U.S. patent application number 17/032657 was filed with the patent office on 2021-10-07 for modified nucleosides, nucleotides, and nucleic acids, and uses thereof.
The applicant listed for this patent is ModernaTX, Inc.. Invention is credited to Antonin DE FOUGEROLLES, Atanu ROY.
Application Number | 20210308283 17/032657 |
Document ID | / |
Family ID | 1000005625436 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210308283 |
Kind Code |
A1 |
DE FOUGEROLLES; Antonin ; et
al. |
October 7, 2021 |
MODIFIED NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS, AND USES
THEREOF
Abstract
The present disclosure provides modified nucleosides,
nucleotides, and nucleic acids, and methods of using them.
Inventors: |
DE FOUGEROLLES; Antonin;
(Waterloo, BE) ; ROY; Atanu; (Stoneham,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
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|
Family ID: |
1000005625436 |
Appl. No.: |
17/032657 |
Filed: |
September 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14987231 |
Jan 4, 2016 |
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17032657 |
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13743518 |
Jan 17, 2013 |
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14987231 |
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13644072 |
Oct 3, 2012 |
9428535 |
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13743518 |
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61542533 |
Oct 3, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/67 20130101;
C07H 21/02 20130101; C12N 15/11 20130101; C07K 14/535 20130101;
A61K 38/193 20130101; A61K 48/0066 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/67 20060101 C12N015/67; C12N 15/11 20060101
C12N015/11; C07H 21/02 20060101 C07H021/02; A61K 38/19 20060101
A61K038/19; C07K 14/535 20060101 C07K014/535 |
Claims
1. An isolated polynucleotide encoding a polypeptide of interest,
said isolated polynucleotide comprising: (a) a sequence of n number
of linked nucleosides or nucleotides comprising at least one
modified nucleoside or nucleotide as compared to the chemical
structure of an A, G, U or C nucleoside or nucleotide, (b) a 5' UTR
comprising at least one Kozak sequence, (c) a 3' UTR, and (d) at
least one 5' cap structure.
2. The isolated polynucleotide of claim 1, further comprising a
poly-A tail.
3. The isolated polynucleotide of claim 2 which is purified.
4. The isolated polynucleotide of claim 3, wherein the at least one
5' cap structure is selected from the group consisting of Cap0,
Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, and 2-azido-guanosine.
5. The isolated polynucleotide of claim 4, wherein the modification
of the nucleoside is located in the nucleoside base and/or sugar
portion of the nucleoside.
6. The isolated polynucleotide of claim 5, wherein the modification
is located in the nucleotide base and the nucleoside base has the
formula: ##STR00241## wherein: denotes a single or double bond; X
is O or S; U and W are each independently C or N; V is O, S, C or
N; wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, 01_6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.c,
--C(O)OR.sup.c, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c; and wherein
when V is O, S, or N then R.sup.1 is absent; R.sup.2 is H,
--OR.sup.c, --SR.sup.c, --NR.sup.aR.sup.b, or halo; or when V is C
then R.sup.1 and R.sup.2 together with the carbon atoms to which
they are attached can form a 5- or 6-membered ring optionally
substituted with 1-4 substituents selected from halo, --OH, --SH,
--NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, C.sub.2-20
alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20 thioalkyl; R.sup.3 is H
or C.sub.1-20 alkyl; R.sup.4 is H or C.sub.1-20 alkyl; wherein when
denotes a double bond then R.sup.4 is absent, or N--R.sup.4, taken
together, forms a positively charged N substituted with C.sub.1-20
alkyl; R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
7. The isolated polynucleotide of claim 6, wherein the modification
is located in the nucleoside base and the nucleoside base has the
formula: ##STR00242## wherein: R.sup.3 is C.sub.1-20 alkyl.
8. The isolated polynucleotide of claim 7, wherein R.sup.3 is
C.sub.1-4 alkyl.
9. The isolated polynucleotide of claim 7, wherein R.sup.3 is
CH.sub.3.
10. The isolated polynucleotide of claim 1, wherein the modified
nucleoside is not pseudouridine (.psi.) or 5-methyl-cytidine
(m.sup.5C).
11. A pharmaceutical composition comprising the isolated
polynucleotide of claim 6 and a pharmaceutically acceptable
excipient.
12. The pharmaceutical composition of claim 11, wherein the
excipient is selected from a solvent, aqueous solvent, non-aqueous
solvent, dispersion media, diluent, dispersion, suspension aid,
surface active agent, isotonic agent, thickening or emulsifying
agent, preservative, lipid, lipidoids liposome, lipid nanoparticle,
core-shell nanoparticles, polymer, lipoplexe peptide, protein,
cell, hyaluronidase, and mixtures thereof.
13. A method of increasing the level of a polypeptide of interest
in a mammalian subject comprising administering to said subject the
isolated polynucleotide of claim 6.
14. The method of claim 13, wherein the polynucleotide is
formulated.
15. The method of claim 13, wherein isolated polynucleotide has a
Protein:Cytokine Ratio of greater than 100 for either TNF-alpha or
IFN-alpha.
16. The method of claim 13, wherein the isolated polynucleotide is
administered at a total daily dose of between 1 ug and 150 ug.
17. The method of claim 16, wherein administration is by
injection.
18. The method of claim 16, wherein administration is intradermal
or subcutaneous or intramuscular.
19. The method of claim 13, wherein levels of the polypeptide of
interest in the serum of the mammal are at least 50 pg/mL at least
two hours after administration.
20. The method of claim 19, wherein the levels of the polypeptide
of interest in the serum of the mammal remain above 50 pg/mL for at
least 72 hours after administration.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/644,072, filed Oct. 3, 2012, entitled
Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses
Thereof which claims priority to U.S. Provisional Patent
Application No. 61/542,533, filed Oct. 3, 2011, entitled Modified
Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof, the
contents of each are herein incorporated by reference in their
entirety.
REFERENCE TO THE SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing file, entitled
M009SQLST.txt, was created on Jan. 17, 2013 and is 9,970 bytes in
size. The information in electronic format of the Sequence Listing
is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present disclosure provides compositions and methods
using modified nucleic acids to modulate cellular function. The
modified nucleic acids of the invention may encode peptides,
polypeptides or multiple proteins. The encoded molecules may be
used as therapeutics and/or diagnostics.
BACKGROUND OF THE INVENTION
[0004] Naturally occurring RNAs are synthesized from four basic
ribonucleotides: ATP, CTP, UTP and GTP, but may contain
post-transcriptionally modified nucleotides. Further, approximately
one hundred different nucleoside modifications have been identified
in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA
Modification Database: 1999 update. Nucl Acids Res 27: 196-197).
The role of nucleoside modifications on the immune-stimulatory
potential and on the translation efficiency of RNA, however, is
unclear.
[0005] There are multiple problems with prior methodologies of
effecting protein expression. For example, heterologous DNA
introduced into a cell can be inherited by daughter cells (whether
or not the heterologous DNA has integrated into the chromosome) or
by offspring. Introduced DNA can integrate into host cell genomic
DNA at some frequency, resulting in alterations and/or damage to
the host cell genomic DNA. In addition, multiple steps must occur
before a protein is made. Once inside the cell, DNA must be
transported into the nucleus where it is transcribed into RNA. The
RNA transcribed from DNA must then enter the cytoplasm where it is
translated into protein. This need for multiple processing steps
creates lag times before the generation of a protein of interest.
Further, it is difficult to obtain DNA expression in cells;
frequently DNA enters cells but is not expressed or not expressed
at reasonable rates or concentrations. This can be a particular
problem when DNA is introduced into cells such as primary cells or
modified cell lines.
[0006] There is a need in the art for biological modalities to
address the modulation of intracellular translation of nucleic
acids.
SUMMARY OF THE INVENTION
[0007] The present disclosure provides, inter alia, modified
nucleosides, modified nucleotides, and modified nucleic acids which
can exhibit a reduced innate immune response when introduced into a
population of cells, both in vivo and ex vivo.
[0008] The present invention provides polynucleotides which may be
isolated or purified. These polynucleotides may encode one or more
polypeptides of interest and comprise a sequence of n number of
linked nucleosides or nucleotides comprising at least one modified
nucleoside or nucleotide as compared to the chemical structure of
an A, G, U or C nucleoside or nucleotide. The polynucleotides may
also contain a 5' UTR comprising at least one Kozak sequence, a 3'
UTR, and at least one 5' cap structure. The isolated
polynucleotides may further contain a poly-A tail and may be
purified.
[0009] The isolated polynucleotides of the invention also comprise
at least one 5' cap structure selected from the group consisting of
Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, and 2-azido-guanosine.
[0010] Modifications of the polynucleotides of the invention may be
on the nucleoside base and/or sugar portion of the nucleosides
which comprise the polynucleotide.
[0011] In some embodiments, the modification is on the nucleobase
and is selected from the group consisting of pseudouridine or
N1-methylpseudouridine.
[0012] In some embodiments, the modified nucleoside is not
pseudouridine (.psi.) or 5-methyl-cytidine (m5C).
[0013] In some embodiments, multiple modifications are included in
the modified nucleic acid or in one or more individual nucleoside
or nucleotide. For example, modifications to a nucleoside may
include one or more modifications to the nucleobase and the
sugar.
[0014] In some embodiments are provided novel building blocks,
e.g., nucleosides and nucleotides for the preparation of modified
polynucleotides and their method of synthesis and manufacture.
[0015] The present invention also provides for pharmaceutical
compositions comprising the modified polynucleotides described
herein. These may also further include one or more pharmaceutically
acceptable excipients selected from a solvent, aqueous solvent,
non-aqueous solvent, dispersion media, diluent, dispersion,
suspension aid, surface active agent, isotonic agent, thickening or
emulsifying agent, preservative, lipid, lipidoids liposome, lipid
nanoparticle, core-shell nanoparticles, polymer, lipoplexe peptide,
protein, cell, hyaluronidase, and mixtures thereof.
[0016] Methods of using the polynucleotides and modified nucleic
acids of the invention are also provided. In this instance, the
polynucleotides may be formulated by any means known in the art or
administered via any of several routes including injection by
intradermal, subcutaneous or intramuscular means.
[0017] Administration of the modified nucleic acids of the
invention may be via two or more equal or unequal split doses. In
some embodiments, the level of the polypeptide produced by the
subject by administering split doses of the polynucleotide is
greater than the levels produced by administering the same total
daily dose of polynucleotide as a single administration.
[0018] Detection of the modified nucleic acids or the encoded
polypeptides may be performed in the bodily fluid of the subject or
patient where the bodily fluid is selected from the group
consisting of peripheral blood, serum, plasma, ascites, urine,
cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial
fluid, aqueous humor, amniotic fluid, cerumen, breast milk,
bronchioalveolar lavage fluid, semen, prostatic fluid, cowper's
fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears,
cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph,
chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit,
vaginal secretions, mucosal secretion, stool water, pancreatic
juice, lavage fluids from sinus cavities, bronchopulmonary
aspirates, blastocyl cavity fluid, and umbilical cord blood.
[0019] In some embodiments, administration is according to a dosing
regimen which occurs over the course of hours, days, weeks, months,
or years and may be achieved by using one or more devices selected
from multi-needle injection systems, catheter or lumen systems, and
ultrasound, electrical or radiation based systems.
[0020] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
disclosure; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0021] Other features and advantages of the present disclosure will
be apparent from the following detailed description and figures,
and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The foregoing and other objects, features and advantages
will be apparent from the following description of particular
embodiments of the invention, as illustrated in the accompanying
drawings in which like reference characters refer to the same parts
throughout the different views. The drawings are not necessarily to
scale, emphasis instead being placed upon illustrating the
principles of various embodiments of the invention.
[0023] FIG. 1 provides the spectrum and graphs of the analytical
results for N4-Me-CTP (NTP of compound 1). FIG. 1A provides the
nuclear magnetic resonance (NMR) spectrum in DMSO and
[0024] FIG. 1B provides the NMR spectrum in D.sub.2O, FIG. 1C
provides the mass spectrometry (MS) results, and FIG. 1D is the
high performance liquid chromatography (HPLC) results for
N4-methylcytidine (N4-Me-cytidine, compound 1).
[0025] FIG. 2 shows the HPLC results for N4-Me-CTP (NTP of compound
1).
[0026] FIG. 3 provides the analytical results for 2'-OMe-N,
N-di-Me-CTP (NTP of compound 2).
[0027] FIG. 3A provides the NMR spectrum. FIG. 3B provides the MS
results. FIG. 3C provides HPLC results for 2'-O-methyl-N.sup.4,
N.sup.4-dimethylcytidine (2'-OMe-N,N-di-Me-cytidine, compound
2).
[0028] FIG. 4 shows the HPLC results for 2'-OMe-N, N-di-Me-CTP (NTP
of compound 2).
[0029] FIG. 5 provides the HPLC results for
5-methoxycarbonylmethoxy-UTP (NTP of compound 3).
[0030] FIG. 6 provides the analytical results of 3-methyl
pseudouridine (compound 4). FIG. 6A provides the NMR spectrum of
3-methyl pseudouridine (compound 4) and FIG. 6B provides the HPLC
results for 3-methyl pseudouridine (compound 4).
[0031] FIG. 7 provides the analytical results of
5-TBDMS-OCH.sub.2-cytidine (compound 6). FIG. 7A provide the NMR
spectrum, FIG. 7B provides the MS results, and FIG. 7C provides the
HPLC results for 5-TBDMS-OCH.sub.2-cytidine (compound 6).
[0032] FIG. 8 provides the analytical results of 5-trifluoromethyl
uridine (compound 8). FIG. 8A provides the NMR spectrum, FIG. 8B
provides MS results, and FIG. 8C provides HPLC results for
5-trifluoromethyl uridine (compound 8).
[0033] FIG. 9 provides the NMR spectrum results for of
5-(methoxycarbonyl) methyl uridine (compound 9).
[0034] FIG. 10 provides a graph showing the variability of protein
(GCSF; line B) and cytokine (interferon-alpha (IFNa); line A and
tumor necrosis factor-alpha (TNFa); line C) expression as function
of percent modification.
DETAILED DESCRIPTION
[0035] The present disclosure provides, inter alia, modified
nucleosides, modified nucleotides, and modified nucleic acids that
exhibit improved therapeutic properties including, but not limited
to, a reduced innate immune response when introduced into a
population of cells.
[0036] As there remains a need in the art for therapeutic
modalities to address the myriad of barriers surrounding the
efficacious modulation of intracellular translation and processing
of nucleic acids encoding polypeptides or fragments thereof, the
inventors have shown that certain modified mRNA sequences have the
potential as therapeutics with benefits beyond just evading,
avoiding or diminishing the immune response.
[0037] The present invention addresses this need by providing
nucleic acid based compounds or polynucleotides which encode a
polypeptide of interest (e.g., modified mRNA) and which have
structural and/or chemical features that avoid one or more of the
problems in the art, for example, features which are useful for
optimizing nucleic acid-based therapeutics while retaining
structural and functional integrity, overcoming the threshold of
expression, improving expression rates, half life and/or protein
concentrations, optimizing protein localization, and avoiding
deleterious bio-responses such as the immune response and/or
degradation pathways.
[0038] Provided herein, in part, are polynucleotides encoding
polypeptides of interest which have been chemically modified to
improve one or more of the stability and/or clearance in tissues,
receptor uptake and/or kinetics, cellular access by the
compositions, engagement with translational machinery, mRNA
half-life, translation efficiency, immune evasion, protein
production capacity, secretion efficiency (when applicable),
accessibility to circulation, protein half-life and/or modulation
of a cell's status, function and/or activity.
[0039] The modified nucleosides, nucleotides and nucleic acids of
the invention, including the combination of modifications taught
herein have superior properties making them more suitable as
therapeutic modalities.
[0040] It has been determined that the "all or none" model in the
art is sorely insufficient to describe the biological phenomena
associated with the therapeutic utility of modified mRNA. The
present inventors have determined that to improve protein
production, one may consider the nature of the modification, or
combination of modifications, the percent modification and survey
more than one cytokine or metric to determine the efficacy and risk
profile of a particular modified mRNA.
[0041] In one aspect of the invention, methods of determining the
effectiveness of a modified mRNA as compared to unmodified involves
the measure and analysis of one or more cytokines whose expression
is triggered by the administration of the exogenous nucleic acid of
the invention. These values are compared to administration of an
unmodified nucleic acid or to a standard metric such as cytokine
response, PolyIC, R-848 or other standard known in the art.
[0042] One example of a standard metric developed herein is the
measure of the ratio of the level or amount of encoded polypeptide
(protein) produced in the cell, tissue or organism to the level or
amount of one or more (or a panel) of cytokines whose expression is
triggered in the cell, tissue or organism as a result of
administration or contact with the modified nucleic acid. Such
ratios are referred to herein as the Protein:Cytokine Ratio or "PC"
Ratio. The higher the PC ratio, the more efficacious the modified
nucleic acid (polynucleotide encoding the protein measured).
Preferred PC Ratios, by cytokine, of the present invention may be
greater than 1, greater than 10, greater than 100, greater than
1000, greater than 10,000 or more. Modified nucleic acids having
higher PC Ratios than a modified nucleic acid of a different or
unmodified construct are preferred.
[0043] The PC ratio may be further qualified by the percent
modification present in the polynucleotide. For example, normalized
to a 100% modified nucleic acid, the protein production as a
function of cytokine (or risk) or cytokine profile can be
determined.
[0044] In one embodiment, the present invention provides a method
for determining, across chemistries, cytokines or percent
modification, the relative efficacy of any particular modified
polynucleotide by comparing the PC Ratio of the modified nucleic
acid (polynucleotide).
[0045] In another embodiment, the chemically modified mRNA are
substantially non toxic and non mutagenic.
[0046] In one embodiment, the modified nucleosides, modified
nucleotides, and modified nucleic acids can be chemically modified
on the major groove face, thereby disrupting major groove binding
partner interactions, which may cause innate immune responses.
Further, these modified nucleosides, modified nucleotides, and
modified nucleic acids can be used to deliver a payload, e.g.,
detectable or therapeutic agent, to a biological target. For
example, the nucleic acids can be covalently linked to a payload,
e.g. a detectable or therapeutic agent, through a linker attached
to the nucleobase or the sugar moiety. The compositions and methods
described herein can be used, in vivo and in vitro, both
extracellarly or intracellularly, as well as in assays such as cell
free assays.
[0047] In some embodiments, the present disclosure provides
compounds comprising a nucleotide that disrupts binding of a major
groove interacting, e.g. binding, partner with a nucleic acid,
wherein the nucleotide has decreased binding affinity to major
groove interacting partners.
[0048] In another aspect, the present disclosure provides
nucleotides that contain chemical modifications, wherein the
nucleotide has altered binding to major groove interacting
partners.
[0049] In some embodiments, the chemical modifications are located
on the major groove face of the nucleobase, and wherein the
chemical modifications can include replacing or substituting an
atom of a pyrimidine nucleobase with an amine, an SH, an alkyl
(e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro).
[0050] In another aspect, the present disclosure provides chemical
modifications located on the sugar moiety of the nucleotide.
[0051] In another aspect, the present disclosure provides chemical
modifications located on the phosphate backbone of the nucleic
acid.
[0052] In some embodiments, the chemical modifications alter the
electrochemistry on the major groove face of the nucleic acid.
[0053] In another aspect, the present disclosure provides
nucleotides that contain chemical modifications, wherein the
nucleotide reduces the cellular innate immune response, as compared
to the cellular innate immune induced by a corresponding unmodified
nucleic acid.
[0054] In another aspect, the present disclosure provides nucleic
acid sequences comprising at least two nucleotides, the nucleic
acid sequence comprising a nucleotide that disrupts binding of a
major groove interacting partner with the nucleic acid sequence,
wherein the nucleotide has decreased binding affinity to the major
groove binding partner.
[0055] In another aspect, the present disclosure provides
compositions comprising a compound as described herein. In some
embodiments, the composition is a reaction mixture. In some
embodiments, the composition is a pharmaceutical composition. In
some embodiments, the composition is a cell culture. In some
embodiments, the composition further comprises an RNA polymerase
and a cDNA template. In some embodiments, the composition further
comprises a nucleotide selected from the group consisting of
adenosine, cytosine, guanosine, and uracil.
[0056] In a further aspect, the present disclosure provides methods
of making a pharmaceutical formulation comprising a physiologically
active secreted protein, comprising transfecting a first population
of human cells with the pharmaceutical nucleic acid made by the
methods described herein, wherein the secreted protein is active
upon a second population of human cells.
[0057] In some embodiments, the secreted protein is capable of
interacting with a receptor on the surface of at least one cell
present in the second population.
[0058] In some embodiments, the secreted protein is
Granulocyte-Colony Stimulating Factor (G-CSF).
[0059] In some embodiments, the second population contains
myeloblast cells that express the G-CSF receptor.
[0060] In certain embodiments, provided herein are combination
therapeutics containing one or more modified nucleic acids
containing translatable regions that encode for a protein or
proteins that boost a mammalian subject's immunity along with a
protein that induces antibody-dependent cellular toxitity. For
example, provided are therapeutics containing one or more nucleic
acids that encode trastuzumab and granulocyte-colony stimulating
factor (G-CSF). In particular, such combination therapeutics are
useful in Her2+ breast cancer patients who develop induced
resistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy.
2(6):795-8 (2010)).
[0061] In one embodiment, it is intended that the compounds of the
present disclosure are stable. It is further appreciated that
certain features of the present disclosure, which are, for clarity,
described in the context of separate embodiments, can also be
provided in combination in a single embodiment. Conversely, various
features of the present disclosure which are, for brevity,
described in the context of a single embodiment, can also be
provided separately or in any suitable subcombination.
Modified Nucleotides, Nucleosides and Polynucleotides of the
Invention
[0062] Herein, in a nucleotide, nucleoside or polynucleotide (such
as the nucleic acids of the invention, e.g., mRNA molecule), the
terms "modification" or, as appropriate, "modified" refer to
modification with respect to A, G, U or C ribonucleotides.
Generally, herein, these terms are not intended to refer to the
ribonucleotide modifications in naturally occurring 5'-terminal
mRNA cap moieties. In a polypeptide, the term "modification" refers
to a modification as compared to the canonical set of 20 amino
acids, moiety)
[0063] The modifications may be various distinct modifications. In
some embodiments, where the nucleic acid is an mRNA, the coding
region, the flanking regions and/or the terminal regions may
contain one, two, or more (optionally different) nucleoside or
nucleotide modifications. In some embodiments, a modified
polynucleotide introduced to a cell may exhibit reduced degradation
in the cell, as compared to an unmodified polynucleotide.
[0064] The polynucleotides can include any useful modification,
such as to the sugar, the nucleobase, or the internucleoside
linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to
the phosphodiester backbone). For example, the major groove of a
polynucleotide, or the major groove face of a nucleobase may
comprise one or more modifications. One or more atoms of a
pyrimidine nucleobase (e.g. on the major groove face) may be
replaced or substituted with optionally substituted amino,
optionally substituted thiol, optionally substituted alkyl (e.g.,
methyl or ethyl), or halo (e.g., chloro or fluoro). In certain
embodiments, modifications (e.g., one or more modifications) are
present in each of the sugar and the internucleoside linkage.
Modifications according to the present invention may be
modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids
(DNAs), e.g., the substitution of the 2'OH of the ribofuranysyl
ring to 2'H, threose nucleic acids (TNAs), glycol nucleic acids
(GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs)
or hybrids thereof). Additional modifications are described
herein.
[0065] As described herein, the polynucleotides of the invention do
not substantially induce an innate immune response of a cell into
which the polynucleotide (e.g., mRNA) is introduced. Features of an
induced innate immune response include 1) increased expression of
pro-inflammatory cytokines, 2) activation of intracellular PRRs
(RIG-I, MDA5, etc, and/or 3) termination or reduction in protein
translation.
[0066] In certain embodiments, it may desirable for a modified
nucleic acid molecule introduced into the cell to be degraded
intracellulary. For example, degradation of a modified nucleic acid
molecule may be preferable if precise timing of protein production
is desired. Thus, in some embodiments, the invention provides a
modified nucleic acid molecule containing a degradation domain,
which is capable of being acted on in a directed manner within a
cell. In another aspect, the present disclosure provides
polynucleotides comprising a nucleoside or nucleotide that can
disrupt the binding of a major groove interacting, e.g. binding,
partner with the polynucleotide (e.g., where the modified
nucleotide has decreased binding affinity to major groove
interacting partner, as compared to an unmodified nucleotide).
[0067] The polynucleotides can optionally include other agents
(e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs,
antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce
triple helix formation, aptamers, vectors, etc.). In some
embodiments, the polynucleotides may include one or more messenger
RNAs (mRNAs) having one or more modified nucleoside or nucleotides
(i.e., modified mRNA molecules). Details for these polynucleotides
follow.
Polynucleotides
[0068] The polynucleotides of the invention includes a first region
of linked nucleosides encoding a polypeptide of interest, a first
flanking region located at the 5' terminus of the first region, and
a second flanking region located at the 3' terminus of the first
region.
[0069] In some embodiments, the polynucleotide (e.g., the first
region, first flanking region, or second flanking region) includes
n number of linked nucleosides having Formula (Ia) or Formula
(Ia-1):
##STR00001##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein U is O, S, N(R.sup.U).sub.nu, or C(R.sup.U).sub.nu, wherein
nu is an integer from 0 to 2 and each R.sup.U is, independently, H,
halo, or optionally substituted alkyl;
[0070] is a single bond or absent;
[0071] each of R.sup.1', R.sup.2', R.sup.1'', R.sup.2'', R.sup.1,
R.sup.2, R.sup.3, R.sup.4, and R.sup.5, if present, is,
independently, H, halo, hydroxy, thiol, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted
alkenyloxy, optionally substituted alkynyloxy, optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy,
optionally substituted hydroxyalkoxy, optionally substituted amino,
azido, optionally substituted aryl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, or absent; wherein the combination of
R.sup.3 with one or more of R.sup.1', R.sup.1'', R.sup.2',
R.sup.2'', or R.sup.5 (e.g., the combination of R.sup.1' and
R.sup.3, the combination of R.sup.1'' and R.sup.3, the combination
of R.sup.2' and R.sup.3, the combination of R.sup.2'' and R.sup.3,
or the combination of R.sup.5 and R.sup.3) can join together to
form optionally substituted alkylene or optionally substituted
heteroalkylene and, taken together with the carbons to which they
are attached, provide an optionally substituted heterocyclyl (e.g.,
a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein the
combination of R.sup.5 with one or more of R.sup.1', R.sup.1'',
R.sup.2', or R.sup.2'' (e.g., the combination of R.sup.1' and
R.sup.5, the combination of R.sup.1'' and R.sup.5, the combination
of R.sup.2' and R.sup.5, or the combination of R.sup.2'' and
R.sup.5) can join together to form optionally substituted alkylene
or optionally substituted heteroalkylene and, taken together with
the carbons to which they are attached, provide an optionally
substituted heterocyclyl (e.g., a bicyclic, tricyclic, or
tetracyclic heterocyclyl); and wherein the combination of R.sup.4
and one or more of R.sup.1', R.sup.1'', R.sup.2', R.sup.2'',
R.sup.3, or R.sup.5 can join together to form optionally
substituted alkylene or optionally substituted heteroalkylene and,
taken together with the carbons to which they are attached, provide
an optionally substituted heterocyclyl (e.g., a bicyclic,
tricyclic, or tetracyclic heterocyclyl);
[0072] each of m' and m'' is, independently, an integer from 0 to 3
(e.g., from 0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);
[0073] each of Y.sup.1, Y.sup.2, and Y.sup.3, is, independently, O,
S, Se, --NR.sup.N1--, optionally substituted alkylene, or
optionally substituted heteroalkylene, wherein R.sup.N1 is H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted aryl, or
absent;
[0074] each Y.sup.4 is, independently, H, hydroxy, thiol, boranyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted thioalkoxy, optionally
substituted alkoxyalkoxy, or optionally substituted amino;
[0075] each Y.sup.5 is, independently, O, S, Se, optionally
substituted alkylene (e.g., methylene), or optionally substituted
heteroalkylene;
[0076] n is an integer from 1 to 100,000; and
[0077] B is a nucleobase (e.g., a purine, a pyrimidine, or
derivatives thereof), wherein the combination of B and R.sup.1',
the combination of B and R.sup.2', the combination of B and
R.sup.1'', or the combination of B and R.sup.2'' can, taken
together with the carbons to which they are attached, optionally
form a bicyclic group (e.g., a bicyclic heterocyclyl) or wherein
the combination of B, R.sup.1'', and R.sup.3 or the combination of
B, R.sup.2'', and R.sup.3 can optionally form a tricyclic or
tetracyclic group (e.g., a tricyclic or tetracyclic heterocyclyl,
such as in Formula (IIo)-(IIp) herein).
[0078] In some embodiments, the polynucleotide includes a modified
ribose. In some embodiments, the polynucleotide (e.g., the first
region, the first flanking region, or the second flanking region)
includes n number of linked nucleosides having Formula
(Ia-2)-(Ia-5) or a pharmaceutically acceptable salt or stereoisomer
thereof.
##STR00002##
[0079] In some embodiments, the polynucleotide (e.g., the first
region, the first flanking region, or the second flanking region)
includes n number of linked nucleosides having Formula (Ib) or
Formula (Ib-1):
##STR00003##
[0080] or a pharmaceutically acceptable salt or stereoisomer
thereof, wherein
[0081] U is O, S, N(R.sup.U).sub.nu, or C(R.sup.U).sub.nu, wherein
nu is an integer from 0 to 2 and each R.sup.U is, independently, H,
halo, or optionally substituted alkyl;
[0082] is a single bond or absent;
[0083] each of R.sup.1, R.sup.3', R.sup.3'', and R.sup.4 is,
independently, H, halo, hydroxy, optionally substituted alkyl,
optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally substituted alkynyloxy, optionally substituted
aminoalkoxy, optionally substituted alkoxyalkoxy, optionally
substituted hydroxyalkoxy, optionally substituted amino, azido,
optionally substituted aryl, optionally substituted aminoalkyl,
optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl, or absent; and wherein the combination of R.sup.1 and
R.sup.3' or the combination of R.sup.1 and R.sup.3'' can be taken
together to form optionally substituted alkylene or optionally
substituted heteroalkylene (e.g., to produce a locked nucleic
acid);
[0084] each R.sup.5 is, independently, H, halo, hydroxy, optionally
substituted alkyl, optionally substituted alkoxy, optionally
substituted alkenyloxy, optionally substituted alkynyloxy,
optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy, or absent;
[0085] each of Y.sup.1, Y.sup.2, and Y.sup.3 is, independently, O,
S, Se, NR.sup.N1--, optionally substituted alkylene, or optionally
substituted heteroalkylene, wherein R.sup.N1 is H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, or optionally substituted aryl;
[0086] each Y.sup.4 is, independently, H, hydroxy, thiol, boranyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted alkoxyalkoxy, or optionally
substituted amino;
[0087] n is an integer from 1 to 100,000; and
[0088] B is a nucleobase.
[0089] In some embodiments, the polynucleotide (e.g., the first
region, first flanking region, or second flanking region) includes
n number of linked nucleosides having Formula (Ic):
##STR00004##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0090] U is O, S, N(R.sup.U).sub.nu, or C(R.sup.U).sub.nu, wherein
nu is an integer from 0 to 2 and each R.sup.U is, independently, H,
halo, or optionally substituted alkyl;
[0091] is a single bond or absent;
[0092] each of B.sup.1, B.sup.2, and B.sup.3 is, independently, a
nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof,
as described herein), H, halo, hydroxy, thiol, optionally
substituted alkyl, optionally substituted alkoxy, optionally
substituted alkenyloxy, optionally substituted alkynyloxy,
optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally
substituted amino, azido, optionally substituted aryl, optionally
substituted aminoalkyl, optionally substituted aminoalkenyl, or
optionally substituted aminoalkynyl, wherein one and only one of
B.sup.1, B.sup.2, and B.sup.3 is a nucleobase;
[0093] each of R.sup.b1, R.sup.b2, R.sup.b3, R.sup.3, and R.sup.5
is, independently, H, halo, hydroxy, thiol, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted
alkenyloxy, optionally substituted alkynyloxy, optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy,
optionally substituted hydroxyalkoxy, optionally substituted amino,
azido, optionally substituted aryl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, or optionally
substituted aminoalkynyl;
[0094] each of Y.sup.1, Y.sup.2, and Y.sup.3, is, independently, O,
S, Se, --NR.sup.N1--, optionally substituted alkylene, or
optionally substituted heteroalkylene, wherein R.sup.N1 is H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, or optionally substituted aryl;
[0095] each Y.sup.4 is, independently, H, hydroxy, thiol, boranyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted thioalkoxy, optionally
substituted alkoxyalkoxy, or optionally substituted amino;
[0096] each Y.sup.5 is, independently, O, S, Se, optionally
substituted alkylene (e.g., methylene), or optionally substituted
heteroalkylene;
[0097] n is an integer from 1 to 100,000; and
[0098] wherein the ring including U can include one or more double
bonds.
[0099] In particular embodiments, the ring including U does not
have a double bond between U--CB.sup.3R.sup.b3 or between
CB.sup.3R.sup.b3--C.sup.B2R.sup.b2.
[0100] In some embodiments, the polynucleotide (e.g., the first
region, first flanking region, or second flanking region) includes
n number of linked nucleosides having Formula (Id):
##STR00005##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein U is O, S, N(R.sup.U).sub.nu, or C(R.sup.U).sub.nu, wherein
nu is an integer from 0 to 2 and each R.sup.U is, independently, H,
halo, or optionally substituted alkyl;
[0101] each R.sup.3 is, independently, H, halo, hydroxy, thiol,
optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally substituted amino, azido, optionally substituted aryl,
optionally substituted aminoalkyl, optionally substituted
aminoalkenyl, or optionally substituted aminoalkynyl;
[0102] each of Y.sup.1, Y.sup.2, and Y.sup.3, is, independently, O,
S, Se, --NR.sup.N1--, optionally substituted alkylene, or
optionally substituted heteroalkylene, wherein R.sup.N1 is H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, or optionally substituted aryl;
[0103] each Y.sup.4 is, independently, H, hydroxy, thiol, boranyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted thioalkoxy, optionally
substituted alkoxyalkoxy, or optionally substituted amino;
[0104] each Y.sup.5 is, independently, O, S, optionally substituted
alkylene (e.g., methylene), or optionally substituted
heteroalkylene;
[0105] n is an integer from 1 to 100,000; and
[0106] B is a nucleobase (e.g., a purine, a pyrimidine, or
derivatives thereof).
[0107] In some embodiments, the polynucleotide (e.g., the first
region, first flanking region, or second flanking region) includes
n number of linked nucleosides having Formula (Ie):
##STR00006##
or a pharmaceutically acceptable salt or stereoisomer thereof,
[0108] wherein each of U' and U'' is, independently, O, S,
N(R.sup.U).sub.nu, or C(R.sup.U).sub.nu, wherein nu is an integer
from 0 to 2 and each R.sup.U is, independently, H, halo, or
optionally substituted alkyl;
[0109] each R.sup.6 is, independently, H, halo, hydroxy, thiol,
optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally substituted amino, azido, optionally substituted aryl,
optionally substituted aminoalkyl, optionally substituted
aminoalkenyl, or optionally substituted aminoalkynyl;
[0110] each Y.sup.5' is, independently, O, S, optionally
substituted alkylene (e.g., methylene or ethylene), or optionally
substituted heteroalkylene;
[0111] n is an integer from 1 to 100,000; and
[0112] B is a nucleobase (e.g., a purine, a pyrimidine, or
derivatives thereof).
[0113] In some embodiments, the polynucleotide (e.g., the first
region, first flanking region, or second flanking region) includes
n number of linked nucleosides having Formula (If) or (If-1):
##STR00007##
or a pharmaceutically acceptable salt or stereoisomer thereof,
[0114] wherein each of U' and U'' is, independently, O, S, N,
N(R.sup.U).sub.nu, or C(R.sup.U).sub.nu, wherein nu is an integer
from 0 to 2 and each R.sup.U is, independently, H, halo, or
optionally substituted alkyl (e.g., U' is O and U'' is N);
[0115] is a single bond or absent;
[0116] each of R.sup.1', R.sup.2', R.sup.1'', R.sup.2'', R.sup.3,
and R.sup.4 is, independently, H, halo, hydroxy, thiol, optionally
substituted alkyl, optionally substituted alkoxy, optionally
substituted alkenyloxy, optionally substituted alkynyloxy,
optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally
substituted amino, azido, optionally substituted aryl, optionally
substituted aminoalkyl, optionally substituted aminoalkenyl,
optionally substituted aminoalkynyl, or absent; and wherein the
combination of R.sup.1' and R.sup.3, the combination of R.sup.1''
and R.sup.3, the combination of R.sup.2' and R.sup.3, or the
combination of R.sup.2'' and R.sup.3 can be taken together to form
optionally substituted alkylene or optionally substituted
heteroalkylene (e.g., to produce a locked nucleic acid); each of m'
and m'' is, independently, an integer from 0 to 3 (e.g., from 0 to
2, from 0 to 1, from 1 to 3, or from 1 to 2);
[0117] each of Y.sup.1, Y.sup.2, and Y.sup.3, is, independently, O,
S, Se, --NR.sup.N1--, optionally substituted alkylene, or
optionally substituted heteroalkylene, wherein R.sup.N1 is H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted aryl, or
absent;
[0118] each Y.sup.4 is, independently, H, hydroxy, thiol, boranyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted thioalkoxy, optionally
substituted alkoxyalkoxy, or optionally substituted amino;
[0119] each Y.sup.5 is, independently, O, S, Se, optionally
substituted alkylene (e.g., methylene), or optionally substituted
heteroalkylene;
[0120] n is an integer from 1 to 100,000; and
[0121] B is a nucleobase (e.g., a purine, a pyrimidine, or
derivatives thereof).
