonary, and to other mucosal surfaces.
[0041] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically
acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk ratio.
[0042] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically
acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such
as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in
carrying or transporting the subject compound from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not injurious to the patient. Some
examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars,
such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose,
and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose
acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as
magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or
polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical
formulations.
[0043] Pharmaceutically acceptable salt: The term "pharmaceutically acceptable salt",
as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical
contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al.
describes pharmaceutically acceptable salts in detail in J . Pharmaceutical Sciences, 66: 1-19
(1977). In some embodiments, pharmaceutically acceptable salt include, but are not limited to,
nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or
with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or
malonic acid or by using other methods used in the art such as ion exchange. In some
embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,
hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate,
malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,
oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, /?-toluenesulfonate,
undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts
include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments,
pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary
ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate,
sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
[0044] Prodrug: A general, a "prodrug," as that term is used herein and as is understood
in the art, is an entity that, when administered to an organism, is metabolized in the body to
deliver an active (e.g., therapeutic or diagnostic) agent of interest. Typically, such metabolism
involves removal of at least one "prodrug moiety" so that the active agent is formed. Various
forms of "prodrugs" are known in the art. For examples of such prodrug moieties, see:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in
Enzymology, 42:309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) Prodrugs and Targeted Delivery, edited by by J . Rautio (Wiley, 201 1);
c) Prodrugs and Targeted Delivery, edited by by J . Rautio (Wiley, 201 1);
d) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen;
e) Bundgaard, Chapter 5 "Design and Application of Prodrugs", by H. Bundgaard, p.
113-191 (1991);
f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);
g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and
h) Kakeya, et al, Chem. Pharm. Bull, 32:692 (1984).
[0045] As with other compounds described herein, prodrugs may be provided in any of a
variety of forms, e.g., crystal forms, salt forms etc. In some embodiments, prodrugs are provided
as pharmaceutically acceptable salts thereof.
[0046] Protecting group: The term "protecting group," as used herein, is well known in
the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W.
Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is
incorporated herein by reference. Also included are those protecting groups specially adapted
for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid
Chemistry, edited by Serge L. Beaucage et al. 06/2012, the entirety of Chapter 2 is incorporated
herein by reference. Suitable amino-protecting groups include methyl carbamate, ethyl
carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-
(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di - t-butyl-[9-( 10,1 0-dioxo-l 0,10,10,10-
tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate
(Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-
phenylethyl carbamate (hZ), l-(l-adamantyl)-l-methylethyl carbamate (Adpoc), 1,1-dimethyl-
2-haloethyl carbamate, l,l-dimethyl-2,2-dibromoethyl carbamate (DB- t-BOC), 1,1-dimethyl-
2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc),
l-(3,5-di - t-butylphenyl)-l-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl
carbamate (Pyoc), 2-(N,N -dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC),
1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl
carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl
carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p—
methoxybenzyl carbamate (Moz), /?-nitobenzyl carbamate, /?-bromobenzyl carbamate, p—
chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate
(Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthio ethyl carbamate, 2-
methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2—( 1,3—
dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-
dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-
triphenylphosphonioisopropyl carbamate (Ppoc), l,l-dimethyl-2-cyanoethyl carbamate, mchloro-
p-acyloxybenzyl carbamate, /?-(dihydroxyboryl)benzyl carbamate, 5-
benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-
dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-
(lO)-carbonyl derivative, N '-/?-toluenesulfonylaminocarbonyl derivative, N -
phenylaminothiocarbonyl derivative, t-amyl carbamate, -benzyl thiocarbamate, /?-cyanobenzyl
carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate,
cyclopropylmethyl carbamate, /?-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl
carbamate, -(N,N -dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3 -(NNdimethylcarboxamido)
propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl
carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl
carbamate, isonicotinyl carbamate, p-(p -methoxyphenylazo)benzyl carbamate, 1-
methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl- 1-cyclopropylmethyl
carbamate, 1-methyl- 1-(3, 5-dimethoxyphenyl)ethyl carbamate, 1-methyl- l-(pphenylazophenyl)
ethyl carbamate, 1-methyl- 1-phenylethyl carbamate, 1-methyl- 1-(4-
pyridyl)ethyl carbamate, phenyl carbamate, /?-(phenylazo)benzyl carbamate, 2,4,6-tri -tbutylphenyl
carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl
carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide,
phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, Nbenzoylphenylalanyl
derivative, benzamide, /?-phenylbenzamide, o-nitophenylacetamide, onitrophenoxyacetamide,
acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(phydroxyphenyl)
propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(onitrophenoxy)
propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-
chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-
one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-
dimethylpyrrole, N-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-
substituted l,3-dimethyl-l,3,5-triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5-
triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(l-isopropyl-4-
nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-
methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-
methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfiuorenylamine (PhF), N-2,7-
dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N -
oxide, N-l,l-dimethylthiomethyleneamine, N-benzylideneamine, N-pmethoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-
pyridyl)mesityljmethyleneamine, N-(N',N'-dimethy laminomethylene)amine, N,N -
isopropylidenediamine, N-/?-nitrobenzylideneamine, N-salicylideneamine, N-5-
chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, Ncyclohexylideneamine,
N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivative,
N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or
tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine,
amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),
diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate,
diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-
dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro—4-
methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),
/?-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-
methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-
4-methoxybenzenesulfonamide (Pme), 2,3 ,5,6-tetramethyl-4-methoxybenzenesulfonamide
(Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-
dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-
sulfonamide (Pmc), methanesulfonamide (Ms), b-trimethylsilylethanesulfonamide (SES), 9-
anthracenesulfonamide, 4-(4 ' ,8 '-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
[0047] Suitably protected carboxylic acids further include, but are not limited to, silyl-,
alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl
groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,
triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, pmethoxybenzyl,
3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples of
suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally
substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include
optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, Onitrobenzyl,
p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-
picolyl.
[0048] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM),
methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),
benzyloxymethyl (BOM), /?-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl
(p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),
siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-
chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-
bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-
methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-
methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4-methyl)phenyl]-4-
methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-
(2-chloroethoxy)ethyl, 1-methyl- 1-methoxyethyl, 1-methyl- 1-benzyloxyethyl, 1-methyl- 1-
benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, tbutyl,
allyl, /?-chlorophenyl, /?-methoxyphenyl, 2,4-dinitrophenyl, benzyl, /?-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, /?-nitrobenzyl, /?-halobenzyl, 2,6-dichlorobenzyl, p -
cyanobenzyl, /?-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,
diphenylmethyl, p,p -dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, anaphthyldiphenylmethyl,
/?-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,
tri(/?-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4 ',4"-
tris(4,5-dichlorophthalimidophenyl)methyl, 4,4 ' ,4 ' '-tris(levulinoyloxyphenyl)methyl, 4,4 ' ,4 " -
tris(benzoyloxyphenyl)methyl, 3-(imidazol- 1-yl)bis(4 ' ,4 ' '-dimethoxyphenyl)methyl, 1,1-
bis(4-methoxyphenyl)-l'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-
oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS),
triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS),
diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), tbutyldiphenylsilyl
(TBDPS), tribenzylsilyl, tri-/?-xylylsilyl, triphenylsilyl, diphenylmethylsilyl
(DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate,
dichloroacetate, trichloroacetate, trifiuoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, /?-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate),
4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-
methoxycrotonate, benzoate, /?-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl
methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-
trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)
ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate,
alkyl vinyl carbonate alkyl allyl carbonate, alkyl /?-nitrophenyl carbonate, alkyl benzyl
carbonate, alkyl /?-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl onitrobenzyl
carbonate, alkyl /?-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-lnapththyl
carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-
methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-
(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-
(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-
( 1, 1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(l ,l-dimethylpropyl)phenoxy acetate,
chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-
(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N ',N '-
tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphmothioyl,
alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and
tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal,
ethylidene acetal, 1- t-butylethylidene ketal, 1-phenylethylidene ketal, (4-
methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene
ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, /?-methoxybenzylidene
acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene
acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-
methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho
ester, a-methoxybenzylidene ortho ester, l -(N,N -dimethylamino)ethylidene derivative, a-
(N,N '-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-tbutylsilylene
group (DTBS), l,3-(l,l,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),
tetra- t-butoxydisiloxane-l,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates,
ethyl boronate, and phenyl boronate.
[0049] In some embodiments, a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl,
methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 2-
trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-
dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,
triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-
fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-
dimethoxytrityl, (DMTr) and 4,4',4"-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-
(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-
nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-
dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl,
butylthiocarbonyl, 4,4',4"-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-
(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-
phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In some
embodiments, each of the hydroxyl protecting groups is, independently selected from acetyl,
benzyl, t- butyldimethylsilyl, t-butyldiphenylsilyl and 4,4'-dimethoxytrityl. In some
embodiments, the hydroxyl protecting group is selected from the group consisting of trityl,
monomethoxytrityl and 4,4'-dimethoxytrityl group.
[0050] In some embodiments, a phosphorous protecting group is a group attached to the
internucleotide phosphorous linkage throughout oligonucleotide synthesis. In some
embodiments, the phosphorous protecting group is attached to the sulfur atom of the
internucleotide phosphorothioate linkage. In some embodiments, the phosphorous protecting
group is attached to the oxygen atom of the internucleotide phosphorothioate linkage. In some
embodiments, the phosphorous protecting group is attached to the oxygen atom of the
internucleotide phosphate linkage. In some embodiments the phosphorous protecting group is 2-
cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, onitrobenzyl,
2-(/?-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-t r t-butylcarboxamido)-
1-propyl, 4-oxopentyl, 4-methylthio-l-butyl, 2-cyano-l,l-dimethylethyl, 4-N-methylaminobutyl,
3-(2-pyridyl)- 1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,Nmethyl)
aminoethyl, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.