[0122] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
the ring including U has one or two double bonds.
[0123] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
each of R.sup.1, R.sup.1', and R.sup.1'', if present, is H. In
further embodiments, each of R.sup.2, R.sup.2', and R.sup.2'', if
present, is, independently, H, halo (e.g., fluoro), hydroxy,
optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally substituted alkoxyalkoxy. In particular embodiments,
alkoxyalkoxy is
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl). In some embodiments, s2
is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C.sub.1-6 alkyl.
[0124] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
each of R.sup.2, R.sup.2', and R.sup.2'', if present, is H. In
further embodiments, each of R.sup.1, R.sup.1', and R.sup.1'', if
present, is, independently, H, halo (e.g., fluoro), hydroxy,
optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally substituted alkoxyalkoxy. In particular embodiments,
alkoxyalkoxy is
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl). In some embodiments, s2
is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C.sub.1-6 alkyl.
[0125] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
each of R.sup.3, R.sup.4, and R.sup.5 is, independently, H, halo
(e.g., fluoro), hydroxy, optionally substituted alkyl, optionally
substituted alkoxy (e.g., methoxy or ethoxy), or optionally
substituted alkoxyalkoxy. In particular embodiments, R.sup.3 is H,
R.sup.4 is H, R.sup.5 is H, or R.sup.3, R.sup.4, and R.sup.5 are
all H. In particular embodiments, R.sup.3 is C.sub.1-6 alkyl,
R.sup.4 is C.sub.1-6 alkyl, R.sup.5 is C.sub.1-6 alkyl, or R.sup.3,
R.sup.4, and R.sup.5 are all C.sub.1-6 alkyl. In particular
embodiments, R.sup.3 and R.sup.4 are both H, and R.sup.5 is
C.sub.1-6 alkyl.
[0126] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
R.sup.3 and R.sup.5 join together to form optionally substituted
alkylene or optionally substituted heteroalkylene and, taken
together with the carbons to which they are attached, provide an
optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic,
or tetracyclic heterocyclyl, such as trans-3',4' analogs, wherein
R.sup.3 and R.sup.5 join together to form heteroalkylene (e.g.,
--(CH.sub.2).sub.b1O(CH.sub.2).sub.b2O(CH.sub.2).sub.b3--, wherein
each of b1, b2, and b3 are, independently, an integer from 0 to
3).
[0127] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
R.sup.3 and one or more of R.sup.1', R.sup.1'', R.sup.2',
R.sup.2'', or R.sup.5 join together to form optionally substituted
alkylene or optionally substituted heteroalkylene and, taken
together with the carbons to which they are attached, provide an
optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic,
or tetracyclic heterocyclyl, R.sup.3 and one or more of R.sup.1',
R.sup.1'', R.sup.2', R.sup.2'', or R.sup.5 join together to form
heteroalkylene (e.g.,
--(CH.sub.2).sub.b1O(CH.sub.2).sub.b2O(CH.sub.2).sub.b3--, wherein
each of b1, b2, and b3 are, independently, an integer from 0 to
3).
[0128] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
R.sup.5 and one or more of R.sup.1', R.sup.1'', R.sup.2', or
R.sup.2'' join together to form optionally substituted alkylene or
optionally substituted heteroalkylene and, taken together with the
carbons to which they are attached, provide an optionally
substituted heterocyclyl (e.g., a bicyclic, tricyclic, or
tetracyclic heterocyclyl, R.sup.5 and one or more of R.sup.1',
R.sup.1'', R.sup.2', or R.sup.2'' join together to form
heteroalkylene (e.g.,
--(CH.sub.2).sub.b1O(CH.sub.2).sub.b2O(CH.sub.2).sub.b3--, wherein
each of b1, b2, and b3 are, independently, an integer from 0 to
3).
[0129] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
each Y.sup.2 is, independently, O, S, or --NR.sup.N1--, wherein
R.sup.N1 is H, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, or optionally substituted
aryl. In particular embodiments, Y.sup.2 is NR.sup.N1--, wherein
R.sup.N1 is H or optionally substituted alkyl (e.g., C.sub.1-6
alkyl, such as methyl, ethyl, isopropyl, or n-propyl).
[0130] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
each Y.sup.3 is, independently, O or S.
[0131] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
R.sup.1 is H; each R.sup.2 is, independently, H, halo (e.g.,
fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or
ethoxy), or optionally substituted alkoxyalkoxy (e.g.,
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, such as wherein s2 is 0,
s1 is 1 or 2, s3 is 0 or 1, and R' is C.sub.1-6 alkyl); each
Y.sup.2 is, independently, 0 or --NR.sup.N1--, wherein R.sup.N1 is
H, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, or optionally substituted aryl
(e.g., wherein R.sup.N1 is H or optionally substituted alkyl (e.g.,
C.sub.1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl));
and each Y.sup.3 is, independently, O or S (e.g., S). In further
embodiments, R.sup.3 is H, halo (e.g., fluoro), hydroxy, optionally
substituted alkyl, optionally substituted alkoxy (e.g., methoxy or
ethoxy), or optionally substituted alkoxyalkoxy. In yet further
embodiments, each Y.sup.1 is, independently, 0 or --NR.sup.N1--,
wherein R.sup.N1 is H, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, or optionally
substituted aryl (e.g., wherein R.sup.N1 is H or optionally
substituted alkyl (e.g., C.sub.1-6 alkyl, such as methyl, ethyl,
isopropyl, or n-propyl)); and each Y.sup.4 is, independently, H,
hydroxy, thiol, optionally substituted alkyl, optionally
substituted alkoxy, optionally substituted thioalkoxy, optionally
substituted alkoxyalkoxy, or optionally substituted amino.
[0132] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each
R.sup.1 is, independently, H, halo (e.g., fluoro), hydroxy,
optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally substituted alkoxyalkoxy (e.g.,
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, such as wherein s2 is 0,
s1 is 1 or 2, s3 is 0 or 1, and R' is C.sub.1-6 alkyl); R.sup.2 is
H; each Y.sup.2 is, independently, 0 or --NR.sup.N1--, wherein
R.sup.N1 is H, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, or optionally substituted
aryl (e.g., wherein R.sup.N1 is H or optionally substituted alkyl
(e.g., C.sub.1-6 alkyl, such as methyl, ethyl, isopropyl, or
n-propyl)); and each Y.sup.3 is, independently, O or S (e.g., S).
In further embodiments, R.sup.3 is H, halo (e.g., fluoro), hydroxy,
optionally substituted alkyl, optionally substituted alkoxy (e.g.,
methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In yet
further embodiments, each Y.sup.1 is, independently, 0 or
--NR.sup.N1--, wherein R.sup.N1 is H, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, or
optionally substituted aryl (e.g., wherein R.sup.N1 is H or
optionally substituted alkyl (e.g., C.sub.1-6 alkyl, such as
methyl, ethyl, isopropyl, or n-propyl)); and each Y.sup.4 is,
independently, H, hydroxy, thiol, optionally substituted alkyl,
optionally substituted alkoxy, optionally substituted thioalkoxy,
optionally substituted alkoxyalkoxy, or optionally substituted
amino.
[0133] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
the ring including U is in the .beta.-D (e.g., .beta.-D-ribo)
configuration.
[0134] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
the ring including U is in the .alpha.-L (e.g., .alpha.-L-ribo)
configuration.
[0135] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
one or more B is not pseudouridine (.psi.) or 5-methyl-cytidine
(m.sup.5C).
[0136] In some embodiments, about 10% to about 100% of n number of
B nucleobases is not .psi. or m.sup.5C (e.g., from 10% to 20%, from
10% to 35%, from 10% to 50%, from 10% to 60%, from 10% to 75%, from
10% to 90%, from 10% to 95%, from 10% to 98%, from 10% to 99%, from
20% to 35%, from 20% to 50%, from 20% to 60%, from 20% to 75%, from
20% to 90%, from 20% to 95%, from 20% to 98%, from 20% to 99%, from
20% to 100%, from 50% to 60%, from 50% to 75%, from 50% to 90%,
from 50% to 95%, from 50% to 98%, from 50% to 99%, from 50% to
100%, from 75% to 90%, from 75% to 95%, from 75% to 98%, from 75%
to 99%, and from 75% to 100% of n number of B is not .psi. or
m.sup.5C). In some embodiments, B is not .psi. or m.sup.5C.
[0137] In some embodiments of the polynucleotides (e.g., Formulas
(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
when B is an unmodified nucleobase selected from cytosine, guanine,
uracil and adenine, then at least one of Y', Y.sup.2, or Y.sup.3 is
not O.
[0138] In some embodiments, the polynucleotide includes a modified
ribose. In some embodiments, the polynucleotide (e.g., the first
region, the first flanking region, or the second flanking region)
includes n number of linked nucleosides having Formula
(IIa)-(IIc):
##STR00008##
or a pharmaceutically acceptable salt or stereoisomer thereof. In
particular embodiments, U is O or C(R.sup.U).sub.nu, wherein nu is
an integer from 0 to 2 and each R.sup.U is, independently, H, halo,
or optionally substituted alkyl (e.g., U is --CH.sub.2-- or
--CH--). In other embodiments, each of R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 is, independently, H, halo, hydroxy, thiol,
optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally substituted amino, azido, optionally substituted aryl,
optionally substituted aminoalkyl, optionally substituted
aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g.,
each R.sup.1 and R.sup.2 is, independently H, halo, hydroxy,
optionally substituted alkyl, or optionally substituted alkoxy;
each R.sup.3 and R.sup.4 is, independently, H or optionally
substituted alkyl; and R.sup.5 is H or hydroxy), and is a single
bond or double bond.
[0139] In particular embodiments, the polynucleotide (e.g., the
first region, the first flanking region, or the second flanking
region) includes n number of linked nucleosides having Formula
(IIb-1)-(IIb-2):
##STR00009##
or a pharmaceutically acceptable salt or stereoisomer thereof. In
some embodiments, U is O or C(R.sup.U).sub.nu, wherein nu is an
integer from 0 to 2 and each R.sup.U is, independently, H, halo, or
optionally substituted alkyl (e.g., U is --CH.sub.2-- or --CH--).
In other embodiments, each of R.sup.1 and R.sup.2 is,
independently, H, halo, hydroxy, thiol, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted
alkenyloxy, optionally substituted alkynyloxy, optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy,
optionally substituted hydroxyalkoxy, optionally substituted amino,
azido, optionally substituted aryl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, or absent (e.g., each R.sup.1 and R.sup.2
is, independently, H, halo, hydroxy, optionally substituted alkyl,
or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or
alkoxy). In particular embodiments, R.sup.2 is hydroxy or
optionally substituted alkoxy (e.g., methoxy, ethoxy, or any
described herein).
[0140] In particular embodiments, the polynucleotide (e.g., the
first region, the first flanking region, or the second flanking
region) includes n number of linked nucleosides having Formula
(IIc-1)-(IIc-4):
##STR00010##
or a pharmaceutically acceptable salt or stereoisomer thereof.
[0141] In some embodiments, U is O or C(R.sup.U).sub.nu, wherein nu
is an integer from 0 to 2 and each R.sup.U is, independently, H,
halo, or optionally substituted alkyl (e.g., U is --CH.sub.2-- or
--CH--). In some embodiments, each of R.sup.1, R.sup.2, and R.sup.3
is, independently, H, halo, hydroxy, thiol, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted
alkenyloxy, optionally substituted alkynyloxy, optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy,
optionally substituted hydroxyalkoxy, optionally substituted amino,
azido, optionally substituted aryl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, or absent (e.g., each R.sup.1 and R.sup.2
is, independently, H, halo, hydroxy, optionally substituted alkyl,
or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or
alkoxy; and each R.sup.3 is, independently, H or optionally
substituted alkyl)). In particular embodiments, R.sup.2 is
optionally substituted alkoxy (e.g., methoxy or ethoxy, or any
described herein). In particular embodiments, R.sup.1 is optionally
substituted alkyl, and R.sup.2 is hydroxy. In other embodiments,
R.sup.1 is hydroxy, and R.sup.2 is optionally substituted alkyl. In
further embodiments, R.sup.3 is optionally substituted alkyl.
[0142] In some embodiments, the polynucleotide includes an acyclic
modified ribose. In some embodiments, the polynucleotide (e.g., the
first region, the first flanking region, or the second flanking
region) includes n number of linked nucleosides having Formula
(IId)-(IIf):
##STR00011##
or a pharmaceutically acceptable salt or stereoisomer thereof.
[0143] In some embodiments, the polynucleotide includes an acyclic
modified hexitol. In some embodiments, the polynucleotide (e.g.,
the first region, the first flanking region, or the second flanking
region) includes n number of linked nucleosides having Formula
(IIg)-(IIj):
##STR00012##
or a pharmaceutically acceptable salt or stereoisomer thereof.
[0144] In some embodiments, the polynucleotide includes a sugar
moiety having a contracted or an expanded ribose ring. In some
embodiments, the polynucleotide (e.g., the first region, the first
flanking region, or the second flanking region) includes n number
of linked nucleosides having Formula (IIk)-(IIm):
##STR00013##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each of R.sup.1', R.sup.1'', R.sup.2', and R.sup.2'' is,
independently, H, halo, hydroxy, optionally substituted alkyl,
optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally substituted alkynyloxy, optionally substituted
aminoalkoxy, optionally substituted alkoxyalkoxy, or absent; and
wherein the combination of R.sup.2' and R.sup.3 or the combination
of R.sup.2'' and R.sup.3 can be taken together to form optionally
substituted alkylene or optionally substituted heteroalkylene.
[0145] In some embodiments, the polynucleotide includes a locked
modified ribose. In some embodiments, the polynucleotide (e.g., the
first region, the first flanking region, or the second flanking
region) includes n number of linked nucleosides having Formula
(IIn):
##STR00014##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein R.sup.3' is O, S, or --NR.sup.N1--, wherein R.sup.N1 is H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, or optionally substituted aryl and
R.sup.3'' is optionally substituted alkylene (e.g., --CH.sub.2--,
--CH.sub.2CH.sub.2--, or --CH.sub.2CH.sub.2CH.sub.2--) or
optionally substituted heteroalkylene (e.g., --CH.sub.2NH--,
--CH.sub.2CH.sub.2NH--, --CH.sub.2OCH.sub.2--, or
--CH.sub.2CH.sub.2OCH.sub.2--) (e.g., R.sup.3' is O and R.sup.3''
is optionally substituted alkylene (e.g., --CH.sub.2--,
--CH.sub.2CH.sub.2--, or --CH.sub.2CH.sub.2CH.sub.2--)).
[0146] In some embodiments, the polynucleotide (e.g., the first
region, the first flanking region, or the second flanking region)
includes n number of linked nucleosides having Formula
(IIn-1)-(II-n2):
##STR00015##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein R.sup.3' is O, S, or --NR.sup.N1--, wherein R.sup.N1 is H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, or optionally substituted aryl and
R.sup.3'' is optionally substituted alkylene (e.g., --CH.sub.2--,
--CH.sub.2CH.sub.2--, or --CH.sub.2CH.sub.2CH.sub.2--) or
optionally substituted heteroalkylene (e.g., --CH.sub.2NH--,
--CH.sub.2CH.sub.2NH--, --CH.sub.2OCH.sub.2--, or
--CH.sub.2CH.sub.2OCH.sub.2--) (e.g., R.sup.3' is O and R.sup.3''
is optionally substituted alkylene (e.g., --CH.sub.2--,
--CH.sub.2CH.sub.2--, or --CH.sub.2CH.sub.2CH.sub.2--)).
[0147] In some embodiments, the polynucleotide includes a locked
modified ribose that forms a tetracyclic heterocyclyl. In some
embodiments, the polynucleotide (e.g., the first region, the first
flanking region, or the second flanking region) includes n number
of linked nucleosides having Formula (IIo):
##STR00016##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein R.sup.12a, R.sup.12c, T.sup.1', T.sup.1'', T.sup.2',
T.sup.2'', V.sup.1, and V.sup.3 are as described herein.
[0148] Any of the formulas for the polynucleotides can include one
or more nucleobases described herein (e.g., Formulas
(b1)-(b43)).
[0149] In one embodiment, the present invention provides methods of
preparing a polynucleotide comprising at least one nucleotide that
disrupts binding of a major groove interacting partner with the
nucleic acid, wherein the polynucleotide comprises n number of
nucleosides having Formula (Ia), as defined herein:
##STR00017##
the method comprising reacting a compound of Formula (IIIa), as
defined herein:
##STR00018##
[0150] with an RNA polymerase, and a cDNA template.
[0151] In a further embodiment, the present invention provides
methods of amplifying a polynucleotide comprising at least one
nucleotide that disrupts binding of a major groove binding partner
with the polynucleotide sequence, the method comprising: reacting a
compound of Formula (IIIa), as defined herein, with a primer, a
cDNA template, and an RNA polymerase.
[0152] In one embodiment, the present invention provides methods of
preparing a polynucleotide comprising at least one nucleotide that
disrupts binding of a major groove interacting partner with the
nucleic acid, wherein the polynucleotide comprises n number of
nucleosides having Formula (Ia-1), as defined herein:
##STR00019##
the method comprising reacting a compound of Formula (IIIa-1), as
defined herein:
##STR00020##
with an RNA polymerase, and a cDNA template.
[0153] In a further embodiment, the present invention provides
methods of amplifying a polynucleotide comprising at least one
nucleotide (e.g., modified mRNA molecule) that disrupts binding of
a major groove binding partner with the polynucleotide sequence,
the method comprising: reacting a compound of Formula (IIIa-1), as
defined herein, with a primer, a cDNA template, and an RNA
polymerase.
[0154] In one embodiment, the present invention provides methods of
preparing a polynucleotide comprising at least one nucleotide that
disrupts binding of a major groove interacting partner with the
nucleic acid sequence, wherein the polynucleotide comprises n
number of nucleosides having Formula (Ia-2), as defined herein:
##STR00021##
the method comprising reacting a compound of Formula (IIIa-2), as
defined herein:
##STR00022##
with an RNA polymerase, and a cDNA template.
[0155] In a further embodiment, the present invention provides
methods of amplifying a polynucleotide comprising at least one
nucleotide (e.g., modified mRNA molecule) that disrupts binding of
a major groove binding partner with the polynucleotide, the method
comprising reacting a compound of Formula (IIIa-2), as defined
herein, with a primer, a cDNA template, and an RNA polymerase.
[0156] In some embodiments, the reaction may be repeated from 1 to
about 7,000 times. In any of the embodiments herein, B may be a
nucleobase of Formula (b1)-(b43).
[0157] The polynucleotides can optionally include 5' and/or 3'
flanking regions, which are described herein.
Modified Nucleotides and Nucleosides
[0158] The present invention also includes the building blocks,
e.g., modified ribonucleosides, modified ribonucleotides, of the
polynucleotides, e.g., modified RNA (or mRNA) molecules. For
example, these building blocks can be useful for preparing the
polynucleotides of the invention.
[0159] In some embodiments, the building block molecule has Formula
(IIIa) or (IIIa-1):
##STR00023##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein the substituents are as described herein (e.g., for Formula
(Ia) and (Ia-1)), and wherein when B is an unmodified nucleobase
selected from cytosine, guanine, uracil and adenine, then at least
one of Y.sup.1, Y.sup.2, or Y.sup.3 is not O.
[0160] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide, has Formula (IVa)-(IVb):
##STR00024##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein B is as described herein (e.g., any one of (b1)-(b43)).
[0161] In particular embodiments, Formula (IVa) or (IVb) is
combined with a modified uracil (e.g., any one of formulas
(b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),
(b8), (b28), (b29), or (b30)). In particular embodiments, Formula
(IVa) or (IVb) is combined with a modified cytosine (e.g., any one
of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as
formula (b10) or (b32)). In particular embodiments, Formula (IVa)
or (IVb) is combined with a modified guanine (e.g., any one of
formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,
Formula (IVa) or (IVb) is combined with a modified adenine (e.g.,
any one of formulas (b18)-(b20) and (b41)-(b43)).
[0162] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide, has Formula (IVc)-(IVk):
##STR00025## ##STR00026##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein B is as described herein (e.g., any one of (b1)-(b43)).
[0163] In particular embodiments, one of Formulas (IVc)-(IVk) is
combined with a modified uracil (e.g., any one of formulas
(b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),
(b8), (b28), (b29), or (b30)).
[0164] In particular embodiments, one of Formulas (IVc)-(IVk) is
combined with a modified cytosine (e.g., any one of formulas
(b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10)
or (b32)).
[0165] In particular embodiments, one of Formulas (IVc)-(IVk) is
combined with a modified guanine (e.g., any one of formulas
(b15)-(b17) and (b37)-(b40)).
[0166] In particular embodiments, one of Formulas (IVc)-(IVk) is
combined with a modified adenine (e.g., any one of formulas
(b18)-(b20) and (b41)-(b43)).
[0167] In other embodiments, the building block molecule, which may
be incorporated into a polynucleotide has Formula (Va) or (Vb):
##STR00027##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein B is as described herein (e.g., any one of (b1)-(b43)).
[0168] In other embodiments, the building block molecule, which may
be incorporated into a polynucleotide has Formula (IXa)-(IXd):
##STR00028##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein B is as described herein (e.g., any one of (b1)-(b43)). In
particular embodiments, one of Formulas (IXa)-(IXd) is combined
with a modified uracil (e.g., any one of formulas (b1)-(b9),
(b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28),
(b29), or (b30)). In particular embodiments, one of Formulas
(IXa)-(IXd) is combined with a modified cytosine (e.g., any one of
formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as
formula (b10) or (b32)). In particular embodiments, one of Formulas
(IXa)-(IXd) is combined with a modified guanine (e.g., any one of
formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,
one of Formulas (IXa)-(IXd) is combined with a modified adenine
(e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[0169] In other embodiments, the building block molecule, which may
be incorporated into a polynucleotide has Formula (IXe)-(IXg):
##STR00029##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein B is as described herein (e.g., any one of (b1)-(b43)).
[0170] In particular embodiments, one of Formulas (IXe)-(IXg) is
combined with a modified uracil (e.g., any one of formulas
(b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),
(b8), (b28), (b29), or (b30)).
[0171] In particular embodiments, one of Formulas (IXe)-(IXg) is
combined with a modified cytosine (e.g., any one of formulas
(b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10)
or (b32)).
[0172] In particular embodiments, one of Formulas (IXe)-(IXg) is
combined with a modified guanine (e.g., any one of formulas
(b15)-(b17) and (b37)-(b40)).
[0173] In particular embodiments, one of Formulas (IXe)-(IXg) is
combined with a modified adenine (e.g., any one of formulas
(b18)-(b20) and (b41)-(b43)).
[0174] In other embodiments, the building block molecule, which may
be incorporated into a polynucleotide has Formula (IXh)-(IXk):
##STR00030##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein B is as described herein (e.g., any one of (b1)-(b43)). In
particular embodiments, one of Formulas (IXh)-(IXk) is combined
with a modified uracil (e.g., any one of formulas (b1)-(b9),
(b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28),
(b29), or (b30)). In particular embodiments, one of Formulas
(IXh)-(IXk) is combined with a modified cytosine (e.g., any one of
formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as
formula (b10) or (b32)).
[0175] In particular embodiments, one of Formulas (IXh)-(IXk) is
combined with a modified guanine (e.g., any one of formulas
(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of
Formulas (IXh)-(IXk) is combined with a modified adenine (e.g., any
one of formulas (b18)-(b20) and (b41)-(b43)).
[0176] In other embodiments, the building block molecule, which may
be incorporated into a polynucleotide has Formula (IXl)-(IXr):
##STR00031##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r1 and r2 is, independently, an integer from 0 to 5
(e.g., from 0 to 3, from 1 to 3, or from 1 to 5) and B is as
described herein (e.g., any one of (b1)-(b43)).
[0177] In particular embodiments, one of Formulas (IXl)-(IXr) is
combined with a modified uracil (e.g., any one of formulas
(b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),
(b8), (b28), (b29), or (b30)).
[0178] In particular embodiments, one of Formulas (IXl)-(IXr) is
combined with a modified cytosine (e.g., any one of formulas
(b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10)
or (b32)).
[0179] In particular embodiments, one of Formulas (IXl)-(IXr) is
combined with a modified guanine (e.g., any one of formulas
(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of
Formulas (IXl)-(IXr) is combined with a modified adenine (e.g., any
one of formulas (b18)-(b20) and (b41)-(b43)).
[0180] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide can be selected from the
group consisting of:
##STR00032## ##STR00033## ##STR00034##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r is, independently, an integer from 0 to 5 (e.g.,
from 0 to 3, from 1 to 3, or from 1 to 5).
[0181] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide can be selected from the
group consisting of:
##STR00035## ##STR00036##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r is, independently, an integer from 0 to 5 (e.g.,
from 0 to 3, from 1 to 3, or from 1 to 5) and s1 is as described
herein.
[0182] In some embodiments, the building block molecule, which may
be incorporated into a nucleic acid (e.g., RNA, mRNA,
polynucleotide), is a modified uridine (e.g., selected from the
group consisting of:
##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041##
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein Y.sup.1, Y.sup.3, Y.sup.4, Y.sup.6, and r are as described
herein (e.g., each r is, independently, an integer from 0 to 5,
such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[0183] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide is a modified cytidine (e.g.,
selected from the group consisting of:
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein Y.sup.1, Y.sup.3, Y.sup.4, Y.sup.6, and r are as described
herein (e.g., each r is, independently, an integer from 0 to 5,
such as from 0 to 3, from 1 to 3, or from 1 to 5)). For example,
the building block molecule, which may be incorporated into a
polynucleotide can be:
##STR00065##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r is, independently, an integer from 0 to 5 (e.g.,
from 0 to 3, from 1 to 3, or from 1 to 5).
[0184] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide is a modified adenosine
(e.g., selected from the group consisting of:
##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070##
##STR00071## ##STR00072## ##STR00073##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein Y.sup.1, Y.sup.3, Y.sup.4, Y.sup.6, and r are as described
herein (e.g., each r is, independently, an integer from 0 to 5,
such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[0185] In some embodiments, the building block molecule, which may
be incorporated into a polynucleotide, is a modified guanosine
(e.g., selected from the group consisting of:
##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079## ##STR00080## ##STR00081##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein Y.sup.1, Y.sup.3, Y.sup.4, Y.sup.6, and r are as described
herein (e.g., each r is, independently, an integer from 0 to 5,
such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[0186] In some embodiments, the major groove chemical modification
can include replacement of C group at C-5 of the ring (e.g., for a
pyrimidine nucleoside, such as cytosine or uracil) with N (e.g.,
replacement of the >CH group at C-5 with >NR.sup.N1 group,
wherein R.sup.N1 is H or optionally substituted alkyl). For
example, the building block molecule, which may be incorporated
into a polynucleotide can be:
##STR00082##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r is, independently, an integer from 0 to 5 (e.g.,
from 0 to 3, from 1 to 3, or from 1 to 5).
[0187] In another embodiment, the major groove chemical
modification can include replacement of the hydrogen at C-5 of
cytosine with halo (e.g., Br, Cl, F, or I) or optionally
substituted alkyl (e.g., methyl). For example, the building block
molecule, which may be incorporated into a polynucleotide can
be:
##STR00083##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r is, independently, an integer from 0 to 5 (e.g.,
from 0 to 3, from 1 to 3, or from 1 to 5).
[0188] In yet a further embodiment, the major groove chemical
modification can include a fused ring that is formed by the
NH.sub.2 at the C-4 position and the carbon atom at the C-5
position. For example, the building block molecule, which may be
incorporated into a polynucleotide can be:
##STR00084##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r is, independently, an integer from 0 to 5 (e.g.,
from 0 to 3, from 1 to 3, or from 1 to 5).
Modifications on the Sugar
[0189] The modified nucleosides and nucleotides (e.g., building
block molecules), which may be incorporated into a polynucleotide
(e.g., RNA or mRNA, as described herein), can be modified on the
sugar of the ribonucleic acid. For example, the 2' hydroxyl group
(OH) can be modified or replaced with a number of different
substituents. Exemplary substitutions at the 2'-position include,
but are not limited to, H, halo, optionally substituted C.sub.1-6
alkyl; optionally substituted C.sub.1-6 alkoxy; optionally
substituted C.sub.6-10 aryloxy; optionally substituted C.sub.3-8
cycloalkyl; optionally substituted C.sub.3-8 cycloalkoxy;
optionally substituted C.sub.6-10 aryloxy; optionally substituted
C.sub.6-10 aryl-C.sub.1-6 alkoxy, optionally substituted C.sub.1-12
(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described
herein); a polyethyleneglycol (PEG),
--O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR, where R is H or
optionally substituted alkyl, and n is an integer from 0 to 20
(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1
to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2
to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4
to 8, from 4 to 10, from 4 to 16, and from 4 to 20); "locked"
nucleic acids (LNA) in which the 2'-hydroxyl is connected by a
C.sub.1-6 alkylene or C.sub.1-6 heteroalkylene bridge to the
4'-carbon of the same ribose sugar, where exemplary bridges
included methylene, propylene, ether, or amino bridges; aminoalkyl,
as defined herein; aminoalkoxy, as defined herein; amino as defined
herein; and amino acid, as defined herein
[0190] Generally, RNA includes the sugar group ribose, which is a
5-membered ring having an oxygen. Exemplary, non-limiting modified
nucleotides include replacement of the oxygen in ribose (e.g., with
S, Se, or alkylene, such as methylene or ethylene); addition of a
double bond (e.g., to replace ribose with cyclopentenyl or
cyclohexenyl); ring contraction of ribose (e.g., to form a
4-membered ring of cyclobutane or oxetane); ring expansion of
ribose (e.g., to form a 6- or 7-membered ring having an additional
carbon or heteroatom, such as for anhydrohexitol, altritol,
mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has
a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and
"unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or
S-GNA, where ribose is replaced by glycol units attached to
phosphodiester bonds), threose nucleic acid (TNA, where ribose is
replace with .alpha.-L-threofuranosyl-(3'.fwdarw.2')), and peptide
nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the
ribose and phosphodiester backbone). The sugar group can also
contain one or more carbons that possess the opposite
stereochemical configuration than that of the corresponding carbon
in ribose. Thus, a polynucleotide molecule can include nucleotides
containing, e.g., arabinose, as the sugar.
Modifications on the Nucleobase
[0191] The present disclosure provides for modified nucleosides and
nucleotides. As described herein "nucleoside" is defined as a
compound containing a sugar molecule (e.g., a pentose or ribose) or
derivative thereof in combination with an organic base (e.g., a
purine or pyrimidine) or a derivative thereof (also referred to
herein as "nucleobase"). As described herein, "nucleotide" is
defined as a nucleoside including a phosphate group. In some
embodiments, the nucleosides and nucleotides described herein are
generally chemically modified on the major groove face. Exemplary
non-limiting modifications include an amino group, a thiol group,
an alkyl group, a halo group, or any described herein. The modified
nucleotides may by synthesized by any useful method, as described
herein (e.g., chemically, enzymatically, or recombinantly to
include one or more modified or non-natural nucleosides).
[0192] The modified nucleotide base pairing encompasses not only
the standard adenosine-thymine, adenosine-uracil, or
guanosine-cytosine base pairs, but also base pairs formed between
nucleotides and/or modified nucleotides comprising non-standard or
modified bases, wherein the arrangement of hydrogen bond donors and
hydrogen bond acceptors permits hydrogen bonding between a
non-standard base and a standard base or between two complementary
non-standard base structures. One example of such non-standard base
pairing is the base pairing between the modified nucleotide inosine
and adenine, cytosine or uracil.
[0193] The modified nucleosides and nucleotides can include a
modified nucleobase. Examples of nucleobases found in RNA include,
but are not limited to, adenine, guanine, cytosine, and uracil.
Examples of nucleobase found in DNA include, but are not limited
to, adenine, guanine, cytosine, and thymine. These nucleobases can
be modified or wholly replaced to provide polynucleotide molecules
having enhanced properties, e.g., resistance to nucleases,
stability, and these properties may manifest through disruption of
the binding of a major groove binding partner. For example, the
nucleosides and nucleotides described can be chemically modified on
the major groove face. In some embodiments, the major groove
chemical modifications can include an amino group, a thiol group,
an alkyl group, or a halo group.
[0194] Table 1 below identifies the chemical faces of each
canonical nucleotide. Circles identify the atoms comprising the
respective chemical regions.
TABLE-US-00001 TABLE 1 Major Minor Watson-Crick Groove Groove
Base-pairing Face Face Face Pyrim- idines Cy- tidine: ##STR00085##
##STR00086## ##STR00087## Uridine: ##STR00088## ##STR00089##
##STR00090## Purines Adeno- sine: ##STR00091## ##STR00092##
##STR00093## Guano- sine: ##STR00094## ##STR00095##
##STR00096##
[0195] In some embodiments, B is a modified uracil. Exemplary
modified uracils include those having Formula (b1)-(b5):
##STR00097##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0196] is a single or double bond;
[0197] each of T.sup.1, T.sup.1'', T.sup.2', and T.sup.2'' is,
independently, H, optionally substituted alkyl, optionally
substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of T.sup.1' and T.sup.1'' or the combination of
T.sup.2' and T.sup.2'' join together (e.g., as in T.sup.2) to form
O (oxo), S (thio), or Se (seleno);
[0198] each of V.sup.1 and V.sup.2 is, independently, O, S,
N(R.sup.Vb).sub.nv, or C(R.sup.Vb).sub.nv, wherein nv is an integer
from 0 to 2 and each R.sup.Vb is, independently, H, halo,
optionally substituted amino acid, optionally substituted alkyl,
optionally substituted haloalkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted hydroxyalkyl, optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,
optionally substituted aminoalkyl (e.g., substituted with an
N-protecting group, such as any described herein, e.g.,
trifluoroacetyl), optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, optionally substituted acylaminoalkyl
(e.g., substituted with an N-protecting group, such as any
described herein, e.g., trifluoroacetyl), optionally substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,
optionally substituted alkoxycarbonylalkynyl, or optionally
substituted alkoxycarbonylalkoxy (e.g., optionally substituted with
any substituent described herein, such as those selected from
(1)-(21) for alkyl);
[0199] R.sup.10 is H, halo, optionally substituted amino acid,
hydroxy, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
aminoalkyl, optionally substituted hydroxyalkyl, optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,
optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl, optionally substituted alkoxy, optionally substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,
optionally substituted alkoxycarbonylalkynyl, optionally
substituted alkoxycarbonylalkoxy, optionally substituted
carboxyalkoxy, optionally substituted carboxyalkyl, or optionally
substituted carbamoylalkyl;
[0200] R.sup.11 is H or optionally substituted alkyl;
[0201] R.sup.12a is H, optionally substituted alkyl, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl,
optionally substituted hydroxyalkynyl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, or optionally
substituted aminoalkynyl, optionally substituted carboxyalkyl
(e.g., optionally substituted with hydroxy), optionally substituted
carboxyalkoxy, optionally substituted carboxyaminoalkyl, or
optionally substituted carbamoylalkyl; and
[0202] R.sup.12c is H, halo, optionally substituted alkyl,
optionally substituted alkoxy, optionally substituted thioalkoxy,
optionally substituted amino, optionally substituted hydroxyalkyl,
optionally substituted hydroxyalkenyl, optionally substituted
hydroxyalkynyl, optionally substituted aminoalkyl, optionally
substituted aminoalkenyl, or optionally substituted
aminoalkynyl.
[0203] Other exemplary modified uracils include those having
Formula (b6)-(b9):
##STR00098##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0204] is a single or double bond;
[0205] each of T.sup.1', T.sup.1'', T.sup.2', and T.sup.2'' is,
independently, H, optionally substituted alkyl, optionally
substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of T.sup.1' and T.sup.1'' join together (e.g., as in
T.sup.1) or the combination of T.sup.2' and T.sup.2'' join together
(e.g., as in T.sup.2) to form O (oxo), S (thio), or Se (seleno), or
each T.sup.1 and T.sup.2 is, independently, O (oxo), S (thio), or
Se (seleno);
[0206] each of W.sup.1 and W.sup.2 is, independently,
N(R.sup.Wa).sub.nw or C(R.sup.Wa).sub.nw, wherein nw is an integer
from 0 to 2 and each ea is, independently, H, optionally
substituted alkyl, or optionally substituted alkoxy;
[0207] each V.sup.3 is, independently, O, S, N(R.sup.Va).sub.nv, or
C(R.sup.Va).sub.nv, wherein nv is an integer from 0 to 2 and each
R.sup.Va is, independently, H, halo, optionally substituted amino
acid, optionally substituted alkyl, optionally substituted
hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally
substituted hydroxyalkynyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted
heterocyclyl, optionally substituted alkheterocyclyl, optionally
substituted alkoxy, optionally substituted alkenyloxy, or
optionally substituted alkynyloxy, optionally substituted
aminoalkyl (e.g., substituted with an N-protecting group, such as
any described herein, e.g., trifluoroacetyl, or sulfoalkyl),
optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,
substituted with an N-protecting group, such as any described
herein, e.g., trifluoroacetyl), optionally substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,
optionally substituted alkoxycarbonylalkynyl, optionally
substituted alkoxycarbonylacyl, optionally substituted
alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,
optionally substituted with hydroxy and/or an O-protecting group),
optionally substituted carboxyalkoxy, optionally substituted
carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,
optionally substituted with any substituent described herein, such
as those selected from (1)-(21) for alkyl), and wherein R.sup.Va
and R.sup.12c taken together with the carbon atoms to which they
are attached can form optionally substituted cycloalkyl, optionally
substituted aryl, or optionally substituted heterocyclyl (e.g., a
5- or 6-membered ring);
[0208] R.sup.12a is H, optionally substituted alkyl, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl,
optionally substituted hydroxyalkynyl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, optionally substituted carboxyalkyl
(e.g., optionally substituted with hydroxy and/or an O-protecting
group), optionally substituted carboxyalkoxy, optionally
substituted carboxyaminoalkyl, optionally substituted
carbamoylalkyl, or absent;
[0209] R.sup.12b is H, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl,
optionally substituted hydroxyalkynyl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, optionally substituted alkaryl,
optionally substituted heterocyclyl, optionally substituted
alkheterocyclyl, optionally substituted amino acid, optionally
substituted alkoxycarbonylacyl, optionally substituted
alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl,
optionally substituted alkoxycarbonylalkenyl, optionally
substituted alkoxycarbonylalkynyl, optionally substituted
alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,
optionally substituted with hydroxy and/or an O-protecting group),
optionally substituted carboxyalkoxy, optionally substituted
carboxyaminoalkyl, or optionally substituted carbamoylalkyl,
[0210] wherein the combination of R.sup.12b and T.sup.1' or the
combination of R.sup.12b and R.sup.12c can join together to form
optionally substituted heterocyclyl; and
[0211] R.sup.12c is H, halo, optionally substituted alkyl,
optionally substituted alkoxy, optionally substituted thioalkoxy,
optionally substituted amino, optionally substituted aminoalkyl,
optionally substituted aminoalkenyl, or optionally substituted
aminoalkynyl.
[0212] Further exemplary modified uracils include those having
Formula (b28)-(b31):
##STR00099##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0213] each of T.sup.1 and T.sup.2 is, independently, O (oxo), S
(thio), or Se (seleno);
[0214] each R.sup.Vb' and R.sup.Vb'' is, independently, H, halo,
optionally substituted amino acid, optionally substituted alkyl,
optionally substituted haloalkyl, optionally substituted
hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally
substituted hydroxyalkynyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkyl (e.g., substituted
with an N-protecting group, such as any described herein, e.g.,
trifluoroacetyl, or sulfoalkyl), optionally substituted
aminoalkenyl, optionally substituted aminoalkynyl, optionally
substituted acylaminoalkyl (e.g., substituted with an N-protecting
group, such as any described herein, e.g., trifluoroacetyl),
optionally substituted alkoxycarbonylalkyl, optionally substituted
alkoxycarbonylalkenyl, optionally substituted
alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl,
optionally substituted alkoxycarbonylalkoxy, optionally substituted
carboxyalkyl (e.g., optionally substituted with hydroxy and/or an
O-protecting group), optionally substituted carboxyalkoxy,
optionally substituted carboxyaminoalkyl, or optionally substituted
carbamoylalkyl (e.g., optionally substituted with any substituent
described herein, such as those selected from (1)-(21) for alkyl)
(e.g., R.sup.Vb' is optionally substituted alkyl, optionally
substituted alkenyl, or optionally substituted aminoalkyl, e.g.,
substituted with an N-protecting group, such as any described
herein, e.g., trifluoroacetyl, or sulfoalkyl);
[0215] R.sup.12a is H, optionally substituted alkyl, optionally
substituted carboxyaminoalkyl, optionally substituted aminoalkyl
(e.g., e.g., substituted with an N-protecting group, such as any
described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally
substituted aminoalkenyl, or optionally substituted aminoalkynyl;
and
[0216] R.sup.12b is H, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl,
optionally substituted hydroxyalkynyl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl (e.g., e.g., substituted with an
N-protecting group, such as any described herein, e.g.,
trifluoroacetyl, or sulfoalkyl), optionally substituted
alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy,
optionally substituted alkoxycarbonylalkyl, optionally substituted
alkoxycarbonylalkenyl, optionally substituted
alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,
optionally substituted carboxyalkoxy, optionally substituted
carboxyalkyl, or optionally substituted carbamoylalkyl.
[0217] In particular embodiments, T.sup.1 is O (oxo), and T.sup.2
is S (thio) or Se (seleno). In other embodiments, T.sup.1 is S
(thio), and T.sup.2 is O (oxo) or Se (seleno). In some embodiments,
R.sup.Vb' is H, optionally substituted alkyl, or optionally
substituted alkoxy.
[0218] In other embodiments, each R.sup.12a and R.sup.12b is,
independently, H, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, or optionally
substituted hydroxyalkyl. In particular embodiments, R.sup.12a is
H. In other embodiments, both R.sup.12a and R.sup.12b are H.
[0219] In some embodiments, each R.sup.Vb' of R.sup.12b is,
independently, optionally substituted aminoalkyl (e.g., substituted
with an N-protecting group, such as any described herein, e.g.,
trifluoroacetyl, or sulfoalkyl), optionally substituted
aminoalkenyl, optionally substituted aminoalkynyl, or optionally
substituted acylaminoalkyl (e.g., substituted with an N-protecting
group, such as any described herein, e.g., trifluoroacetyl). In
some embodiments, the amino and/or alkyl of the optionally
substituted aminoalkyl is substituted with one or more of
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted sulfoalkyl, optionally substituted carboxy
(e.g., substituted with an O-protecting group), optionally
substituted hydroxy (e.g., substituted with an O-protecting group),
optionally substituted carboxyalkyl (e.g., substituted with an
O-protecting group), optionally substituted alkoxycarbonylalkyl
(e.g., substituted with an O-protecting group), or N-protecting
group. In some embodiments, optionally substituted aminoalkyl is
substituted with an optionally substituted sulfoalkyl or optionally
substituted alkenyl. In particular embodiments, R.sup.12a and
R.sup.Vb'' are both H. In particular embodiments, T.sup.1 is O
(oxo), and T.sup.2 is S (thio) or Se (seleno).
[0220] In some embodiments, R is optionally substituted
alkoxycarbonylalkyl or optionally substituted carbamoylalkyl.
[0221] In particular embodiments, the optional substituent for
R.sup.12a, R.sup.12b, R.sup.12c, or R.sup.Va is a polyethylene
glycol group (e.g.,
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl); or an
amino-polyethylene glycol group (e.g.,
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl).
[0222] In some embodiments, B is a modified cytosine. Exemplary
modified cytosines include compounds of Formula (b10)-(b14):
##STR00100##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0223] each of T.sup.3' and T.sup.3'' is, independently, H,
optionally substituted alkyl, optionally substituted alkoxy, or
optionally substituted thioalkoxy, or the combination of T.sup.3'
and T.sup.3'' join together (e.g., as in T.sup.3) to form O (oxo),
S (thio), or Se (seleno);
[0224] each V.sup.4 is, independently, O, S, N(R.sup.Vc).sub.nv, or
C(R.sup.Vc).sub.nv, wherein nv is an integer from 0 to 2 and each
R.sup.Vc is, independently, H, halo, optionally substituted amino
acid, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
heterocyclyl, optionally substituted alkheterocyclyl, or optionally
substituted alkynyloxy (e.g., optionally substituted with any
substituent described herein, such as those selected from (1)-(21)
for alkyl), wherein the combination of R.sup.13b and R.sup.Vc can
be taken together to form optionally substituted heterocyclyl;
[0225] each V.sup.5 is, independently, N(R.sup.Vd).sub.nv, or
C(R.sup.Vd).sub.nv, wherein nv is an integer from 0 to 2 and each
R.sup.Vd is, independently, H, halo, optionally substituted amino
acid, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, optionally substituted
heterocyclyl, optionally substituted alkheterocyclyl, or optionally
substituted alkynyloxy (e.g., optionally substituted with any
substituent described herein, such as those selected from (1)-(21)
for alkyl) (e.g., V.sup.5 is --CH or N);
[0226] each of R.sup.13a and R.sup.13b is, independently, H,
optionally substituted acyl, optionally substituted acyloxyalkyl,
optionally substituted alkyl, or optionally substituted alkoxy,
wherein the combination of R.sup.13b and R.sup.14 can be taken
together to form optionally substituted heterocyclyl;
[0227] each R.sup.14 is, independently, H, halo, hydroxy, thiol,
optionally substituted acyl, optionally substituted amino acid,
optionally substituted alkyl, optionally substituted haloalkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted hydroxyalkyl (e.g., substituted with an
O-protecting group), optionally substituted hydroxyalkenyl,
optionally substituted hydroxyalkynyl, optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted alkoxyalkoxy, optionally substituted acyloxyalkyl,
optionally substituted amino (e.g., --NHR, wherein R is H, alkyl,
aryl, or phosphoryl), azido, optionally substituted aryl,
optionally substituted heterocyclyl, optionally substituted
alkheterocyclyl, optionally substituted aminoalkyl, optionally
substituted aminoalkenyl, or optionally substituted aminoalkynyl;
and
[0228] each of R.sup.15 and R.sup.16 is, independently, H,
optionally substituted alkyl, optionally substituted alkenyl, or
optionally substituted alkynyl.
[0229] Further exemplary modified cytosines include those having
Formula (b32)-(b35):
##STR00101##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0230] each of T.sup.1 and T.sup.3 is, independently, O (oxo), S
(thio), or Se (seleno);
[0231] each of R.sup.13a and R.sup.13b is, independently, H,
optionally substituted acyl, optionally substituted acyloxyalkyl,
optionally substituted alkyl, or optionally substituted alkoxy,
wherein the combination of R.sup.13b and R.sup.14 can be taken
together to form optionally substituted heterocyclyl;
[0232] each R.sup.14 is, independently, H, halo, hydroxy, thiol,
optionally substituted acyl, optionally substituted amino acid,
optionally substituted alkyl, optionally substituted haloalkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted hydroxyalkyl (e.g., substituted with an
O-protecting group), optionally substituted hydroxyalkenyl,
optionally substituted hydroxyalkynyl, optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted alkoxyalkoxy, optionally substituted acyloxyalkyl,
optionally substituted amino (e.g., --NHR, wherein R is H, alkyl,
aryl, or phosphoryl), azido, optionally substituted aryl,
optionally substituted heterocyclyl, optionally substituted
alkheterocyclyl, optionally substituted aminoalkyl (e.g.,
hydroxyalkyl, alkyl, alkenyl, or alkynyl), optionally substituted
aminoalkenyl, or optionally substituted aminoalkynyl; and
[0233] each of R.sup.15 and R.sup.16 is, independently, H,
optionally substituted alkyl, optionally substituted alkenyl, or
optionally substituted alkynyl (e.g., R.sup.15 is H, and R.sup.16
is H or optionally substituted alkyl).
[0234] In some embodiments, R.sup.15 is H, and R.sup.16 is H or
optionally substituted alkyl. In particular embodiments, R.sup.14
is H, acyl, or hydroxyalkyl. In some embodiments, R.sup.14 is halo.
In some embodiments, both R.sup.14 and R.sup.15 are H. In some
embodiments, both R.sup.15 and R.sup.16 are H. In some embodiments,
each of R.sup.14 and R.sup.15 and R.sup.16 is H. In further
embodiments, each of R.sup.13a and R.sup.13b is independently, H or
optionally substituted alkyl.
[0235] Further non-limiting examples of modified cytosines include
compounds of Formula (b36):
##STR00102##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0236] each R.sup.13b is, independently, H, optionally substituted
acyl, optionally substituted acyloxyalkyl, optionally substituted
alkyl, or optionally substituted alkoxy, wherein the combination of
R.sup.13b and R.sup.14b can be taken together to form optionally
substituted heterocyclyl;
[0237] each R.sup.14a and R.sup.14b is, independently, H, halo,
hydroxy, thiol, optionally substituted acyl, optionally substituted
amino acid, optionally substituted alkyl, optionally substituted
haloalkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted hydroxyalkyl (e.g., substituted
with an O-protecting group), optionally substituted hydroxyalkenyl,
optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally substituted alkynyloxy, optionally substituted
aminoalkoxy, optionally substituted alkoxyalkoxy, optionally
substituted acyloxyalkyl, optionally substituted amino (e.g.,
--NHR, wherein R is H, alkyl, aryl, phosphoryl, optionally
substituted aminoalkyl, or optionally substituted
carboxyaminoalkyl), azido, optionally substituted aryl, optionally
substituted heterocyclyl, optionally substituted alkheterocyclyl,
optionally substituted aminoalkyl, optionally substituted
aminoalkenyl, or optionally substituted aminoalkynyl; and
[0238] each of R.sup.15 is, independently, H, optionally
substituted alkyl, optionally substituted alkenyl, or optionally
substituted alkynyl.
[0239] In particular embodiments, R.sup.14b is an optionally
substituted amino acid (e.g., optionally substituted lysine). In
some embodiments, R.sup.14a is H.
[0240] In some embodiments, B is a modified guanine. Exemplary
modified guanines include compounds of Formula (b15)-(b17):
##STR00103##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0241] Each of T.sup.4', T.sup.4'', T.sup.5', T.sup.5'', T.sup.6',
and T.sup.6'' is, independently, H, optionally substituted alkyl,
or optionally substituted alkoxy, and wherein the combination of
T.sup.4' and T.sup.4'' (e.g., as in T.sup.4) or the combination of
T.sup.5' and T.sup.5'' (e.g., as in T.sup.5) or the combination of
T.sup.6' and T.sup.6'' join together (e.g., as in T.sup.6) form O
(oxo), S (thio), or Se (seleno);
[0242] each of V.sup.5 and V.sup.6 is, independently, O, S,
N(R.sup.Vd).sub.nv, or C(R.sup.Vd).sub.nv, wherein nv is an integer
from 0 to 2 and each R.sup.Vd is, independently, H, halo, thiol,
optionally substituted amino acid, cyano, amidine, optionally
substituted aminoalkyl, optionally substituted aminoalkenyl,
optionally substituted aminoalkynyl, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally substituted alkynyloxy (e.g., optionally substituted
with any substituent described herein, such as those selected from
(1)-(21) for alkyl), optionally substituted thioalkoxy, or
optionally substituted amino; and
[0243] each of R.sup.17, R.sup.18, R.sup.19a, R.sup.19b, R.sup.21,
R.sup.22, R.sup.23, and R.sup.24 is, independently, H, halo, thiol,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted thioalkoxy,
optionally substituted amino, or optionally substituted amino
acid.
[0244] Exemplary modified guanosines include compounds of Formula
(b37)-(b40):
##STR00104##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0245] each of T.sup.4' is, independently, H, optionally
substituted alkyl, or optionally substituted alkoxy, and each
T.sup.4 is, independently, O (oxo), S (thio), or Se (seleno);
[0246] each of R.sup.18, R.sup.19a, R.sup.19b, and R.sup.21 is,
independently, H, halo, thiol, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted thioalkoxy, optionally substituted amino, or
optionally substituted amino acid.
[0247] In some embodiments, R.sup.18 is H or optionally substituted
alkyl. In further embodiments, T.sup.4 is oxo. In some embodiments,
each of R.sup.19a and R.sup.19b is, independently, H or optionally
substituted alkyl.
[0248] In some embodiments, B is a modified adenine. Exemplary
modified adenines include compounds of Formula (b18)-(b20):
##STR00105##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0249] each V.sup.7 is, independently, O, S, N(R.sup.Ve).sub.nv, or
C(R.sup.Ve).sub.nv, wherein nv is an integer from 0 to 2 and each
R.sup.Ve is, independently, H, halo, optionally substituted amino
acid, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy,
optionally substituted alkenyloxy, or optionally substituted
alkynyloxy (e.g., optionally substituted with any substituent
described herein, such as those selected from (1)-(21) for
alkyl);
[0250] each R.sup.25 is, independently, H, halo, thiol, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted thioalkoxy, or
optionally substituted amino;
[0251] each of R.sup.26a and R.sup.26b is, independently, H,
optionally substituted acyl, optionally substituted amino acid,
optionally substituted carbamoylalkyl, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted hydroxyalkyl, optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,
optionally substituted alkoxy, or polyethylene glycol group (e.g.,
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl); or an
amino-polyethylene glycol group (e.g.,
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl);
[0252] each R.sup.27 is, independently, H, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted alkoxy, optionally substituted
thioalkoxy, or optionally substituted amino;
[0253] each R.sup.28 is, independently, H, optionally substituted
alkyl, optionally substituted alkenyl, or optionally substituted
alkynyl; and
[0254] each R.sup.29 is, independently, H, optionally substituted
acyl, optionally substituted amino acid, optionally substituted
carbamoylalkyl, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl,
optionally substituted alkoxy, or optionally substituted amino.
[0255] Exemplary modified adenines include compounds of Formula
(b41)-(b43):
##STR00106##
or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
[0256] each R.sup.25 is, independently, H, halo, thiol, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted thioalkoxy, or
optionally substituted amino;
[0257] each of R.sup.26a and R.sup.26b is, independently, H,
optionally substituted acyl, optionally substituted amino acid,
optionally substituted carbamoylalkyl, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted hydroxyalkyl, optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,
optionally substituted alkoxy, or polyethylene glycol group (e.g.,
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl); or an
amino-polyethylene glycol group (e.g.,
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl); and
[0258] each R.sup.27 is, independently, H, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted alkoxy, optionally substituted
thioalkoxy, or optionally substituted amino.
[0259] In some embodiments, R.sup.26a is H, and R.sup.26b is
optionally substituted alkyl. In some embodiments, each of
R.sup.26a and R.sup.26b is, independently, optionally substituted
alkyl. In particular embodiments, R.sup.27 is optionally
substituted alkyl, optionally substituted alkoxy, or optionally
substituted thioalkoxy. In other embodiments, R.sup.25 is
optionally substituted alkyl, optionally substituted alkoxy, or
optionally substituted thioalkoxy.
[0260] In particular embodiments, the optional substituent for
R.sup.26a, R.sup.26b, or R.sup.29 is a polyethylene glycol group
(e.g.,
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl); or an
amino-polyethylene glycol group (e.g.,
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl).
[0261] In some embodiments, B may have Formula (b21):
##STR00107##
wherein X.sup.12 is, independently, O, S, optionally substituted
alkylene (e.g., methylene), or optionally substituted
heteroalkylene, xa is an integer from 0 to 3, and R.sup.12a and
T.sup.2 are as described herein.
[0262] In some embodiments, B may have Formula (b22):
##STR00108##
wherein R.sup.10' is, independently, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted heterocyclyl,
optionally substituted aminoalkyl, optionally substituted
aminoalkenyl, optionally substituted aminoalkynyl, optionally
substituted alkoxy, optionally substituted alkoxycarbonylalkyl,
optionally substituted alkoxycarbonylalkenyl, optionally
substituted alkoxycarbonylalkynyl, optionally substituted
alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy,
optionally substituted carboxyalkyl, or optionally substituted
carbamoylalkyl, and R.sup.11, R.sup.12a, T.sup.1, and T.sup.2 are
as described herein.
[0263] In some embodiments, B may have Formula (b23):
##STR00109##
wherein R.sup.10 is optionally substituted heterocyclyl (e.g.,
optionally substituted furyl, optionally substituted thienyl, or
optionally substituted pyrrolyl), optionally substituted aryl
(e.g., optionally substituted phenyl or optionally substituted
naphthyl), or any substituent described herein (e.g., for)
R.sup.10; and wherein R.sup.11 (e.g., H or any substituent
described herein), R.sup.12a (e.g., H or any substituent described
herein), T.sup.1 (e.g., oxo or any substituent described herein),
and T.sup.2 (e.g., oxo or any substituent described herein) are as
described herein.
[0264] In some embodiments, B may have Formula (b24):
##STR00110##
wherein R.sup.14' is, independently, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted heterocyclyl,
optionally substituted alkaryl, optionally substituted
alkheterocyclyl, optionally substituted aminoalkyl, optionally
substituted aminoalkenyl, optionally substituted aminoalkynyl,
optionally substituted alkoxy, optionally substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,
optionally substituted alkoxycarbonylalkynyl, optionally
substituted alkoxycarbonylalkoxy, optionally substituted
carboxyalkoxy, optionally substituted carboxyalkyl, or optionally
substituted carbamoylalkyl, and R.sup.13a, R.sup.13b, R.sup.15, and
T.sup.3 are as described herein.
[0265] In some embodiments, B may have Formula (b25):
##STR00111##
wherein R.sup.14' is optionally substituted heterocyclyl (e.g.,
optionally substituted furyl, optionally substituted thienyl, or
optionally substituted pyrrolyl), optionally substituted aryl
(e.g., optionally substituted phenyl or optionally substituted
naphthyl), or any substituent described herein (e.g., for R.sup.14
or R.sup.14'); and wherein R.sup.13a (e.g., H or any substituent
described herein), R.sup.13b (e.g., H or any substituent described
herein), R.sup.15 (e.g., H or any substituent described herein),
and T.sup.3 (e.g., oxo or any substituent described herein) are as
described herein.
[0266] In some embodiments, B is a nucleobase selected from the
group consisting of cytosine, guanine, adenine, and uracil. In some
embodiments, B may be:
##STR00112##
[0267] In some embodiments, the modified nucleobase is a modified
uracil. Exemplary nucleobases and nucleosides having a modified
uracil include pseudouridine (.psi.), pyridin-4-one ribonucleoside,
5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine
(s.sup.2U), 4-thio-uridine (s.sup.4U), 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxy-uridine (ho.sup.5U),
5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or
5-bromo-uridine), 3-methyl-uridine (m.sup.3U), 5-methoxy-uridine
(mo.sup.5U), uridine 5-oxyacetic acid (cmo.sup.5U), uridine
5-oxyacetic acid methyl ester (mcmo.sup.5U),
5-carboxymethyl-uridine (cm.sup.5U), 1-carboxymethyl-pseudouridine,
5-carboxyhydroxymethyl-uridine (chm.sup.5U),
5-carboxyhydroxymethyl-uridine methyl ester (mchm.sup.5U),
5-methoxycarbonylmethyl-uridine (mcm.sup.5U),
5-methoxycarbonylmethyl-2-thio-uridine (mcm.sup.5s.sup.2U),
5-aminomethyl-2-thio-uridine (nm.sup.5s.sup.2U),
5-methylaminomethyl-uridine (mnm.sup.5U),
5-methylaminomethyl-2-thio-uridine (mnm.sup.5s.sup.2U),
5-methylaminomethyl-2-seleno-uridine (mnm.sup.5se.sup.2U),
5-carbamoylmethyl-uridine (ncm.sup.5U),
5-carboxymethylaminomethyl-uridine (cmnm.sup.5U),
5-carboxymethylaminomethyl-2-thio-uridine (cmnm.sup.5s.sup.2U),
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyl-uridine (mcm.sup.5U),
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine
(.tau.m.sup.5s.sup.2U), 1-taurinomethyl-4-thio-pseudouridine,
5-methyl-uridine (m.sup.5U, i.e., having the nucleobase
deoxythymine), 1-methyl-pseudouridine (m.sup.1.psi.),
5-methyl-2-thio-uridine (m.sup.5s.sup.2U),
1-methyl-4-thio-pseudouridine) (m.sup.1s.sup.4.psi.),
4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine
(m.sup.3.psi.), 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),
dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine
(m.sup.5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine,
2-methoxy-uridine, 2-methoxy-4-thio-uridine,
4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,
N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine
(acp.sup.3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine
(acp.sup.3.psi., 5-(isopentenylaminomethyl)uridine (inm.sup.5U),
5-(isopentenylaminomethyl)-2-thio-uridine (inm.sup.5s.sup.2U),
.alpha.-thio-uridine, 2'-O-methyl-uridine (Um),
5,2'-0-dimethyl-uridine (m.sup.5Um), 2'-O-methyl-pseudouridine
(.psi.m), 2-thio-2'-O-methyl-uridine (s.sup.2Um),
5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm.sup.5Um),
5-carbamoylmethyl-2'-O-methyl-uridine (ncm.sup.5Um),
5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm.sup.5Um),
3,2'-O-dimethyl-uridine (m.sup.3Um), and
5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm.sup.5Um),
1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine,
2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and
5-[3-(1-E-propenylamino)uridine.
[0268] In some embodiments, the modified nucleobase is a modified
cytosine. Exemplary nucleobases and nucleosides having a modified
cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine (m.sup.3C), N4-acetyl-cytidine (act),
5-formyl-cytidine (f.sup.5C), N4-methyl-cytidine (m.sup.4C),
5-methyl-cytidine (m.sup.5C), 5-halo-cytidine (e.g.,
5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm.sup.5C),
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine (s.sup.2C),
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,
lysidine (k.sub.2C), .alpha.-thio-cytidine, 2'-O-methyl-cytidine
(Cm), 5,2'-O-dimethyl-cytidine (m.sup.5Cm),
N4-acetyl-2'-O-methyl-cytidine (ac.sup.4Cm),
N4,2'-O-dimethyl-cytidine (m.sup.4Cm),
5-formyl-2'-O-methyl-cytidine (f.sup.5Cm),
N4,N4,2'-O-trimethyl-cytidine (m.sup.4.sub.2Cm), 1-thio-cytidine,
2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.
[0269] In some embodiments, the modified nucleobase is a modified
adenine. Exemplary nucleobases and nucleosides having a modified
adenine include 2-amino-purine, 2, 6-diaminopurine,
2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine),
6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine,
8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine,
7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine,
7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,
1-methyl-adenosine (m'A), 2-methyl-adenine (m.sup.2A),
N6-methyl-adenosine (m.sup.6A), 2-methylthio-N6-methyl-adenosine
(ms.sup.2 m.sup.6A), N6-isopentenyl-adenosine (i.sup.6A),
2-methylthio-N6-isopentenyl-adenosine (ms.sup.2i.sup.6A),
N6-(cis-hydroxyisopentenyl)adenosine (io.sup.6A),
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine
(ms.sup.2io.sup.6A), N6-glycinylcarbamoyl-adenosine (g.sup.6A),
N6-threonylcarbamoyl-adenosine (t.sup.6A),
N6-methyl-N6-threonylcarbamoyl-adenosine (m.sup.6.sub.2A),
2-methylthio-N6-threonylcarbamoyl-adenosine (ms.sup.2g.sup.6A),
N6,N6-dimethyl-adenosine (m.sup.6.sub.2A),
N6-hydroxynorvalylcarbamoyl-adenosine (hn.sup.6A),
2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine
(ms.sup.2hn.sup.6A), N6-acetyl-adenosine (ac.sup.6A),
7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine,
.alpha.-thio-adenosine, 2'-O-methyl-adenosine (Am),
N6,2'-O-dimethyl-adenosine (m.sup.6Am),
N6,N6,2'-O-trimethyl-adenosine (m.sup.6.sub.2Am),
1,2'-O-dimethyl-adenosine (m.sup.1Am), 2'-O-ribosyladenosine
(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,
8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine,
2'-OH-ara-adenosine, and
N6-(19-amino-pentaoxanonadecyl)-adenosine.
[0270] In some embodiments, the modified nucleobase is a modified
guanine. Exemplary nucleobases and nucleosides having a modified
guanine include inosine (I), 1-methyl-inosine (m.sup.iI), wyosine
(imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14),
isowyosine (imG2), wybutosine (yW), peroxywybutosine (o.sub.2yW),
hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*),
7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ),
galactosyl-queuosine (galQ), mannosyl-queuosine (manQ),
7-cyano-7-deaza-guanosine (preQ.sub.0),
7-aminomethyl-7-deaza-guanosine (preQ.sub.1), archaeosine
(G.sup.+), 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine (m.sup.7G), 6-thio-7-methyl-guanosine,
7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m'G),
N2-methyl-guanosine (m.sup.2G), N2,N2-dimethyl-guanosine
(m.sup.2.sub.2G), N2,7-dimethyl-guanosine (m.sup.2,7G), N2,
N2,7-dimethyl-guanosine (m.sup.2,2,7G), 8-oxo-guanosine,
7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,
N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,
.alpha.-thio-guanosine, 2'-O-methyl-guanosine (Gm),
N2-methyl-2'-O-methyl-guanosine (m.sup.2Gm),
N2,N2-dimethyl-2'-O-methyl-guanosine (m.sup.2.sub.2Gm),
1-methyl-2'-O-methyl-guanosine (m.sup.1Gm),
N2,7-dimethyl-2'-O-methyl-guanosine (m.sup.2'.sup.7Gm),
2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m'Im),
2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine,
O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.
[0271] In some embodiments, the nucleotide can be modified on the
major groove face. For example, such modifications include
replacing hydrogen on C-5 of uracil or cytosine with alkyl (e.g.,
methyl) or halo.
[0272] The nucleobase of the nucleotide can be independently
selected from a purine, a pyrimidine, a purine or pyrimidine
analog. For example, the nucleobase can each be independently
selected from adenine, cytosine, guanine, uracil, or hypoxanthine.
In another embodiment, the nucleobase can also include, for
example, naturally-occurring and synthetic derivatives of a base,
including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil
and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine,
7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine,
3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5
triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines,
thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine,
pyridazine; and 1,3,5 triazine. When the nucleotides are depicted
using the shorthand A, G, C, T or U, each letter refers to the
representative base and/or derivatives thereof, e.g., A includes
adenine or adenine analogs, e.g., 7-deaza adenine).
[0273] In some embodiments, the modified nucleotide is a compound
of Formula XI:
##STR00113##
[0274] wherein:
[0275] denotes a single or a double bond;
[0276] denotes an optional single bond;
[0277] U is O, S, --NR.sup.a--, or --CR.sup.aR.sup.b-- when denotes
a single bond, or U is --CR.sup.a-- when denotes a double bond;
[0278] Z is H, C.sub.1-12 alkyl, or C.sub.6-20 aryl, or Z is absent
when denotes a double bond; and
[0279] Z can be --CR.sup.aR.sup.b-- and form a bond with A;
[0280] A is H, OH, NHR wherein R=alkyl or aryl or phosphoryl,
sulfate, --NH.sub.2, N.sub.3, azido, --SH, N an amino acid, or a
peptide comprising 1 to 12 amino acids;
[0281] D is H, OH, NHR wherein R=alkyl or aryl or phosphoryl,
--NH.sub.2, --SH, an amino acid, a peptide comprising 1 to 12 amino
acids, or a group of Formula XII:
##STR00114##
[0282] or A and D together with the carbon atoms to which they are
attached form a 5-membered ring;
[0283] X is O or S;
[0284] each of Y.sup.1 is independently selected from and --Se;
[0285] each of Y.sup.2 and Y.sup.3 are independently selected from
O, --CR.sup.aR.sup.b--, NR.sup.c, S or a linker comprising one or
more atoms selected from the group consisting of C, O, N, and
S;
[0286] n is 0, 1, 2, or 3;
[0287] m is 0, 1, 2 or 3;
[0288] B is nucleobase;
[0289] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20
aryl;
[0290] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group;
[0291] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0292] --OR.sup.c1 is OH at a pH of about 1 or --OR.sup.c1 is
O.sup.- at physiological pH;
[0293] provided that the ring encompassing the variables A, B, D,
U, Z, Y.sup.2 and Y.sup.3 cannot be ribose.
[0294] In some embodiments, B is a nucleobase selected from the
group consisting of cytosine, guanine, adenine, and uracil.
[0295] In some embodiments, the nucleobase is a pyrimidine or
derivative thereof.
[0296] In some embodiments, the modified nucleotides are a compound
of Formula XI-a:
##STR00115##
[0297] In some embodiments, the modified nucleotides are a compound
of Formula XI-b:
##STR00116##
[0298] In some embodiments, the modified nucleotides are a compound
of Formula XI-c1, XI-c2, or XI-c3:
##STR00117##
[0299] In some embodiments, the modified nucleotides are a compound
of Formula XI:
##STR00118##
[0300] wherein:
[0301] denotes a single or a double bond;
[0302] denotes an optional single bond;
[0303] U is O, S, --NR.sup.a--, or --CR.sup.aR.sup.b-- when denotes
a single bond, or U is --CR.sup.a-- when denotes a double bond;
[0304] Z is H, C.sub.1-12 alkyl, or C.sub.6-20 aryl, or Z is absent
when denotes a double bond; and
[0305] Z can be --CR.sup.aR.sup.b-- and form a bond with A;
[0306] A is H, OH, sulfate, --NH.sub.2, --SH, an amino acid, or a
peptide comprising 1 to 12 amino acids;
[0307] D is H, OH, --NH.sub.2, --SH, an amino acid, a peptide
comprising 1 to 12 amino acids, or a group of Formula XII:
##STR00119##
[0308] or A and D together with the carbon atoms to which they are
attached form a 5-membered ring;
[0309] X is O or S;
[0310] each of Y.sup.1 is independently selected from and --Se;
[0311] each of Y.sup.2 and Y.sup.3 are independently selected from
O, --CR.sup.aR.sup.b--, NR.sup.c, S or a linker comprising one or
more atoms selected from the group consisting of C, O, N, and
S;
[0312] n is 0, 1, 2, or 3;
[0313] m is 0, 1, 2 or 3;
[0314] B is a nucleobase of Formula XIII:
##STR00120##
[0315] wherein:
[0316] V is N or positively charged NR.sup.c;
[0317] R.sup.3 is NR.sup.cR.sup.d, --OR.sup.a, or --SR.sup.a;
[0318] R.sup.4 is H or can optionally form a bond with Y.sup.3;
[0319] R.sup.5 is H, --NR.sup.cR.sup.d, or --OR.sup.a;
[0320] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20
aryl;
[0321] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group;
[0322] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0323] --OR.sup.c1 is OH at a pH of about 1 or --OR.sup.c1 is
O.sup.- at physiological pH.
[0324] In some embodiments, B is:
##STR00121##
[0325] wherein R.sup.3 is --OH, --SH, or
##STR00122##
[0326] In some embodiments, B is:
##STR00123##
[0327] In some embodiments, B is:
##STR00124##
[0328] In some embodiments, the modified nucleotides are a compound
of Formula I-d:
##STR00125##
[0329] In some embodiments, the modified nucleotides are a compound
selected from the group consisting of:
##STR00126## ##STR00127## ##STR00128##
or a pharmaceutically acceptable salt thereof.
[0330] In some embodiments, the modified nucleotides are a compound
selected from the group consisting of:
##STR00129## ##STR00130##
or a pharmaceutically acceptable salt thereof.
Modifications on the Internucleoside Linkage
[0331] The modified nucleotides, which may be incorporated into a
polynucleotide molecule, can be modified on the internucleoside
linkage (e.g., phosphate backbone). Herein, in the context of the
polynucleotide backbone, the phrases "phosphate" and
"phosphodiester" are used interchangeably. Backbone phosphate
groups can be modified by replacing one or more of the oxygen atoms
with a different substituent. Further, the modified nucleosides and
nucleotides can include the wholesale replacement of an unmodified
phosphate moiety with another internucleoside linkage as described
herein. Examples of modified phosphate groups include, but are not
limited to, phosphorothioate, phosphoroselenates, boranophosphates,
boranophosphate esters, hydrogen phosphonates, phosphoramidates,
phosphorodiamidates, alkyl or aryl phosphonates, and
phosphotriesters. Phosphorodithioates have both non-linking oxygens
replaced by sulfur. The phosphate linker can also be modified by
the replacement of a linking oxygen with nitrogen (bridged
phosphoramidates), sulfur (bridged phosphorothioates), and carbon
(bridged methylene-phosphonates).
[0332] The .alpha.-thio substituted phosphate moiety is provided to
confer stability to RNA and DNA polymers through the unnatural
phosphorothioate backbone linkages. Phosphorothioate DNA and RNA
have increased nuclease resistance and subsequently a longer
half-life in a cellular environment. While not wishing to be bound
by theory, phosphorothioate linked polynucleotide molecules are
expected to also reduce the innate immune response through weaker
binding/activation of cellular innate immune molecules.
[0333] In specific embodiments, a modified nucleoside includes an
alpha-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine,
5'-O-(1-thiophosphate)-cytidine (.alpha.-thio-cytidine),
5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine,
or 5'-O-(1-thiophosphate)-pseudouridine).
[0334] Other internucleoside linkages that may be employed
according to the present invention, including internucleoside
linkages which do not contain a phosphorous atom, are described
herein below.
Combinations of Modified Sugars, Nucleobases, and Internucleoside
Linkages
[0335] The polynucleotides of the invention can include a
combination of modifications to the sugar, the nucleobase, and/or
the internucleoside linkage. These combinations can include any one
or more modifications described herein. For examples, any of the
nucleotides described herein in Formulas (Ia), (Ia-1)-(Ia-3),
(Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IVl), and (IXa)-(IXr) can be combined with any of
the nucleobases described herein (e.g., in Formulas (b1)-(b43) or
any other described herein).
Synthesis of Polynucleotide Molecules
[0336] The polynucleotide molecules for use in accordance with the
invention may be prepared according to any useful technique, as
described herein. The modified nucleosides and nucleotides used in
the synthesis of polynucleotide molecules disclosed herein can be
prepared from readily available starting materials using the
following general methods and procedures. Where typical or
preferred process conditions (e.g., reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are provided,
a skilled artisan would be able to optimize and develop additional
process conditions. Optimum reaction conditions may vary with the
particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization
procedures.