[0051] Protein: As used herein, the term "protein" refers to a polypeptide {i.e., a string
of at least two amino acids linked to one another by peptide bonds). In some embodiments,
proteins include only naturally-occurring amino acids. In some embodiments, proteins include
one or more non-naturally-occurring amino acids (e.g., moieties that form one or more peptide
bonds with adjacent amino acids). In some embodiments, one or more residues in a protein
chain contain a non-amino-acid moiety (e.g., a glycan, etc). In some embodiments, a protein
includes more than one polypeptide chain, for example linked by one or more disulfide bonds or
associated by other means. In some embodiments, proteins contain L-amino acids, D-amino
acids, or both; in some embodiments, proteins contain one or more amino acid modifications or
analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation,
methylation, etc. The term "peptide" is generally used to refer to a polypeptide having a length
of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or
less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments,
biologically active portions thereof, and/or characteristic portions thereof.
[0052] Sample: A "sample" as used herein is a specific organism or material obtained
therefrom. In some embodiments, a sample is a biological sample obtained or derived from a
source of interest, as described herein, . In some embodiments, a source of interest comprises an
organism, such as an animal or human. In some embodiments, a biological sample comprises
biological tissue or fluid. In some embodiments, a biological sample is or comprises bone
marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body
fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid;
pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal
swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates;
scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other
body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a
biological sample is or comprises cells obtained from an individual. In some embodiments, a
sample is a "primary sample" obtained directly from a source of interest by any appropriate
means. For example, in some embodiments, a primary biological sample is obtained by methods
selected from the group consisting of biopsy {e.g., fine needle aspiration or tissue biopsy),
surgery, collection of body fluid {e.g., blood, lymph, feces etc.), etc. In some embodiments, as
will be clear from context, the term "sample" refers to a preparation that is obtained by
processing (e.g., by removing one or more components of and/or by adding one or more agents
to) a primary sample. For example, filtering using a semi-permeable membrane. Such a
"processed sample" may comprise, for example nucleic acids or proteins extracted from a sample
or obtained by subjecting a primary sample to techniques such as amplification or reverse
transcription of mR A, isolation and/or purification of certain components, etc. In some
embodiments, a sample is an organism. In some embodiments, a sample is a plant. In some
embodiments, a sample is an animal. In some embodiments, a sample is a human. In some
embodiments, a sample is an organism other than a human.
[0053] Stereochemically isomeric forms: The phrase "stereochemically isomeric forms,"
as used herein, refers to different compounds made up of the same atoms bonded by the same
sequence of bonds but having different three-dimensional structures which are not
interchangeable. In some embodiments of the invention, provided chemical compositions may
be or include pure preparations of individual stereochemically isomeric forms of a compound; in
some embodiments, provided chemical compositions may be or include mixtures of two or more
stereochemically isomeric forms of the compound. In certain embodiments, such mixtures
contain equal amounts of different stereochemically isomeric forms; in certain embodiments,
such mixtures contain different amounts of at least two different stereochemically isomeric
forms. In some embodiments, a chemical composition may contain all diastereomers and/or
enantiomers of the compound. In some embodiments, a chemical composition may contain less
than all diastereomers and/or enantiomers of a compound. In some embodiments, if a particular
enantiomer of a compound of the present invention is desired, it may be prepared, for example,
by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting
diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired
enantiomers. Alternatively, where the molecule contains a basic functional group, such as
amino, diastereomeric salts are formed with an appropriate optically-active acid, and resolved,
for example, by fractional crystallization.
[0054] Subject: As used herein, the term "subject" or "test subject" refers to any
organism to which a provided compound or composition is administered in accordance with the
present invention e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
Typical subjects include animals {e.g., mammals such as mice, rats, rabbits, non-human
primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be
suffering from, and/or susceptible to a disease, disorder, and/or condition.
[0055] Substantially: As used herein, the term "substantially" refers to the qualitative
condition of exhibiting total or near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will understand that biological and chemical
phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used herein to capture the potential lack
of completeness inherent in many biological and/or chemical phenomena.
[0056] Suffering from: An individual who is "suffering from" a disease, disorder, and/or
condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder,
and/or condition.
[0057] Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or
condition is one who has a higher risk of developing the disease, disorder, and/or condition than
does a member of the general public. In some embodiments, an individual who is susceptible to
a disease, disorder and/or condition may not have been diagnosed with the disease, disorder,
and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder,
and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some
embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not
exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or condition will develop the disease,
disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder, and/or condition.
[0058] Systemic: The phrases "systemic administration," "administered systemically,"
"peripheral administration," and "administered peripherally" as used herein have their artunderstood
meaning referring to administration of a compound or composition such that it enters
the recipient's system.
[0059] Tautomeric forms: The phrase "tautomeric forms," as used herein, is used to
describe different isomeric forms of organic compounds that are capable of facile
interconversion. Tautomers may be characterized by the formal migration of a hydrogen atom or
proton, accompanied by a switch of a single bond and adjacent double bond. In some
embodiments, tautomers may result from prototropic tautomerism (i.e., the relocation of a
proton). In some embodiments, tautomers may result from valence tautomerism (i.e., the rapid
reorganization of bonding electrons). All such tautomeric forms are intended to be included
within the scope of the present invention. In some embodiments, tautomeric forms of a
compound exist in mobile equilibrium with each other, so that attempts to prepare the separate
substances results in the formation of a mixture. In some embodiments, tautomeric forms of a
compound are separable and isolatable compounds. In some embodiments of the invention,
chemical compositions may be provided that are or include pure preparations of a single
tautomeric form of a compound. In some embodiments of the invention, chemical compositions
may be provided as mixtures of two or more tautomeric forms of a compound. In certain
embodiments, such mixtures contain equal amounts of different tautomeric forms; in certain
embodiments, such mixtures contain different amounts of at least two different tautomeric forms
of a compound. In some embodiments of the invention, chemical compositions may contain all
tautomeric forms of a compound. In some embodiments of the invention, chemical compositions
may contain less than all tautomeric forms of a compound. In some embodiments of the
invention, chemical compositions may contain one or more tautomeric forms of a compound in
amounts that vary over time as a result of interconversion. In some embodiments of the
invention, the tautomerism is keto-enol tautomerism. One of skill in the chemical arts would
recognize that a keto-enol tautomer can be "trapped" (i.e., chemically modified such that it
remains in the "enol" form) using any suitable reagent known in the chemical arts in to provide
an enol derivative that may subsequently be isolated using one or more suitable techniques
known in the art. Unless otherwise indicated, the present invention encompasses all tautomeric
forms of relevant compounds, whether in pure form or in admixture with one another.
[0060] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any
agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired
biological and/or pharmacological effect. In some embodiments, a therapeutic agent is any
substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of,
reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease,
disorder, and/or condition.
[0061] Therapeutically effective amount: As used herein, the term "therapeutically
effective amount" means an amount of a substance {e.g., a therapeutic agent, composition, and/or
formulation) that elicits a desired biological response when administered as part of a therapeutic
regimen. In some embodiments, a therapeutically effective amount of a substance is an amount
that is sufficient, when administered to a subject suffering from or susceptible to a disease,
disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease,
disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the
effective amount of a substance may vary depending on such factors as the desired biological
endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective
amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount
that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or
reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
In some embodiments, a therapeutically effective amount is administered in a single dose; in
some embodiments, multiple unit doses are required to deliver a therapeutically effective
amount.
[0062] Treat: As used herein, the term "treat," "treatment," or "treating" refers to any
method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset
of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease,
disorder, and/or condition. Treatment may be administered to a subject who does not exhibit
signs of a disease, disorder, and/or condition. In some embodiments, treatment may be
administered to a subject who exhibits only early signs of the disease, disorder, and/or condition,
for example for the purpose of decreasing the risk of developing pathology associated with the
disease, disorder, and/or condition.
[0063] Unsaturated: The term "unsaturated," as used herein, means that a moiety has
one or more units of unsaturation.
[0064] Unit dose: The expression "unit dose" as used herein refers to an amount
administered as a single dose and/or in a physically discrete unit of a pharmaceutical
composition. In many embodiments, a unit dose contains a predetermined quantity of an active
agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some
embodiments, more than one unit dose is administered to achieve a total single dose. In some
embodiments, administration of multiple unit doses is required, or expected to be required, in
order to achieve an intended effect. A unit dose may be, for example, a volume of liquid (e.g.,
an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a
predetermined amount of one or more therapeutic agents in solid form, a sustained release
formulation or drug delivery device containing a predetermined amount of one or more
therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation
that includes any of a variety of components in addition to the therapeutic agent(s). For example,
acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers,
preservatives, etc., may be included as described infra. It will be appreciated by those skilled in
the art, in many embodiments, a total appropriate daily dosage of a particular therapeutic agent
may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the
attending physician within the scope of sound medical judgment. In some embodiments, the
specific effective dose level for any particular subject or organism may depend upon a variety of
factors including the disorder being treated and the severity of the disorder; activity of specific
active compound employed; specific composition employed; age, body weight, general health,
sex and diet of the subject; time of administration, and rate of excretion of the specific active
compound employed; duration of the treatment; drugs and/or additional therapies used in
combination or coincidental with specific compound(s) employed, and like factors well known in
the medical arts.
[0065] Wild-type: As used herein, the term "wild-type" has its art-understood meaning
that refers to an entity having a structure and/or activity as found in nature in a "normal" (as
contrasted with mutant, diseased, altered, etc) state or context. Those of ordinary skill in the art
will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g.,
alleles).
[0066] Nucleic acid: The term "nucleic acid" includes any nucleotides, analogs thereof,
and polymers thereof. The term "polynucleotide" as used herein refer to a polymeric form of
nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These
terms refer to the primary structure of the molecules and, thus, include double- and singlestranded
DNA, and double- and single-stranded RNA. These terms include, as equivalents,
analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides
such as, though not limited to, methylated, protected and/or capped nucleotides or
polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligodeoxyribonucleotides
(DNA); RNA or DNA derived from N-glycosides or C-glycosides of
nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified
sugars; and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom
bridges (also referred to herein as "internucleotide linkages"). The term encompasses nucleic
acids containing any combinations of nucleobases, modified nucleobases, sugars, modified
sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not
limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose
moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing
ribose and modified ribose moieties. The prefix poly- refers to a nucleic acid containing 2 to
about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid
containing 2 to about 200 nucleotide monomer units.