[0337] The processes described herein can be monitored according to
any suitable method known in the art. For example, product
formation can be monitored by spectroscopic means, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C)
infrared spectroscopy, spectrophotometry (e.g., UV-visible), or
mass spectrometry, or by chromatography such as high performance
liquid chromatography (HPLC) or thin layer chromatography.
[0338] Preparation of polynucleotide molecules of the present
invention can involve the protection and deprotection of various
chemical groups. The need for protection and deprotection, and the
selection of appropriate protecting groups can be readily
determined by one skilled in the art. The chemistry of protecting
groups can be found, for example, in Greene, et al., Protective
Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which
is incorporated herein by reference in its entirety.
[0339] The reactions of the processes described herein can be
carried out in suitable solvents, which can be readily selected by
one of skill in the art of organic synthesis. Suitable solvents can
be substantially nonreactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, i.e., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected.
[0340] Resolution of racemic mixtures of modified polynucleotides
or nucleic acids (e.g., polynucleotides or modified mRNA molecules)
can be carried out by any of numerous methods known in the art. An
example method includes fractional recrystallization using a
"chiral resolving acid" which is an optically active, salt-forming
organic acid. Suitable resolving agents for fractional
recrystallization methods are, for example, optically active acids,
such as the D and L forms of tartaric acid, diacetyltartaric acid,
dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or
the various optically active camphorsulfonic acids. Resolution of
racemic mixtures can also be carried out by elution on a column
packed with an optically active resolving agent (e.g.,
dinitrobenzoylphenylglycine). Suitable elution solvent composition
can be determined by one skilled in the art.
[0341] Modified nucleosides and nucleotides (e.g., building block
molecules) can be prepared according to the synthetic methods
described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009);
Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et
al., Biochemistry, 1(4): 563-568 (1962); and Xu et al.,
Tetrahedron, 48(9): 1729-1740 (1992), each of which are
incorporated by reference in their entirety.
[0342] The polynucleotides of the invention may or may not be
uniformly modified along the entire length of the molecule. For
example, one or more or all types of nucleotide (e.g., purine or
pyrimidine, or any one or more or all of A, G, U, C) may or may not
be uniformly modified in a polynucleotide of the invention, or in a
given predetermined sequence region thereof. In some embodiments,
all nucleotides X in a polynucleotide of the invention (or in a
given sequence region thereof) are modified, wherein X may any one
of nucleotides A, G, U, C, or any one of the combinations A+G, A+U,
A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
[0343] Different sugar modifications, nucleotide modifications,
and/or internucleoside linkages (e.g., backbone structures) may
exist at various positions in the polynucleotide. One of ordinary
skill in the art will appreciate that the nucleotide analogs or
other modification(s) may be located at any position(s) of a
polynucleotide such that the function of the polynucleotide is not
substantially decreased. A modification may also be a 5' or 3'
terminal modification. The polynucleotide may contain from about 1%
to about 100% modified nucleotides (either in relation to overall
nucleotide content, or in relation to one or more types of
nucleotide, i.e. any one or more of A, G, U or C) or any
intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from
1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1%
to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10%
to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%
to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from
20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from
20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%,
from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%,
from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to
95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80%
to 100%, from 90% to 95%, from 90% to 100%, and from 95% to
100%).
[0344] In some embodiments, the polynucleotide includes a modified
pyrimidine (e.g., a modified uracil/uridine/U or modified
cytosine/cytidine/C). In some embodiments, the uracil or uridine
(generally: U) in the polynucleotide molecule may be replaced with
from about 1% to about 100% of a modified uracil or modified
uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from
1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1%
to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10%
to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10%
to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from
20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from
20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,
from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to
100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70%
to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from
90% to 95%, from 90% to 100%, and from 95% to 100% of a modified
uracil or modified uridine). The modified uracil or uridine can be
replaced by a compound having a single unique structure or by a
plurality of compounds having different structures (e.g., 2, 3, 4
or more unique structures, as described herein). In some
embodiments, the cytosine or cytidine (generally: C) in the
polynucleotide molecule may be replaced with from about 1% to about
100% of a modified cytosine or modified cytidine (e.g., from 1% to
20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to
70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to
20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to
70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to
100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20%
to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20%
to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from
50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%,
from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to
90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90%
to 100%, and from 95% to 100% of a modified cytosine or modified
cytidine). The modified cytosine or cytidine can be replaced by a
compound having a single unique structure or by a plurality of
compounds having different structures (e.g., 2, 3, 4 or more unique
structures, as described herein).
[0345] In some embodiments, the present disclosure provides methods
of synthesizing a polynucleotide (e.g., the first region, first
flanking region, or second flanking region) including n number of
linked nucleosides having Formula (Ia-1):
##STR00131##
comprising:
[0346] a) reacting a nucleotide of Formula (IV-1):
##STR00132##
[0347] with a phosphoramidite compound of Formula (V-1):
##STR00133##
[0348] wherein Y.sup.9 is H, hydroxy, phosphoryl, pyrophosphate,
sulfate, amino, thiol, optionally substituted amino acid, or a
peptide (e.g., including from 2 to 12 amino acids); and each
P.sup.1, P.sup.2, and P.sup.3 is, independently, a suitable
protecting group; and
##STR00134##
denotes a solid support;
[0349] to provide a polynucleotide of Formula (VI-1):
##STR00135##
and
[0350] b) oxidizing or sulfurizing the polynucleotide of Formula
(V) to yield a polynucleotide of Formula (VII-1):
##STR00136##
and
[0351] c) removing the protecting groups to yield the
polynucleotide of Formula (Ia).
[0352] In some embodiments, steps a) and b) are repeated from 1 to
about 10,000 times. In some embodiments, the methods further
comprise a nucleotide selected from the group consisting of A, C, G
and U adenosine, cytosine, guanosine, and uracil. In some
embodiments, the nucleobase may be a pyrimidine or derivative
thereof. In some embodiments, the polynucleotide is
translatable.
[0353] Other components of polynucleotides are optional, and are
beneficial in some embodiments. For example, a 5' untranslated
region (UTR) and/or a 3'UTR are provided, wherein either or both
may independently contain one or more different nucleotide
modifications. In such embodiments, nucleotide modifications may
also be present in the translatable region. Also provided are
polynucleotides containing a Kozak sequence.
Combinations of Nucleotides
[0354] Further examples of modified nucleotides and modified
nucleotide combinations are provided below in Table 2. These
combinations of modified nucleotides can be used to form the
polynucleotides of the invention. Unless otherwise noted, the
modified nucleotides may be completely substituted for the natural
nucleotides of the polynucleotides of the invention. As a
non-limiting example, the natural nucleotide uridine may be
substituted with a modified nucleoside described herein. In another
non-limiting example, the natural nucleotide uridine may be
partially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 99.9%) with at least one of the modified nucleoside
disclosed herein.
TABLE-US-00002 TABLE 2 Modified Nucleotide Modified Nucleotide
Combination .alpha.-thio-cytidine
.alpha.-thio-cytidine/5-iodo-uridine
.alpha.-thio-cytidine/N1-methyl-pseudo-uridine
.alpha.-thio-cytidine/.alpha.-thio-uridine
.alpha.-thio-cytidine/5-methyl-uridine
.alpha.-thio-cytidine/pseudo-uridine about 50% of the cytosines are
.alpha.-thio-cytidine pseudoisocytidine
pseudoisocytidine/5-iodo-uridine
pseudoisocytidine/N1-methyl-pseudouridine
pseudoisocytidine/.alpha.-thio-uridine
pseudoisocytidine/5-methyl-uridine pseudoisocytidine/pseudouridine
about 25% of cytosines are pseudoisocytidine
pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine
and about 50% of uridines are pseudouridine pseudoisocytidine/about
25% of uridines are N1-methyl-pseudouridine and about 25% of
uridines are pseudouridine (e.g., 25% N1-methyl- pseudouridine/75%
pseudouridine) pyrrolo-cytidine pyrrolo-cytidine/5-iodo-uridine
pyrrolo-cytidine/N1-methyl-pseudouridine
pyrrolo-cytidine/.alpha.-thio-uridine
pyrrolo-cytidine/5-methyl-uridine pyrrolo-cytidine/pseudouridine
about 50% of the cytosines are pyrrolo-cytidine 5-methyl-cytidine
5-methyl-cytidine/5-iodo-uridine
5-methyl-cytidine/N1-methyl-pseudouridine
5-methyl-cytidine/.alpha.-thio-uridine
5-methyl-cytidine/5-methyl-uridine 5-methyl-cytidine/pseudouridine
about 25% of cytosines are 5-methyl-cytidine about 50% of cytosines
are 5-methyl-cytidine 5-methyl-cytidine/5-methoxy-uridine
5-methyl-cytidine/5-bromo-uridine 5-methyl-cytidine/2-thio-uridine
5-methyl-cytidine/about 50% of uridines are 2-thio-uridine about
50% of uridines are 5-methyl-cytidine/ about 50% of uridines are
2-thio-uridine N4-acetyl-cytidine N4-acetyl-cytidine/5-iodo-uridine
N4-acetyl-cytidine/N1-methyl-pseudouridine
N4-acetyl-cytidine/.alpha.-thio-uridine
N4-acetyl-cytidine/5-methyl-uridine
N4-acetyl-cytidine/pseudouridine about 50% of cytosines are
N4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidine
N4-acetyl-cytidine/5-methoxy-uridine
N4-acetyl-cytidine/5-bromo-uridine
N4-acetyl-cytidine/2-thio-uridine about 50% of cytosines are
N4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine
[0355] Certain modified nucleotides and nucleotide combinations
have been explored by the current inventors. These findings are
described in U.S. Provisional Application No. 61/404,413, filed on
Oct. 1, 2010, entitled Engineered Nucleic Acids and Methods of Use
Thereof, U.S. patent application Ser. No. 13/251,840, filed on Oct.
3, 2011, entitled Modified Nucleotides, and Nucleic Acids, and Uses
Thereof, now abandoned, U.S. patent application Ser. No.
13/481,127, filed on May 25, 2012, entitled Modified Nucleotides,
and Nucleic Acids, and Uses Thereof, International Patent
Publication No WO2012045075, filed on Oct. 3, 2011, entitled
Modified Nucleosides, Nucleotides, And Nucleic Acids, and Uses
Thereof, U.S. Patent Publication No US20120237975 filed on Oct. 3,
2011, entitled Engineered Nucleic Acids and Method of Use Thereof,
and International Patent Publication No WO2012045082, which are
incorporated by reference in their entireties.
[0356] Further examples of modified nucleotide combinations are
provided below in Table 3. These combinations of modified
nucleotides can be used to form the polynucleotides of the
invention.
TABLE-US-00003 TABLE 3 Modified Nucleotide Modified Nucleotide
Combination modified cytidine having one or more modified cytidine
with (b10)/pseudouridine nucleobases of Formula (b10) modified
cytidine with (b10)/N1-methyl-pseudouridine modified cytidine with
(b10)/5-methoxy-uridine modified cytidine with
(b10)/5-methyl-uridine modified cytidine with (b10)/5-bromo-uridine
modified cytidine with (b10)/2-thio-uridine about 50% of cytidine
substituted with modified cytidine (b10)/about 50% of uridines are
2-thio-uridine modified cytidine having one or more modified
cytidine with (b32)/pseudouridine nucleobases of Formula (b32)
modified cytidine with (b32)/N1-methyl-pseudouridine modified
cytidine with (b32)/5-methoxy-uridine modified cytidine with
(b32)/5-methyl-uridine modified cytidine with (b32)/5-bromo-uridine
modified cytidine with (b32)/2-thio-uridine about 50% of cytidine
substituted with modified cytidine (b32)/about 50% of uridines are
2-thio-uridine modified uridine having one or more modified uridine
with (b1)/N4-acetyl-cytidine nucleobases of Formula (b1) modified
uridine with (b1)/5-methyl-cytidine modified uridine having one or
more modified uridine with (b8)/N4-acetyl-cytidine nucleobases of
Formula (b8) modified uridine with (b8)/5-methyl-cytidine modified
uridine having one or more modified uridine with
(b28)/N4-acetyl-cytidine nucleobases of Formula (b28) modified
uridine with (b28)/5-methyl-cytidine modified uridine having one or
more modified uridine with (b29)/N4-acetyl-cytidine nucleobases of
Formula (b29) modified uridine with (b29)/5-methyl-cytidine
modified uridine having one or more modified uridine with
(b30)/N4-acetyl-cytidine nucleobases of Formula (b30) modified
uridine with (b30)/5-methyl-cytidine
[0357] In some embodiments, at least 25% of the cytosines are
replaced by a compound of Formula (b10)-(b14), (b24), (b25), or
(b32)-(b35) (e.g., at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100% of, e.g., a compound
of Formula (b10) or (b32)).
[0358] In some embodiments, at least 25% of the uracils are
replaced by a compound of Formula (b1)-(b9), (b21)-(b23), or
(b28)-(b31) (e.g., at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100% of, e.g., a compound
of Formula (b1), (b8), (b28), (b29), or (b30)).
[0359] In some embodiments, at least 25% of the cytosines are
replaced by a compound of Formula (b10)-(b14), (b24), (b25), or
(b32)-(b35) (e.g. Formula (b10) or (b32)), and at least 25% of the
uracils are replaced by a compound of Formula (b1)-(b9),
(b21)-(b23), or (b28)-(b31) (e.g. Formula (b1), (b8), (b28), (b29),
or (b30)) (e.g., at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100%).
Modifications Including Linker and a Payload
[0360] The nucleobase of the nucleotide can be covalently linked at
any chemically appropriate position to a payload, e.g., detectable
agent or therapeutic agent. For example, the nucleobase can be
deaza-adenosine or deaza-guanosine and the linker can be attached
at the C-7 or C-8 positions of the deaza-adenosine or
deaza-guanosine. In other embodiments, the nucleobase can be
cytosine or uracil and the linker can be attached to the N-3 or C-5
positions of cytosine or uracil. Scheme 1 below depicts an
exemplary modified nucleotide wherein the nucleobase, adenine, is
attached to a linker at the C-7 carbon of 7-deaza adenine. In
addition, Scheme 1 depicts the modified nucleotide with the linker
and payload, e.g., a detectable agent, incorporated onto the 3' end
of the mRNA. Disulfide cleavage and 1,2-addition of the thiol group
onto the propargyl ester releases the detectable agent. The
remaining structure (depicted, for example, as pApC5Parg in Scheme
1) is the inhibitor. The rationale for the structure of the
modified nucleotides is that the tethered inhibitor sterically
interferes with the ability of the polymerase to incorporate a
second base. Thus, it is critical that the tether be long enough to
affect this function and that the inhibiter be in a stereochemical
orientation that inhibits or prohibits second and follow on
nucleotides into the growing polynucleotide strand.
##STR00137##
Linker
[0361] The term "linker" as used herein refers to a group of atoms,
e.g., 10-1,000 atoms, and can be comprised of the atoms or groups
such as, but not limited to, carbon, amino, alkylamino, oxygen,
sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be
attached to a modified nucleoside or nucleotide on the nucleobase
or sugar moiety at a first end, and to a payload, e.g., detectable
or therapeutic agent, at a second end. The linker is of sufficient
length as to not interfere with incorporation into a nucleic acid
sequence.
[0362] Examples of chemical groups that can be incorporated into
the linker include, but are not limited to, an alkyl, alkene, an
alkyne, an amido, an ether, a thioether, an or an ester group. The
linker chain can also comprise part of a saturated, unsaturated or
aromatic ring, including polycyclic and heteroaromatic rings
wherein the heteroaromatic ring is an aryl group containing from
one to four heteroatoms, N, O or S. Specific examples of linkers
include, but are not limited to, unsaturated alkanes, polyethylene
glycols, and dextran polymers.
[0363] For example, the linker can include ethylene or propylene
glycol monomeric units, e.g., diethylene glycol, dipropylene
glycol, triethylene glycol, tripropylene glycol, tetraethylene
glycol, or tetraethylene glycol. In some embodiments, the linker
can include a divalent alkyl, alkenyl, and/or alkynyl moiety. The
linker can include an ester, amide, or ether moiety.
[0364] Other examples include cleavable moieties within the linker,
such as, for example, a disulfide bond (--S--S--) or an azo bond
(--N.dbd.N--), which can be cleaved using a reducing agent or
photolysis. A cleavable bond incorporated into the linker and
attached to a modified nucleotide, when cleaved, results in, for
example, a short "scar" or chemical modification on the nucleotide.
For example, after cleaving, the resulting scar on a nucleotide
base, which formed part of the modified nucleotide, and is
incorporated into a polynucleotide strand, is unreactive and does
not need to be chemically neutralized. This increases the ease with
which a subsequent nucleotide can be incorporated during sequencing
of a nucleic acid polymer template. For example, conditions include
the use of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol
(DTT) and/or other reducing agents for cleavage of a disulfide
bond. A selectively severable bond that includes an amido bond can
be cleaved for example by the use of TCEP or other reducing agents,
and/or photolysis. A selectively severable bond that includes an
ester bond can be cleaved for example by acidic or basic
hydrolysis.
Payload
[0365] The methods and compositions described herein are useful for
delivering a payload to a biological target. The payload can be
used, e.g., for labeling (e.g., a detectable agent such as a
fluorophore), or for therapeutic purposes (e.g., a cytotoxin or
other therapeutic agent).
Payload: Therapeutic Agents
[0366] In some embodiments the payload is a therapeutic agent such
as a cytotoxin, radioactive ion, chemotherapeutic, or other
therapeutic agent. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, puromycin,
maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020),
CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and
analogs or homologs thereof. Radioactive ions include, but are not
limited to iodine (e.g., iodine 125 or iodine 131), strontium 89,
phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium
90, Samarium 153 and praseodymium. Other therapeutic agents
include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, CC-1065, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine, vinblastine, taxol and maytansinoids).
Payload: Detectable Agents
[0367] Examples of detectable substances include various organic
small molecules, inorganic compounds, nanoparticles, enzymes or
enzyme substrates, fluorescent materials, luminescent materials,
bioluminescent materials, chemiluminescent materials, radioactive
materials, and contrast agents. Such optically-detectable labels
include for example, without limitation,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5'
5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red);
N,N,N',Nletramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3
(Cy3); Cyanine-5 (Cy5); Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD
700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and
naphthalo cyanine. In some embodiments, the detectable label is a
fluorescent dye, such as Cy5 and Cy3.
[0368] Examples luminescent material includes luminol; examples of
bioluminescent materials include luciferase, luciferin, and
aequorin.
[0369] Examples of suitable radioactive material include .sup.18F,
.sup.67Ga, .sup.81mKr, .sup.82Rb, .sup.111In, .sup.123I,
.sup.133Xe, .sup.201Tl, .sup.125I, .sup.35S, .sup.14C, or .sup.3H,
.sup.99mTc (e.g., as pertechnetate (technetate(VII),
TcO.sub.4.sup.-) either directly or indirectly, or other
radioisotope detectable by direct counting of radioemission or by
scintillation counting.
[0370] In addition, contrast agents, e.g., contrast agents for MRI
or NMR, for X-ray CT, Raman imaging, optical coherence tomography,
absorption imaging, ultrasound imaging, or thermal imaging can be
used. Exemplary contrast agents include gold (e.g., gold
nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,
superparamagnetic iron oxide (SPIO), monocrystalline iron oxide
nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide
(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate,
iodinated contrast media (iohexol), microbubbles, or
perfluorocarbons can also be used.
[0371] In some embodiments, the detectable agent is a
non-detectable pre-cursor that becomes detectable upon activation.
Examples include fluorogenic tetrazine-fluorophore constructs
(e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or
tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents
(e.g., PROSENSE (VisEn Medical)).
[0372] When the compounds are enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, the enzymatic label is detected by determination of
conversion of an appropriate substrate to product.
[0373] In vitro assays in which these compositions can be used
include enzyme linked immunosorbent assays (ELISAs),
immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA),
radioimmunoassay (RIA), and Western blot analysis.
[0374] Labels other than those described herein are contemplated by
the present disclosure, including other optically-detectable
labels. Labels can be attached to the modified nucleotide of the
present disclosure at any position using standard chemistries such
that the label can be removed from the incorporated base upon
cleavage of the cleavable linker.
[0375] Payload: Cell Penetrating Payloads
[0376] In some embodiments, the modified nucleotides and modified
nucleic acids can also include a payload that can be a cell
penetrating moiety or agent that enhances intracellular delivery of
the compositions. For example, the compositions can include a
cell-penetrating peptide sequence that facilitates delivery to the
intracellular space, e.g., HIV-derived TAT peptide, penetratins,
transportans, or hCT derived cell-penetrating peptides, see, e.g.,
Caron et al., (2001) Mol Ther. 3(3):310-8; Langel, Cell-Penetrating
Peptides: Processes and Applications (CRC Press, Boca Raton Fla.
2002); El-Andaloussi et al., (2005) Curr Pharm Des.
11(28):3597-611; and Deshayes et al., (2005) Cell Mol Life Sci.
62(16):1839-49. The compositions can also be formulated to include
a cell penetrating agent, e.g., liposomes, which enhance delivery
of the compositions to the intracellular space.
Payload: Biological Targets
[0377] The modified nucleotides and modified nucleic acids
described herein can be used to deliver a payload to any biological
target for which a specific ligand exists or can be generated. The
ligand can bind to the biological target either covalently or
non-covalently.
[0378] Exemplary biological targets include biopolymers, e.g.,
antibodies, nucleic acids such as RNA and DNA, proteins, enzymes;
exemplary proteins include enzymes, receptors, and ion channels. In
some embodiments the target is a tissue- or cell-type specific
marker, e.g., a protein that is expressed specifically on a
selected tissue or cell type. In some embodiments, the target is a
receptor, such as, but not limited to, plasma membrane receptors
and nuclear receptors; more specific examples include
G-protein-coupled receptors, cell pore proteins, transporter
proteins, surface-expressed antibodies, HLA proteins, MHC proteins
and growth factor receptors.
Synthesis of Modified Nucleotides
[0379] The modified nucleosides and nucleotides disclosed herein
can be prepared from readily available starting materials using the
following general methods and procedures. It is understood that
where typical or preferred process conditions (i.e., reaction
temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given; other process conditions can also be used unless
otherwise stated. Optimum reaction conditions may vary with the
particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization
procedures.
[0380] The processes described herein can be monitored according to
any suitable method known in the art. For example, product
formation can be monitored by spectroscopic means, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C)
infrared spectroscopy, spectrophotometry (e.g., UV-visible), or
mass spectrometry, or by chromatography such as high performance
liquid chromatography (HPLC) or thin layer chromatography.
[0381] Preparation of modified nucleosides and nucleotides can
involve the protection and deprotection of various chemical groups.
The need for protection and deprotection, and the selection of
appropriate protecting groups can be readily determined by one
skilled in the art. The chemistry of protecting groups can be
found, for example, in Greene, et al., Protective Groups in Organic
Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated
herein by reference in its entirety.
[0382] The reactions of the processes described herein can be
carried out in suitable solvents, which can be readily selected by
one of skill in the art of organic synthesis. Suitable solvents can
be substantially nonreactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, i.e., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected.
[0383] Resolution of racemic mixtures of modified nucleosides and
nucleotides can be carried out by any of numerous methods known in
the art. An example method includes fractional recrystallization
using a "chiral resolving acid" which is an optically active,
salt-forming organic acid. Suitable resolving agents for fractional
recrystallization methods are, for example, optically active acids,
such as the D and L forms of tartaric acid, diacetyltartaric acid,
dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or
the various optically active camphorsulfonic acids. Resolution of
racemic mixtures can also be carried out by elution on a column
packed with an optically active resolving agent (e.g.,
dinitrobenzoylphenylglycine). Suitable elution solvent composition
can be determined by one skilled in the art.
[0384] Exemplary syntheses of modified nucleotides, which are
incorporated into a polynucleotides, e.g., RNA or mRNA, are
provided below in Scheme 2 through Scheme 12. Scheme 2 provides a
general method for phosphorylation of nucleosides, including
modified nucleosides.
##STR00138##
[0385] Various protecting groups may be used to control the
reaction. For example, Scheme 3 provides the use of multiple
protecting and deprotecting steps to promote phosphorylation at the
5' position of the sugar, rather than the 2' and 3' hydroxyl
groups.
##STR00139##
[0386] Modified nucleotides can be synthesized in any useful
manner. Schemes 4, 5, and 8 provide exemplary methods for
synthesizing modified nucleotides having a modified purine
nucleobase; and Schemes 6 and 7 provide exemplary methods for
synthesizing modified nucleotides having a modified pseudouridine
or pseudoisocytidine, respectively.
##STR00140##
##STR00141##
##STR00142##
##STR00143##
##STR00144##
[0387] Schemes 9 and 10 provide exemplary syntheses of modified
nucleotides. Scheme 11 provides a non-limiting biocatalytic method
for producing nucleotides.
##STR00145##
##STR00146##
##STR00147##
[0388] Scheme 12 provides an exemplary synthesis of a modified
uracil, where the N1 position on the major groove face is modified
with R.sup.12b, as provided elsewhere, and the 5'-position of
ribose is phosphorylated. T.sup.1, T.sup.2, R.sup.12a, R.sup.12b,
and r are as provided herein. This synthesis, as well as optimized
versions thereof, can be used to modify the major groove face of
other pyrimidine nucleobases and purine nucleobases (see e.g.,
Formulas (b1)-(b43)) and/or to install one or more phosphate groups
(e.g., at the 5' position of the sugar). This alkylating reaction
can also be used to include one or more optionally substituted
alkyl group at any reactive group (e.g., amino group) in any
nucleobase described herein (e.g., the amino groups in the
Watson-Crick base-pairing face for cytosine, uracil, adenine, and
guanine).
##STR00148##
[0389] Modified nucleosides and nucleotides can also be prepared
according to the synthetic methods described in Ogata et al.
Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al.
Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al.
Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9):
1729-1740, 1992, each of which are incorporated by reference in
their entirety.
Modified Nucleic Acids
[0390] The present disclosure provides nucleic acids (or
polynucleotides), including RNAs such as mRNAs that contain one or
more modified nucleosides (termed "modified nucleic acids") or
nucleotides as described herein, which have useful properties
including the lack of a substantial induction of the innate immune
response of a cell into which the mRNA is introduced. Because these
modified nucleic acids enhance the efficiency of protein
production, intracellular retention of nucleic acids, and viability
of contacted cells, as well as possess reduced immunogenicity,
these nucleic acids having these properties are also termed
"enhanced nucleic acids" herein.
[0391] In addition, the present disclosure provides nucleic acids,
which have decreased binding affinity to a major groove
interacting, e.g. binding, partner. For example, the nucleic acids
are comprised of at least one nucleotide that has been chemically
modified on the major groove face as described herein.
[0392] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that is or can be incorporated into an
oligonucleotide chain. In this context, the term nucleic acid is
used synonymously with polynucleotide. Exemplary nucleic acids for
use in accordance with the present disclosure include, but are not
limited to, one or more of DNA, RNA including messenger mRNA
(mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs,
shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that
induce triple helix formation, aptamers, vectors, etc., described
in detail herein.
[0393] Provided are modified nucleic acids containing a
translatable region and one, two, or more than two different
nucleoside modifications. In some embodiments, the modified nucleic
acid exhibits reduced degradation in a cell into which the nucleic
acid is introduced, relative to a corresponding unmodified nucleic
acid. Exemplary nucleic acids include ribonucleic acids (RNAs),
deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol
nucleic acids (GNAs), or a hybrid thereof. In preferred
embodiments, the modified nucleic acid includes messenger RNAs
(mRNAs). As described herein, the nucleic acids of the present
disclosure do not substantially induce an innate immune response of
a cell into which the mRNA is introduced.
[0394] In certain embodiments, it is desirable to intracellularly
degrade a modified nucleic acid introduced into the cell, for
example if precise timing of protein production is desired. Thus,
the present disclosure provides a modified nucleic acid containing
a degradation domain, which is capable of being acted on in a
directed manner within a cell.
[0395] Other components of nucleic acid are optional, and are
beneficial in some embodiments. For example, a 5' untranslated
region (UTR) and/or a 3'UTR are provided, wherein either or both
may independently contain one or more different nucleoside
modifications. In such embodiments, nucleoside modifications may
also be present in the translatable region. Also provided are
nucleic acids containing a Kozak sequence.
[0396] Additionally, provided are nucleic acids containing one or
more intronic nucleotide sequences capable of being excised from
the nucleic acid.
[0397] Further, provided are nucleic acids containing an internal
ribosome entry site (IRES). An IRES may act as the sole ribosome
binding site, or may serve as one of multiple ribosome binding
sites of an mRNA. An mRNA containing more than one functional
ribosome binding site may encode several peptides or polypeptides
that are translated independently by the ribosomes ("multicistronic
mRNA"). When nucleic acids are provided with an IRES, further
optionally provided is a second translatable region. Examples of
IRES sequences that can be used according to the present disclosure
include without limitation, those from picornaviruses (e.g. FMDV),
pest viruses (CFFV), polio viruses (PV), encephalomyocarditis
viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C
viruses (HCV), classical swine fever viruses (CSFV), murine
leukemia virus (MLV), simian immune deficiency viruses (SIV) or
cricket paralysis viruses (CrPV).
[0398] In another aspect, the present disclosure provides for
nucleic acid sequences comprising at least two nucleotides, the
nucleic acid sequence comprising a nucleotide that disrupts binding
of a major groove binding partner with the nucleic acid sequence,
wherein the nucleotide has decreased binding affinity to the major
groove binding partner.
[0399] In some embodiments, the nucleic acid is a compound of
Formula XI-a:
##STR00149##
[0400] wherein:
[0401] denotes an optional double bond;
[0402] denotes an optional single bond;
[0403] U is O, S, --NR.sup.a--, or --CR.sup.aR.sup.b-- when denotes
a single bond, or U is --CR.sup.a-- when denotes a double bond;
[0404] A is H, OH, phosphoryl, pyrophosphate, sulfate, --NH.sub.2,
--SH, an amino acid, a peptide comprising 2 to 12 amino acids;
[0405] X is O or S;
[0406] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.a1;
[0407] each of Y.sup.2 and Y.sup.3 are independently selected from
O, --CR.sup.aR.sup.b--, NR.sup.c, S or a linker comprising one or
more atoms selected from the group consisting of C, O, N, and
S;
[0408] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20
aryl;
[0409] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group;
[0410] R.sup.a1 and R.sup.b1 are each independently H or a
counterion;
[0411] --OR.sup.c1 is OH at a pH of about 1 or --OR.sup.c1 is
O.sup.- at physiological pH; and
[0412] B is nucleobase;
[0413] provided that the ring encompassing the variables A, B, D,
U, Z, Y.sup.2 and Y.sup.3 cannot be ribose.
[0414] In some embodiments, B is a nucleobase of Formula XII-a,
XII-b, or XII-c:
##STR00150##
[0415] wherein:
[0416] denotes a single or double bond;
[0417] X is O or S;
[0418] U and W are each independently C or N;
[0419] V is O, S, C or N;
[0420] wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl,
C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each
optionally substituted with --OH, --NR.sup.aR.sup.b, --SH,
--C(O)R.sup.c, --C(O)OR.sup.c, --NHC(O)R.sup.c, or
--NHC(O)OR.sup.c;
[0421] and wherein when V is O, S, or N then R.sup.1 is absent;
[0422] R.sup.2 is H, --OR.sup.c, --SR.sup.c, --NR.sup.aR.sup.b, or
halo;
[0423] or when V is C then R.sup.1 and R.sup.2 together with the
carbon atoms to which they are attached can form a 5- or 6-membered
ring optionally substituted with 1-4 substituents selected from
halo, --OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0424] R.sup.3 is H or C.sub.1-20 alkyl;
[0425] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0426] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0427] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0428] In some embodiments, B is a nucleobase of Formula XII-a1,
XII-a2, XII-a3, XII-a4, or XII-a5:
##STR00151##
[0429] In some embodiments, the nucleobase is a pyrimidine or
derivative thereof.
[0430] In some embodiments, the nucleic acid contains a plurality
of structurally unique compounds of Formula XI-a.
[0431] In some embodiments, at least 25% of the cytosines are
replaced by a compound of Formula XI-a (e.g., at least about 30%,
at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
about 100%).
[0432] In some embodiments, at least 25% of the uracils are
replaced by a compound of Formula XI-a (e.g., at least about 30%,
at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
about 100%).
[0433] In some embodiments, at least 25% of the cytosines and 25%
of the uracils are replaced by a compound of Formula XI-a (e.g., at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or about 100%).
[0434] In some embodiments, the nucleic acid is translatable.
[0435] In some embodiments, when the nucleic acid includes a
nucleotide modified with a linker and payload, for example, as
described herein, the nucleotide modified with a linker and payload
is on the 3' end of the nucleic acid.
Major Groove Interacting Partners
[0436] As described herein, the phrase "major groove interacting
partner" refers RNA recognition receptors that detect and respond
to RNA ligands through interactions, e.g. binding, with the major
groove face of a nucleotide or nucleic acid. As such, RNA ligands
comprising modified nucleotides or nucleic acids as described
herein decrease interactions with major groove binding partners,
and therefore decrease an innate immune response, or expression and
secretion of pro-inflammatory cytokines, or both.
[0437] Example major groove interacting, e.g. binding, partners
include, but are not limited to the following nucleases and
helicases. Within membranes, TLRs (Toll-like Receptors) 3, 7, and 8
can respond to single- and double-stranded RNAs. Within the
cytoplasm, members of the superfamily 2 class of DEX(D/H) helicases
and ATPases can sense RNAs to initiate antiviral responses. These
helicases include the RIG-I (retinoic acid-inducible gene I) and
MDA5 (melanoma differentiation-associated gene 5). Other examples
include laboratory of genetics and physiology 2 (LGP2), HIN-200
domain containing proteins, or Helicase-domain containing
proteins.
Prevention or Reduction of Innate Cellular Immune Response
[0438] The term "innate immune response" includes a cellular
response to exogenous single stranded nucleic acids, generally of
viral or bacterial origin, which involves the induction of cytokine
expression and release, particularly the interferons, and cell
death. Protein synthesis is also reduced during the innate cellular
immune response. While it is advantageous to eliminate the innate
immune response in a cell which is triggered by introduction of
exogenous nucleic acids, the present disclosure provides modified
nucleic acids such as mRNAs that substantially reduce the immune
response, including interferon signaling, without entirely
eliminating such a response. In some embodiments, the immune
response is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 99%, 99.9%, or greater than 99.9% as compared to the immune
response induced by a corresponding unmodified nucleic acid. Such a
reduction can be measured by expression or activity level of Type 1
interferons or the expression of interferon-regulated genes such as
the toll-like receptors (e.g., TLR7 and TLR8). Reduction or lack of
induction of innate immune response can also be measured by
decreased cell death following one or more administrations of
modified RNAs to a cell population; e.g., cell death is 10%, 25%,
50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death
frequency observed with a corresponding unmodified nucleic acid.
Moreover, cell death may affect fewer than 50%, 40%, 30%, 20%, 10%,
5%, 1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the
modified nucleic acids.
[0439] In some embodiments, the modified nucleic acids, including
polynucleotides and/or mRNA molecules are modified in such a way as
to not induce, or induce only minimally, an immune response by the
recipient cell or organism. Such evasion or avoidance of an immune
response trigger or activation is a novel feature of the modified
polynucleotides of the present invention.
[0440] The present disclosure provides for the repeated
introduction (e.g., transfection) of modified nucleic acids into a
target cell population, e.g., in vitro, ex vivo, or in vivo. The
step of contacting the cell population may be repeated one or more
times (such as two, three, four, five or more than five times). In
some embodiments, the step of contacting the cell population with
the modified nucleic acids is repeated a number of times sufficient
such that a predetermined efficiency of protein translation in the
cell population is achieved. Given the reduced cytotoxicity of the
target cell population provided by the nucleic acid modifications,
such repeated transfections are achievable in a diverse array of
cell types in vitro and/or in vivo.
Polypeptide Variants
[0441] Provided are nucleic acids that encode variant polypeptides,
which have a certain identity with a reference polypeptide
sequence. The term "identity" as known in the art, refers to a
relationship between the sequences of two or more peptides, as
determined by comparing the sequences. In the art, "identity" also
means the degree of sequence relatedness between peptides, as
determined by the number of matches between strings of two or more
amino acid residues. "Identity" measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model or
computer program (i.e., "algorithms"). Identity of related peptides
can be readily calculated by known methods. Such methods include,
but are 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 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
[0442] In some embodiments, the polypeptide variant has the same or
a similar activity as the reference polypeptide. Alternatively, the
variant has an altered activity (e.g., increased or decreased)
relative to a reference polypeptide. Generally, variants of a
particular polynucleotide or polypeptide of the present disclosure
will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to that particular reference polynucleotide or
polypeptide as determined by sequence alignment programs and
parameters described herein and known to those skilled in the
art.
[0443] As recognized by those skilled in the art, protein
fragments, functional protein domains, and homologous proteins are
also considered to be within the scope of this present disclosure.
For example, provided herein is any protein fragment of a reference
protein (meaning a polypeptide sequence at least one amino acid
residue shorter than a reference polypeptide sequence but otherwise
identical) 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than
100 amino acids in length In another example, any protein that
includes a stretch of about 20, about 30, about 40, about 50, or
about 100 amino acids which are about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 95%, or about 100% identical
to any of the sequences described herein can be utilized in
accordance with the present disclosure. In certain embodiments, a
protein sequence to be utilized in accordance with the present
disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations
as shown in any of the sequences provided or referenced herein.
Polypeptide Libraries
[0444] Also provided are polynucleotide libraries containing
nucleoside modifications, wherein the polynucleotides individually
contain a first nucleic acid sequence encoding a polypeptide, such
as an antibody, protein binding partner, scaffold protein, and
other polypeptides known in the art. Preferably, the
polynucleotides are mRNA in a form suitable for direct introduction
into a target cell host, which in turn synthesizes the encoded
polypeptide.
[0445] In certain embodiments, multiple variants of a protein, each
with different amino acid modification(s), are produced and tested
to determine the best variant in terms of pharmacokinetics,
stability, biocompatibility, and/or biological activity, or a
biophysical property such as expression level. Such a library may
contain 10, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, or over 10.sup.9 possible variants
(including substitutions, deletions of one or more residues, and
insertion of one or more residues).
Polypeptide-Nucleic Acid Complexes
[0446] Proper protein translation involves the physical aggregation
of a number of polypeptides and nucleic acids associated with the
mRNA. Provided by the present disclosure are protein-nucleic acid
complexes, containing a translatable mRNA having one or more
nucleoside modifications (e.g., at least two different nucleoside
modifications) and one or more polypeptides bound to the mRNA.
Generally, the proteins are provided in an amount effective to
prevent or reduce an innate immune response of a cell into which
the complex is introduced.
Untranslatable Modified Nucleic Acids
[0447] As described herein, provided are mRNAs having sequences
that are substantially not translatable. Such mRNA is effective as
a vaccine when administered to a mammalian subject.
[0448] Also provided are modified nucleic acids that contain one or
more noncoding regions. Such modified nucleic acids are generally
not translated, but are capable of binding to and sequestering one
or more translational machinery component such as a ribosomal
protein or a transfer RNA (tRNA), thereby effectively reducing
protein expression in the cell. The modified nucleic acid may
contain a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small
interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
Synthesis of Modified Nucleic Acids
[0449] Nucleic acids for use in accordance with the present
disclosure may be prepared according to any available technique
including, but not limited to chemical synthesis, enzymatic
synthesis, which is generally termed in vitro transcription,
enzymatic or chemical cleavage of a longer precursor, etc. Methods
of synthesizing RNAs are known in the art (see, e.g., Gait, M. J.
(ed.) Oligonucleotide synthesis: a practical approach, Oxford
[Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P.
(ed.) Oligonucleotide synthesis: methods and applications, Methods
in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana
Press, 2005; both of which are incorporated herein by
reference).
[0450] Modified nucleic acids need not be uniformly modified along
the entire length of the molecule. Different nucleotide
modifications and/or backbone structures may exist at various
positions in the nucleic acid. One of ordinary skill in the art
will appreciate that the nucleotide analogs or other
modification(s) may be located at any position(s) of a nucleic acid
such that the function of the nucleic acid is not substantially
decreased. A modification may also be a 5' or 3' terminal
modification. The nucleic acids may contain at a minimum one and at
maximum 100% modified nucleotides, or any intervening percentage,
such as at least 5% modified nucleotides, at least 10% modified
nucleotides, at least 25% modified nucleotides, at least 50%
modified nucleotides, at least 80% modified nucleotides, or at
least 90% modified nucleotides. For example, the nucleic acids may
contain a modified pyrimidine such as uracil or cytosine. In some
embodiments, at least 5%, at least 10%, at least 25%, at least 50%,
at least 80%, at least 90% or 100% of the uracil in the nucleic
acid is replaced with a modified uracil. The modified uracil can be
replaced by a compound having a single unique structure, or can be
replaced by a plurality of compounds having different structures
(e.g., 2, 3, 4 or more unique structures). In some embodiments, at
least 5%, at least 10%, at least 25%, at least 50%, at least 80%,
at least 90% or 100% of the cytosine in the nucleic acid is
replaced with a modified cytosine. The modified cytosine can be
replaced by a compound having a single unique structure, or can be
replaced by a plurality of compounds having different structures
(e.g., 2, 3, 4 or more unique structures).
[0451] Generally, the shortest length of a modified mRNA of the
present disclosure can be the length of an mRNA sequence that is
sufficient to encode for a dipeptide. In another embodiment, the
length of the mRNA sequence is sufficient to encode for a
tripeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a tetrapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
pentapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a hexapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
heptapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for an octapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
nonapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a decapeptide.
[0452] Examples of dipeptides that the modified nucleic acid
sequences can encode for include, but are not limited to, carnosine
and anserine.
[0453] In a further embodiment, the mRNA is greater than 30
nucleotides in length. In another embodiment, the RNA molecule is
greater than 35 nucleotides in length. In another embodiment, the
length is at least 40 nucleotides. In another embodiment, the
length is at least 45 nucleotides. In another embodiment, the
length is at least 55 nucleotides. In another embodiment, the
length is at least 60 nucleotides. In another embodiment, the
length is at least 60 nucleotides. In another embodiment, the
length is at least 80 nucleotides. In another embodiment, the
length is at least 90 nucleotides. In another embodiment, the
length is at least 100 nucleotides. In another embodiment, the
length is at least 120 nucleotides. In another embodiment, the
length is at least 140 nucleotides. In another embodiment, the
length is at least 160 nucleotides. In another embodiment, the
length is at least 180 nucleotides. In another embodiment, the
length is at least 200 nucleotides. In another embodiment, the
length is at least 250 nucleotides. In another embodiment, the
length is at least 300 nucleotides. In another embodiment, the
length is at least 350 nucleotides. In another embodiment, the
length is at least 400 nucleotides. In another embodiment, the
length is at least 450 nucleotides. In another embodiment, the
length is at least 500 nucleotides. In another embodiment, the
length is at least 600 nucleotides. In another embodiment, the
length is at least 700 nucleotides. In another embodiment, the
length is at least 800 nucleotides. In another embodiment, the
length is at least 900 nucleotides. In another embodiment, the
length is at least 1000 nucleotides. In another embodiment, the
length is at least 1100 nucleotides. In another embodiment, the
length is at least 1200 nucleotides. In another embodiment, the
length is at least 1300 nucleotides. In another embodiment, the
length is at least 1400 nucleotides. In another embodiment, the
length is at least 1500 nucleotides. In another embodiment, the
length is at least 1600 nucleotides. In another embodiment, the
length is at least 1800 nucleotides. In another embodiment, the
length is at least 2000 nucleotides. In another embodiment, the
length is at least 2500 nucleotides. In another embodiment, the
length is at least 3000 nucleotides. In another embodiment, the
length is at least 4000 nucleotides. In another embodiment, the
length is at least 5000 nucleotides, or greater than 5000
nucleotides.
[0454] For example, the modified nucleic acids described herein can
be prepared using methods that are known to those skilled in the
art of nucleic acid synthesis.
[0455] In some embodiments, the present disclosure provides
methods, e.g., enzymatic, of preparing a nucleic acid sequence
comprising a nucleotide that disrupts binding of a major groove
binding partner with the nucleic acid sequence, wherein the nucleic
acid sequence comprises a compound of Formula XI-a:
##STR00152##
[0456] wherein:
[0457] the nucleotide has decreased binding affinity to the major
groove binding partner;
[0458] denotes an optional double bond;
[0459] denotes an optional single bond;
[0460] U is O, S, --NR.sup.a--, or --CR.sup.aR.sup.b-- when denotes
a single bond, or U is --CR.sup.a-- when denotes a double bond;
[0461] A is H, OH, phosphoryl, pyrophosphate, sulfate, --NH.sub.2,
--SH, an amino acid, a peptide comprising 2 to 12 amino acids;
[0462] X is O or S;
[0463] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.a1;
[0464] each of Y.sup.2 and Y.sup.3 are independently selected from
O, --CR.sup.aR.sup.b--, NR.sup.c, S or a linker comprising one or
more atoms selected from the group consisting of C, O, N, and
S;
[0465] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20
aryl;
[0466] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group;
[0467] R.sup.a1 and R.sup.b1 are each independently H or a
counterion;
[0468] --OR.sup.c1 is OH at a pH of about 1 or --OR.sup.c1 is
O.sup.- at physiological pH; and
[0469] B is nucleobase;
[0470] provided that the ring encompassing the variables A, B, D,
U, Z, Y.sup.2 and Y.sup.3 cannot be ribose the method comprising
reacting a compound of Formula XIII:
##STR00153##
[0471] with an RNA polymerase, and a cDNA template.
[0472] In some embodiments, the reaction is repeated from 1 to
about 7,000 times.
[0473] In some embodiments, B is a nucleobase of Formula XII-a,
XII-b, or XII-c:
##STR00154##
[0474] wherein:
[0475] denotes a single or double bond;
[0476] X is O or S;
[0477] U and W are each independently C or N;
[0478] V is O, S, C or N;
[0479] wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl,
C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each
optionally substituted with --OH, --NR.sup.aR.sup.b, --SH,
--C(O)R.sup.c, --C(O)OR.sup.c, --NHC(O)R.sup.c, or
--NHC(O)OR.sup.c;
[0480] and wherein when V is O, S, or N then R.sup.1 is absent;
[0481] R.sup.2 is H, --OR.sup.c, --SR.sup.c, --NR.sup.aR.sup.b, or
halo;
[0482] or when V is C then R.sup.1 and R.sup.2 together with the
carbon atoms to which they are attached can form a 5- or 6-membered
ring optionally substituted with 1-4 substituents selected from
halo, --OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0483] R.sup.3 is H or C.sub.1-20 alkyl;
[0484] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0485] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0486] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0487] In some embodiments, B is a nucleobase of Formula XII-a1,
XII-a2, XII-a3, XII-a4, or XII-a5:
##STR00155##
[0488] In some embodiments, the methods further comprise a
nucleotide selected from the group consisting of adenosine,
cytosine, guanosine, and uracil.
[0489] In some embodiments, the nucleobase is a pyrimidine or
derivative thereof.
[0490] In another aspect, the present disclosure provides for
methods of amplifying a nucleic acid sequence comprising a
nucleotide that disrupts binding of a major groove binding partner
with the nucleic acid sequence, the method comprising:
[0491] reacting a compound of Formula XI-d:
##STR00156##
[0492] wherein:
[0493] the nucleotide has decreased binding affinity to the major
groove binding partner;
[0494] denotes a single or a double bond;
[0495] denotes an optional single bond;
[0496] U is O, S, --NR.sup.a--, or --CR.sup.aR.sup.b-- when denotes
a single bond, or U is --CR.sup.a-- when denotes a double bond;
[0497] Z is H, C.sub.1-12 alkyl, or C.sub.6-20 aryl, or Z is absent
when denotes a double bond; and
[0498] Z can be --CR.sup.aR.sup.b-- and form a bond with A;
[0499] A is H, OH, phosphoryl, pyrophosphate, sulfate, --NH.sub.2,
--SH, an amino acid, or a peptide comprising 1 to 12 amino
acids;
[0500] X is O or S;
[0501] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.a1;
[0502] each of Y.sup.2 and Y.sup.3 are independently selected from
O, --CR.sup.aR.sup.b--, NR.sup.c, S or a linker comprising one or
more atoms selected from the group consisting of C, O, N, and
S;
[0503] n is 0, 1, 2, or 3;
[0504] m is 0, 1, 2 or 3;
[0505] B is nucleobase;
[0506] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20
aryl;
[0507] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group;
[0508] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0509] --OR.sup.c1 is OH at a pH of about 1 or --OR.sup.c1 is
O.sup.- at physiological pH;
[0510] provided that the ring encompassing the variables A, B, D,
U, Z, Y.sup.2 and Y.sup.3 cannot be ribose with a primer, a cDNA
template, and an RNA polymerase.
[0511] In some embodiments, B is a nucleobase of Formula XII-a,
XII-b, or XII-c:
##STR00157##
[0512] wherein:
[0513] denotes a single or double bond;
[0514] X is O or S;
[0515] U and W are each independently C or N;
[0516] V is O, S, C or N;
[0517] wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl,
C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each
optionally substituted with --OH, --NR.sup.aR.sup.b, --SH,
--C(O)R.sup.c, --C(O)OR.sup.c, --NHC(O)R.sup.c, or
--NHC(O)OR.sup.c;
[0518] and wherein when V is O, S, or N then R.sup.1 is absent;
[0519] R.sup.2 is H, --OR.sup.c, --SR.sup.c, --NR.sup.aR.sup.b, or
halo;
[0520] or when V is C then R.sup.1 and R.sup.2 together with the
carbon atoms to which they are attached can form a 5- or 6-membered
ring optionally substituted with 1-4 substituents selected from
halo, --OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0521] R.sup.3 is H or C.sub.1-20 alkyl;
[0522] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0523] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0524] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0525] In some embodiments, B is a nucleobase of Formula XII-a1,
XII-a2, XII-a3, XII-a4, or XII-a5:
##STR00158##
[0526] In some embodiments, the methods further comprise a
nucleotide selected from the group consisting of adenosine,
cytosine, guanosine, and uracil.
[0527] In some embodiments, the nucleobase is a pyrimidine or
derivative thereof.
[0528] In some embodiments, the present disclosure provides for
methods of synthesizing a pharmaceutical nucleic acid, comprising
the steps of:
[0529] a) providing a complementary deoxyribonucleic acid (cDNA)
that encodes a pharmaceutical protein of interest;
[0530] b) selecting a nucleotide that is known to disrupt a binding
of a major groove binding partner with a nucleic acid, wherein the
nucleotide has decreased binding affinity to the major groove
binding partner; and
[0531] c) contacting the provided cDNA and the selected nucleotide
with an RNA polymerase, under conditions such that the
pharmaceutical nucleic acid is synthesized.
[0532] In further embodiments, the pharmaceutical nucleic acid is a
ribonucleic acid (RNA).
[0533] In still a further aspect of the present disclosure, the
modified nucleic acids can be prepared using solid phase synthesis
methods.
[0534] In some embodiments, the present disclosure provides methods
of synthesizing a nucleic acid comprising a compound of Formula
XI-a:
##STR00159##
[0535] wherein:
[0536] denotes an optional double bond;
[0537] denotes an optional single bond;
[0538] U is O, S, --NR.sup.a--, or --CR.sup.aR.sup.b-- when denotes
a single bond, or U is --CR.sup.a-- when denotes a double bond;
[0539] A is H, OH, phosphoryl, pyrophosphate, sulfate, --NH.sub.2,
--SH, an amino acid, a peptide comprising 2 to 12 amino acids;
[0540] X is O or S;
[0541] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --Se;
[0542] each of Y.sup.2 and Y.sup.3 are independently selected from
O, --CR.sup.aR.sup.b--, NR.sup.c, S or a linker comprising one or
more atoms selected from the group consisting of C, O, N, and
S;
[0543] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20
aryl;
[0544] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group;
[0545] R.sup.a1 and R.sup.b1 are each independently H or a
counterion;
[0546] --OR.sup.c1 is OH at a pH of about 1 or --OR.sup.c1 is
O.sup.- at physiological pH; and
[0547] B is nucleobase;
[0548] provided that the ring encompassing the variables A, B, U,
Z, Y.sup.2 and Y.sup.3 cannot be ribose;
[0549] comprising:
[0550] a) reacting a nucleotide of Formula XIII-a:
##STR00160##
[0551] with a phosphoramidite compound of Formula XIII-b:
##STR00161##
[0552] wherein:
##STR00162##
denotes a solid support; and
[0553] P.sup.1, P.sup.2 and P.sup.3 are each independently suitable
protecting groups;
[0554] to provide a nucleic acid of Formula XIV-a:
##STR00163##
XIV-a and b) oxidizing or sulfurizing the nucleic acid of Formula
XIV-a to yield a nucleic acid of Formula XIVb:
##STR00164##
[0555] and c) removing the protecting groups to yield the nucleic
acid of Formula XI-a.
[0556] In some embodiments, the methods further comprise a
nucleotide selected from the group consisting of adenosine,
cytosine, guanosine, and uracil.
[0557] In some embodiments, B is a nucleobase of Formula XIII:
##STR00165##
[0558] wherein:
[0559] V is N or positively charged NR.sup.c;
[0560] R.sup.3 is NR.sup.cR.sup.d, --OR.sup.a, or --SR.sup.a;
[0561] R.sup.4 is H or can optionally form a bond with Y.sup.3;
[0562] R.sup.5 is H, --NR.sup.cR.sup.d, or --OR.sup.a;
[0563] R.sup.a and R.sup.b are each independently H, C.sub.1-12
alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or C.sub.6-20 aryl;
and
[0564] R.sup.c is H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0565] In some embodiments, steps a) and b) are repeated from 1 to
about 10,000 times.
Uses of Modified Nucleic Acids
Therapeutic Agents
[0566] The modified nucleic acids described herein can be used as
therapeutic agents. For example, a modified nucleic acid described
herein can be administered to an animal or subject, wherein the
modified nucleic acid is translated in vivo to produce a
therapeutic peptide in the animal or subject. Accordingly, provided
herein are compositions, methods, kits, and reagents for treatment
or prevention of disease or conditions in humans and other mammals.
The active therapeutic agents of the present disclosure include
modified nucleic acids, cells containing modified nucleic acids or
polypeptides translated from the modified nucleic acids,
polypeptides translated from modified nucleic acids, cells
contacted with cells containing modified nucleic acids or
polypeptides translated from the modified nucleic acids, tissues
containing cells containing modified nucleic acids and organs
containing tissues containing cells containing modified nucleic
acids.
[0567] Provided are methods of inducing translation of a synthetic
or recombinant polynucleotide to produce a polypeptide in a cell
population using the modified nucleic acids described herein. Such
translation can be in vivo, ex vivo, in culture, or in vitro. The
cell population is contacted with an effective amount of a
composition containing a nucleic acid that has at least one
nucleoside modification, and a translatable region encoding the
polypeptide. The population is contacted under conditions such that
the nucleic acid is localized into one or more cells of the cell
population and the recombinant polypeptide is translated in the
cell from the nucleic acid.
[0568] An effective amount of the composition is provided based, at
least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the nucleic acid (e.g.,
size, and extent of modified nucleosides), and other determinants.
In general, an effective amount of the composition provides
efficient protein production in the cell, preferably more efficient
than a composition containing a corresponding unmodified nucleic
acid. Increased efficiency may be demonstrated by increased cell
transfection (i.e., the percentage of cells transfected with the
nucleic acid), increased protein translation from the nucleic acid,
decreased nucleic acid degradation (as demonstrated, e.g., by
increased duration of protein translation from a modified nucleic
acid), or reduced innate immune response of the host cell or
improve therapeutic utility.
[0569] Aspects of the present disclosure are directed to methods of
inducing in vivo translation of a recombinant polypeptide in a
mammalian subject in need thereof. Therein, an effective amount of
a composition containing a nucleic acid that has at least one
nucleoside modification and a translatable region encoding the
polypeptide is administered to the subject using the delivery
methods described herein. The nucleic acid is provided in an amount
and under other conditions such that the nucleic acid is localized
into a cell or cells of the subject and the recombinant polypeptide
is translated in the cell from the nucleic acid. The cell in which
the nucleic acid is localized, or the tissue in which the cell is
present, may be targeted with one or more than one rounds of
nucleic acid administration.
[0570] Other aspects of the present disclosure relate to
transplantation of cells containing modified nucleic acids to a
mammalian subject. Administration of cells to mammalian subjects is
known to those of ordinary skill in the art, such as local
implantation (e.g., topical or subcutaneous administration), organ
delivery or systemic injection (e.g., intravenous injection or
inhalation), as is the formulation of cells in pharmaceutically
acceptable carrier. Compositions containing modified nucleic acids
are formulated for administration intramuscularly, transarterially,
intraperitoneally, intravenously, intranasally, subcutaneously,
endoscopically, transdermally, or intrathecally. In some
embodiments, the composition is formulated for extended
release.
[0571] The subject to whom the therapeutic agent is administered
suffers from or is at risk of developing a disease, disorder, or
deleterious condition. Provided are methods of identifying,
diagnosing, and classifying subjects on these bases, which may
include clinical diagnosis, biomarker levels, genome-wide
association studies (GWAS), and other methods known in the art.
[0572] In certain embodiments, the administered modified nucleic
acid directs production of one or more recombinant polypeptides
that provide a functional activity which is substantially absent in
the cell in which the recombinant polypeptide is translated. For
example, the missing functional activity may be enzymatic,
structural, or gene regulatory in nature.
[0573] In other embodiments, the administered modified nucleic acid
directs production of one or more recombinant polypeptides that
replace a polypeptide (or multiple polypeptides) that is
substantially absent in the cell in which the recombinant
polypeptide is translated. Such absence may be due to genetic
mutation of the encoding gene or regulatory pathway thereof. In
other embodiments, the administered modified nucleic acid directs
production of one or more recombinant polypeptides to supplement
the amount of polypeptide (or multiple polypeptides) that is
present in the cell in which the recombinant polypeptide is
translated. Alternatively, the recombinant polypeptide functions to
antagonize the activity of an endogenous protein present in, on the
surface of, or secreted from the cell. Usually, the activity of the
endogenous protein is deleterious to the subject, for example, due
to mutation of the endogenous protein resulting in altered activity
or localization. Additionally, the recombinant polypeptide
antagonizes, directly or indirectly, the activity of a biological
moiety present in, on the surface of, or secreted from the cell.
Examples of antagonized biological moieties include lipids (e.g.,
cholesterol), a lipoprotein (e.g., low density lipoprotein), a
nucleic acid, a carbohydrate, or a small molecule toxin.
[0574] The recombinant proteins described herein are engineered for
localization within the cell, potentially within a specific
compartment such as the nucleus, or are engineered for secretion
from the cell or translocation to the plasma membrane of the
cell.
[0575] As described herein, a useful feature of the modified
nucleic acids of the present disclosure is the capacity to reduce,
evade, avoid or eliminate the innate immune response of a cell to
an exogenous nucleic acid. Provided are methods for performing the
titration, reduction or elimination of the immune response in a
cell or a population of cells. In some embodiments, the cell is
contacted with a first composition that contains a first dose of a
first exogenous nucleic acid including a translatable region and at
least one nucleoside modification, and the level of the innate
immune response of the cell to the first exogenous nucleic acid is
determined. Subsequently, the cell is contacted with a second
composition, which includes a second dose of the first exogenous
nucleic acid, the second dose containing a lesser amount of the
first exogenous nucleic acid as compared to the first dose.
Alternatively, the cell is contacted with a first dose of a second
exogenous nucleic acid. The second exogenous nucleic acid may
contain one or more modified nucleosides, which may be the same or
different from the first exogenous nucleic acid or, alternatively,
the second exogenous nucleic acid may not contain modified
nucleosides. The steps of contacting the cell with the first
composition and/or the second composition may be repeated one or
more times. Additionally, efficiency of protein production (e.g.,
protein translation) in the cell is optionally determined, and the
cell may be re-transfected with the first and/or second composition
repeatedly until a target protein production efficiency is
achieved.
Therapeutics for Diseases and Conditions
[0576] Provided are methods for treating or preventing a symptom of
diseases characterized by missing or aberrant protein activity, by
replacing the missing protein activity or overcoming the aberrant
protein activity. Because of the rapid initiation of protein
production following introduction of modified mRNAs, as compared to
viral DNA vectors, the compounds of the present disclosure are
particularly advantageous in treating acute diseases such as
sepsis, stroke, and myocardial infarction. Moreover, the lack of
transcriptional regulation of the modified mRNAs of the present
disclosure is advantageous in that accurate titration of protein
production is achievable. Multiple diseases are characterized by
missing (or substantially diminished such that proper protein
function does not occur) protein activity. Such proteins may not be
present, are present in very low quantities or are essentially
non-functional. The present disclosure provides a method for
treating such conditions or diseases in a subject by introducing
nucleic acid or cell-based therapeutics containing the modified
nucleic acids provided herein, wherein the modified nucleic acids
encode for a protein that replaces the protein activity missing
from the target cells of the subject.
[0577] Diseases characterized by dysfunctional or aberrant protein
activity include, but not limited to, cancer and proliferative
diseases, genetic diseases (e.g., cystic fibrosis), autoimmune
diseases, diabetes, neurodegenerative diseases, cardiovascular
diseases, and metabolic diseases. The present disclosure provides a
method for treating such conditions or diseases in a subject by
introducing nucleic acid or cell-based therapeutics containing the
modified nucleic acids provided herein, wherein the modified
nucleic acids encode for a protein that antagonizes or otherwise
overcomes the aberrant protein activity present in the cell of the
subject.
[0578] Specific examples of a dysfunctional protein are the
missense or nonsense mutation variants of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which produce a
dysfunctional or nonfunctional, respectively, protein variant of
CFTR protein, which causes cystic fibrosis.
[0579] Thus, provided are methods of treating cystic fibrosis in a
mammalian subject by contacting a cell of the subject with a
modified nucleic acid having a translatable region that encodes a
functional CFTR polypeptide, under conditions such that an
effective amount of the CTFR polypeptide is present in the cell.
Preferred target cells are epithelial cells, such as the lung, and
methods of administration are determined in view of the target
tissue; i.e., for lung delivery, the RNA molecules are formulated
for administration by inhalation.
[0580] In another embodiment, the present disclosure provides a
method for treating hyperlipidemia in a subject, by introducing
into a cell population of the subject with a modified mRNA molecule
encoding Sortilin, a protein recently characterized by genomic
studies, thereby ameliorating the hyperlipidemia in a subject. The
SORT1 gene encodes a trans-Golgi network (TGN) transmembrane
protein called Sortilin. Genetic studies have shown that one of
five individuals has a single nucleotide polymorphism, rs12740374,
in the 1p13 locus of the SORT1 gene that predisposes them to having
low levels of low-density lipoprotein (LDL) and very-low-density
lipoprotein (VLDL). Each copy of the minor allele, present in about
30% of people, alters LDL cholesterol by 8 mg/dL, while two copies
of the minor allele, present in about 5% of the population, lowers
LDL cholesterol 16 mg/dL. Carriers of the minor allele have also
been shown to have a 40% decreased risk of myocardial infarction.
Functional in vivo studies in mice describes that overexpression of
SORT1 in mouse liver tissue led to significantly lower
LDL-cholesterol levels, as much as 80% lower, and that silencing
SORT1 increased LDL cholesterol approximately 200% (Musunuru K et
al. From noncoding variant to phenotype via SORT1 at the 1p13
cholesterol locus. Nature 2010; 466: 714-721).
Methods of Cellular Nucleic Acid Delivery
[0581] Methods of the present disclosure enhance nucleic acid
delivery into a cell population, in vivo, ex vivo, or in culture.
For example, a cell culture containing a plurality of host cells
(e.g., eukaryotic cells such as yeast or mammalian cells) is
contacted with a composition that contains an enhanced nucleic acid
having at least one nucleoside modification and, optionally, a
translatable region. The composition also generally contains a
transfection reagent or other compound that increases the
efficiency of enhanced nucleic acid uptake into the host cells. The
enhanced nucleic acid exhibits enhanced retention in the cell
population, relative to a corresponding unmodified nucleic acid.
The retention of the enhanced nucleic acid is greater than the
retention of the unmodified nucleic acid. In some embodiments, it
is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than
200% greater than the retention of the unmodified nucleic acid.
Such retention advantage may be achieved by one round of
transfection with the enhanced nucleic acid, or may be obtained
following repeated rounds of transfection.
[0582] In some embodiments, the enhanced nucleic acid is delivered
to a target cell population with one or more additional nucleic
acids. Such delivery may be at the same time, or the enhanced
nucleic acid is delivered prior to delivery of the one or more
additional nucleic acids. The additional one or more nucleic acids
may be modified nucleic acids or unmodified nucleic acids. It is
understood that the initial presence of the enhanced nucleic acids
does not substantially induce an innate immune response of the cell
population and, moreover, that the innate immune response will not
be activated by the later presence of the unmodified nucleic acids.
In this regard, the enhanced nucleic acid may not itself contain a
translatable region, if the protein desired to be present in the
target cell population is translated from the unmodified nucleic
acids.
Targeting Moieties
[0583] In embodiments of the present disclosure, modified nucleic
acids are provided to express a protein-binding partner or a
receptor on the surface of the cell, which functions to target the
cell to a specific tissue space or to interact with a specific
moiety, either in vivo or in vitro. Suitable protein-binding
partners include antibodies and functional fragments thereof,
scaffold proteins, or peptides. Additionally, modified nucleic
acids can be employed to direct the synthesis and extracellular
localization of lipids, carbohydrates, or other biological
moieties.
Permanent Gene Expression Silencing
[0584] A method for epigenetically silencing gene expression in a
mammalian subject, comprising a nucleic acid where the translatable
region encodes a polypeptide or polypeptides capable of directing
sequence-specific histone H3 methylation to initiate
heterochromatin formation and reduce gene transcription around
specific genes for the purpose of silencing the gene. For example,
a gain-of-function mutation in the Janus Kinase 2 gene is
responsible for the family of Myeloproliferative Diseases.
Delivery of a Detectable or Therapeutic Agent to a Biological
Target
[0585] The modified nucleosides, modified nucleotides, and modified
nucleic acids described herein can be used in a number of different
scenarios in which delivery of a substance (the "payload") to a
biological target is desired, for example delivery of detectable
substances for detection of the target, or delivery of a
therapeutic agent. Detection methods can include both imaging in
vitro and in vivo imaging methods, e.g., immunohistochemistry,
bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI),
positron emission tomography (PET), electron microscopy, X-ray
computed tomography, Raman imaging, optical coherence tomography,
absorption imaging, thermal imaging, fluorescence reflectance
imaging, fluorescence microscopy, fluorescence molecular
tomographic imaging, nuclear magnetic resonance imaging, X-ray
imaging, ultrasound imaging, photoacoustic imaging, lab assays, or
in any situation where tagging/staining/imaging is required.
[0586] For example, the modified nucleosides, modified nucleotides,
and modified nucleic acids described herein can be used in
reprogramming induced pluripotent stem cells (iPS cells), which can
then be used to directly track cells that are transfected compared
to total cells in the cluster. In another example, a drug that is
attached to the modified nucleic acid via a linker and is
fluorescently labeled can be used to track the drug in vivo, e.g.
intracellularly. Other examples include the use of a modified
nucleic acid in reversible drug delivery into cells.
[0587] The modified nucleosides, modified nucleotides, and modified
nucleic acids described herein can be used in intracellular
targeting of a payload, e.g., detectable or therapeutic agent, to
specific organelle. Exemplary intracellular targets can include the
nuclear localization for advanced mRNA processing, or a nuclear
localization sequence (NLS) linked to the mRNA containing an
inhibitor.
[0588] In addition, the modified nucleosides, modified nucleotides,
and modified nucleic acids described herein can be used to deliver
therapeutic agents to cells or tissues, e.g., in living animals.
For example, the modified nucleosides, modified nucleotides, and
modified nucleic acids described herein can be used to deliver
highly polar chemotherapeutics agents to kill cancer cells. The
modified nucleic acids attached to the therapeutic agent through a
linker can facilitate member permeation allowing the therapeutic
agent to travel into a cell to reach an intracellular target.
[0589] In another example, the modified nucleosides, modified
nucleotides, and modified nucleic acids can be attached to a viral
inhibitory peptide (VIP) through a cleavable linker. The cleavable
linker will release the VIP and dye into the cell. In another
example, the modified nucleosides, modified nucleotides, and
modified nucleic acids can be attached through the linker to a
ADP-ribosylate, which is responsible for the actions of some
bacterial toxins, such as cholera toxin, diphtheria toxin, and
pertussis toxin. These toxin proteins are ADP-ribosyltransferases
that modify target proteins in human cells. For example, cholera
toxin ADP-ribosylates G proteins, causing massive fluid secretion
from the lining of the small intestine, resulting in
life-threatening diarrhea.
Pharmaceutical Compositions
[0590] The present disclosure provides proteins generated from
modified mRNAs. Pharmaceutical compositions may optionally comprise
one or more additional therapeutically active substances. In
accordance with some embodiments, a method of administering
pharmaceutical compositions comprising a modified nucleic acid
encoding one or more proteins to be delivered to a subject in need
thereof is provided. In some embodiments, compositions are
administered to humans. For the purposes of the present disclosure,
the phrase "active ingredient" generally refers to a protein,
protein encoding or protein-containing complex as described
herein.
[0591] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions is contemplated include, but are not
limited to, humans and/or other primates; mammals, including
commercially relevant mammals such as cattle, pigs, horses, sheep,
cats, dogs, mice, and/or rats; and/or birds, including commercially
relevant birds such as chickens, ducks, geese, and/or turkeys.
[0592] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with an excipient and/or one or more other accessory ingredients,
and then, if necessary and/or desirable, shaping and/or packaging
the product into a desired single- or multi-dose unit.
[0593] A pharmaceutical composition in accordance with the present
disclosure may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" is discrete amount of the pharmaceutical
composition comprising a predetermined amount of the active
ingredient. The amount of the active ingredient is generally equal
to the dosage of the active ingredient which would be administered
to a subject and/or a convenient fraction of such a dosage such as,
for example, one-half or one-third of such a dosage.
[0594] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
present disclosure will vary, depending upon the identity, size,
and/or condition of the subject treated and further depending upon
the route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100% (w/w)
active ingredient.
[0595] Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used herein,
includes any and all solvents, dispersion media, diluents, or other
liquid vehicles, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's The Science and
Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro (Lippincott,
Williams & Wilkins, Baltimore, Md., 2006; incorporated herein
by reference) discloses various excipients used in formulating
pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional excipient
medium is incompatible with a substance or its derivatives, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutical composition, its use is contemplated to be
within the scope of this present disclosure.
[0596] In some embodiments, a pharmaceutically acceptable excipient
is at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% pure. In some embodiments, an excipient is approved
for use in humans and for veterinary use. In some embodiments, an
excipient is approved by United States Food and Drug
Administration. In some embodiments, an excipient is pharmaceutical
grade. In some embodiments, an excipient meets the standards of the
United States Pharmacopoeia (USP), the European Pharmacopoeia (EP),
the British Pharmacopoeia, and/or the International
Pharmacopoeia.
[0597] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, dispersing and/or granulating agents,
surface active agents and/or emulsifiers, disintegrating agents,
binding agents, preservatives, buffering agents, lubricating
agents, and/or oils. Such excipients may optionally be included in
pharmaceutical formulations. Excipients such as cocoa butter and
suppository waxes, coloring agents, coating agents, sweetening,
flavoring, and/or perfuming agents can be present in the
composition, according to the judgment of the formulator.
[0598] Exemplary diluents include, but are not limited to, calcium
carbonate, sodium carbonate, calcium phosphate, dicalcium
phosphate, calcium sulfate, calcium hydrogen phosphate, sodium
phosphate lactose, sucrose, cellulose, microcrystalline cellulose,
kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch,
cornstarch, powdered sugar, etc., and/or combinations thereof.
[0599] Exemplary granulating and/or dispersing agents include, but
are not limited to, potato starch, corn starch, tapioca starch,
sodium starch glycolate, clays, alginic acid, guar gum, citrus
pulp, agar, bentonite, cellulose and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds,
etc., and/or combinations thereof
[0600] Exemplary surface active agents and/or emulsifiers include,
but are not limited to, natural emulsifiers (e.g. acacia, agar,
alginic acid, sodium alginate, tragacanth, chondrux, cholesterol,
xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol,
wax, and lecithin), colloidal clays (e.g. bentonite [aluminum
silicate] and Veegum.RTM. [magnesium aluminum silicate]), long
chain amino acid derivatives, high molecular weight alcohols (e.g.
stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin
monostearate, ethylene glycol distearate, glyceryl monostearate,
and propylene glycol monostearate, polyvinyl alcohol), carbomers
(e.g. carboxy polymethylene, polyacrylic acid, acrylic acid
polymer, and carboxyvinyl polymer), carrageenan, cellulosic
derivatives (e.g. carboxymethylcellulose sodium, powdered
cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty
acid esters (e.g. polyoxyethylene sorbitan monolaurate
[Tween.RTM.20], polyoxyethylene sorbitan [Tween.RTM.60],
polyoxyethylene sorbitan monooleate [Tween.RTM.80], sorbitan
monopalmitate [Span.RTM.40], sorbitan monostearate [Span.RTM.60],
sorbitan tristearate [Span.RTM.65], glyceryl monooleate, sorbitan
monooleate [Span.RTM. 80]), polyoxyethylene esters (e.g.
polyoxyethylene monostearate [Myrj.RTM. 45], polyoxyethylene
hydrogenated castor oil, polyethoxylated castor oil,
polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,
polyethylene glycol fatty acid esters (e.g. Cremophor.RTM.),
polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether
[Brij.RTM. 30]), poly(vinyl-pyrrolidone), diethylene glycol
monolaurate, triethanolamine oleate, sodium oleate, potassium
oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl
sulfate, Pluronic.RTM.F 68, Poloxamer.RTM. 188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, etc. and/or combinations thereof.
[0601] Exemplary binding agents include, but are not limited to,
starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g.
sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol,
mannitol,); natural and synthetic gums (e.g. acacia, sodium
alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage
of isapol husks, carboxymethylcellulose, methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, microcrystalline cellulose,
cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum
silicate (Veegum.RTM.), and larch arabogalactan); alginates;
polyethylene oxide; polyethylene glycol; inorganic calcium salts;
silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and
combinations thereof.