[0067] Nucleotide: The term "nucleotide" as used herein refers to a monomeric unit of a
polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups or
phosphorus-containing internucleotidic linkages. The naturally occurring bases, (guanine, (G),
adenine, (A), cytosine, (C ), thymine, (T), and uracil (U)) are derivatives of purine or pyrimidine,
though it should be understood that naturally and non-naturally occurring base analogs are also
included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which
forms DNA) or ribose (which forms RNA), though it should be understood that naturally and
non-naturally occurring sugar analogs are also included. Nucleotides are linked via
internucleotidic linkages to form nucleic acids, or polynucleotides. Many internucleotidic
linkages are known in the art (such as, though not limited to, phosphate, phosphorothioates,
boranophosphates and the like). Artificial nucleic acids include PNAs (peptide nucleic acids),
phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates,
methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the
phosphate backbone of native nucleic acids, such as those described herein.
[0068] Nucleoside: The term "nucleoside" refers to a moiety wherein a nucleobase or a
modified nucleobase is covalently bound to a sugar or modified sugar.
[0069] Sugar: The term "sugar" refers to a monosaccharide in closed and/or open form.
Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and
hexopyranose moieties. As used herein, the term also encompasses structural analogs used in
lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of
the nucleic acid analog, glycol nucleic acid ("GNA").
[0070] Modified sugar. The term "modified sugar" refers to a moiety that can replace a
sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other
physicochemical property of a sugar.
[0071] Nucleobase: The term "nucleobase" refers to the parts of nucleic acids that are
involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary
strand in a sequence specific manner. The most common naturally-occurring nucleobases are
adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the
naturally-occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In
some embodiments, the naturally-occurring nucleobases are methylated adenine, guanine, uracil,
cytosine, or thymine. In some embodiments, a nucleobase is a "modified nucleobase," e.g., a
nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In
some embodiments, the modified nucleobases are methylated adenine, guanine, uracil, cytosine,
or thymine. In some embodiments, the modified nucleobase mimics the spatial arrangement,
electronic properties, or some other physicochemical property of the nucleobase and retains the
property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific
manner. In some embodiments, a modified nucleobase can pair with all of the five naturally
occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting
the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide
duplex.
[0072] Chiral ligand: The term "chiral ligand" or "chiral auxiliary" refers to a moiety
that is chiral and can be incorporated into a reaction so that the reaction can be carried out with
certain stereoselectivity.
[0073] Condensing reagent: In a condensation reaction, the term "condensing reagent"
refers to a reagent that activates a less reactive site and renders it more susceptible to attack by
another reagent. In some embodiments, such another reagent is a nucleophile.
[0074] Blocking group: The term "blocking group" refers to a group that masks the
reactivity of a functional group. The functional group can be subsequently unmasked by
removal of the blocking group. In some embodiments, a blocking group is a protecting group.
[0075] Moiety: The term "moiety" refers to a specific segment or functional group of a
molecule. Chemical moieties are often recognized chemical entities embedded in or appended to
a molecule.
[0076] Solid support: The term "solid support" refers to any support which enables
synthesis of nucleic acids. In some embodiments, the term refers to a glass or a polymer, that is
insoluble in the media employed in the reaction steps performed to synthesize nucleic acids, and
is derivatized to comprise reactive groups. In some embodiments, the solid support is Highly
Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). In some embodiments, the
solid support is Controlled Pore Glass (CPG). In some embodiments, the solid support is hybrid
support of Controlled Pore Glass (CPG) and Highly Cross-linked Polystyrene (HCP).
[0077] Linking moiety: The term "linking moiety" refers to any moiety optionally
positioned between the terminal nucleoside and the solid support or between the terminal
nucleoside and another nucleoside, nucleotide, or nucleic acid.
[0078] DNA molecule: A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form
or a double-stranded helix. This term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms. Thus, this term includes doublestranded
DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA
molecules, sequences can be described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand
having a sequence homologous to the mRNA).
[0079] Coding sequence: A DNA "coding sequence" or "coding region" is a doublestranded
DNA sequence which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate expression control sequences. The boundaries of the
coding sequence (the "open reading frame" or "ORF") are determined by a start codon at the 5'
(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence
can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA
sequences. A polyadenylation signal and transcription termination sequence is, usually, be
located 3' to the coding sequence. The term "non-coding sequence" or "non-coding region"
refers to regions of a polynucleotide sequence that are not translated into amino acids (e.g. 5' and
3' un-translated regions).
[0080] Reading frame: The term "reading frame" refers to one of the six possible
reading frames, three in each direction, of the double stranded DNA molecule. The reading
frame that is used determines which codons are used to encode amino acids within the coding
sequence of a DNA molecule.
[0081] Antisense: As used herein, an "antisense" nucleic acid molecule comprises a
nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA molecule, complementary to an
mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense
nucleic acid molecule can associate via hydrogen bonds to a sense nucleic acid molecule.
[0082] Wobbleposition: As used herein, a "wobble position" refers to the third position
of a codon. Mutations in a DNA molecule within the wobble position of a codon, in some
embodiments, result in silent or conservative mutations at the amino acid level. For example,
there are four codons that encode Glycine, i.e., GGU, GGC, GGA and GGG, thus mutation of
any wobble position nucleotide, to any other nucleotide selected from A, U , C and G, does not
result in a change at the amino acid level of the encoded protein and, therefore, is a silent
substitution.
[0083] Silent substitution: a "silent substitution" or "silent mutation" is one in which a
nucleotide within a codon is modified, but does not result in a change in the amino acid residue
encoded by the codon. Examples include mutations in the third position of a codon, as well in
the first position of certain codons such as in the codon "CGG" which, when mutated to AGG,
still encodes Arg.
[0084] Gene: The terms "gene," "recombinant gene" and "gene construct" as used
herein, refer to a DNA molecule, or portion of a DNA molecule, that encodes a protein or a
portion thereof. The DNA molecule can contain an open reading frame encoding the protein (as
exon sequences) and can further include intron sequences. The term "intron" as used herein,
refers to a DNA sequence present in a given gene which is not translated into protein and is
found in some, but not all cases, between exons. It can be desirable for the gene to be operably
linked to, (or it can comprise), one or more promoters, enhancers, repressors and/or other
regulatory sequences to modulate the activity or expression of the gene, as is well known in the
art.
[0085] Complementary DNA: As used herein, a "complementary DNA" or "cDNA"
includes recombinant polynucleotides synthesized by reverse transcription of mRNA and from
which intervening sequences (introns) have been removed.
[0086] Homology: "Homology" or "identity" or "similarity" refers to sequence similarity
between two nucleic acid molecules. Homology and identity can each be determined by
comparing a position in each sequence which can be aligned for purposes of comparison. When
an equivalent position in the compared sequences is occupied by the same base, then the
molecules are identical at that position; when the equivalent site occupied by the same or a
similar nucleic acid residue (e.g., similar in steric and/or electronic nature), then the molecules
can be referred to as homologous (similar) at that position. Expression as a percentage of
homology/similarity or identity refers to a function of the number of identical or similar nucleic
acids at positions shared by the compared sequences. A sequence which is "unrelated" or "non
homologous" shares less than 40% identity, less than 35% identity, less than 30%> identity, or
less than 25% identity with a sequence described herein. In comparing two sequences, the
absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases
the identity and homology/similarity.
[0087] In some embodiments, the term "homology" describes a mathematically based
comparison of sequence similarities which is used to identify genes with similar functions or
motifs. The nucleic acid sequences described herein can be used as a "query sequence" to
perform a search against public databases, for example, to identify other family members, related
sequences or homologs. In some embodiments, such searches can be performed using the
NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J . Mol. Biol. 215:403-
10. In some embodiments, BLAST nucleotide searches can be performed with the NBLAST
program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid
molecules of the invention. In some embodiments, to obtain gapped alignments for comparison
purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and BLAST) can be used (See
www.ncbi.nlm.nih.gov).
[0088] Identity: As used herein, "identity" means the percentage of identical nucleotide
residues at corresponding positions in two or more sequences when the sequences are aligned to
maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be
readily calculated by known methods, including but not limited to those described in
(Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New
York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M
Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J . Applied Math., 48:
1073 (1988). Methods to determine identity are designed to give the largest match between the
sequences tested. Moreover, methods to determine identity are codified in publicly available
computer programs. Computer program methods to determine identity between two sequences
include, but are not limited to, the GCG program package (Devereux, J., et al, Nucleic Acids
Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J . Molec.
Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The
BLAST X program is publicly available from NCBI and other sources (BLAST Manual,
Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al, J . Mol. Biol. 215:
403-410 (1990). The well-known Smith Waterman algorithm can also be used to determine
identity.
[0089] Heterologous: A "heterologous" region of a DNA sequence is an identifiable
segment of DNA within a larger DNA sequence that is not found in association with the larger
sequence in nature. Thus, when the heterologous region encodes a mammalian gene, the gene
can usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome
of the source organism. Another example of a heterologous coding sequence is a sequence
where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding
sequence contains introns or synthetic sequences having codons or motifs different than the
unmodified gene). Allelic variations or naturally-occurring mutational events do not give rise to
a heterologous region of DNA as defined herein.
[0090] Transition mutation: The term "transition mutations" refers to base changes in a
DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T) is replaced by another
pyrimidine, or a purine (adenosine (A) or guanosine (G) is replaced by another purine.
[0091] Transversion mutation: The term "transversion mutations" refers to base changes
in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T) is replaced by a purine
(adenosine (A) or guanosine (G), or a purine is replaced by a pyrimidine.