[0602] Exemplary preservatives may include, but are not limited to,
antioxidants, chelating agents, antimicrobial preservatives,
antifungal preservatives, alcohol preservatives, acidic
preservatives, and/or other preservatives. Exemplary antioxidants
include, but are not limited to, alpha tocopherol, ascorbic acid,
acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and/or sodium sulfite. Exemplary chelating
agents include ethylenediaminetetraacetic acid (EDTA), citric acid
monohydrate, disodium edetate, dipotassium edetate, edetic acid,
fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric
acid, and/or trisodium edetate. Exemplary antimicrobial
preservatives include, but are not limited to, benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and/or thimerosal.
Exemplary antifungal preservatives include, but are not limited to,
butyl paraben, methyl paraben, ethyl paraben, propyl paraben,
benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium
sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
Exemplary alcohol preservatives include, but are not limited to,
ethanol, polyethylene glycol, phenol, phenolic compounds,
bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl
alcohol. Exemplary acidic preservatives include, but are not
limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric
acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid,
and/or phytic acid. Other preservatives include, but are not
limited to, tocopherol, tocopherol acetate, deteroxime mesylate,
cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened
(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl
ether sulfate (SLES), sodium bisulfite, sodium metabisulfite,
potassium sulfite, potassium metabisulfite, Glydant Plus.RTM.,
Phenonip.RTM., methylparaben, Germall.RTM. 115, Germaben.RTM.II,
Neolone.TM., Kathon.TM., and/or Euxyl.RTM..
[0603] Exemplary buffering agents include, but are not limited to,
citrate buffer solutions, acetate buffer solutions, phosphate
buffer solutions, ammonium chloride, calcium carbonate, calcium
chloride, calcium citrate, calcium glubionate, calcium gluceptate,
calcium gluconate, d-gluconic acid, calcium glycerophosphate,
calcium lactate, propanoic acid, calcium levulinate, pentanoic
acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium
phosphate, calcium hydroxide phosphate, potassium acetate,
potassium chloride, potassium gluconate, potassium mixtures,
dibasic potassium phosphate, monobasic potassium phosphate,
potassium phosphate mixtures, sodium acetate, sodium bicarbonate,
sodium chloride, sodium citrate, sodium lactate, dibasic sodium
phosphate, monobasic sodium phosphate, sodium phosphate mixtures,
tromethamine, magnesium hydroxide, aluminum hydroxide, alginic
acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl
alcohol, etc., and/or combinations thereof.
[0604] Exemplary lubricating agents include, but are not limited
to, magnesium stearate, calcium stearate, stearic acid, silica,
talc, malt, glyceryl behanate, hydrogenated vegetable oils,
polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate,
etc., and combinations thereof.
[0605] Exemplary oils include, but are not limited to, almond,
apricot kernel, avocado, babassu, bergamot, black current seed,
borage, cade, camomile, canola, caraway, carnauba, castor,
cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton
seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol,
gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba,
kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary oils include, but are not limited
to, butyl stearate, caprylic triglyceride, capric triglyceride,
cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl
myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone
oil, and/or combinations thereof.
[0606] Liquid dosage forms for oral and parenteral administration
include, but are not limited to, pharmaceutically acceptable
emulsions, microemulsions, solutions, suspensions, syrups, and/or
elixirs. In addition to active ingredients, liquid dosage forms may
comprise inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, oral compositions can include adjuvants
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, and/or perfuming agents. In certain
embodiments for parenteral administration, compositions are mixed
with solubilizing agents such as Cremophor.RTM., alcohols, oils,
modified oils, glycols, polysorbates, cyclodextrins, polymers,
and/or combinations thereof.
[0607] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing agents, wetting agents,
and/or suspending agents. Sterile injectable preparations may be
sterile injectable solutions, suspensions, and/or emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for
example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P., and isotonic sodium chloride solution. Sterile,
fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. Fatty acids such as
oleic acid can be used in the preparation of injectables.
[0608] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, and/or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0609] In order to prolong the effect of an active ingredient, it
is often desirable to slow the absorption of the active ingredient
from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution
which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered
drug form is accomplished by dissolving or suspending the drug in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the drug in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body
tissues.
[0610] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing
compositions with suitable non-irritating excipients such as cocoa
butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore
melt in the rectum or vaginal cavity and release the active
ingredient.
[0611] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
an active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient such as sodium citrate or
dicalcium phosphate and/or fillers or extenders (e.g. starches,
lactose, sucrose, glucose, mannitol, and silicic acid), binders
(e.g. carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.
glycerol), disintegrating agents (e.g. agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate), solution retarding agents (e.g. paraffin),
absorption accelerators (e.g. quaternary ammonium compounds),
wetting agents (e.g. cetyl alcohol and glycerol monostearate),
absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g.
talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate), and mixtures thereof. In the case
of capsules, tablets and pills, the dosage form may comprise
buffering agents.
[0612] Solid compositions of a similar type may be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. Solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally comprise opacifying agents and can be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type may be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polyethylene glycols and the
like.
[0613] Dosage forms for topical and/or transdermal administration
of a composition may include ointments, pastes, creams, lotions,
gels, powders, solutions, sprays, inhalants and/or patches.
Generally, an active ingredient is admixed under sterile conditions
with a pharmaceutically acceptable excipient and/or any needed
preservatives and/or buffers as may be required. Additionally, the
present disclosure contemplates the use of transdermal patches,
which often have the added advantage of providing controlled
delivery of a compound to the body. Such dosage forms may be
prepared, for example, by dissolving and/or dispensing the compound
in the proper medium. Alternatively or additionally, rate may be
controlled by either providing a rate controlling membrane and/or
by dispersing the compound in a polymer matrix and/or gel.
[0614] Suitable devices for use in delivering intradermal
pharmaceutical compositions described herein include short needle
devices such as those described in U.S. Pat. Nos. 4,886,499;
5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496;
and 5,417,662. Intradermal compositions may be administered by
devices which limit the effective penetration length of a needle
into the skin, such as those described in PCT publication WO
99/34850 and functional equivalents thereof. Jet injection devices
which deliver liquid compositions to the dermis via a liquid jet
injector and/or via a needle which pierces the stratum corneum and
produces a jet which reaches the dermis are suitable. Jet injection
devices are described, for example, in U.S. Pat. Nos. 5,480,381;
5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;
5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;
5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460;
and PCT publications WO 97/37705 and WO 97/13537. Ballistic
powder/particle delivery devices which use compressed gas to
accelerate vaccine in powder form through the outer layers of the
skin to the dermis are suitable. Alternatively or additionally,
conventional syringes may be used in the classical mantoux method
of intradermal administration.
[0615] Formulations suitable for topical administration include,
but are not limited to, liquid and/or semi liquid preparations such
as liniments, lotions, oil in water and/or water in oil emulsions
such as creams, ointments and/or pastes, and/or solutions and/or
suspensions. Topically-administrable formulations may, for example,
comprise from about 1% to about 10% (w/w) active ingredient,
although the concentration of active ingredient may be as high as
the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0616] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for pulmonary administration
via the buccal cavity. Such a formulation may comprise dry
particles which comprise the active ingredient and which have a
diameter in the range from about 0.5 nm to about 7 nm or from about
1 nm to about 6 nm. Such compositions are conveniently in the form
of dry powders for administration using a device comprising a dry
powder reservoir to which a stream of propellant may be directed to
disperse the powder and/or using a self propelling solvent/powder
dispensing container such as a device comprising the active
ingredient dissolved and/or suspended in a low-boiling propellant
in a sealed container. Such powders comprise particles wherein at
least 98% of the particles by weight have a diameter greater than
0.5 nm and at least 95% of the particles by number have a diameter
less than 7 nm. Alternatively, at least 95% of the particles by
weight have a diameter greater than 1 nm and at least 90% of the
particles by number have a diameter less than 6 nm. Dry powder
compositions may include a solid fine powder diluent such as sugar
and are conveniently provided in a unit dose form.
[0617] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50% to 99.9%
(w/w) of the composition, and active ingredient may constitute 0.1%
to 20% (w/w) of the composition. A propellant may further comprise
additional ingredients such as a liquid non-ionic and/or solid
anionic surfactant and/or a solid diluent (which may have a
particle size of the same order as particles comprising the active
ingredient).
[0618] Pharmaceutical compositions formulated for pulmonary
delivery may provide an active ingredient in the form of droplets
of a solution and/or suspension. Such formulations may be prepared,
packaged, and/or sold as aqueous and/or dilute alcoholic solutions
and/or suspensions, optionally sterile, comprising active
ingredient, and may conveniently be administered using any
nebulization and/or atomization device. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, a flavoring agent such as saccharin sodium, a
volatile oil, a buffering agent, a surface active agent, and/or a
preservative such as methylhydroxybenzoate. Droplets provided by
this route of administration may have an average diameter in the
range from about 0.1 nm to about 200 nm.
[0619] Formulations described herein as being useful for pulmonary
delivery are useful for intranasal delivery of a pharmaceutical
composition. Another formulation suitable for intranasal
administration is a coarse powder comprising the active ingredient
and having an average particle from about 0.2 .mu.m to 500 .mu.m.
Such a formulation is administered in the manner in which snuff is
taken, i.e. by rapid inhalation through the nasal passage from a
container of the powder held close to the nose.
[0620] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of active ingredient, and may comprise one or more of
the additional ingredients described herein. A pharmaceutical
composition may be prepared, packaged, and/or sold in a formulation
suitable for buccal administration. Such formulations may, for
example, be in the form of tablets and/or lozenges made using
conventional methods, and may, for example, 0.1% to 20% (w/w)
active ingredient, the balance comprising an orally dissolvable
and/or degradable composition and, optionally, one or more of the
additional ingredients described herein. Alternately, formulations
suitable for buccal administration may comprise a powder and/or an
aerosolized and/or atomized solution and/or suspension comprising
active ingredient. Such powdered, aerosolized, and/or aerosolized
formulations, when dispersed, may have an average particle and/or
droplet size in the range from about 0.1 nm to about 200 nm, and
may further comprise one or more of any additional ingredients
described herein.
[0621] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in the form
of eye drops including, for example, a 0.1/1.0% (w/w) solution
and/or suspension of the active ingredient in an aqueous or oily
liquid excipient. Such drops may further comprise buffering agents,
salts, and/or one or more other of any additional ingredients
described herein. Other opthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form and/or in a liposomal preparation. Ear
drops and/or eye drops are contemplated as being within the scope
of this present disclosure.
[0622] General considerations in the formulation and/or manufacture
of pharmaceutical agents may be found, for example, in Remington:
The Science and Practice of Pharmacy 21.sup.st ed., Lippincott
Williams & Wilkins, 2005 (incorporated herein by
reference).
Administration
[0623] The present disclosure provides methods comprising
administering proteins or complexes in accordance with the present
disclosure to a subject in need thereof. Proteins or complexes, or
pharmaceutical, imaging, diagnostic, or prophylactic compositions
thereof, may be administered to a subject using any amount and any
route of administration effective for preventing, treating,
diagnosing, or imaging a disease, disorder, and/or condition (e.g.,
a disease, disorder, and/or condition relating to working memory
deficits). The exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the disease, the particular
composition, its mode of administration, its mode of activity, and
the like. Compositions in accordance with the present disclosure
are typically formulated in dosage unit form for ease of
administration and uniformity of dosage. It will be understood,
however, that the total daily usage of the compositions of the
present disclosure will be decided by the attending physician
within the scope of sound medical judgment. The specific
therapeutically effective, prophylactically effective, or
appropriate imaging dose level for any particular patient will
depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed; and like factors well known in the
medical arts.
[0624] Proteins to be delivered and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof may be
administered to animals, such as mammals (e.g., humans,
domesticated animals, cats, dogs, mice, rats, etc.). In some
embodiments, pharmaceutical, prophylactic, diagnostic, or imaging
compositions thereof are administered to humans.
[0625] Proteins to be delivered and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof in
accordance with the present disclosure may be administered by any
route. In some embodiments, proteins and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof, are
administered by one or more of a variety of routes, including oral,
intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, subcutaneous, intraventricular, transdermal,
interdermal, rectal, intravaginal, intraperitoneal, topical (e.g.
by powders, ointments, creams, gels, lotions, and/or drops),
mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual;
by intratracheal instillation, bronchial instillation, and/or
inhalation; as an oral spray, nasal spray, and/or aerosol, and/or
through a portal vein catheter. In some embodiments, proteins or
complexes, and/or pharmaceutical, prophylactic, diagnostic, or
imaging compositions thereof, are administered by systemic
intravenous injection. In specific embodiments, proteins or
complexes and/or pharmaceutical, prophylactic, diagnostic, or
imaging compositions thereof may be administered intravenously
and/or orally. In specific embodiments, proteins or complexes,
and/or pharmaceutical, prophylactic, diagnostic, or imaging
compositions thereof, may be administered in a way which allows the
protein or complex to cross the blood-brain barrier, vascular
barrier, or other epithelial barrier.
[0626] However, the present disclosure encompasses the delivery of
proteins or complexes, and/or pharmaceutical, prophylactic,
diagnostic, or imaging compositions thereof, by any appropriate
route taking into consideration likely advances in the sciences of
drug delivery.
[0627] In general the most appropriate route of administration will
depend upon a variety of factors including the nature of the
protein or complex comprising proteins associated with at least one
agent to be delivered (e.g., its stability in the environment of
the gastrointestinal tract, bloodstream, etc.), the condition of
the patient (e.g., whether the patient is able to tolerate
particular routes of administration), etc. The present disclosure
encompasses the delivery of the pharmaceutical, prophylactic,
diagnostic, or imaging compositions by any appropriate route taking
into consideration likely advances in the sciences of drug
delivery.
[0628] In certain embodiments, compositions in accordance with the
present disclosure may be administered at dosage levels sufficient
to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about
0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40
mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01
mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or
from about 1 mg/kg to about 25 mg/kg, of subject body weight per
day, one or more times a day, to obtain the desired therapeutic,
diagnostic, prophylactic, or imaging effect. The desired dosage may
be delivered three times a day, two times a day, once a day, every
other day, every third day, every week, every two weeks, every
three weeks, or every four weeks. In certain embodiments, the
desired dosage may be delivered using multiple administrations
(e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, or more administrations).
[0629] Proteins or complexes may be used in combination with one or
more other therapeutic, prophylactic, diagnostic, or imaging
agents. By "in combination with," it is not intended to imply that
the agents must be administered at the same time and/or formulated
for delivery together, although these methods of delivery are
within the scope of the present disclosure. Compositions can be
administered concurrently with, prior to, or subsequent to, one or
more other desired therapeutics or medical procedures. In general,
each agent will be administered at a dose and/or on a time schedule
determined for that agent. In some embodiments, the present
disclosure encompasses the delivery of pharmaceutical,
prophylactic, diagnostic, or imaging compositions in combination
with agents that improve their bioavailability, reduce and/or
modify their metabolism, inhibit their excretion, and/or modify
their distribution within the body.
[0630] It will further be appreciated that therapeutically,
prophylactically, diagnostically, or imaging active agents utilized
in combination may be administered together in a single composition
or administered separately in different compositions. In general,
it is expected that agents utilized in combination with be utilized
at levels that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0631] The particular combination of therapies (therapeutics or
procedures) to employ in a combination regimen will take into
account compatibility of the desired therapeutics and/or procedures
and the desired therapeutic effect to be achieved. It will also be
appreciated that the therapies employed may achieve a desired
effect for the same disorder (for example, a composition useful for
treating cancer in accordance with the present disclosure may be
administered concurrently with a chemotherapeutic agent), or they
may achieve different effects (e.g., control of any adverse
effects).
Kits
[0632] The present disclosure provides a variety of kits for
conveniently and/or effectively carrying out methods of the present
disclosure. Typically kits will comprise sufficient amounts and/or
numbers of components to allow a user to perform multiple
treatments of a subject(s) and/or to perform multiple
experiments.
[0633] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a nucleic acid modification, wherein the
nucleic acid is capable of evading or avoiding induction of an
innate immune response of a cell into which the first isolated
nucleic acid is introduced, and packaging and instructions.
[0634] In one aspect, the disclosure provides kits for protein
production, comprising: a first isolated modified nucleic acid
comprising a translatable region, provided in an amount effective
to produce a desired amount of a protein encoded by the
translatable region when introduced into a target cell; a second
nucleic acid comprising an inhibitory nucleic acid, provided in an
amount effective to substantially inhibit the innate immune
response of the cell; and packaging and instructions.
[0635] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a nucleoside modification, wherein the
nucleic acid exhibits reduced degradation by a cellular nuclease,
and packaging and instructions.
[0636] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and at least two different nucleoside
modifications, wherein the nucleic acid exhibits reduced
degradation by a cellular nuclease, and packaging and
instructions.
[0637] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and at least one nucleoside modification,
wherein the nucleic acid exhibits reduced degradation by a cellular
nuclease; a second nucleic acid comprising an inhibitory nucleic
acid; and packaging and instructions.
[0638] In some embodiments, the first isolated nucleic acid
comprises messenger RNA (mRNA). In some embodiments the mRNA
comprises at least one nucleoside selected from the group
consisting of pyridin-4-one ribonucleoside, 5-aza-uridine,
2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,
5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyluridine, 1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine,
5-methyl-uridine, 1-methyl-pseudouridine,
4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine or any disclosed herein.
[0639] In some embodiments, the mRNA comprises at least one
nucleoside selected from the group consisting of 5-aza-cytidine,
pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,
5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine
or any disclosed herein.
[0640] In some embodiments, the mRNA comprises at least one
nucleoside selected from the group consisting of 2-aminopurine, 2,
6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,
7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,
7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,
1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine,
2-methoxy-adenine or any disclosed herein.
[0641] In some embodiments, the mRNA comprises at least one
nucleoside selected from the group consisting of inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine,
N2,N2-dimethyl-6-thio-guanosine or any disclosed herein.
[0642] In another aspect, the disclosure provides compositions for
protein production, comprising a first isolated nucleic acid
comprising a translatable region and a nucleoside modification,
wherein the nucleic acid exhibits reduced degradation by a cellular
nuclease, and a mammalian cell suitable for translation of the
translatable region of the first nucleic acid.
Definitions
[0643] At various places in the present specification, substituents
of compounds of the present disclosure are disclosed in groups or
in ranges. It is specifically intended that the present disclosure
include each and every individual subcombination of the members of
such groups and ranges. For example, the term "C.sub.1-6 alkyl" is
specifically intended to individually disclose methyl, ethyl,
C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, and C.sub.6 alkyl.
[0644] About: As used herein, the term "about" means+/-10% of the
recited value.
[0645] Administered in combination: As used herein, the term
"administered in combination" or "combined administration" means
that two or more agents are administered to a subject at the same
time or within an interval such that there may be an overlap of an
effect of each agent on the patient. In some embodiments, they are
administered within about 60, 30, 15, 10, 5, or 1 minute of one
another. In some embodiments, the administrations of the agents are
spaced sufficiently closely together such that a combinatorial
(e.g., a synergistic) effect is achieved.
[0646] 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, or a pig). In some embodiments, animals include,
but are not limited to, mammals, birds, reptiles, amphibians, fish,
and worms. In some embodiments, the animal is a transgenic animal,
genetically-engineered animal, or a clone.
[0647] Antigens of interest or desired antigens: As used herein,
the terms "antigens of interest" or "desired antigens" include
those proteins and other biomolecules provided herein that are
immunospecifically bound by the antibodies and fragments, mutants,
variants, and alterations thereof described herein. Examples of
antigens of interest include, but are not limited to, insulin,
insulin-like growth factor, hGH, tPA, cytokines, such as
interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or
IFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF
beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
[0648] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0649] Associated with: As used herein, the terms "associated
with," "conjugated," "linked," "attached," and "tethered," when
used with respect to two or more moieties, means that the moieties
are physically associated or connected with one another, either
directly or via one or more additional moieties that serves as a
linking agent, to form a structure that is sufficiently stable so
that the moieties remain physically associated under the conditions
in which the structure is used, e.g., physiological conditions. An
"association" need not be strictly through direct covalent chemical
bonding. It may also suggest ionic or hydrogen bonding or a
hybridization based connectivity sufficiently stable such that the
"associated" entities remain physically associated.
[0650] Biocompatible: As used herein, the term "biocompatible"
means compatible with living cells, tissues, organs or systems
posing little to no risk of injury, toxicity or rejection by the
immune system.
[0651] Biodegradable: As used herein, the term "biodegradable"
means capable of being broken down into innocuous products by the
action of living things.
[0652] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any substance
that has activity in a biological system and/or organism. For
instance, a substance that, when administered to an organism, has a
biological effect on that organism, is considered to be
biologically active. In particular embodiments, a polynucleotide of
the present invention may be considered biologically active if even
a portion of the polynucleotide is biologically active or mimics an
activity considered biologically relevant.
[0653] Chemical terms: The following provides the definition of
various chemical terms from "acyl" to "thiol."
[0654] The term "acyl," as used herein, represents a hydrogen or an
alkyl group (e.g., a haloalkyl group), as defined herein, that is
attached to the parent molecular group through a carbonyl group, as
defined herein, and is exemplified by formyl (i.e., a
carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl,
butanoyl and the like. Exemplary unsubstituted acyl groups include
from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In some
embodiments, the alkyl group is further substituted with 1, 2, 3,
or 4 substituents as described herein.
[0655] The term "acylamino," as used herein, represents an acyl
group, as defined herein, attached to the parent molecular group
though an amino group, as defined herein (i.e.,
--N(R.sup.N1)--C(O)--R, where R is H or an optionally substituted
C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl group (e.g., haloalkyl)
and R.sup.N1 is as defined herein). Exemplary unsubstituted
acylamino groups include from 1 to 41 carbons (e.g., from 1 to 7,
from 1 to 13, from 1 to 21, from 2 to 7, from 2 to 13, from 2 to
21, or from 2 to 41 carbons). In some embodiments, the alkyl group
is further substituted with 1, 2, 3, or 4 substituents as described
herein, and/or the amino group is --NH.sub.2 or --NHR.sup.N1,
wherein R.sup.N1 is, independently, OH, NO.sub.2, NH.sub.2,
NR.sup.N2.sub.2, SO.sub.2OR.sup.N2, SO.sub.2R.sup.N2, SOR.sup.N2,
alkyl, aryl, acyl (e.g., acetyl, trifluoroacetyl, or others
described herein), or alkoxycarbonylalkyl, and each R.sup.N2 can be
H, alkyl, or aryl.
[0656] The term "acylaminoalkyl," as used herein, represents an
acyl group, as defined herein, attached to an amino group that is
in turn attached to the parent molecular group though an alkyl
group, as defined herein (i.e., -alkyl-N(R.sup.N1)--C(O)--R, where
R is H or an optionally substituted C.sub.1-6, C.sub.1-10, or
C.sub.1-20 alkyl group (e.g., haloalkyl) and R.sup.N1 is as defined
herein). Exemplary unsubstituted acylamino groups include from 1 to
41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21, from 2
to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). In some
embodiments, the alkyl group is further substituted with 1, 2, 3,
or 4 substituents as described herein, and/or the amino group is
--NH.sub.2 or --NHR.sup.N1, wherein R.sup.N1 is, independently, OH,
NO.sub.2, NH.sub.2, NR.sup.N2.sub.2, SO.sub.2OR.sup.N2,
SO.sub.2R.sup.N2, SOR.sup.N2, alkyl, aryl, acyl (e.g., acetyl,
trifluoroacetyl, or others described herein), or
alkoxycarbonylalkyl, and each R.sup.N2 can be H, alkyl, or
aryl.
[0657] The term "acyloxy," as used herein, represents an acyl
group, as defined herein, attached to the parent molecular group
though an oxygen atom (i.e., --O--C(O)--R, where R is H or an
optionally substituted C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl
group). Exemplary unsubstituted acyloxy groups include from 1 to 21
carbons (e.g., from 1 to 7 or from 1 to 11 carbons). In some
embodiments, the alkyl group is further substituted with 1, 2, 3,
or 4 substituents as described herein.
[0658] The term "acyloxyalkyl," as used herein, represents an acyl
group, as defined herein, attached to an oxygen atom that in turn
is attached to the parent molecular group though an alkyl group
(i.e., -alkyl-O--C(O)--R, where R is H or an optionally substituted
C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl group). Exemplary
unsubstituted acyloxyalkyl groups include from 1 to 21 carbons
(e.g., from 1 to 7 or from 1 to 11 carbons). In some embodiments,
the alkyl group is, independently, further substituted with 1, 2,
3, or 4 substituents as described herein.
[0659] The term "alkaryl," as used herein, represents an aryl
group, as defined herein, attached to the parent molecular group
through an alkylene group, as defined herein. Exemplary
unsubstituted alkaryl groups are from 7 to 30 carbons (e.g., from 7
to 16 or from 7 to 20 carbons, such as C.sub.1-6 alk-C.sub.6-10
aryl, C.sub.1-10 alk-C.sub.6-10 aryl, or C.sub.1-20 alk-C.sub.6-10
aryl). In some embodiments, the alkylene and the aryl each can be
further substituted with 1, 2, 3, or 4 substituent groups as
defined herein for the respective groups. Other groups preceded by
the prefix "alk-" are defined in the same manner, where "alk"
refers to a C.sub.1-6 alkylene, unless otherwise noted, and the
attached chemical structure is as defined herein.
[0660] The term "alkcycloalkyl" represents a cycloalkyl group, as
defined herein, attached to the parent molecular group through an
alkylene group, as defined herein (e.g., an alkylene group of from
1 to 4, from 1 to 6, from 1 to 10, or form 1 to 20 carbons). In
some embodiments, the alkylene and the cycloalkyl each can be
further substituted with 1, 2, 3, or 4 substituent groups as
defined herein for the respective group.
[0661] The term "alkenyl," as used herein, represents monovalent
straight or branched chain groups of, unless otherwise specified,
from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons)
containing one or more carbon-carbon double bonds and is
exemplified by ethenyl, 1-propenyl, 2-propenyl,
2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyls
include both cis and trans isomers. Alkenyl groups may be
optionally substituted with 1, 2, 3, or 4 substituent groups that
are selected, independently, from amino, aryl, cycloalkyl, or
heterocyclyl (e.g., heteroaryl), as defined herein, or any of the
exemplary alkyl substituent groups described herein.
[0662] The term "alkenyloxy" represents a chemical substituent of
formula --OR, where R is a C.sub.2-20 alkenyl group (e.g.,
C.sub.2-6 or C.sub.2-10 alkenyl), unless otherwise specified.
Exemplary alkenyloxy groups include ethenyloxy, propenyloxy, and
the like. In some embodiments, the alkenyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
(e.g., a hydroxy group).
[0663] The term "alkheteroaryl" refers to a heteroaryl group, as
defined herein, attached to the parent molecular group through an
alkylene group, as defined herein. Exemplary unsubstituted
alkheteroaryl groups are from 2 to 32 carbons (e.g., from 2 to 22,
from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to
14, from 2 to 13, or from 2 to 12 carbons, such as C.sub.1-6
alk-C.sub.1-12 heteroaryl, C.sub.1-10 alk-C.sub.1-12 heteroaryl, or
C.sub.1-20 alk-C.sub.1-12 heteroaryl). In some embodiments, the
alkylene and the heteroaryl each can be further substituted with 1,
2, 3, or 4 substituent groups as defined herein for the respective
group. Alkheteroaryl groups are a subset of alkheterocyclyl
groups.
[0664] The term "alkheterocyclyl" represents a heterocyclyl group,
as defined herein, attached to the parent molecular group through
an alkylene group, as defined herein. Exemplary unsubstituted
alkheterocyclyl groups are from 2 to 32 carbons (e.g., from 2 to
22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2
to 14, from 2 to 13, or from 2 to 12 carbons, such as C.sub.1-6
alk-C.sub.1-12 heterocyclyl, C.sub.1-10 alk-C.sub.1-12
heterocyclyl, or C.sub.1-20 alk-C.sub.1-12 heterocyclyl). In some
embodiments, the alkylene and the heterocyclyl each can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
for the respective group.
[0665] The term "alkoxy" represents a chemical substituent of
formula --OR, where R is a C.sub.1-20 alkyl group (e.g., C.sub.1-6
or C.sub.1-10 alkyl), unless otherwise specified. Exemplary alkoxy
groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and
isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl
group can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein (e.g., hydroxy or alkoxy).
[0666] The term "alkoxyalkoxy" represents an alkoxy group that is
substituted with an alkoxy group. Exemplary unsubstituted
alkoxyalkoxy groups include between 2 to 40 carbons (e.g., from 2
to 12 or from 2 to 20 carbons, such as C.sub.1-6 alkoxy-C.sub.1-6
alkoxy, C.sub.1-10 alkoxy-C.sub.1-10 alkoxy, or C.sub.1-20
alkoxy-C.sub.1-20 alkoxy). In some embodiments, the each alkoxy
group can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein.
[0667] The term "alkoxyalkyl" represents an alkyl group that is
substituted with an alkoxy group. Exemplary unsubstituted
alkoxyalkyl groups include between 2 to 40 carbons (e.g., from 2 to
12 or from 2 to 20 carbons, such as C.sub.1-6 alkoxy-C.sub.1-6
alkyl, C.sub.1-10 alkoxy-C.sub.1-10 alkyl, or C.sub.1-20
alkoxy-C.sub.1-20 alkyl). In some embodiments, the alkyl and the
alkoxy each can be further substituted with 1, 2, 3, or 4
substituent groups as defined herein for the respective group.
[0668] The term "alkoxycarbonyl," as used herein, represents an
alkoxy, as defined herein, attached to the parent molecular group
through a carbonyl atom (e.g., --C(O)--OR, where R is H or an
optionally substituted C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl
group). Exemplary unsubstituted alkoxycarbonyl include from 1 to 21
carbons (e.g., from 1 to 11 or from 1 to 7 carbons). In some
embodiments, the alkoxy group is further substituted with 1, 2, 3,
or 4 substituents as described herein.
[0669] The term "alkoxycarbonylacyl," as used herein, represents an
acyl group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., --C(O)-alkyl-C(O)--
OR, where R is an optionally substituted C.sub.1-6, C.sub.1-10, or
C.sub.1-20 alkyl group). Exemplary unsubstituted alkoxycarbonylacyl
include from 3 to 41 carbons (e.g., from 3 to 10, from 3 to 13,
from 3 to 17, from 3 to 21, or from 3 to 31 carbons, such as
C.sub.1-6 alkoxycarbonyl-C.sub.1-6 acyl, C.sub.1-10
alkoxycarbonyl-C.sub.1-10 acyl, or C.sub.1-20
alkoxycarbonyl-C.sub.1-20 acyl). In some embodiments, each alkoxy
and alkyl group is further independently substituted with 1, 2, 3,
or 4 substituents, as described herein (e.g., a hydroxy group) for
each group.
[0670] The term "alkoxycarbonylalkoxy," as used herein, represents
an alkoxy group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., --O-alkyl-C(O)--OR,
where R is an optionally substituted C.sub.1-6, C.sub.1-10, or
C.sub.1-20 alkyl group). Exemplary unsubstituted
alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g., from 3 to
10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31
carbons, such as C.sub.1-6 alkoxycarbonyl-C.sub.1-6 alkoxy,
C.sub.1-10 alkoxycarbonyl-C.sub.1-10 alkoxy, or C.sub.1-20
alkoxycarbonyl-C.sub.1-20 alkoxy). In some embodiments, each alkoxy
group is further independently substituted with 1, 2, 3, or 4
substituents, as described herein (e.g., a hydroxy group).
[0671] The term "alkoxycarbonylalkyl," as used herein, represents
an alkyl group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., -alkyl-C(O)--OR,
where R is an optionally substituted C.sub.1-20, C.sub.1-10, or
C.sub.1-6 alkyl group). Exemplary unsubstituted alkoxycarbonylalkyl
include from 3 to 41 carbons (e.g., from 3 to 10, from 3 to 13,
from 3 to 17, from 3 to 21, or from 3 to 31 carbons, such as
C.sub.1-6 alkoxycarbonyl-C.sub.1-6 alkyl, C.sub.1-10
alkoxycarbonyl-C.sub.1-10 alkyl, or C.sub.1-20
alkoxycarbonyl-C.sub.1-20 alkyl). In some embodiments, each alkyl
and alkoxy group is further independently substituted with 1, 2, 3,
or 4 substituents as described herein (e.g., a hydroxy group).
[0672] The term "alkoxycarbonylalkenyl," as used herein, represents
an alkenyl group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., -alkenyl-C(O)--OR,
where R is an optionally substituted C.sub.1-20, C.sub.1-10, or
C.sub.1-6 alkyl group). Exemplary unsubstituted
alkoxycarbonylalkenyl include from 4 to 41 carbons (e.g., from 4 to
10, from 4 to 13, from 4 to 17, from 4 to 21, or from 4 to 31
carbons, such as C.sub.1-6 alkoxycarbonyl-C.sub.2-6 alkenyl,
C.sub.1-10 alkoxycarbonyl-C.sub.2-10 alkenyl, or C.sub.1-20
alkoxycarbonyl-C.sub.2-20 alkenyl). In some embodiments, each
alkyl, alkenyl, and alkoxy group is further independently
substituted with 1, 2, 3, or 4 substituents as described herein
(e.g., a hydroxy group).
[0673] The term "alkoxycarbonylalkynyl," as used herein, represents
an alkynyl group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., -alkynyl-C(O)--OR,
where R is an optionally substituted C.sub.1-20, C.sub.1-10, or
C.sub.1-6 alkyl group). Exemplary unsubstituted
alkoxycarbonylalkynyl include from 4 to 41 carbons (e.g., from 4 to
10, from 4 to 13, from 4 to 17, from 4 to 21, or from 4 to 31
carbons, such as C.sub.1-6 alkoxycarbonyl-C.sub.2-6 alkynyl,
C.sub.1-10 alkoxycarbonyl-C.sub.2-10 alkynyl, or C.sub.1-20
alkoxycarbonyl-C.sub.2-20 alkynyl). In some embodiments, each
alkyl, alkynyl, and alkoxy group is further independently
substituted with 1, 2, 3, or 4 substituents as described herein
(e.g., a hydroxy group).
[0674] The term "alkyl," as used herein, is inclusive of both
straight chain and branched chain saturated groups from 1 to 20
carbons (e.g., from 1 to 10 or from 1 to 6), unless otherwise
specified. Alkyl groups are exemplified by methyl, ethyl, n- and
iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like,
and may be optionally substituted with one, two, three, or, in the
case of alkyl groups of two carbons or more, four substituents
independently selected from the group consisting of: (1) C.sub.1-6
alkoxy; (2) C.sub.1-6 alkylsulfinyl; (3) amino, as defined herein
(e.g., unsubstituted amino (i.e., --NH.sub.2) or a substituted
amino (i.e., --N(R.sup.N1).sub.2, where R.sup.N1 is as defined for
amino); (4) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (5) azido; (6) halo;
(7) (C.sub.2-9 heterocyclyl)oxy; (8) hydroxy, optionally
substituted with an O-protecting group; (9) nitro; (10) oxo (e.g.,
carboxyaldehyde or acyl); (11) C.sub.1-7 spirocyclyl; (12)
thioalkoxy; (13) thiol; (14) --CO.sub.2R.sup.A', optionally
substituted with an O-protecting group and where R.sup.A' is
selected from the group consisting of (a) C.sub.1-20 alkyl (e.g.,
C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6 alkenyl),
(c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6 alk-C.sub.6-10
aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (15)
--C(O)NR.sup.B'R.sup.C', where each of R.sup.B' and R.sup.C' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (16) --SO.sub.2R.sup.D', where R.sup.D' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) C.sub.1-6 alk-C.sub.6-10 aryl, and (d)
hydroxy; (17) --SO.sub.2NR.sup.E'R.sup.F', where each of R.sup.E'
and R.sup.F' is, independently, selected from the group consisting
of (a) hydrogen, (b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18) --C(O)R.sup.G', where R.sup.G'
is selected from the group consisting of (a) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6
alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (19)
--NR.sup.H'C(O)R.sup.I', wherein R.sup.H' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.I' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (20)
--NR.sup.J'C(O)OR.sup.K', wherein R.sup.J' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.K' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; and (21)
amidine. In some embodiments, each of these groups can be further
substituted as described herein. For example, the alkylene group of
a C.sub.1-alkaryl can be further substituted with an oxo group to
afford the respective aryloyl substituent.
[0675] The term "alkylene" and the prefix "alk-," as used herein,
represent a saturated divalent hydrocarbon group derived from a
straight or branched chain saturated hydrocarbon by the removal of
two hydrogen atoms, and is exemplified by methylene, ethylene,
isopropylene, and the like. The term "C.sub.x-y alkylene" and the
prefix "C.sub.x-y alk-" represent alkylene groups having between x
and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and
exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, or 20 (e.g., C.sub.1-6, C.sub.1-10, C.sub.2-20, C.sub.2-6,
C.sub.2-10, or C.sub.2-20 alkylene). In some embodiments, the
alkylene can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein for an alkyl group.
[0676] The term "alkylsulfinyl," as used herein, represents an
alkyl group attached to the parent molecular group through an
--S(O)-- group. Exemplary unsubstituted alkylsulfinyl groups are
from 1 to 6, from 1 to 10, or from 1 to 20 carbons. In some
embodiments, the alkyl group can be further substituted with 1, 2,
3, or 4 substituent groups as defined herein.