[0092] Oligonucleotide: the term "oligonucleotide" refers to a polymer or oligomer of
nucleotide monomers, containing any combination of nucleobases, modified nucleobases, sugars,
modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to
herein as "internucleotidic linkage", defined further herein).
[0093] Oligonucleotides can be single-stranded or double-stranded. As used herein, the
term "oligonucleotide strand" encompasses a single-stranded oligonucleotide. 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, Ul
adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators,
immuno-stimulatory oligonucleotides, and decoy oligonucleotides.
[0094] 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.
In some embodiments, 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.
[0095] Oligonucleotides of the present invention can be of various lengths. In particular
embodiments, oligonucleotides can range from about 2 to about 200 nucleotides in length. In
various related embodiments, oligonucleotides, single-stranded, double-stranded, and triplestranded,
can range in length from about 4 to about 10 nucleotides, 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 some embodiments, the oligonucleotide is from
about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotide is at least 4
nucleotides in length. In some embodiments, the oligonucleotide is at least 5 nucleotides in
length. In some embodiments, the oligonucleotide is at least 6 nucleotides in length. In some
embodiments, the oligonucleotide is at least 7 nucleotides in length. In some embodiments, the
oligonucleotide is at least 8 nucleotides in length. In some embodiments, the oligonucleotide is
at least 9 nucleotides in length. In some embodiments, the oligonucleotide is at least 10
nucleotides in length. In some embodiments, the oligonucleotide is at least 11 nucleotides in
length. In some embodiments, the oligonucleotide is at least 12 nucleotides in length. In some
embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the
oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is
at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30
nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary
strands of at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a
duplex of complementary strands of at least 2 1 nucleotides in length.
[0096] Internucleotidic linkage: As used herein, the phrase "internucleotidic linkage"
refers generally to the phosphorus-containing linkage between nucleotide units of an
oligonucleotide, and is interchangeable with "inter-sugar linkage" and "phosphorus atom
bridge," as used above and herein. In some embodiments, an internucleotidic linkage is a
phosphodiester linkage, as found in naturally occurring DNA and RNA molecules. In some
embodiments, an mtemucleotidic linkage is a "modified mtemucleotidic linkage" wherein each
oxygen atom of the phosphodiester linkage is optionally and independently replaced by an
organic or inorganic moiety. In some embodiments, such an organic or inorganic moiety is
selected from but not limited to =S, =Se, =NR', -SR', -SeR', -N(R') 2, B(R') 3, -S- -Se- and -
N(R')-, wherein each R' is independently as defined and described below. In some
embodiments, an mtemucleotidic linkage is a phosphotriester linkage, phosphorothioate diester
linkage or modified phosphorothioate triester linkage. It is understood by a
person of ordinary skill in the art that the mtemucleotidic linkage may exist as an anion or cation
at a given pH due to the existence of acid or base moieties in the linkage.
[0097] Unless otherwise specified, when used with an oligonucleotide sequence, each of
s, si, s2, s3, s4, s5, s6 and s7 independently represents the following modified mtemucleotidic
linkage as illustrated in Table 1, below.
[0098] Table 1. Exemplary Modified Internucleotidic Linkage.

For instance, (Rp, Sp)-ATsCslGA has 1) a phosphorothioate internucleotidic
O
- pI
-_
o -
¾
-
) between phosphorothioate triester internucleotidic
linkage having the structure of between C and G. Unless otherwise
specified, the Rp Sp designations preceding an oligonucleotide sequence describe the
configurations of chiral linkage phosphorus atoms in the internucleotidic linkages sequentially
from 5' to 3' of the oligonucleotide sequence. For instance, in (Rp, ,Sp)-ATsCslGA, the
phosphorus in the "s" linkage between T and C has Rp configuration and the phosphorus in "si"
linkage between C and G has Sp configuration. In some embodiments, "All-( p)" or "All -(S p)"
is used to indicate that all chiral linkage phosphorus atoms in oligonucleotide have the same Rp
or Sp configuration, respectively. For instance, All-( p)-
GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linkage
phosphorus atoms in the oligonucleotide have Rp configuration; A 11- ( r)-
GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linkage
phosphorus atoms in the oligonucleotide have Sp configuration.
[00100] Oligonucleotide type: As used herein, the phrase "oligonucleotide type" is used to
define an oligonucleotide that has a particular base sequence, pattern of backbone linkages (i.e.,
pattern of internucleotidic linkage types, for example, phosphate, phosphorothioate, etc), pattern
of backbone chiral centers (i.e. pattern of linkage phosphorus stereochemistry (Rp/Sp)), and
pattern of backbone phosphorus modifications (e.g., pattern of "-XLR 1" groups in formula I).
Oligonucleotides of a common designated "type" are structurally identical to one another.
[00101] One of skill in the art will appreciate that synthetic methods of the present
invention provide for a degree of control during the synthesis of an oligonucleotide strand such
that each nucleotide unit of the oligonucleotide strand can be designed and/or selected in advance
to have a particular stereochemistry at the linkage phosphorus and/or a particular modification at
the linkage phosphorus, and/or a particular base, and/or a particular sugar. In some
embodiments, an oligonucleotide strand is designed and/or selected in advance to have a
particular combination of stereocenters at the linkage phosphorus. In some embodiments, an
oligonucleotide strand is designed and/or determined to have a particular combination of
modifications at the linkage phosphorus. In some embodiments, an oligonucleotide strand is
designed and/or selected to have a particular combination of bases. In some embodiments, an
oligonucleotide strand is designed and/or selected to have a particular combination of one or
more of the above structural characteristics. The present invention provides compositions
comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirally controlled
oligonucleotide compositions). In some embodiments, all such molecules are of the same type
(i.e., are structurally identical to one another). In many embodiments, however, provided
compositions comprise a plurality of oligonucleotides of different types, typically in pre
determined relative amounts.
[00102] Chiral control: As used herein, "chiral control" refers to an ability to control the
stereochemical designation of every chiral linkage phosphorus within an oligonucleotide strand.
The phrase "chirally controlled oligonucleotide" refers to an oligonucleotide which exists in a
single diastereomeric form with respect to the chiral linkage phosphorus.
[00103] Chirally controlled oligonucleotide composition: As used herein, the phrase
"chirally controlled oligonucleotide composition" refers to an oligonucleotide composition that
contains predetermined levels of individual oligonucleotide types. For instance, in some
embodiments a chirally controlled oligonucleotide composition comprises one oligonucleotide
type. In some embodiments, a chirally controlled oligonucleotide composition comprises more
than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide
composition comprises a mixture of multiple oligonucleotide types. Exemplary chirally
controlled oligonucleotide compositions are described further herein.
[00104] Chirally pure: as used herein, the phrase "chirally pure" is used to describe a
chirally controlled oligonucleotide composition in which all of the oligonucleotides exist in a
single diastereomeric form with respect to the linkage phosphorus.
[00105] Chirally uniform: as used herein, the phrase "chirally uniform" is used to describe
an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry
at the linkage phosphorus. For instance, an oligonucleotide whose nucleotide units all have Rp
stereochemistry at the linkage phosphorus is chirally uniform. Likewise, an oligonucleotide
whose nucleotide units all have S stereochemistry at the linkage phosphorus is chirally uniform.
[00106] Predetermined: By predetermined is meant deliberately selected, for example as
opposed to randomly occurring or achieved. Those of ordinary skill in the art, reading the
present specification, will appreciate that the present invention provides new and surprising
technologies that permit selection of particular oligonucleotide types for preparation and/or
inclusion in provided compositions, and further permits controlled preparation of precisely the
selected particular types, optionally in selected particular relative amounts, so that provided
compositions are prepared. Such provided compositions are "predetermined" as described
herein. Compositions that may contain certain individual oligonucleotide types because they
happen to have been generated through a process that cannot be controlled to intentionally
generate the particular oligonucleotide types is not a "predetermined" composition. In some
embodiments, a predetermined composition is one that can be intentionally reproduced (e.g.,
through repetition of a controlled process).
[00107] Linkage phosphorus: as defined herein, the phrase "linkage phosphorus" is used
to indicate that the particular phosphorus atom being referred to is the phosphorus atom present
in the internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a
phosphodiester of an internucleotidic linkage as occurs in naturally occurring DNA and R A. In
some embodiments, a linkage phosphorus atom is in a modified internucleotidic linkage, wherein
each oxygen atom of a phosphodiester linkage is optionally and independently replaced by an
organic or inorganic moiety. In some embodiments, a linkage phosphorus atom is P* of formula
I . In some embodiments, a linkage phosphorus atom is chiral. In some embodiments, a chiral
linkage phosphorus atom is P* of formula I .
[00108] P-modification: as used herein, the term "P-modification" refers to any
modification at the linkage phosphorus other than a stereochemical modification. In some
embodiments, a P-modification comprises addition, substitution, or removal of a pendant moiety
covalently attached to a linkage phosphorus. In some embodiments, the "P-modification" is -XL-
R 1 wherein each of X, L and R1 is independently as defined and described herein and below.
[00109] Blockmer: the term "blockmer," as used herein, refers to an oligonucleotide strand
whose pattern of structural features characterizing each individual nucleotide unit is
characterized by the presence of at least two consecutive nucleotide units sharing a common
structural feature at the internucleotidic phosphorus linkage. By common structural feature is
meant common stereochemistry at the linkage phosphorus or a common modification at the
linkage phosphorus. In some embodiments, the at least two consecutive nucleotide units sharing
a common structure feature at the internucleotidic phosphours linkage are referred to as a
"block".
[00110] In some embodiments, a blockmer is a "stereoblockmer," e.g., at least two
consecutive nucleotide units have the same stereochemistry at the linkage phosphorus. Such at
lest two consecutive nucleotide units form a "stereoblock." For instance, (Sp, 5p)-ATsCslGA is
a stereoblockmer because at least two consecutive nucleotide units, the Ts and the Csl, have the
same stereochemistry at the linkage phosphorus (both Sp). In the same oligonucleotide (Sp, Sp)-
ATsCslGA, TsCsl forms a block, and it is a stereoblock.
[00111] In some embodiments, a blockmer is a "P-modification blockmer," e.g., at least
two consecutive nucleotide units have the same modification at the linkage phosphorus. Such at
lest two consecutive nucleotide units form a "P-modification block". For instance, (Rp, Sp)-
ATsCsGA is a P-modification blockmer because at least two consecutive nucleotide units, the Ts
and the Cs, have the same P-modification (i.e., both are a phosphorothioate diester). In the same
oligonucleotide of (Rp, 5p)-ATsCsGA, TsCs forms a block, and it is a P-modification block.
[00112] In some embodiments, a blockmer is a "linkage blockmer," e.g., at least two
consecutive nucleotide units have identical stereochemistry and identical modifications at the
linkage phosphorus. At least two consecutive nucleotide units form a "linkage block". For
instance, (Rp, i?p)-ATsCsGA is a linkage blockmer because at least two consecutive nucleotide
units, the Ts and the Cs, have the same stereochemistry (both Rp) and P-modification (both
phosphorothioate). In the same oligonucleotide of (Rp, i?p)-ATsCsGA, TsCs forms a block, and
it is a linkage block.
[00113] In some embodiments, a blockmer comprises one or more blocks independently
selected from a stereoblock, a P-modification block and a linkage block. In some embodiments,
a blockmer is a stereoblockmer with respect to one block, and/or a P-modification blockmer with
respect to another block, and/or a linkage blockmer with respect to yet another block. For
instance, (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp)-AAsTsCsGsAslTslCslGslATCG is a
stereoblockmer with respect to the stereoblock AsTsCsGsAsl (all Rp at linkage phosphorus) or
TslCslGsl (all Sp at linkage phosphorus), a P-modification blockmer with respect to the Pmodification
block AsTsCsGs (all s linkage) or AslTslCslGsl (all si linkage), or a linkage
blockmer with respect to the linkage block AsTsCsGs (all Rp at linkage phosphorus and all s
linkage) or TslCslGsl (all Sp at linkage phosphorus and all si linkage).
[00114] Altmer: the term "altmer," as used herein, refers to an oligonucleotide strand
whose pattern of structural features characterizing each individual nucleotide unit is
characterized in that no two consecutive nucleotide units of the oligonucleotide strand share a
particular structural feature at the internucleotidic phosphorus linkage. In some embodiments, an
altmer is designed such that it comprises a repeating pattern. In some embodiments, an altmer is
designed such that it does not comprise a repeating pattern.
[00115] In some embodiments, an altmer is a "stereoaltmer," e.g., no two consecutive
nucleotide units have the same stereochemistry at the linkage phosphorus. For instance, (Rp, Sp,
Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-
GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.
[00116] In some embodiments, an altmer is a "P-modification altmer" e.g., no two
consecutive nucleotide units have the same modification at the linkage phosphorus. For instance,
All-(Sp)-CAslGsT, in which each linkage phosphorus has a different P-modification than the
others.
[00117] In some embodiments, an altmer is a "linkage altmer," e.g., no two consecutive
nucleotide units have identical stereochemistry or identical modifications at the linkage
phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,
Rp)-GsCs 1CsTs 1CsAs1GsTs 1CsTs 1GsCs1TsTs2CsGs3CsAs4CsC.
[00118] Unimer: the term "unimer," as used herein, refers to an oligonucleotide strand
whose pattern of structural features characterizing each individual nucleotide unit is such that all
nucleotide units within the strand share at least one common structural feature at the
mtemucleotidic phosphorus linkage. By common structural feature is meant common
stereochemistry at the linkage phosphorus or a common modification at the linkage phosphorus.
[00119] In some embodiments, a unimer is a "stereounimer," e.g., all nucleotide units
have the same stereochemistry at the linkage phosphorus. For instance, All-(Sp)-CsAslGsT, in
which all the linkages have Sp phosphorus.
[00120] In some embodiments, a unimer is a "P-modification unimer", e.g., all nucleotide
units have the same modification at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp,
Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-
GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, in which all the mtemucleotidic linkages
are phosphorothioate diester.
[00121] In some embodiments, a unimer is a "linkage unimer," e.g., all nucleotide units
have the same stereochemistry and the same modifications at the linkage phosphorus. For
instance, All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, in which all the
mtemucleotidic linkages are phosphorothioate diester having Sp linkage phosphorus.
[00122] Gapmer. as used herein, the term "gapmer" refers to an oligonucleotide strand
characterized in that at least one mtemucleotidic phosphorus linkage of the oligonucleotide
strand is a phosphate diester linkage, for example such as those found in naturally occurring
DNA or RNA. In some embodiments, more than one mtemucleotidic phosphorus linkage of the
oligonucleotide strand is a phosphate diester linkage such as those found in naturally occurring
DNA or RNA. For instance, All-(Sp)-CAslGsT, in which the mtemucleotidic linkage between
C and A is a phosphate diester linkage.
[00123] Skipmer. as used herein, the term "skipmer" refers to a type of gapmer in which
every other mtemucleotidic phosphorus linkage of the oligonucleotide strand is a phosphate
diester linkage, for example such as those found in naturally occurring DNA or RNA, and every
other mtemucleotidic phosphorus linkage of the oligonucleotide strand is a modified
mtemucleotidic linkage. For instance, All-(Sp)-AsTCslGAs2TCs3G.
[00124] For purposes of this invention, the chemical elements are identified in accordance
with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th
Ed., 1986-87, inside cover.
[00125] The methods and structures described herein relating to compounds and
compositions of the invention also apply to the pharmaceutically acceptable acid or base addition
salts and all stereoisomeric forms of these compounds and compositions.
Brief Description of the Drawing
[00126] Figure 1. Reverse phase HPLCs after incubation with rat liver homogenate.
Total amounts of oligonucleotides remaining when incubated with rat whole liver homogenate at
37°C at different days were measured. The in-vitro metabolic stability of ONT-154 was found to
be similar to ONT-87 which has 2'-MOE wings while both have much better stability than 2'-
MOE gapmer which is stereorandom (ONT-41, Mipomersen). The amount of full length
oligomer remaining was measured by reverse phase HPLC where peak area of the peak of
interest was normalized with internal standard.
[00127] Figure 2. Degradation of various chirally pure analogues of Mipomersen (ONT-
41) in rat whole liver homogenate. Total amounts of oligonucleotide remaining when incubated
with rat whole liver homogenate at 37°C at different days were measured. The in-vitro metabolic
stability of chirally pure diastereomers of human ApoB sequence ONT-41 (Mipomersen) was
found to increase with increased S internucleotidic linkages. The amount of full length
oligomer remaining was measured by reverse phase HPLC where peak area of the peak of
interest was normalized with internal standard.
[00128] Figure 3. Degradation of various chirally pure analogues of mouse ApoB
sequence (ISIS 147764, ONT-83) in rat whole liver homogenate. Total amounts of
oligonucleotide remaining when incubated with rat whole liver homogenate at 37°C at different
days were measured. The in-vitro metabolic stability of chirally pure diastereomers of murine
ApoB sequence (ONT-83, 2'-MOE gapmer, stereorandom phosphorothioate) was found to
increase with increased S internucleotidic linkages. The amount of full length oligomer
remaining was measured by reverse phase HPLC where peak area of the peak of interest was
normalized with internal standard.
[00129] Figure 4. Degradation of Mipomersen analogue ONT-75 in rat whole liver
homogenate over a period of 24hrs. This figure illustrates stability of ONT-75 in rate whole
liver homogenate.
[00130] Figure 5. Degradation of Mipomersen analogue ONT-81 in rat whole liver
homogenate over a period of 24hrs. This figure illustrates stability of ONT-81 in rate whole
liver homogenate.
[00131] Figure 6. Durations of knockdown for ONT-87, ONT-88, and ONT-89.
Stereoisomers can exhibit substantially different durations of knockdown. ONT-87 results in
substantially more durable suppression than other stereoisomers. Increased duration of action of
ONT-87 in multiple in vivo studies were observed. ONT-88 showed similar efficacy and
recovery profile as ONT-41 (Mipomersen) in certain in-vivo studies. Hu ApoB transgenic mice,
n=4, were dosed with 10 mpk IP bolus, 2X/week for three weeks. The mice were randomized to
study groups, and dosed intraperitoneally (IP) at 10 mg/kg on Days 1, 4, 8, 11, 15, 18, and 22,
based on individual mouse body weight measured prior to dosing on each dosing day. Blood
was collected on days 0, 17, 24, 31, 38, 45 and 52 by submandibular (cheek) bleed and at
sacrifice on Day 52 by cardiac puncture and then processed to serum. ApoB was measured by
ELISA. Highlighted: 72% vs. 35% knock-down maintained at 3 weeks postdose.
[00132] Figure 7. HPLC profiles exhibiting the difference in metabolic stability
determined in Human Serum for siRNA duplexes having several R , S or stereorandom
phosphorothioate linkages.
[00133] Figure 8. Effect of stereochemistry on RNase H activity. Oligonucleotides were
hybridized with RNA and then incubated with RNase H at 37°C in the presence of IX RNase H
buffer. From top to bottom at 120 min: ONT-89, ONT-77, ONT-81, ONT-80, ONT-75, ONT-
41, ONT-88, ONT-154, ONT-87, with ONT-77/154 very close to each other.
[00134] Figure 9. Analysis of human RNase HI cleavage of a 20-mer RNA when
hybridized with different preparations of stereoisomers of phosphorothioate oligonucleotides
targeting the same region of human ApoB mRNA. Specific sites of cleavage are strongly
influenced by the distinct stereochemistries. Arrows represent position of cleavage (cleavage
sites). Products were analyzed by UPLC/MS. The length of the arrow signifies the amount of
products present in the reaction mixture which was determined from the ratio of UV peak area to
theoretical extinction coefficient of that fragment (the larger the arrow, the more the detected
cleavage products). (A): Legend for cleavage maps. (B) and (C): cleavage maps of
oligonucleotides.