[0677] The term "alkylsulfinylalkyl," as used herein, represents an
alkyl group, as defined herein, substituted by an alkylsulfinyl
group. Exemplary unsubstituted alkylsulfinylalkyl groups are from 2
to 12, from 2 to 20, or from 2 to 40 carbons. In some embodiments,
each alkyl group can be further substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0678] The term "alkynyl," as used herein, represents monovalent
straight or branched chain groups from 2 to 20 carbon atoms (e.g.,
from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a
carbon-carbon triple bond and is exemplified by ethynyl,
1-propynyl, and the like. Alkynyl groups may be optionally
substituted with 1, 2, 3, or 4 substituent groups that are
selected, independently, from aryl, cycloalkyl, or heterocyclyl
(e.g., heteroaryl), as defined herein, or any of the exemplary
alkyl substituent groups described herein.
[0679] The term "alkynyloxy" represents a chemical substituent of
formula --OR, where R is a C.sub.2-20 alkynyl group (e.g.,
C.sub.2-6 or C.sub.2-10 alkynyl), unless otherwise specified.
Exemplary alkynyloxy groups include ethynyloxy, propynyloxy, and
the like. In some embodiments, the alkynyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
(e.g., a hydroxy group).
[0680] The term "amidine," as used herein, represents a
--C(.dbd.NH)NH.sub.2 group.
[0681] The term "amino," as used herein, represents
--N(R.sup.N1).sub.2, wherein each R.sup.N1 is, independently, H,
OH, NO.sub.2, N(R.sup.N2).sub.2, SO.sub.2OR.sup.N2,
SO.sub.2R.sup.N2, SOR.sup.N2, an N-protecting group, alkyl,
alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl,
carboxyalkyl (e.g., optionally substituted with an O-protecting
group, such as optionally substituted arylalkoxycarbonyl groups or
any described herein), sulfoalkyl, acyl (e.g., acetyl,
trifluoroacetyl, or others described herein), alkoxycarbonylalkyl
(e.g., optionally substituted with an O-protecting group, such as
optionally substituted arylalkoxycarbonyl groups or any described
herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,
alkheteroaryl), wherein each of these recited R.sup.N1 groups can
be optionally substituted, as defined herein for each group; or two
R.sup.N1 combine to form a heterocyclyl or an N-protecting group,
and wherein each R.sup.N2 is, independently, H, alkyl, or aryl. The
amino groups of the invention can be an unsubstituted amino (i.e.,
--NH.sub.2) or a substituted amino (i.e., --N(R.sup.N1).sub.2). In
a preferred embodiment, amino is --NH.sub.2 or --NHR.sup.N1,
wherein R.sup.N1 is, independently, OH, NO.sub.2, NH.sub.2,
NR.sup.N2.sub.2, SO.sub.2OR.sup.N2, SO.sub.2R.sup.N2, SOR.sup.N2,
alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl,
trifluoroacetyl, or others described herein), alkoxycarbonylalkyl
(e.g., t-butoxycarbonylalkyl) or aryl, and each R.sup.N2 can be H,
C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl), or C.sub.6-10 aryl.
[0682] The term "amino acid," as described herein, refers to a
molecule having a side chain, an amino group, and an acid group
(e.g., a carboxy group of --CO.sub.2H or a sulfo group of
--SO.sub.3H), wherein the amino acid is attached to the parent
molecular group by the side chain, amino group, or acid group
(e.g., the side chain). In some embodiments, the amino acid is
attached to the parent molecular group by a carbonyl group, where
the side chain or amino group is attached to the carbonyl group.
Exemplary side chains include an optionally substituted alkyl,
aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl,
carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include
alanine, arginine, asparagine, aspartic acid, cysteine, glutamic
acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine,
leucine, lysine, methionine, norvaline, ornithine, phenylalanine,
proline, pyrrolysine, selenocysteine, serine, taurine, threonine,
tryptophan, tyrosine, and valine. Amino acid groups may be
optionally substituted with one, two, three, or, in the case of
amino acid groups of two carbons or more, four substituents
independently selected from the group consisting of: (1) C.sub.1-6
alkoxy; (2) C.sub.1-6 alkylsulfinyl; (3) amino, as defined herein
(e.g., unsubstituted amino (i.e., --NH.sub.2) or a substituted
amino (i.e., --N(R.sup.N1).sub.2, where R.sup.N1 is as defined for
amino); (4) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (5) azido; (6) halo;
(7) (C.sub.2-9 heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo
(e.g., carboxyaldehyde or acyl); (11) C.sub.1-7 spirocyclyl; (12)
thioalkoxy; (13) thiol; (14) --CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-20 alkyl (e.g.,
C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6 alkenyl),
(c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6 alk-C.sub.6-10
aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (15)
--C(O)NR.sup.B'R.sup.C', where each of R.sup.B' and R.sup.C' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (16) --SO.sub.2R.sup.D', where R.sup.D' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) C.sub.1-6 alk-C.sub.6-10 aryl, and (d)
hydroxy; (17) --SO.sub.2NR.sup.E'R.sup.F', where each of R.sup.E'
and R.sup.F' is, independently, selected from the group consisting
of (a) hydrogen, (b).sub.1-6 alkyl, (c) C.sub.6-10 aryl and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18) --C(O)R.sup.G', where R.sup.G'
is selected from the group consisting of (a) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6
alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (19)
--NR.sup.H'C(O)R.sup.I', wherein R.sup.H' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.I' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (20)
--NR.sup.J'C(O)OR.sup.K', wherein R.sup.J' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.K' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; and (21)
amidine. In some embodiments, each of these groups can be further
substituted as described herein.
[0683] The term "aminoalkoxy," as used herein, represents an alkoxy
group, as defined herein, substituted by an amino group, as defined
herein. The alkyl and amino each can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for the
respective group (e.g., CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6 alk-C.sub.6-10
aryl, e.g., carboxy).
[0684] The term "aminoalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by an amino group, as defined
herein. The alkyl and amino each can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for the
respective group (e.g., CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6 alk-C.sub.6-10
aryl, e.g., carboxy, and/or an N-protecting group).
[0685] The term "aminoalkenyl," as used herein, represents an
alkenyl group, as defined herein, substituted by an amino group, as
defined herein. The alkenyl and amino each can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein for the respective group (e.g., CO.sub.2R.sup.A', where
R.sup.A' is selected from the group consisting of (a) C.sub.1-6
alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6
alk-C.sub.6-10 aryl, e.g., carboxy, and/or an N-protecting
group).
[0686] The term "aminoalkynyl," as used herein, represents an
alkynyl group, as defined herein, substituted by an amino group, as
defined herein. The alkynyl and amino each can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein for the respective group (e.g., CO.sub.2R.sup.A', where
R.sup.A' is selected from the group consisting of (a) C.sub.1-6
alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6
alk-C.sub.6-10 aryl, e.g., carboxy, and/or an N-protecting
group).
[0687] The term "aryl," as used herein, represents a mono-,
bicyclic, or multicyclic carbocyclic ring system having one or two
aromatic rings and is exemplified by phenyl, naphthyl,
1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl,
phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and may
be optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from the group consisting of: (1) C.sub.1-7
acyl (e.g., carboxyaldehyde); (2) C.sub.1-20 alkyl (e.g., C.sub.1-6
alkyl, C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6
alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6 alkyl,
azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.1-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.1-6
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) alkyl, (b) C.sub.6-10 aryl, and (c)
alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (25)
C.sub.1-6 alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6
alk-C.sub.1-12 heteroaryl); (26) C.sub.2-20 alkenyl; and (27)
C.sub.2-20 alkynyl. In some embodiments, each of these groups can
be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl
can be further substituted with an oxo group to afford the
respective aryloyl and (heterocyclyl)oyl substituent group.
[0688] The term "arylalkoxy," as used herein, represents an alkaryl
group, as defined herein, attached to the parent molecular group
through an oxygen atom. Exemplary unsubstituted arylalkoxy groups
include from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20
carbons, such as C.sub.6-10 aryl-C.sub.1-6 alkoxy, C.sub.6-10
aryl-C.sub.1-10 alkoxy, or C.sub.6-10 aryl-C.sub.1-20 alkoxy). In
some embodiments, the arylalkoxy group can be substituted with 1,
2, 3, or 4 substituents as defined herein
[0689] The term "arylalkoxycarbonyl," as used herein, represents an
arylalkoxy group, as defined herein, attached to the parent
molecular group through a carbonyl (e.g., --C(O)--O-alkyl-aryl).
Exemplary unsubstituted arylalkoxy groups include from 8 to 31
carbons (e.g., from 8 to 17 or from 8 to 21 carbons, such as
C.sub.6-10 aryl-C.sub.1-6 alkoxy-carbonyl, C.sub.6-10
aryl-C.sub.1-10 alkoxy-carbonyl, or C.sub.6-10 aryl-C.sub.1-20
alkoxy-carbonyl). In some embodiments, the arylalkoxycarbonyl group
can be substituted with 1, 2, 3, or 4 substituents as defined
herein.
[0690] The term "aryloxy" represents a chemical substituent of
formula --OR', where R' is an aryl group of 6 to 18 carbons, unless
otherwise specified. In some embodiments, the aryl group can be
substituted with 1, 2, 3, or 4 substituents as defined herein.
[0691] The term "aryloyl," as used herein, represents an aryl
group, as defined herein, that is attached to the parent molecular
group through a carbonyl group. Exemplary unsubstituted aryloyl
groups are of 7 to 11 carbons. In some embodiments, the aryl group
can be substituted with 1, 2, 3, or 4 substituents as defined
herein.
[0692] The term "azido" represents an --N.sub.3 group, which can
also be represented as --N.dbd.N.dbd.N.
[0693] The term "bicyclic," as used herein, refer to a structure
having two rings, which may be aromatic or non-aromatic. Bicyclic
structures include spirocyclyl groups, as defined herein, and two
rings that share one or more bridges, where such bridges can
include one atom or a chain including two, three, or more atoms.
Exemplary bicyclic groups include a bicyclic carbocyclyl group,
where the first and second rings are carbocyclyl groups, as defined
herein; a bicyclic aryl groups, where the first and second rings
are aryl groups, as defined herein; bicyclic heterocyclyl groups,
where the first ring is a heterocyclyl group and the second ring is
a carbocyclyl (e.g., aryl) or heterocyclyl (e.g., heteroaryl)
group; and bicyclic heteroaryl groups, where the first ring is a
heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)
or heterocyclyl (e.g., heteroaryl) group. In some embodiments, the
bicyclic group can be substituted with 1, 2, 3, or 4 substituents
as defined herein for cycloalkyl, heterocyclyl, and aryl
groups.
[0694] The term "boranyl," as used herein, represents
--B(R.sup.B1).sub.3, where each R.sup.B1 is, independently,
selected from the group consisting of H and optionally substituted
alkyl. In some embodiments, the boranyl group can be substituted
with 1, 2, 3, or 4 substituents as defined herein for alkyl.
[0695] The terms "carbocyclic" and "carbocyclyl," as used herein,
refer to an optionally substituted C.sub.3-12 monocyclic, bicyclic,
or tricyclic structure in which the rings, which may be aromatic or
non-aromatic, are formed by carbon atoms. Carbocyclic structures
include cycloalkyl, cycloalkenyl, and aryl groups.
[0696] The term "carbamoyl," as used herein, represents
--C(O)--N(R.sup.N1).sub.2, where the meaning of each R.sup.N1 is
found in the definition of "amino" provided herein.
[0697] The term "carbamoylalkyl," as used herein, represents an
alkyl group, as defined herein, substituted by a carbamoyl group,
as defined herein. The alkyl group can be further substituted with
1, 2, 3, or 4 substituent groups as described herein.
[0698] The term "carbamyl," as used herein, refers to a carbamate
group having the structure --NR.sup.N1C(.dbd.O)OR or
--OC(.dbd.O)N(R.sup.N1).sub.2, where the meaning of each R.sup.N1
is found in the definition of "amino" provided herein, and R is
alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl
(e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as
defined herein.
[0699] The term "carbonyl," as used herein, represents a C(O)
group, which can also be represented as C.dbd.O.
[0700] The term "carboxyaldehyde" represents an acyl group having
the structure --CHO.
[0701] The term "carboxy," as used herein, means --CO.sub.2H.
[0702] The term "carboxyalkoxy," as used herein, represents an
alkoxy group, as defined herein, substituted by a carboxy group, as
defined herein. The alkoxy group can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for the alkyl
group, and the carboxy group can be optionally substituted with one
or more O-protecting groups.
[0703] The term "carboxyalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a carboxy group, as
defined herein. The alkyl group can be further substituted with 1,
2, 3, or 4 substituent groups as described herein, and the carboxy
group can be optionally substituted with one or more O-protecting
groups.
[0704] The term "carboxyaminoalkyl," as used herein, represents an
aminoalkyl group, as defined herein, substituted by a carboxy, as
defined herein. The carboxy, alkyl, and amino each can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein for the respective group (e.g., CO.sub.2R.sup.A', where
R.sup.A' is selected from the group consisting of (a) C.sub.1-6
alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6
alk-C.sub.6-10 aryl, e.g., carboxy, and/or an N-protecting group,
and/or an 0-protecting group).
[0705] The term "cyano," as used herein, represents an --CN
group.
[0706] The term "cycloalkoxy" represents a chemical substituent of
formula --OR, where R is a C.sub.3-8 cycloalkyl group, as defined
herein, unless otherwise specified. The cycloalkyl group can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein. Exemplary unsubstituted cycloalkoxy groups are
from 3 to 8 carbons. In some embodiment, the cycloalkyl group can
be further substituted with 1, 2, 3, or 4 substituent groups as
described herein.
[0707] The term "cycloalkyl," as used herein represents a
monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon
group from three to eight carbons, unless otherwise specified, and
is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, bicycle heptyl, and the like. When the cycloalkyl
group includes one carbon-carbon double bond, the cycloalkyl group
can be referred to as a "cycloalkenyl" group. Exemplary
cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the
like. The cycloalkyl groups of this invention can be optionally
substituted with: (1) C.sub.1-7 acyl (e.g., carboxyaldehyde); (2)
C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl, C.sub.1-6 alkoxy-C.sub.1-6
alkyl, C.sub.1-6 alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6
alkyl, azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.1-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.6-10
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) C.sub.6-10 alkyl, (b) C.sub.6-10 aryl, and (c)
C.sub.1-6 alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.6-10 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (25)
C.sub.1-6 alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6
alk-C.sub.1-12 heteroaryl); (26) oxo; (27) C.sub.2-20 alkenyl; and
(28) C.sub.2-20 alkynyl. In some embodiments, each of these groups
can be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl
can be further substituted with an oxo group to afford the
respective aryloyl and (heterocyclyl)oyl substituent group.
[0708] The term "diastereomer," as used herein means stereoisomers
that are not mirror images of one another and are
non-superimposable on one another.
[0709] The term "effective amount" of an agent, as used herein, is
that amount sufficient to effect beneficial or desired results, for
example, clinical results, and, as such, an "effective amount"
depends upon the context in which it is being applied. For example,
in the context of administering an agent that treats cancer, an
effective amount of an agent is, for example, an amount sufficient
to achieve treatment, as defined herein, of cancer, as compared to
the response obtained without administration of the agent.
[0710] The term "enantiomer," as used herein, means each individual
optically active form of a compound of the invention, having an
optical purity or enantiomeric excess (as determined by methods
standard in the art) of at least 80% (i.e., at least 90% of one
enantiomer and at most 10% of the other enantiomer), preferably at
least 90% and more preferably at least 98%.
[0711] The term "halo," as used herein, represents a halogen
selected from bromine, chlorine, iodine, or fluorine.
[0712] The term "haloalkoxy," as used herein, represents an alkoxy
group, as defined herein, substituted by a halogen group (i.e., F,
Cl, Br, or I). A haloalkoxy may be substituted with one, two,
three, or, in the case of alkyl groups of two carbons or more, four
halogens. Haloalkoxy groups include perfluoroalkoxys (e.g.,
--OCF.sub.3), --OCHF.sub.2, --OCH.sub.2F, --OCCl.sub.3,
--OCH.sub.2CH.sub.2Br, --OCH.sub.2CH(CH.sub.2CH.sub.2Br)CH.sub.3,
and --OCHICH.sub.3. In some embodiments, the haloalkoxy group can
be further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkyl groups.
[0713] The term "haloalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a halogen group (i.e., F,
Cl, Br, or I). A haloalkyl may be substituted with one, two, three,
or, in the case of alkyl groups of two carbons or more, four
halogens. Haloalkyl groups include perfluoroalkyls (e.g.,
--CF.sub.3), --CHF.sub.2, --CH.sub.2F, --CCl.sub.3,
--CH.sub.2CH.sub.2Br, --CH.sub.2CH(CH.sub.2CH.sub.2Br)CH.sub.3, and
--CHICH.sub.3. In some embodiments, the haloalkyl group can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkyl groups.
[0714] The term "heteroalkylene," as used herein, refers to an
alkylene group, as defined herein, in which one or two of the
constituent carbon atoms have each been replaced by nitrogen,
oxygen, or sulfur. In some embodiments, the heteroalkylene group
can be further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkylene groups.
[0715] The term "heteroaryl," as used herein, represents that
subset of heterocyclyls, as defined herein, which are aromatic:
i.e., they contain 4n+2 pi electrons within the mono- or
multicyclic ring system. Exemplary unsubstituted heteroaryl groups
are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2
to 10, or 2 to 9) carbons. In some embodiment, the heteroaryl is
substituted with 1, 2, 3, or 4 substituents groups as defined for a
heterocyclyl group.
[0716] The term "heterocyclyl," as used herein represents a 5-, 6-
or 7-membered ring, unless otherwise specified, containing one,
two, three, or four heteroatoms independently selected from the
group consisting of nitrogen, oxygen, and sulfur. The 5-membered
ring has zero to two double bonds, and the 6- and 7-membered rings
have zero to three double bonds. Exemplary unsubstituted
heterocyclyl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9,
2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term
"heterocyclyl" also represents a heterocyclic compound having a
bridged multicyclic structure in which one or more carbons and/or
heteroatoms bridges two non-adjacent members of a monocyclic ring,
e.g., a quinuclidinyl group. The term "heterocyclyl" includes
bicyclic, tricyclic, and tetracyclic groups in which any of the
above heterocyclic rings is fused to one, two, or three carbocyclic
rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring,
a cyclopentane ring, a cyclopentene ring, or another monocyclic
heterocyclic ring, such as indolyl, quinolyl, isoquinolyl,
tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples
of fused heterocyclyls include tropanes and
1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl,
pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,
imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,
homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl,
thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,
isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,
quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,
phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,
benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,
triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl),
purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl),
tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
dihydrothienyl, dihydroindolyl, dihydroquinolyl,
tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl,
pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,
isobenzofuranyl, benzothienyl, and the like, including dihydro and
tetrahydro forms thereof, where one or more double bonds are
reduced and replaced with hydrogens. Still other exemplary
heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl;
2,3-dihydro-2-oxo-1H-imidazolyl;
2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,
2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);
2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,
2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);
2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,
2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);
4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino
5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,
1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);
2,6-dioxo-piperidinyl (e.g.,
2,6-dioxo-3-ethyl-3-phenylpiperidinyl);
1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,
2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);
1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,
1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);
1,6-dihydro-6-oxo-pyridazinyl (e.g.,
1,6-dihydro-6-oxo-3-ethylpyridazinyl);
1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g.,
1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);
2,3-dihydro-2-oxo-1H-indolyl (e.g.,
3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and
2,3-dihydro-2-oxo-3,3'-spiropropane-1H-indol-1-yl);
1,3-dihydro-1-oxo-2H-iso-indolyl;
1,3-dihydro-1,3-dioxo-2H-iso-indolyl; 1H-benzopyrazolyl (e.g.,
1-(ethoxycarbonyl)-1H-benzopyrazolyl);
2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,
3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);
2,3-dihydro-2-oxo-benzoxazolyl (e.g.,
5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);
2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;
1,4-benzodioxanyl; 1,3-benzodioxanyl;
2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;
3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,
2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);
1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,
1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);
1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,
1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);
1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,
1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);
2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl;
and 1,8-naphthylenedicarboxamido. Additional heterocyclics include
3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and
2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or
diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl,
benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and
thiocanyl. Heterocyclic groups also include groups of the
formula
##STR00166##
where
[0717] E' is selected from the group consisting of --N-- and
--CH--; F' is selected from the group consisting of --N.dbd.CH--,
--NH--CH.sub.2--, --NH--C(O)--, --NH--, --CH.dbd.N--,
--CH.sub.2--NH--, --C(O)--NH--, --CH.dbd.CH--, --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2O--, --OCH.sub.2--, --O--, and
--S--; and G' is selected from the group consisting of --CH-- and
--N--. Any of the heterocyclyl groups mentioned herein may be
optionally substituted with one, two, three, four or five
substituents independently selected from the group consisting of:
(1) C.sub.1-7 acyl (e.g., carboxyaldehyde); (2) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl, C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6
alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6 alkyl,
azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.2-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2)--CO.sub.2R.sup.A', where q is an integer from zero to
four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.1-6
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, and (c)
C.sub.1-6 alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) arylalkoxy; (25) C.sub.1-6
alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6 alk-C.sub.1-12
heteroaryl); (26) oxo; (27) (C.sub.1-12 heterocyclyl)imino; (28)
C.sub.2-20 alkenyl; and (29) C.sub.2-20 alkynyl. In some
embodiments, each of these groups can be further substituted as
described herein. For example, the alkylene group of a
C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl can be further
substituted with an oxo group to afford the respective aryloyl and
(heterocyclyl)oyl substituent group.
[0718] The term "(heterocyclyl) imino," as used herein, represents
a heterocyclyl group, as defined herein, attached to the parent
molecular group through an imino group. In some embodiments, the
heterocyclyl group can be substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0719] The term "(heterocyclyl)oxy," as used herein, represents a
heterocyclyl group, as defined herein, attached to the parent
molecular group through an oxygen atom. In some embodiments, the
heterocyclyl group can be substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0720] The term "(heterocyclyl)oyl," as used herein, represents a
heterocyclyl group, as defined herein, attached to the parent
molecular group through a carbonyl group. In some embodiments, the
heterocyclyl group can be substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0721] The term "hydrocarbon," as used herein, represents a group
consisting only of carbon and hydrogen atoms.
[0722] The term "hydroxy," as used herein, represents an --OH
group. In some embodiments, the hydroxy group can be substituted
with 1, 2, 3, or 4 substituent groups (e.g., O-protecting groups)
as defined herein for an alkyl.
[0723] The term "hydroxyalkenyl," as used herein, represents an
alkenyl group, as defined herein, substituted by one to three
hydroxy groups, with the proviso that no more than one hydroxy
group may be attached to a single carbon atom of the alkyl group,
and is exemplified by dihydroxypropenyl, hydroxyisopentenyl, and
the like. In some embodiments, the hydroxyalkenyl group can be
substituted with 1, 2, 3, or 4 substituent groups (e.g.,
O-protecting groups) as defined herein for an alkyl.
[0724] The term "hydroxyalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by one to three hydroxy
groups, with the proviso that no more than one hydroxy group may be
attached to a single carbon atom of the alkyl group, and is
exemplified by hydroxymethyl, dihydroxypropyl, and the like. In
some embodiments, the hydroxyalkyl group can be substituted with 1,
2, 3, or 4 substituent groups (e.g., O-protecting groups) as
defined herein for an alkyl.
[0725] The term "hydroxyalkynyl," as used herein, represents an
alkynyl group, as defined herein, substituted by one to three
hydroxy groups, with the proviso that no more than one hydroxy
group may be attached to a single carbon atom of the alkyl group.
In some embodiments, the hydroxyalkynyl group can be substituted
with 1, 2, 3, or 4 substituent groups (e.g., O-protecting groups)
as defined herein for an alkyl.
[0726] The term "isomer," as used herein, means any tautomer,
stereoisomer, enantiomer, or diastereomer of any compound of the
invention. It is recognized that the compounds of the invention can
have one or more chiral centers and/or double bonds and, therefore,
exist as stereoisomers, such as double-bond isomers (i.e.,
geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e.,
(+) or (-)) or cis/trans isomers). According to the invention, the
chemical structures depicted herein, and therefore the compounds of
the invention, encompass all of the corresponding stereoisomers,
that is, both the stereomerically pure form (e.g., geometrically
pure, enantiomerically pure, or diastereomerically pure) and
enantiomeric and stereoisomeric mixtures, e.g., racemates.
Enantiomeric and stereoisomeric mixtures of compounds of the
invention can typically be resolved into their component
enantiomers or stereoisomers by well-known methods, such as
chiral-phase gas chromatography, chiral-phase high performance
liquid chromatography, crystallizing the compound as a chiral salt
complex, or crystallizing the compound in a chiral solvent.
Enantiomers and stereoisomers can also be obtained from
stereomerically or enantiomerically pure intermediates, reagents,
and catalysts by well-known asymmetric synthetic methods.
[0727] The term "N-protected amino," as used herein, refers to an
amino group, as defined herein, to which is attached one or two
N-protecting groups, as defined herein.
[0728] The term "N-protecting group," as used herein, represents
those groups intended to protect an amino group against undesirable
reactions during synthetic procedures. Commonly used N-protecting
groups are disclosed in Greene, "Protective Groups in Organic
Synthesis," 3.sup.rd Edition (John Wiley & Sons, New York,
1999), which is incorporated herein by reference. N-protecting
groups include acyl, aryloyl, or carbamyl groups such as formyl,
acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,
2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,
o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl,
4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral
auxiliaries such as protected or unprotected D, L or D, L-amino
acids such as alanine, leucine, phenylalanine, and the like;
sulfonyl-containing groups such as benzenesulfonyl,
p-toluenesulfonyl, and the like; carbamate forming groups such as
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxy carbonyl, t-butyloxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxy carbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like, alkaryl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, and the like and silyl groups, such as
trimethylsilyl, and the like. Preferred N-protecting groups are
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl,
phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and
benzyloxycarbonyl (Cbz).
[0729] The term "nitro," as used herein, represents an --NO.sub.2
group.
[0730] The term "O-protecting group," as used herein, represents
those groups intended to protect an oxygen containing (e.g.,
phenol, hydroxyl, or carbonyl) group against undesirable reactions
during synthetic procedures. Commonly used O-protecting groups are
disclosed in Greene, "Protective Groups in Organic Synthesis,"
3.sup.rd Edition (John Wiley & Sons, New York, 1999), which is
incorporated herein by reference. Exemplary O-protecting groups
include acyl, aryloyl, or carbamyl groups, such as formyl, acetyl,
propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl,
trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
.alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,
t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl,
4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl,
4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl;
alkylcarbonyl groups, such as acyl, acetyl, propionyl, pivaloyl,
and the like; optionally substituted arylcarbonyl groups, such as
benzoyl; silyl groups, such as trimethylsilyl (TMS),
tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl
(TOM), triisopropylsilyl (TIPS), and the like; ether-forming groups
with the hydroxyl, such methyl, methoxymethyl, tetrahydropyranyl,
benzyl, p-methoxybenzyl, trityl, and the like; alkoxycarbonyls,
such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,
n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl,
sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl,
cyclohexyloxycarbonyl, methyloxycarbonyl, and the like;
alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,
ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl,
2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl,
2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl,
propargyloxycarbonyl, 2-butenoxycarbonyl,
3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls,
such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl,
2,2,2-trichloroethoxycarbonyl, and the like; optionally substituted
arylalkoxycarbonyl groups, such as benzyloxycarbonyl,
p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl,
3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-bromobenzyloxy-carbonyl, fluorenylmethyloxycarbonyl, and the
like; and optionally substituted aryloxycarbonyl groups, such as
phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl,
2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl,
m-methylphenoxycarbonyl, o-bromophenoxycarbonyl,
3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl,
2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl,
aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl;
methoxymethyl; benzyloxymethyl; siloxymethyl;
2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl;
ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl;
2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl,
p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and
nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;
triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;
t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and
diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,
9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl;
2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl;
methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl);
carbonyl-protecting groups (e.g., acetal and ketal groups, such as
dimethyl acetal, 1,3-dioxolane, and the like; acylal groups; and
dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, and the
like); carboxylic acid-protecting groups (e.g., ester groups, such
as methyl ester, benzyl ester, t-butyl ester, orthoesters, and the
like; and oxazoline groups.
[0731] The term "oxo" as used herein, represents .dbd.O.
[0732] The term "perfluoroalkyl," as used herein, represents an
alkyl group, as defined herein, where each hydrogen radical bound
to the alkyl group has been replaced by a fluoride radical.
Perfluoroalkyl groups are exemplified by trifluoromethyl,
pentafluoroethyl, and the like.
[0733] The term "perfluoroalkoxy," as used herein, represents an
alkoxy group, as defined herein, where each hydrogen radical bound
to the alkoxy group has been replaced by a fluoride radical.
Perfluoroalkoxy groups are exemplified by trifluoromethoxy,
pentafluoroethoxy, and the like.
[0734] The term "spirocyclyl," as used herein, represents a
C.sub.2-7 alkylene diradical, both ends of which are bonded to the
same carbon atom of the parent group to form a spirocyclic group,
and also a C.sub.1-6 heteroalkylene diradical, both ends of which
are bonded to the same atom. The heteroalkylene radical forming the
spirocyclyl group can containing one, two, three, or four
heteroatoms independently selected from the group consisting of
nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl
group includes one to seven carbons, excluding the carbon atom to
which the diradical is attached. The spirocyclyl groups of the
invention may be optionally substituted with 1, 2, 3, or 4
substituents provided herein as optional substituents for
cycloalkyl and/or heterocyclyl groups.
[0735] The term "stereoisomer," as used herein, refers to all
possible different isomeric as well as conformational forms which a
compound may possess (e.g., a compound of any formula described
herein), in particular all possible stereochemically and
conformationally isomeric forms, all diastereomers, enantiomers
and/or conformers of the basic molecular structure. Some compounds
of the present invention may exist in different tautomeric forms,
all of the latter being included within the scope of the present
invention.
[0736] The term "sulfoalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a sulfo group of
--SO.sub.3H. In some embodiments, the alkyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein, and the sulfo group can be further substituted with one or
more O-protecting groups (e.g., as described herein).
[0737] The term "sulfonyl," as used herein, represents an
--S(O).sub.2-- group.
[0738] The term "thioalkaryl," as used herein, represents a
chemical substituent of formula --SR, where R is an alkaryl group.
In some embodiments, the alkaryl group can be further substituted
with 1, 2, 3, or 4 substituent groups as described herein.
[0739] The term "thioalkheterocyclyl," as used herein, represents a
chemical substituent of formula --SR, where R is an alkheterocyclyl
group. In some embodiments, the alkheterocyclyl group can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein.
[0740] The term "thioalkoxy," as used herein, represents a chemical
substituent of formula --SR, where R is an alkyl group, as defined
herein. In some embodiments, the alkyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein.
[0741] Compound: As used herein, the term "compound," is meant to
include all stereoisomers, geometric isomers, tautomers, and
isotopes of the structures depicted.
[0742] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Compounds of the present disclosure that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present disclosure. Cis and trans geometric
isomers of the compounds of the present disclosure are described
and may be isolated as a mixture of isomers or as separated
isomeric forms.
[0743] Compounds of the present disclosure also include tautomeric
forms. Tautomeric forms result from the swapping of a single bond
with an adjacent double bond and the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge. Examples prototropic tautomers include ketone--enol
pairs, amide--imidic acid pairs, lactam--lactim pairs,
amide--imidic acid pairs, enamine--imine pairs, and annular forms
where a proton can occupy two or more positions of a heterocyclic
system, such as, 1H- and 3H-imidazole, 1H-, 2H- and
4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.
Tautomeric forms can be in equilibrium or sterically locked into
one form by appropriate substitution.
[0744] Compounds of the present disclosure also include all of the
isotopes of the atoms occurring in the intermediate or final
compounds. "Isotopes" refers to atoms having the same atomic number
but different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium.
[0745] The compounds and salts of the present disclosure can be
prepared in combination with solvent or water molecules to form
solvates and hydrates by routine methods.
[0746] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
polypeptide sequence, respectively, that are those that occur
unaltered in the same position of two or more sequences being
compared. Nucleotides or amino acids that are relatively conserved
are those that are conserved amongst more related sequences than
nucleotides or amino acids appearing elsewhere in the
sequences.
[0747] In some embodiments, two or more sequences are said to be
"completely conserved" if they are 100% identical to one another.
In some embodiments, two or more sequences are said to be "highly
conserved" if they are at least 70% identical, at least 80%
identical, at least 90% identical, or at least 95% identical to one
another. In some embodiments, two or more sequences are said to be
"highly conserved" if they are about 70% identical, about 80%
identical, about 90% identical, about 95%, about 98%, or about 99%
identical to one another. In some embodiments, two or more
sequences are said to be "conserved" if they are at least 30%
identical, at least 40% identical, at least 50% identical, at least
60% identical, at least 70% identical, at least 80% identical, at
least 90% identical, or at least 95% identical to one another. In
some embodiments, two or more sequences are said to be "conserved"
if they are about 30% identical, about 40% identical, about 50%
identical, about 60% identical, about 70% identical, about 80%
identical, about 90% identical, about 95% identical, about 98%
identical, or about 99% identical to one another. Conservation of
sequence may apply to the entire length of an oligonucleotide or
polypeptide or may apply to a portion, region or feature
thereof.
[0748] Cyclic or Cyclized: As used herein, the term "cyclic" refers
to the presence of a continuous loop. Cyclic molecules need not be
circular, only joined to form an unbroken chain of subunits. Cyclic
molecules such as the mRNA of the present invention may be single
units or multimers or comprise one or more components of a complex
or higher order structure.
[0749] Cytostatic: As used herein, "cytostatic" refers to
inhibiting, reducing, suppressing the growth, division, or
multiplication of a cell (e.g., a mammalian cell (e.g., a human
cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a
combination thereof.
[0750] Cytotoxic: As used herein, "cytotoxic" refers to killing or
causing injurious, toxic, or deadly effect on a cell (e.g., a
mammalian cell (e.g., a human cell)), bacterium, virus, fungus,
protozoan, parasite, prion, or a combination thereof.
[0751] Delivery: As used herein, "delivery" refers to the act or
manner of delivering a compound, substance, entity, moiety, cargo
or payload.
[0752] Delivery Agent: As used herein, "delivery agent" refers to
any substance which facilitates, at least in part, the in vivo
delivery of a polynucleotide to targeted cells.
[0753] Destabilized: As used herein, the term "destable,"
"destabilize," or "destabilizing region" means a region or molecule
that is less stable than a starting, wild-type or native form of
the same region or molecule.
[0754] Detectable label: As used herein, "detectable label" refers
to one or more markers, signals, or moieties which are attached,
incorporated or associated with another entity that is readily
detected by methods known in the art including radiography,
fluorescence, chemiluminescence, enzymatic activity, absorbance and
the like. Detectable labels include radioisotopes, fluorophores,
chromophores, enzymes, dyes, metal ions, ligands such as biotin,
avidin, streptavidin and haptens, quantum dots, and the like.
Detectable labels may be located at any position in the peptides or
proteins disclosed herein. They may be within the amino acids, the
peptides, or proteins, or located at the N- or C-termini.
[0755] Digest: As used herein, the term "digest" means to break
apart into smaller pieces or components. When referring to
polypeptides or proteins, digestion results in the production of
peptides.
[0756] Distal: As used herein, the term "distal" means situated
away from the center or away from a point or region of
interest.
[0757] Encoded protein cleavage signal: As used herein, "encoded
protein cleavage signal" refers to the nucleotide sequence which
encodes a protein cleavage signal.
[0758] Engineered: As used herein, embodiments of the invention are
"engineered" when they are designed to have a feature or property,
whether structural or chemical, that varies from a starting point,
wild type or native molecule.
[0759] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end processing); (3)
translation of an RNA into a polypeptide or protein; and (4)
post-translational modification of a polypeptide or protein.
[0760] Feature: As used herein, a "feature" refers to a
characteristic, a property, or a distinctive element.
[0761] Formulation: As used herein, a "formulation" includes at
least a polynucleotide and a delivery agent.
[0762] Fragment: A "fragment," as used herein, refers to a portion.
For example, fragments of proteins may comprise polypeptides
obtained by digesting full-length protein isolated from cultured
cells.
[0763] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized.
[0764] Homology: As used herein, the term "homology" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. In some embodiments,
polymeric molecules are considered to be "homologous" to one
another if their sequences are at least 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical
or similar. The term "homologous" necessarily refers to a
comparison between at least two sequences (polynucleotide or
polypeptide sequences). In accordance with the invention, two
polynucleotide sequences are considered to be homologous if the
polypeptides they encode are at least about 50%, 60%, 70%, 80%,
90%, 95%, or even 99% for at least one stretch of at least about 20
amino acids. In some embodiments, homologous polynucleotide
sequences are characterized by the ability to encode a stretch of
at least 4-5 uniquely specified amino acids. For polynucleotide
sequences less than 60 nucleotides in length, homology is
determined by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. In accordance with the invention,
two protein sequences are considered to be homologous if the
proteins are at least about 50%, 60%, 70%, 80%, or 90% identical
for at least one stretch of at least about 20 amino acids.
[0765] Identity: As used herein, the term "identity" refers to the
overall relatedness between polymeric molecules, e.g., between
oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of the percent
identity of two polynucleotide sequences, for example, can be
performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. For example, the percent identity between two nucleotide
sequences can be determined using methods such as 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;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991; each of which is incorporated
herein by reference. For example, the percent identity between two
nucleotide sequences can be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4:11-17), which has been
incorporated into the ALIGN program (version 2.0) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The percent identity between two nucleotide sequences can,
alternatively, be determined using the GAP program in the GCG
software package using an NWSgapdna.CMP matrix. Methods commonly
employed to determine percent identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, 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 (1990)).