[00135] Figure 10. Cleavage maps of different oligonucleotide compositions ((A)-(C)).
These three sequences target different regions in FOXOl mRNA. Each sequence was studied
with five different chemistries. Cleavage maps are derived from reaction mixtures obtained after
30 minutes of incubation of respective duplexes with R ase H1C in the presence of 1XPBS
buffer at 37°C. Arrows indicate sites of cleavage (- ) indicates that both fragments, 5'-
phosphate specie as well as 5'-OH 3'-OH specie were identified in reaction mixtures. ( )
indicates that only 5'-phosphate specie was detected and ( ) indicates that 5'-OH 3'-OH
component was detected in mass spectrometry analysis. The length of the arrow signifies the
amount of products present in the reaction mixture which was determined from the ratio of UV
peak area to theoretical extinction coefficient of that fragment (the larger the arrow, the more the
detectable cleavage products). Only in the cases where 5'-OH 3'-OH was not detected in the
reaction mixture, 5'-phosphate specie peak was used for quantification. Cleavage rates were
determined by measuring amount of full length R A remaining in the reaction mixtures by
reverse phase HPLC. Reactions were quenched at fixed time points by 30mM Na2EDTA.
[00136] Figure 11. Cleavage maps of oligonucleotide compositions having different
common base sequences and lengths ((A)-(B)). The maps show a comparison of stereorandom
DNA compositions (top panel) with three distinct and stereochemically pure oligonucleotide
compositions. Data compare results of chirally controlled oligonucleotide compositions with
two stereorandom phosphorothioate oligonucleotide compositions (ONT-366 and ONT-367)
targeting different regions in FOXOl mRNA. Each panel shows a comparison of setreorandom
DNA (top panel) with three distinct and stereochemically pure oligonucleotide preparaitons.
Cleavage maps were derived from reaction mixtures obtained after 30 minutes of incubation of
respective duplexes with RNase H1C in the presence of 1XPBS buffer at 37°C. Arrows indicate
sites of cleavage (-p) indicates that both fragments, 5'-phosphate specie as well as 5'-OH 3'-OH
specie were identified in reaction mixtures. ( ) indicates that only 5'-phosphate specie was
detected and ( ) indicates that 5'-OH 3'-OH component was detected in mass spectrometry
analysis. The length of the arrow signifies the amount of metabolite present in the reaction
mixture which was determined from the ratio of UV peak area to theoretical extinction
coefficient of that fragment (the larger the arrow, the more the detectable cleavage products).
Only in the cases where 5'-OH 3'-OH was not detected in the reaction mixture, 5'-phosphate
specie peak was used for quantification.
[00137] Figure 12. Effect of stereochemistry on RNase H activity. In two independent
experiments, antisense oligonucleotides targeting an identical region of FOXOl mRNA were
hybridized with RNA and then incubated with RNase H at 37°C in the presence of IX RNase H
buffer. Disappearance of full length RNA was measured from its peak area at 254nm using RPHPLC.
(A): from top to bottom at 60 min: ONT-355, ONT-316, ONT-367, ONT-392, ONT-393
and ONT-394 (ONT-393 and ONT-394 about the same at 60 min; ONT-393 had higher %RNA
substrate remaining at 5 min). (B): from top to bottom at 60 min: ONT-315, ONT-354, ONT-
366, ONT-391, ONT-389 and ONT-390. Cleavage rates were determined by measuring amount
of full length RNA remaining in the reaction mixtures by reverse phase HPLC. Reactions were
quenched at fixed time points by 30mM Na2EDTA.
[00138] Figure 13. Turnover of antisense oligonucleotides. The duplexes were made
with each DNA strand concentration equal to 6 mM and RNA being 100 mM. These duplexes
were incubated with 0.02 mM RNase H enzyme and disappearance of full length RNA was
measured from its peak area at 254 nm using RP-HPLC. Cleavage rates were determined by
measuring amount of full length RNA remaining in the reaction mixtures by reverse phase
HPLC. Reactions were quenched at fixed time points by 30 mM Na2EDTA. From top to bottom
at 40 min: ONT-316, ONT-367 and ONT-392.
[00139] Figure 14. Cleavage map comparing a stereorandom phosphorothioate
oligonucleotide with six distinct and stereochemically pure oligonucleotide preparations
targeting the same FOXOl mRNA region.
[00140] Figure 15. Effect of stereochemistry on RNase H activity. Antisense
oligonucleotides were hybridized with RNA and then incubated with RNase H at 37°C in the
presence of IX RNase H buffer. Dependence of stereochemistry upon RNase H activity was
observed. Also evident in comparing ONT-367 (stereorandom DNA) and ONT-316 (5-10-5 2'-
MOE Gapmer) is the strong dependence of compositional chemistry upon RNase H activity.
From top to bottom at 40 min: ONT-316, ONT-421, ONT-367, ONT-392, ONT-394, ONT-415,
and ONT-422 (ONT-394/4 15/422 have similar levels at 40 min; at 5 min, ONT-422 > ONT-394
> ONT-415 in % RNA remaining in DNA/RNA duplex).
[00141] Figure 16. Effect of stereochemistry on RNase H activity. Antisense
oligonucleotides targeting an identical region of FOXOl mRNA were hybridized with RNA and
then incubated with RNase H at 37°C in the presence of IX RNase H buffer. Dependence of
stereochemistry upon RNase H activity was observed. Form top to bottom at 40 min: ONT-396,
ONT-409, ONT-414, ONT-408 (ONT-396/409/414/408 have similar levels at 40 min), ONT-
404, ONT-410, ONT-402 (ONT-404/4 10/408 have similar levels at 40 min), ONT-403, ONT-
407, ONT-405, ONT-401, ONT-406 and ONT-400 (ONT-40 1/405/406/400 have similar levels
at 40 min).
[00142] Figure 17. Effect of stereochemistry on RNase H activity. Antisense
oligonucleotides targeting an identical region of FOXOl mRNA were hybridized with RNA and
then incubated with RNase H at 37°C in the presence of IX RNase H buffer. Dependence of
stereochemistry upon RNase H activity was observed. ONT-406 was observed to elicit cleavage
of duplexed RNA at a rate in slight excess of that of the phosphodiester oligonucleotide ONT-
415. From top to bottom at 40 min: ONT-396, ONT-421, ONT-392, ONT-394, ONT-415 ONT-
406, and ONT-422 (ONT-394/4 15/406 have similar levels at 40 min; at 5 min, ONT-394 >
ONT-415 > ONT-406 in % RNA remaining in DNA/RNA duplex).
[00143] Figure 18. Exemplary UV chromatograms of RNA cleavage products obtained
when RNA (ONT-388) was duplexed with stereorandom DNA, ONT-367 (top) and stereopure
DNA with repeat triplet motif-3'-SSR-5', ONT-394 (bottom). ) . 2.35min: 7mer; 3.16min: 8mer
and p-6mer; 4.48min: P-7mer; 5.83min: P-8mer; 6.88min: 12mer; 9.32min: 13mer; 10.13min: Pl
lmer; l l.Omin: P-12mer and 14mer; 11.93min: P-13mer; 13.13min: P-14mer. ONT-394 (on the
bottom) peak assignment: 4.55min: p-7mer; 4.97min: lOmer; 9.53min: 13mer.
[00144] Figure 19. Electrospray Ionization Spectrum of RNA cleavage products. RNA
fragments obtained from the duplex ONT-387, RNA/ONT-354, (7-6-7, DNA-2'-OMe-DNA) on
the top and ONT-387, RNA/ONT-315, (5-10-5,2'-MOE Gapmer) at the bottom when these
duplexes were incubated with RNase H for 30min in the presence of IX RNse H buffer.
[00145] Figure 20. UV Chromatogram and TIC of ONT-406 and ONT-388 duplex after
30 minutes of incubation with RNase H.
[00146] Figure 21. An exemplary proposed cleavage. Provided chirally controlled
oligonucleotide compositions are capable of cleaving targets as depicted.
[00147] Figure 22. Exemplary allele specific cleavage targeting mutant Huntingtin
mRNA. (A) and (B): exemplary oligonucleotides. (C)-(E): cleavage maps. (F)-(H): RNA
cleavage. Stereorandom and chirally controlled oligonucleotide compositions were prepared to
target single nucleotide polymorphisms for allele selective suppression of mutant Huntingtin.
ONT-450 (stereorandom) targeting ONT-453 (muHTT) and ONT-454 (wtHTT) showed
marginal differentiation in RNA cleavage and their cleavage maps. Chirally controlled ONT-
451 with selective placement of 3'-SSR-5' motif in RNase H recognition site targeting ONT-453
(muHTT) and ONT-454 (wtHTT) showed large differentiation in RNA cleavage rate. From the
cleavage map, it is notable that 3'-SSR-5' motif is placed to direct the cleavage between
positions 8 and 9 which is after the mismatch if read from 5'-end of RNA. ONT-452 with
selective placement of 3'-SSR-5' motif in RNase H recognition site targeting ONT-453
(muHTT) and ONT-454 (wtHTT) showed moderate differentiation in RNA cleavage rate. 3'-
SSR-5' motif was placed to direct the cleavage at positions 7 and 8 which is before the mismatch
if read from 5'-end of RNA. Exemplary data illustrate significance of position in placement of
3'-SSR-5' motif to achieve enhanced discrimination for allele specific cleavage. All cleavage
maps are derived from the reaction mixtures obtained after 5 minutes of incubation of respective
duplexes with RNase H1C in the presence of 1XPBS buffer at 37°C. Arrows indicate sites of
cleavage (-p) indicates that both fragments, 5'-phosphate specie as well as 5'-OH 3'-OH specie
were identified in reaction mixtures. ( ) indicates that only 5'-phosphate specie was detected
and ( ) indicates that 5'-OH 3'-OH component was detected in mass spectrometry analysis. The
length of the arrow signifies the amount of metabolite present in the reaction mixture which was
determined from the ratio of UV peak area to theoretical extinction coefficient of that fragment.
Only in the cases where 5'-OH 3'-OH was not detected in the reaction mixture, 5'-phosphate
specie peak was used for quantification.
[00148] Figure 23. (A)-(C): exemplary allele specific cleavage targeting FOXOl mRNA.
[00149] Figure 24. In vitro dose response silencing of ApoB mRNA after treatment with
ApoB oligonucleotides. Stereochemically pure diasetereomers with and without 2'-MOE wings
show similar efficacy as ONT-41 (Mipomersen).
[00150] Figure 25. Comparison of RNase H cleavage maps (A) and RNA cleavage rates
(B) for stereorandom composition (ONT-367) and chirally controlled oligonucleotide
compositions (ONT-421, all S and ONT-455, all R ) and DNA (ONT-415). These sequences
target the same region in FOXOl mRNA. Cleavage maps were derived from the reaction
mixtures obtained after 5 minutes of incubation of respective duplexes with R ase H1C in the
presence of IXPBS buffer at 37°C. Arrows indicate sites of cleavage (- ) indicates that both
fragments, 5'-phosphate specie as well as 5'-OH 3'-OH specie were identified in reaction
mixtures. ( ) indicates that only 5'-phosphate specie was detected and ( ) indicates that 5'-OH
3'-OH component was detected in mass spectrometry analysis. The length of the arrow signifies
the amount of metabolite present in the reaction mixture which was determined from the ratio of
UV peak area to theoretical extinction coefficient of that fragment. Only in the cases where 5'-
OH 3'-OH was not detected in the reaction mixture, 5'-phosphate specie peak was used for
quantification. Cleavage rates were determined by measuring amount of full length RNA
remaining in the reaction mixtures by reverse phase HPLC. Reactions are quenched at fixed
time points by 30mM Na2EDTA.
[00151] Figure 26. Comparison of cleavage maps of sequences containing one Rp with
change of position starting from 3'-end of DNA. These sequences target the same region in
FOXOl mRNA. Cleavage maps are derived from the reaction mixtures obtained after 5 minutes
of incubation of respective duplexes with RNase H1C in the presence of IXPBS buffer at 37°C.
Arrows indicate sites of cleavage (- ) indicates that both fragments, 5'-phosphate specie as well
as 5'-OH 3'-OH specie were identified in reaction mixtures. ( ) indicates that only 5'-
phosphate specie was detected and ( ) indicates that 5'-OH 3'-OH component was detected in
mass spectrometry analysis. The length of the arrow signifies the amount of metabolite present
in the reaction mixture which was determined from the ratio of UV peak area to theoretical
extinction coefficient of that fragment. Only in the cases where 5'-OH 3'-OH was not detected
in the reaction mixture, 5'-phosphate specie peak was used for quantification.
[00152] Figure 27. (A) Comparison of RNase H cleavage rates for stereopure
oligonucleotides (ONT-406), (ONT-401), (ONT-404) and (ONT-408). All four sequences are
stereopure phosphorothioates with one Rp linkage. These sequences target the same region in
FOXOl mRNA. All duplexes were incubation with RNase H1C in the presence of IXPBS
buffer at 37°C. Reactions were quenched at fixed time points by 30mM Na2EDTA. Cleavage
rates were determined by measuring amount of full length RNA remaining in the reaction
mixtures by reverse phase HPLC. ONT-406 and ONT-401 were found to have superior cleavage
rates. (B) Correlation between %RNA cleaved in RNase H assay (10 mM oligonucleotide) and
%mR A knockdown in in vitro assay (20 nM oligonucleotide). All sequences target the same
region of mR A in the FOXOl target. The quantity of RNA remaining is determined by UV
peak area for RNA when normalized to DNA in the same reaction mixture. All of the above
maps are derived from the reaction mixture obtained after 5 minutes of incubation of respective
duplexes with RNase H1C in the presence of 1XPBS buffer at 37°C. All sequences from ONT-
396 to ONT-414 have one Rp phosphorothioate and they vary in the position of Rp. ONT-421
(All Sp) phosphorothioate was inactive in-vitro assay. It relates poor cleavage rate of RNA in
RNase H assay when ONT-421 is duplexed with complementary RNA.
[00153] Figure 28. Serum stability assay of single Rp walk PS DNA (ONT-396-ONT-
414), stereorandom PS DNA(ONT-367), all-Sp PS DNA (ONT-421) and all-Rp PS DNA (ONT-
455) in rat serum for 2 days. Note ONT-396 and ONT-455 decomposed at tested time point.
[00154] Figure 29. Exemplary oligonucleotides including hemimers. (A): cleavage maps.
(B): RNA cleavage assay. (C): FOXOl mRNA knockdown. In some embodiments, introduction
of 2'-modifications on 5'-end of the sequences increases stability for binding to target RNA
while maintaining RNase H activity. ONT-367 (stereorandom phosphorothioate DNA) and
ONT-440 (5-15, 2'-F-DNA) have similar cleavage maps and similar rate of RNA cleavage in
RNase H assay (10 mM oligonucleotide). In some embodiments, ONT-440 (5-1 1, 2'-F-DNA)
sequence can have better cell penetration properties. In some embodiments, asymmetric 2'-
modifications provide Tm advantage while maintaining RNase H activity. Introduction of RSS
motifs can further enhance RNase H efficiency in the hemimers. Cleavage maps are derived
from the reaction mixtures obtained after 5 minutes of incubation of respective duplexes with
RNase H1C in the presence of 1XPBS buffer at 37°C. Arrows indicate sites of cleavage (- )
indicates that both fragments, 5'-phosphate specie as well as 5'-OH 3'-OH specie were identified
in reaction mixtures. ( ) indicates that only 5'-phosphate specie was detected and ( ) indicates
that 5'-OH 3'-OH component was detected in mass spectrometry analysis. The length of the
arrow signifies the amount of metabolite present in the reaction mixture which was determined
from the ratio of UV peak area to theoretical extinction coefficient of that fragment. Only in the
cases where 5'-OH 3'-OH was not detected in the reaction mixture, 5'-phosphate specie peak
was used for quantification.

Claims
1. A chirally controlled oligonucleotide composition comprising oligonucleotides defined
by having:
1) a common base sequence and length;
2) a common pattern of backbone linkages; and
3) a common pattern of backbone chiral centers,
which composition is a substantially pure preparation of a single oligonucleotide in that at least
about 10% of the oligonucleotides in the composition have the common base sequence and
length, the common pattern of backbone linkages, and the common pattern of backbone chiral
centers; wherein:
the common base sequence has at least 1 bases; or
the single oligonucleotide comprises 11 or more chiral, modified phosphate linkages.
2. The composition of claim 1, wherein each chiral, modified phosphate linkage
independent has the structure of formula I :
(I)
wherein:
P* is an asymmetric phosphorus atom and is either R or Sp;
W is O, S or Se;
each of X, Y and Z is independently -0-, -S-, -Ni-L-R 1)-, or L;
L is a covalent bond or an optionally substituted, linear or branched Ci-Cio alkylene, wherein
one or more methylene units of L are optionally and independently replaced by an optionally
substituted Ci-C alkylene, Ci-C alkenylene, CºC—, -C(R') 2- -Cy- -0-, -S-, -S-S-,
-N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(0)N(R')-, -N(R')C(0)N(R')-, -N(R')C(0)-, -
N(R')C(0)0- -OC(0)N(R>, -S(O)-, -S(0) 2- -S(0) 2N(R')-, -N(R')S(0) 2- -SC(O)-, -
C(0)S- -OC(O)-, or -C(0)0-;
R1 is halogen, R, or an optionally substituted C1-C50 aliphatic wherein one or more methylene
units are optionally and independently replaced by an optionally substituted Ci-C 6 alkylene,
Ci-C alkenylene, CºC—, -C(R') 2- -Cy- -0-, -S-, -S-S-, -N(R')-, -C(0)-, -C(S)-, -
C(NR')-, -C(0)N(R')-, -N(R')C(0)N(R')-, -N(R')C(0)-, -N(R')C(0)0-, -OC(0)N(R')-, -
S(0)-, -S(0) 2- , -S(0) 2N(R')-, -N(R')S(0) 2- -SC(O)-, -C(0)S-, -0C(0)-, or -C(0)0-;
each R' is independently -R, -C(0)R, -C0 2R, or -S0 2R, or:
two R' on the same nitrogen are taken together with their intervening atoms to form an
optionally substituted heterocyclic or heteroaryl ring, or
two R' on the same carbon are taken together with their intervening atoms to form an
optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene,
heteroarylene, or heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from Ci-C
aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and
each independently represents a connection to a nucleoside.
3. The composition of claim 2, wherein each chiral, modified phosphate linkage is a
phosphorothioate linkage.
4. The composition of claim 2, wherein X is -S- and -L-R 1 is not hydrogen.
5. The composition of any one of claims 1-4, wherein the common pattern of backbone
chiral centers comprises from 5' to 3' (Rp)n(Sp)m, wherein m is 2, 3, 4, 5, 6, 7 or 8 and n is 1, 2,
3, 4, 5, 6, 7 or 8.
6. The composition of any one of claims 1-4, wherein the common pattern of backbone
chiral centers comprises from 5' to 3' R (S ) , wherein m is 2, 3, 4, 5, 6, 7 or 8.
7. The composition of any one of claims 1-4, wherein the common pattern of backbone
chiral centers comprises from 5' to 3' i p( p)2.
8. The composition of any one of claims 1-4, wherein the pattern of backbone chiral centers
comprises from 5' to 3' (N p)t(R p)n(Sp)m, wherein each n and t is independently 1, 2, 3, 4, 5, 6, 7
or 8, m is 2, 3, 4, 5, 6, 7 or 8, and each Np is independent Rp or S .
9. The composition of claim 8, wherein n is 1.
10. The composition of claim 9, wherein t is 2, 3, 4, 5, 6, 7 or 8.
11. The composition of claim 10, wherein m is 2, 3, 4, 5, 6, 7 or 8.
12. The composition of claim 8, wherein at least one of t and m is greater than 5.
13. The composition of any one of claims 1-12, wherein oligonucleotides of the particular
oligonucleotide type are antisense oligonucleotide, antagomir, microRNA, pre-microRNs,
antimir, supermir, ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide,
triplex forming oligonucleotide, aptamer or adjuvant.
14. A method for controlled cleavage of a nucleic acid polymer, the method comprising steps
of:
contacting a nucleic acid polymer whose nucleotide sequence comprises a target
sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of
a particular oligonucleotide type characterized by:
1) a common base sequence and length, wherein the common base sequence is or
comprises a sequence that is complementary to a target sequence found in the nucleic
acid polymer;
2) a common pattern of backbone linkages; and
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the particular base sequence and length, for
oligonucleotides of the particular oligonucleotide type.
15. The method of claim 14, wherein the cleavage occurs with a cleavage pattern that differs
from a reference cleavage pattern observed when the nucleic acid polymer is contacted under
comparable conditions with a reference oligonucleotide composition.
16. A method for altering a cleavage pattern observed when a nucleic acid polymer whose
nucleotide sequence includes a target sequence is contacted with a reference oligonucleotide
composition that comprises oligonucleotides having a particular base sequence and length, which
particular base sequence is or comprises a sequence that is complementary to the target sequence,
the method comprising:
contacting the nucleic acid polymer with a chirally controlled oligonucleotide
composition of oligonucleotides having the particular base sequence and length, which
composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the particular base sequence and length, for
oligonucleotides of a single oligonucleotide type characterized by:
1) the particular base sequence and length;
2) a particular pattern of backbone linkages; and
3) a particular pattern of backbone chiral centers.
17. The method of claim 15 or 16, wherein the reference oligonucleotide composition is a
substantially racemic preparation of oligonucleotides that share the common sequence and length.
18. The method of any one of claims 15-17, wherein the cleavage pattern provided by the
chirally controlled oligonucleotide composition differs from a reference cleavage pattern in that
it has fewer cleavage sites within the target sequence found in the nucleic acid polymer than the
reference cleavage pattern.
19. The method of claim 18, wherein the cleavage pattern provided by the chirally controlled
oligonucleotide composition has a single cleavage site within the target sequence found in the
nucleic acid polymer.
20. The method of any one of claims 14-19, wherein the cleavage pattern provided by the
chirally controlled oligonucleotide composition differs from a reference cleavage pattern in that
it increases cleavage percentage at a cleavage site.
2 1. The method of any one of claims 14-20, wherein the chirally controlled oligonucleotide
composition provides a higher cleavage rate of the target nucleic acid polymer than a reference
oligonucleotide composition.
22. The method of any one of claims 14-21, wherein the chirally controlled oligonucleotide
composition provides a lower level of remaining un-cleaved target nucleic acid polymer than a
reference oligonucleotide composition.
23. A method for allele-specific suppression of a transcript from a target nucleic acid
sequence for which a plurality of alleles exist within a population, each of which contains a
specific nucleotide characteristic sequence element that defines the allele relative to other alleles
of the same target nucleic acid sequence, the method comprising steps of:
contacting a sample comprising transcripts of the target nucleic acid sequence with a
chirally controlled oligonucleotide composition comprising oligonucleotides of a particular
oligonucleotide type characterized by:
1) a common base sequence and length;
2) a common pattern of backbone linkages;
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence and length, for oligonucleotides
of the particular oligonucleotide type;
wherein the common base sequence for the oligonucleotides of the particular oligonucleotide
type is or comprises a sequence that is complementary to the characteristic sequence element that
defines a particular allele, the composition being characterized in that, when it is contacted with
a system comprising transcripts of both the target allele and another allele of the same nucleic
acid sequence, transcripts of the particular allele are suppressed at a greater level than a level of
suppression observed for another allele of the same nucleic acid sequence.
24. A method for allele-specific suppression of a transcript from a target gene for which a
plurality of alleles exist within a population, each of which contains a specific nucleotide
characteristic sequence element that defines the allele relative to other alleles of the same target
gene, the method comprising steps of:
contacting a sample comprising transcripts of the target gene with a chirally controlled
oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type
characterized by:
1) a common base sequence and length;
2) a common pattern of backbone linkages;
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence and length, for oligonucleotides
of the particular oligonucleotide type;
wherein the common base sequence for the oligonucleotides of the particular oligonucleotide
type is or comprises a sequence that is complementary to the characteristic sequence element that
defines a particular allele, the composition being characterized in that, when it is contacted with
a system comprising transcripts of both the target allele and another allele of the same gene,
transcripts of the particular allele are suppressed at a level at least 2 fold greater than a level of
suppression observed for another allele of the same gene.
25. The method of claim 23 or 24, the contacting being performed under conditions
determined to permit the composition to suppress transcripts of the particular allele.
26. A method for allele-specific suppression of a transcript from a target gene for which a
plurality of alleles exist within a population, each of which contains a specific nucleotide
characteristic sequence element that defines the allele relative to other alleles of the same target
gene, the method comprising steps of:
contacting a sample comprising transcripts of the target gene with a chirally controlled
oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type
characterized by:
1) a common base sequence and length;
2) a common pattern of backbone linkages;
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence and length, for oligonucleotides
of the particular oligonucleotide type;
wherein the common base sequence for the oligonucleotides of the particular oligonucleotide
type is or comprises a sequence that is complementary to the characteristic sequence element that
defines a particular allele, the composition being characterized in that, when it is contacted with
a system expressing transcripts of both the target allele and another allele of the same gene,
transcripts of the particular allele are suppressed at a level at least 2 fold greater than a level of
suppression observed for another allele of the same gene.
27. A method for allele-specific suppression of a transcript from a target nucleic acid
sequence for which a plurality of alleles exist within a population, each of which contains a
specific nucleotide characteristic sequence element that defines the allele relative to other alleles
of the same target nucleic acid sequence, the method comprising steps of:
contacting a sample comprising transcripts of the target nucleic acid sequence with a
chirally controlled oligonucleotide composition comprising oligonucleotides of a particular
oligonucleotide type characterized by:
1) a common base sequence and length;
2) a common pattern of backbone linkages;
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence and length, for oligonucleotides
of the particular oligonucleotide type;
wherein the common base sequence for the oligonucleotides of the particular oligonucleotide
type is or comprises a sequence that is complementary to the characteristic sequence element that
defines a particular allele, the composition being characterized in that, when it is contacted with
a system comprising transcripts of the same target nucleic acid sequence, it shows suppression of
transcripts of the particular allele at a level that is:
a) greater than when the composition is absent;
b) greater than a level of suppression observed for another allele of the same nucleic acid
sequence; or
c) both greater than when the composition is absent, and greater than a level of
suppression observed for another allele of the same nucleic acid sequence.
28. A method for allele-specific suppression of a transcript from a target gene for which a
plurality of alleles exist within a population, each of which contains a specific nucleotide
characteristic sequence element that defines the allele relative to other alleles of the same target
gene, the method comprising steps of:
contacting a sample comprising transcripts of the target gene with a chirally controlled
oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type
characterized by:
1) a common base sequence and length;
2) a common pattern of backbone linkages;
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence and length, for oligonucleotides
of the particular oligonucleotide type;
wherein the common base sequence for the oligonucleotides of the particular oligonucleotide
type is or comprises a sequence that is complementary to the characteristic sequence element that
defines a particular allele, the composition being characterized in that, when it is contacted with
a system expressing transcripts of the target gene, it shows suppression of expression of
transcripts of the particular allele at a level that is:
a) at least 2 fold in that transcripts from the particular allele are detected in amounts that
are 2 fold lower when the composition is present relative to when it is absent;
b) at least 2 fold greater than a level of suppression observed for another allele of the
same gene; or
c) both at least 2 fold in that transcripts from the particular allele are detected in amounts
that are 2 fold lower when the composition is present relative to when it is absent, and at least 2
fold greater than a level of suppression observed for another allele of the same gene.
29. A method for allele-specific suppression of a transcript from a target gene for which a
plurality of alleles exist within a population, each of which contains a specific nucleotide
characteristic sequence element that defines the allele relative to other alleles of the same target
gene, the method comprising steps of:
contacting a sample comprising transcripts of the target gene with a chirally controlled
oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type
characterized by:
1) a common base sequence and length;
2) a common pattern of backbone linkages;
3) a common pattern of backbone chiral centers;
which composition is chirally controlled in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence and length, for oligonucleotides
of the particular oligonucleotide type;
wherein the common base sequence for the oligonucleotides of the particular oligonucleotide
type is or comprises a sequence that is complementary to the characteristic sequence element that
defines a particular allele, the composition being characterized in that, when it is contacted with
a system expressing transcripts of the target gene, it shows suppression of expression of
transcripts of the particular allele at a level that is:
a) at least 2 fold in that transcripts from the particular allele are detected in amounts that
are 2 fold lower when the composition is present relative to when it is absent;
b) at least 2 fold greater than a level of suppression observed for another allele of the
same gene; or
c) both at least 2 fold in that transcripts from the particular allele are detected in amounts
that are 2 fold lower when the composition is present relative to when it is absent, and at least 2
fold greater than a level of suppression observed for another allele of the same gene.
30. The method of any one of claims 23-29, wherein the specific nucleotide characteristic
sequence element is present within an intron of the target nucleic acid sequence or gene.
31. The method of any one of claims 23-29, wherein the specific nucleotide characteristic
sequence element is present within an exon of the target nucleic acid sequence or gene.
32. The method of any one of claims 23-29, wherein the specific nucleotide characteristic
sequence element spans an exon and an intron of the target nucleic acid sequence or gene.
33. The method of any one of claims 23-29, wherein the specific nucleotide characteristic
sequence element comprises a mutation.
34. The method of any one of claims 23-29, wherein the specific nucleotide characteristic
sequence element comprises a SNP.
35. The method of any one of claims 14-34, wherein the chirally controlled oligonucleotide
composition is a composition of claim 8.