[0766] Inhibit expression of a gene: As used herein, the phrase
"inhibit expression of a gene" means to cause a reduction in the
amount of an expression product of the gene. The expression product
can be an RNA transcribed from the gene (e.g., an mRNA) or a
polypeptide translated from an mRNA transcribed from the gene.
Typically a reduction in the level of an mRNA results in a
reduction in the level of a polypeptide translated therefrom. The
level of expression may be determined using standard techniques for
measuring mRNA or protein.
[0767] 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, in a Petri dish, etc.,
rather than within an organism (e.g., animal, plant, or
microbe).
[0768] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or microbe or
cell or tissue thereof).
[0769] Isolated: As used herein, the term "isolated" refers to a
substance or entity that has been separated from at least some of
the components with which it was associated (whether in nature or
in an experimental setting). Isolated substances may have varying
levels of purity in reference to the substances from which they
have been associated. Isolated substances and/or entities may be
separated from at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, or more of
the other components with which they were initially associated. In
some embodiments, isolated agents are more than about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, or more than about
99% pure. As used herein, a substance is "pure" if it is
substantially free of other components. Substantially isolated: By
"substantially isolated" is meant that the compound is
substantially separated from the environment in which it was formed
or detected. Partial separation can include, for example, a
composition enriched in the compound of the present disclosure.
Substantial separation can include compositions containing at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95%, at least about 97%, or
at least about 99% by weight of the compound of the present
disclosure, or salt thereof. Methods for isolating compounds and
their salts are routine in the art.
[0770] Linker: As used herein, a linker refers to a group of atoms,
e.g., 10-1,000 atoms, and can be comprised of the atoms or groups
such as, but not limited to, carbon, amino, alkylamino, oxygen,
sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be
attached to a modified nucleoside or nucleotide on the nucleobase
or sugar moiety at a first end, and to a payload, e.g., a
detectable or therapeutic agent, at a second end. The linker may be
of sufficient length as to not interfere with incorporation into a
nucleic acid sequence. The linker can be used for any useful
purpose, such as to form multimers (e.g., through linkage of two or
more polynucleotides) or conjugates, as well as to administer a
payload, as described herein. Examples of chemical groups that can
be incorporated into the linker include, but are not limited to,
alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester,
alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can
be optionally substituted, as described herein. Examples of linkers
include, but are not limited to, unsaturated alkanes, polyethylene
glycols (e.g., ethylene or propylene glycol monomeric units, e.g.,
diethylene glycol, dipropylene glycol, triethylene glycol,
tripropylene glycol, tetraethylene glycol, or tetraethylene
glycol), and dextran polymers, Other examples include, but are not
limited to, cleavable moieties within the linker, such as, for
example, a disulfide bond (--S--S--) or an azo bond (--N.dbd.N--),
which can be cleaved using a reducing agent or photolysis.
Non-limiting examples of a selectively cleavable bond include an
amido bond can be cleaved for example by the use of
tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,
and/or photolysis, as well as an ester bond can be cleaved for
example by acidic or basic hydrolysis.
[0771] Modified: As used herein "modified" refers to a changed
state or structure of a molecule of the invention. Molecules may be
modified in many ways including chemically, structurally, and
functionally. In one embodiment, the mRNA molecules of the present
invention are modified by the introduction of non-natural
nucleosides and/or nucleotides, e.g., as it relates to the natural
ribonucleotides A, U, G, and C. Noncanonical nucleotides such as
the cap structures are not considered "modified" although they
differ from the chemical structure of the A, C, G, U
ribonucleotides.
[0772] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid.
[0773] Non-human vertebrate: As used herein, a "non human
vertebrate" includes all vertebrates except Homo sapiens, including
wild and domesticated species. Examples of non-human vertebrates
include, but are not limited to, mammals, such as alpaca, banteng,
bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea
pig, horse, llama, mule, pig, rabbit, reindeer, sheep water
buffalo, and yak.
[0774] Off-target: As used herein, "off target" refers to any
unintended effect on any one or more target, gene, or cellular
transcript.
[0775] Open reading frame: As used herein, "open reading frame" or
"ORF" refers to a sequence which does not contain a stop codon in a
given reading frame.
[0776] Operably linked: As used herein, the phrase "operably
linked" refers to a functional connection between two or more
molecules, constructs, transcripts, entities, moieties or the
like.
[0777] Paratope: As used herein, a "paratope" refers to the
antigen-binding site of an antibody.
[0778] Patient: As used herein, "patient" refers to a subject who
may seek or be in need of treatment, requires treatment, is
receiving treatment, will receive treatment, or a subject who is
under care by a trained professional for a particular disease or
condition.
[0779] Optionally substituted: Herein a phrase of the form
"optionally substituted X" (e.g., optionally substituted alkyl) is
intended to be equivalent to "X, wherein X is optionally
substituted" (e.g., "alkyl, wherein said alkyl is optionally
substituted"). It is not intended to mean that the feature "X"
(e.g. alkyl) per se is optional.
[0780] Peptide: As used herein, "peptide" is less than or equal to
50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids long.
[0781] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0782] Pharmaceutically acceptable excipients: The phrase
"pharmaceutically acceptable excipient," as used herein, refers any
ingredient other than the compounds described herein (for example,
a vehicle capable of suspending or dissolving the active compound)
and having the properties of being substantially nontoxic and
non-inflammatory in a patient. Excipients may include, for example:
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers
(diluents), film formers or coatings, flavors, fragrances, glidants
(flow enhancers), lubricants, preservatives, printing inks,
sorbents, suspensing or dispersing agents, sweeteners, and waters
of hydration. Exemplary excipients include, but are not limited to:
butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,
ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol,
methionine, methylcellulose, methyl paraben, microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
and xylitol.
[0783] Pharmaceutically acceptable salts: The present disclosure
also includes pharmaceutically acceptable salts of the compounds
described herein. As used herein, "pharmaceutically acceptable
salts" refers to derivatives of the disclosed compounds wherein the
parent compound is modified by converting an existing acid or base
moiety to its salt form (e.g., by reacting the free base group with
a suitable organic acid). Examples of pharmaceutically acceptable
salts include, but are not limited to, mineral or organic acid
salts of basic residues such as amines; alkali or organic salts of
acidic residues such as carboxylic acids; and the like.
Representative acid addition salts include acetate, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,
hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,
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, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine, and the like. The pharmaceutically
acceptable salts of the present disclosure include the conventional
non-toxic salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. The pharmaceutically
acceptable salts of the present disclosure can be synthesized from
the parent compound which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts can be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two;
generally, nonaqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile are preferred. Lists of suitable salts
are found in Remington's Pharmaceutical Sciences, 17.sup.th ed.,
Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical
Salts: Properties, Selection, and Use, P. H. Stahl and C. G.
Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of
Pharmaceutical Science, 66, 1-19 (1977), each of which is
incorporated herein by reference in its entirety.
[0784] Pharmacokinetic: As used herein, "pharmacokinetic" refers to
any one or more properties of a molecule or compound as it relates
to the determination of the fate of substances administered to a
living organism. Pharmacokinetics is divided into several areas
including the extent and rate of absorption, distribution,
metabolism and excretion. This is commonly referred to as ADME
where: (A) Absorption is the process of a substance entering the
blood circulation; (D) Distribution is the dispersion or
dissemination of substances throughout the fluids and tissues of
the body; (M) Metabolism (or Biotransformation) is the irreversible
transformation of parent compounds into daughter metabolites; and
(E) Excretion (or Elimination) refers to the elimination of the
substances from the body. In rare cases, some drugs irreversibly
accumulate in body tissue.
[0785] Pharmaceutically acceptable solvate: The term
"pharmaceutically acceptable solvate," as used herein, means a
compound of the invention wherein molecules of a suitable solvent
are incorporated in the crystal lattice. A suitable solvent is
physiologically tolerable at the dosage administered. For example,
solvates may be prepared by crystallization, recrystallization, or
precipitation from a solution that includes organic solvents,
water, or a mixture thereof. Examples of suitable solvents are
ethanol, water (for example, mono-, di-, and tri-hydrates),
N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),
N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC),
1,3-dimethyl-2-imidazolidinone (DMEU),
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water
is the solvent, the solvate is referred to as a "hydrate."
[0786] Physicochemical: As used herein, "physicochemical" means of
or relating to a physical and/or chemical property.
[0787] Preventing: As used herein, the term "preventing" refers to
partially or completely delaying onset of an infection, disease,
disorder and/or condition; partially or completely delaying onset
of one or more symptoms, features, or clinical manifestations of a
particular infection, disease, disorder, and/or condition;
partially or completely delaying onset of one or more symptoms,
features, or manifestations of a particular infection, disease,
disorder, and/or condition; partially or completely delaying
progression from an infection, a particular disease, disorder
and/or condition; and/or decreasing the risk of developing
pathology associated with the infection, the disease, disorder,
and/or condition.
[0788] Prodrug: The present disclosure also includes prodrugs of
the compounds described herein. As used herein, "prodrugs" refer to
any substance, molecule or entity which is in a form predicate for
that substance, molecule or entity to act as a therapeutic upon
chemical or physical alteration. Prodrugs may by covalently bonded
or sequestered in some way and which release or are converted into
the active drug moiety prior to, upon or after administered to a
mammalian subject. Prodrugs can be prepared by modifying functional
groups present in the compounds in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compounds. Prodrugs include compounds wherein
hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any
group that, when administered to a mammalian subject, cleaves to
form a free hydroxyl, amino, sulfhydryl, or carboxyl group
respectively. Preparation and use of prodrugs is discussed in T.
Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol.
14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in
Drug Design, ed. Edward B. Roche, American Pharmaceutical
Association and Pergamon Press, 1987, both of which are hereby
incorporated by reference in their entirety.
[0789] Proliferate: As used herein, the term "proliferate" means to
grow, expand or increase or cause to grow, expand or increase
rapidly. "Proliferative" means having the ability to proliferate.
"Anti-proliferative" means having properties counter to or
inapposite to proliferative properties.
[0790] Protein cleavage site: As used herein, "protein cleavage
site" refers to a site where controlled cleavage of the amino acid
chain can be accomplished by chemical, enzymatic or photochemical
means.
[0791] Protein cleavage signal: As used herein "protein cleavage
signal" refers to at least one amino acid that flags or marks a
polypeptide for cleavage.
[0792] Protein of interest: As used herein, the terms "proteins of
interest" or "desired proteins" include those provided herein and
fragments, mutants, variants, and alterations thereof.
[0793] Proximal: As used herein, the term "proximal" means situated
nearer to the center or to a point or region of interest.
[0794] Purified: As used herein, "purify," "purified,"
"purification" means to make substantially pure or clear from
unwanted components, material defilement, admixture or
imperfection.
[0795] Sample: As used herein, the term "sample" or "biological
sample" refers to a subset of its tissues, cells or component parts
(e.g. body fluids, including but not limited to blood, mucus,
lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,
amniotic fluid, amniotic cord blood, urine, vaginal fluid and
semen). A sample further may include a homogenate, lysate or
extract prepared from a whole organism or a subset of its tissues,
cells or component parts, or a fraction or portion thereof,
including but not limited to, for example, plasma, serum, spinal
fluid, lymph fluid, the external sections of the skin, respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, organs. A sample further refers to a medium, such as
a nutrient broth or gel, which may contain cellular components,
such as proteins or nucleic acid molecule.
[0796] Signal Sequences: As used herein, the phrase "signal
sequences" refers to a sequence which can direct the transport or
localization of a protein.
[0797] Significant or Significantly: As used herein, the terms
"significant" or "significantly" are used synonymously with the
term "substantially."
[0798] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administered in one dose/at one time/single
route/single point of contact, i.e., single administration
event.
[0799] Similarity: As used herein, the term "similarity" refers to
the overall relatedness between polymeric molecules, e.g. between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of percent
similarity of polymeric molecules to one another can be performed
in the same manner as a calculation of percent identity, except
that calculation of percent similarity takes into account
conservative substitutions as is understood in the art.
[0800] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[0801] Stable: As used herein "stable" refers to a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and preferably capable of
formulation into an efficacious therapeutic agent.
[0802] Stabilized: As used herein, the term "stabilize",
"stabilized," "stabilized region" means to make or become
stable.
[0803] Subject: As used herein, the term "subject" or "patient"
refers to any organism to which a composition in accordance with
the invention may be administered, 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) and/or plants.
[0804] 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 chemical phenomena.
[0805] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[0806] Substantially simultaneously: As used herein and as it
relates to plurality of doses, the term means within 2 seconds.
[0807] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more symptoms of a disease, disorder, and/or
condition.
[0808] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with
and/or may not exhibit symptoms of the disease, disorder, and/or
condition but harbors a propensity to develop a disease or its
symptoms. In some embodiments, an individual who is susceptible to
a disease, disorder, and/or condition (for example, cancer) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development 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.
[0809] Synthetic: The term "synthetic" means produced, prepared,
and/or manufactured by the hand of man. Synthesis of
polynucleotides or polypeptides or other molecules of the present
invention may be chemical or enzymatic.
[0810] Targeted Cells: As used herein, "targeted cells" refers to
any one or more cells of interest. The cells may be found in vitro,
in vivo, in situ or in the tissue or organ of an organism. The
organism may be an animal, preferably a mammal, more preferably a
human and most preferably a patient.
[0811] Therapeutic Agent: The term "therapeutic agent" refers to
any agent that, when administered to a subject, has a therapeutic,
diagnostic, and/or prophylactic effect and/or elicits a desired
biological and/or pharmacological effect.
[0812] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., nucleic acid, drug, therapeutic agent,
diagnostic agent, prophylactic agent, etc.) that is sufficient,
when administered to a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0813] Therapeutically effective outcome: As used herein, the term
"therapeutically effective outcome" means an outcome that is
sufficient in a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0814] Total daily dose: As used herein, a "total daily dose" is an
amount given or prescribed in 24 hr period. It may be administered
as a single unit dose.
[0815] Transcription factor: As used herein, the term
"transcription factor" refers to a DNA-binding protein that
regulates transcription of DNA into RNA, for example, by activation
or repression of transcription. Some transcription factors effect
regulation of transcription alone, while others act in concert with
other proteins. Some transcription factor can both activate and
repress transcription under certain conditions. In general,
transcription factors bind a specific target sequence or sequences
highly similar to a specific consensus sequence in a regulatory
region of a target gene. Transcription factors may regulate
transcription of a target gene alone or in a complex with other
molecules.
[0816] Treating: As used herein, the term "treating" refers to
partially or completely alleviating, ameliorating, improving,
relieving, delaying onset of, inhibiting progression of, reducing
severity of, and/or reducing incidence of one or more symptoms or
features of a particular infection, disease, disorder, and/or
condition. For example, "treating" cancer may refer to inhibiting
survival, growth, and/or spread of a tumor. Treatment may be
administered to a subject who does not exhibit signs of a disease,
disorder, and/or condition and/or to a subject who exhibits only
early signs of a disease, disorder, and/or condition for the
purpose of decreasing the risk of developing pathology associated
with the disease, disorder, and/or condition.
[0817] Unmodified: As used herein, "unmodified" refers to any
substance, compound or molecule prior to being changed in any way.
Unmodified may, but does not always, refer to the wild type or
native form of a biomolecule. Molecules may undergo a series of
modifications whereby each modified molecule may serve as the
"unmodified" starting molecule for a subsequent modification.
Equivalents and Scope
[0818] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0819] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0820] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0821] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0822] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0823] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
EXAMPLES
[0824] The present disclosure is further described in the following
examples, which do not limit the scope of the disclosure described
in the claims.
Example 1. Modified mRNA In Vitro Transcription
A. Materials and Methods
[0825] Modified mRNAs according to the invention are made using
standard laboratory methods and materials for in vitro
transcription with the exception that the nucleotide mix contains
modified nucleotides. The open reading frame (ORF) of the gene of
interest is flanked by a 5' untranslated region (UTR) containing a
strong Kozak translational initiation signal and an alpha-globin 3'
UTR terminating with an oligo(dT) sequence for templated addition
of a polyA tail for mRNAs not incorporating adenosine analogs.
Adenosine-containing mRNAs are synthesized without an oligo (dT)
sequence to allow for post-transcription poly (A) polymerase
poly-(A) tailing.
[0826] The modified mRNAs may be modified to reduce the cellular
innate immune response. The modifications to reduce the cellular
response may include pseudouridine (.psi.) and 5-methyl-cytidine
(5meC, 5mc or m.sup.5C). (See, Kariko K et al. Immunity 23:165-75
(2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson B R et
al. NAR (2010); herein incorporated by reference).
[0827] The ORF may also include various upstream or downstream
additions (such as, but not limited to, .beta.-globin, tags, etc.)
may be ordered from an optimization service such as, but limited
to, DNA2.0 (Menlo Park, Calif.) and may contain multiple cloning
sites which may have XbaI recognition. Upon receipt of the
construct, it may be reconstituted and transformed into chemically
competent E. coli.
[0828] For the present invention, NEB DH5-alpha Competent E. coli
are used. Transformations are performed according to NEB
instructions using 100 ng of plasmid. The protocol is as
follows:
[0829] Thaw a tube of NEB 5-alpha Competent E. coli cells on ice
for 10 minutes.
[0830] Add 1-5 .mu.l containing 1 pg-100 ng of plasmid DNA to the
cell mixture. Carefully flick the tube 4-5 times to mix cells and
DNA. Do not vortex.
[0831] Place the mixture on ice for 30 minutes. Do not mix.
[0832] Heat shock at 42.degree. C. for exactly 30 seconds. Do not
mix.
[0833] Place on ice for 5 minutes. Do not mix.
[0834] Pipette 950 .mu.l of room temperature SOC into the
mixture.
[0835] Place at 37.degree. C. for 60 minutes. Shake vigorously (250
rpm) or rotate.
[0836] Warm selection plates to 37.degree. C.
[0837] Mix the cells thoroughly by flicking the tube and
inverting.
[0838] Spread 50-100 .mu.l of each dilution onto a selection plate
and incubate overnight at 37.degree. C. Alternatively, incubate at
30.degree. C. for 24-36 hours or 25.degree. C. for 48 hours.
[0839] A single colony is then used to inoculate 5 ml of LB growth
media using the appropriate antibiotic and then allowed to grow
(250 RPM, 37.degree. C.) for 5 hours. This is then used to
inoculate a 200 ml culture medium and allowed to grow overnight
under the same conditions.
[0840] To isolate the plasmid (up to 850 .mu.g), a maxi prep is
performed using the Invitrogen PURELINK.TM. HiPure Maxiprep Kit
(Carlsbad, Calif.), following the manufacturer's instructions.
[0841] In order to generate cDNA for In Vitro Transcription (IVT),
the plasmid (an Example of which is shown in FIG. 3) is first
linearized using a restriction enzyme such as XbaI. A typical
restriction digest with XbaI will comprise the following: Plasmid
1.0 .mu.g; 10.times. Buffer 1.0 .mu.l; XbaI 1.5 .mu.l; dH.sub.2O up
to 10 .mu.l; incubated at 37.degree. C. for 1 hr. If performing at
lab scale (<5 .mu.g), the reaction is cleaned up using
Invitrogen's PURELINK.TM. PCR Micro Kit (Carlsbad, Calif.) per
manufacturer's instructions. Larger scale purifications may need to
be done with a product that has a larger load capacity such as
Invitrogen's standard PURELINK.TM. PCR Kit (Carlsbad, Calif.).
Following the cleanup, the linearized vector is quantified using
the NanoDrop and analyzed to confirm linearization using agarose
gel electrophoresis.
B. Agarose Gel Electrophoresis of Modified mRNA
[0842] Individual modified mRNAs (200-400 ng in a 20 .mu.l volume)
are loaded into a well on a non-denaturing 1.2% Agarose E-Gel
(Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes according
to the manufacturer protocol.
C. Agarose Gel Electrophoresis of RT-PCR Products
[0843] Individual reverse transcribed-PCR products (200-400 ng) are
loaded into a well of a non-denaturing 1.2% Agarose E-Gel
(Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes according
to the manufacturer protocol.
D. Nanodrop Modified mRNA Quantification and UV Spectral Data
[0844] Modified mRNAs in TE buffer (1 .mu.l) are used for Nanodrop
UV absorbance readings to quantitate the yield of each modified
mRNA from an in vitro transcription reaction (UV absorbance traces
are not shown).
Example 2. Modified mRNA Transfection
A. Reverse Transfection
[0845] For experiments performed in a 24-well collagen-coated
tissue culture plate, Keratinocytes are seeded at a cell density of
1.times.10.sup.5. For experiments performed in a 96-well
collagen-coated tissue culture plate, Keratinocytes are seeded at a
cell density of 0.5.times.10.sup.5. For each modified mRNA to be
transfected, modified mRNA: RNAIMAX.TM. are prepared as described
and mixed with the cells in the multi-well plate within 6 hours of
cell seeding before cells had adhered to the tissue culture
plate.
B. Forward Transfection
[0846] In a 24-well collagen-coated tissue culture plate,
Keratinocytes are seeded at a cell density of 0.7.times.10.sup.5.
For experiments performed in a 96-well collagen-coated tissue
culture plate, Keratinocytes are seeded at a cell density of
0.3.times.10.sup.5. Keratinocytes are then grown to a confluency of
>70% for over 24 hours. For each modified mRNA to be
transfected, modified mRNA: RNAIMAX.TM. are prepared as described
and transfected onto the cells in the multi-well plate over 24
hours after cell seeding and adherence to the tissue culture
plate.
C. Modified mRNA Translation Screen: G-CSF ELISA
[0847] Keratinocytes are grown in EpiLife medium with Supplement S7
from Invitrogen at a confluence of >70%. Keratinocytes are
reverse transfected with 300 ng of the indicated chemically
modified mRNA complexed with RNAIMAX.TM. from Invitrogen.
Alternatively, keratinocytes are forward transfected with 300 ng
modified mRNA complexed with RNAIMAX.TM. from Invitrogen. The RNA:
RNAIMAX.TM. complex is formed by first incubating the RNA with
Supplement-free EPILIFE.RTM. media in a 5.times. volumetric
dilution for 10 minutes at room temperature.
[0848] In a second vial, RNAIMAX.TM. reagent is incubated with
Supplement-free EPILIFE.RTM. Media in a 10.times. volumetric
dilution for 10 minutes at room temperature. The RNA vial is then
mixed with the RNAIMAX.TM. vial and incubated for 20-30 at room
temperature before being added to the cells in a drop-wise fashion.
Secreted huG-CSF concentration in the culture medium is measured at
18 hours post-transfection for each of the chemically modified
mRNAs in triplicate. Secretion of Human Granulocyte-Colony
Stimulating Factor (G-CSF) from transfected human keratinocytes is
quantified using an ELISA kit from Invitrogen or R&D Systems
(Minneapolis, Minn.) following the manufacturers recommended
instructions.
D. Modified mRNA Dose and Duration: G-CSF ELISA
[0849] Keratinocytes are grown in EPILIFE.RTM. medium with
Supplement S7 from Invitrogen at a confluence of >70%.
Keratinocytes are reverse transfected with Ong, 46.875 ng, 93.75
ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng modified mRNA complexed
with RNAIMAX.TM. from Invitrogen. The modified mRNA: RNAIMAX.TM.
complex is formed as described. Secreted huG-CSF concentration in
the culture medium is measured at 0, 6, 12, 24, and 48 hours
post-transfection for each concentration of each modified mRNA in
triplicate. Secretion of Human Granulocyte-Colony Stimulating
Factor (G-CSF) from transfected human keratinocytes is quantified
using an ELISA kit from Invitrogen or R&D Systems following the
manufacturers recommended instructions.
Example 3. Cellular Innate Immune Response to Modified Nucleic
Acids: IFN-Beta ELISA and TNF-Alpha ELISA
[0850] An enzyme-linked immunosorbent assay (ELISA) for Human Tumor
Necrosis Factor-.alpha. (TNF-.alpha.), Human Interferon-.beta.
(IFN-.beta.) and Human Granulocyte-Colony Stimulating Factor
(G-CSF) secreted from in vitro-transfected Human Keratinocyte cells
is tested for the detection of a cellular innate immune
response.
[0851] Keratinocytes are grown in EPILIFE.RTM. medium with Human
Keratinocyte Growth Supplement in the absence of hydrocortisone
from Invitrogen at a confluence of >70%. Keratinocytes are
reverse transfected with Ong, 93.75 ng, 187.5 ng, 375 ng, 750 ng,
1500 ng or 3000 ng of the indicated chemically modified mRNA
complexed with RNAIMAX.TM. from Invitrogen as described in
triplicate. Secreted TNF-.alpha. in the culture medium is measured
24 hours post-transfection for each of the chemically modified
mRNAs using an ELISA kit from Invitrogen according to the
manufacturer protocols.
[0852] Secreted IFN-.beta. is measured 24 hours post-transfection
for each of the chemically modified mRNAs using an ELISA kit from
Invitrogen according to the manufacturer protocols. Secreted
hu-G-CSF concentration is measured at 24 hours post-transfection
for each of the chemically modified mRNAs. Secretion of Human
Granulocyte-Colony Stimulating Factor (G-CSF) from transfected
human keratinocytes is quantified using an ELISA kit from
Invitrogen or R&D Systems (Minneapolis, Minn.) following the
manufacturers recommended instructions. These data indicate which
modified mRNA are capable eliciting a reduced cellular innate
immune response in comparison to natural and other chemically
modified polynucleotides or reference compounds by measuring
exemplary type 1 cytokines TNF-alpha and IFN-beta.
Example 4. Human Granulocyte-Colony Stimulating Factor-Modified
mRNA-Induced Cell Proliferation Assay
[0853] Human keratinocytes are grown in EPILIFE.RTM. medium with
Supplement S7 from Invitrogen at a confluence of >70% in a
24-well collagen-coated TRANSWELL.RTM. (Corning, Lowell, Mass.)
co-culture tissue culture plate. Keratinocytes are reverse
transfected with 750 ng of the indicated chemically modified mRNA
complexed with RNAIMAX.TM. from Invitrogen as described in
triplicate. The modified mRNA: RNAIMAX.TM. complex is formed as
described. Keratinocyte media is exchanged 6-8 hours
post-transfection. 42-hours post-transfection, the 24-well
TRANSWELL.RTM. plate insert with a 0.4 .mu.m-pore semi-permeable
polyester membrane is placed into the hu-G-CSF modified
mRNA-transfected keratinocyte containing culture plate.
[0854] Human myeloblast cells, Kasumi-1 cells or KG-1
(0.2.times.10.sup.5 cells), are seeded into the insert well and
cell proliferation is quantified 42 hours post-co-culture
initiation using the CyQuant Direct Cell Proliferation Assay
(Invitrogen) in a 100-120 .mu.l volume in a 96-well plate. modified
mRNA-encoding hu-G-CSF-induced myeloblast cell proliferation is
expressed as a percent cell proliferation normalized to
untransfected keratinocyte/myeloblast co-culture control wells.
Secreted hu-G-CSF concentration in both the keratinocyte and
myeloblast insert co-culture wells is measured at 42 hours
post-co-culture initiation for each modified mRNA in duplicate.
Secretion of Human Granulocyte-Colony Stimulating Factor (G-CSF) is
quantified using an ELISA kit from Invitrogen following the
manufacturers recommended instructions.
[0855] Transfected hu-G-CSF modified mRNA in human keratinocyte
feeder cells and untransfected human myeloblast cells are detected
by RT-PCR. Total RNA from sample cells is extracted and lysed using
RNAEASY.RTM. kit (Qiagen, Valencia, Calif.) according to the
manufacturer instructions. Extracted total RNA is submitted to
RT-PCR for specific amplification of modified mRNA-G-CSF using
PROTOSCRIPT.RTM. M-MuLV Taq RT-PCR kit (New England BioLabs,
Ipswich, Mass.) according to the manufacturer instructions with
hu-G-CSF-specific primers. RT-PCR products are visualized by 1.2%
agarose gel electrophoresis.
Example 5. Cytotoxicity and Apoptosis
[0856] This experiment demonstrates cellular viability, cytotoxity
and apoptosis for distinct modified mRNA-in vitro transfected Human
Keratinocyte cells. Keratinocytes are grown in EPILIFE.RTM. medium
with Human Keratinocyte Growth Supplement in the absence of
hydrocortisone from Invitrogen at a confluence of >70%.
Keratinocytes are reverse transfected with Ong, 46.875 ng, 93.75
ng, 187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of
modified mRNA complexed with RNAIMAX.TM. from Invitrogen. The
modified mRNA: RNAIMAX.TM. complex is formed. Secreted huG-CSF
concentration in the culture medium is measured at 0, 6, 12, 24,
and 48 hours post-transfection for each concentration of each
modified mRNA in triplicate. Secretion of Human Granulocyte-Colony
Stimulating Factor (G-CSF) from transfected human keratinocytes is
quantified using an ELISA kit from Invitrogen or R&D Systems
following the manufacturers recommended instructions. Cellular
viability, cytotoxicity and apoptosis is measured at 0, 12, 48, 96,
and 192 hours post-transfection using the APOTOX-GLO.TM. kit from
Promega (Madison, Wis.) according to manufacturer instructions.
Example 6. Co-Culture Environment
[0857] The modified mRNA comprised of chemically-distinct modified
nucleotides encoding human Granulocyte-Colony Stimulating Factor
(G-CSF) may stimulate the cellular proliferation of a transfection
incompetent cell in co-culture environment. The co-culture includes
a highly transfectable cell type such as a human keratinocyte and a
transfection incompetent cell type such as a white blood cell
(WBC). The modified mRNA encoding G-CSF may be transfected into the
highly transfectable cell allowing for the production and secretion
of G-CSF protein into the extracellular environment where G-CSF
acts in a paracrine-like manner to stimulate the white blood cell
expressing the G-CSF receptor to proliferate. The expanded WBC
population may be used to treat immune-compromised patients or
partially reconstitute the WBC population of an immunosuppressed
patient and thus reduce the risk of opportunistic infections.
[0858] In another example, a highly transfectable cell such as a
fibroblast are transfected with certain growth factors to support
and simulate the growth, maintenance, or differentiation of poorly
transfectable embryonic stem cells or induced pluripotent stem
cells.
Example 7. 5'-Guanosine Capping on Modified Nucleic Acids (modified
mRNAs)
A. Materials and Methods
[0859] The cloning, gene synthesis and vector sequencing was
performed by DNA2.0 Inc. (Menlo Park, Calif.). The ORF was
restriction digested using XbaI and used for cDNA synthesis using
tailed-or tail-less-PCR. The tailed-PCR cDNA product was used as
the template for the modified mRNA synthesis reaction using 25 mM
each modified nucleotide mix (all modified nucleotides were custom
synthesized or purchased from TriLink Biotech, San Diego, Calif.
except pyrrolo-C triphosphate purchased from Glen Research,
Sterling Va.; unmodifed nucleotides were purchased from Epicenter
Biotechnologies, Madison, Wis.) and CellScript MEGASCRIPT.TM.
(Epicenter Biotechnologies, Madison, Wis.) complete mRNA synthesis
kit. The in vitro transcription reaction was run for 4 hours at
37.degree. C. Modified mRNAs incorporating adenosine analogs were
poly (A) tailed using yeast Poly (A) Polymerase (Affymetrix, Santa
Clara, Calif.). PCR reaction used HiFi PCR 2.times. MASTER MIX.TM.
(Kapa Biosystems, Woburn, Mass.). Modified mRNAs were
post-transcriptionally capped using recombinant Vaccinia Virus
Capping Enzyme (New England BioLabs, Ipswich, Mass.) and a
recombinant 2'-o-methyltransferase (Epicenter Biotechnologies,
Madison, Wis.) to generate the 5'-guanosine Cap1 structure. Cap 2
structure and Cap 2 structures may be generated using additional
2'-o-methyltransferases. The In vitro transcribed mRNA product was
run on an agarose gel and visualized. Modified mRNA was purified
with Ambion/Applied Biosystems (Austin, Tex.) MEGAClear RNA.TM.
purification kit. PCR used PURELINK.TM. PCR purification kit
(Invitrogen, Carlsbad, Calif.). The product was quantified on
NANODROP.TM. UV Absorbance (ThermoFisher, Waltham, Mass.). Quality,
UV absorbance quality and visualization of the product was
performed on an 1.2% agarose gel. The product was resuspended in TE
buffer.
B. 5' Capping Modified Nucleic Acid (mRNA) Structure
[0860] 5'-capping of modified mRNA may be completed concomitantly
during the in vitro-transcription reaction using the following
chemical RNA cap analogs to generate the 5'-guanosine cap structure
according to manufacturer protocols: 3'-0-Me-m.sup.7G(5')ppp(5')G
(the ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m.sup.7G(5')ppp(5')A;
m.sup.7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.).
5'-capping of modified mRNA may be completed post-transcriptionally
using a Vaccinia Virus Capping Enzyme to generate the "Cap 0"
structure: m.sup.7G(5')ppp(5')G (New England BioLabs, Ipswich,
Mass.). Cap 1 structure may be generated using both Vaccinia Virus
Capping Enzyme and a 2'-0 methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from
the Cap 1 structure followed by the 2'-o-methylation of the
5'-antepenultimate nucleotide using a 2'-0 methyl-transferase. Cap
3 structure may be generated from the Cap 2 structure followed by
the 2'-o-methylation of the 5'-preantepenultimate nucleotide using
a 2'-0 methyl-transferase. Enzymes are preferably derived from a
recombinant source.
[0861] When transfected into mammalian cells, the modified mRNAs
have a stability of 12-18 hours or more than 18 hours, e.g., 24,
36, 48, 60, 72 or greater than 72 hours.
Example 8. Synthesis of N4-methyl Cytidine (Compound 1) and
N4-methyl CTP (NTP of Said Compound)
##STR00167##
[0863] Uridine was silylated to provide a trisilylated compound,
which was purified by column, activated with re-distilled
POCl.sub.3/triazole under anhydrous condition, and then followed by
nucleophilic substitution with 40% methylamine aqueous solution.
N4-Methyl-2',3',5'-tri-O-TBDMS-cytidine was thus obtained after
chromatographic purification. The resultant product was deprotected
with TBAF and then purified with an ethanol-ethyl acetate (3:1)
solvent system to obtain compound 1. The final product was
characterized by NMR (in DMSO); MS: 258 (M+H).sup.+, 280
(M+Na).sup.+, and 296 (M+H).sup.+; and HPLC: purity, 99.35% (FIGS.
1A-1D). HPLC, purity 98% (FIG. 2).
Example 9. Synthesis of 2'-OMe-N,N-di-Me-cytidine (Compound 2) and
2'-OMe-N,N-di-Me-CTP (NTP of Said Compound)
##STR00168##
[0865] 2'-O-Methyluridine was silylated to give the di-silylated
compound. Purified 2'-O-methyl-3',5'-di-O-TBDMS uridine was
activated with re-distilled POCl.sub.3 and imidazole under
anhydrous condition, followed by the nucleophilic substitution with
dimethylamine hydrochloride under triethylamine environment to trap
HCl. Intermediate compound N4,N4,2'-tri-O-methyl-3',5'-bis-O-TBDMS
uridine was purified by flash chromatography and obtained as a
white foam. The resultant compound was de-protected with TBAF and
then purified to provide .about.400 mg final product compound 2 as
white foam. ES MS: m/z 308 (M+Na).sup.+, 386 (M+H).sup.+; HPLC:
purity, 99.49% (FIGS. 3A-3C).
[0866] To synthesize the corresponding NTP, 70 mg of nucleoside
compound 2 provided 23 mg of 2'-OMe-N,N-di-Me-CTP after
purification via ion-exchange and reverse phase columns. HPLC:
purity, 95% (FIG. 4).
Example 10. Synthesis of 5-methoxycarbonylmethoxy Uridine (Compound
3) and 5-methoxycarbonylmethoxy-UTP (NTP of Said Compound)
##STR00169##
[0868] Uridine 3-a in water was treated with excess amount of
bromine and then flushed with air to remove bromine. The reaction
mixture was treated with pyridine at a controlled speed and
temperature. During the reaction, unstable bromo-intermediate 3-b
gradually converted to di-hydroxyl intermediate 3-c, which
presumably dehydrated to the stable 5-hydroxyuridine 3-d. Then, the
5-hydroxyuridine was protected with a 2',3'-isopropylidene group to
provide compound 3-g. Reaction with compound 3-f provided compound
3.
[0869] 60-70 mg of the nucleoside provided >21 mg of the desired
triphosphate after two HPLC column purification and two
lyophilization steps. HPLC: purity, 98% (FIG. 5).
Example 11. Synthesis of 3-methyl Pseudouridine (Compound 4) and
3-methyl pseudo-UTP (NTP of Said Compound)
##STR00170##
[0871] Pseudouridine 4-a was reacted with Ac.sub.2O to provide
acetyl-protected pseudouridine 4-b. Then, N1 was selectively
protected with POM to provide compound 4-c. Methylation of N3,
followed by deprotected, provided compound 4 (.about.400 mg).
Molecular formula: C10H14N2O6, molecular weight: 258.23 g/mol;
appearance: white solid; storage conditions: store at 25.degree.
C.; HPLC: purity, 98.51%; .sup.1H NMR (DMSO-d.sub.6): .delta. 11.17
(d, 1H, J=3.0 Hz), 7.56 (d, 1H, J=3.6 Hz), 4.91 (d, 1H, J=3.6 Hz),
4.79 (t, 1H, J=4.2 Hz), 4.70 (d, 1H, J=4.2 Hz), 4.49 (d, 1H, J=3.0
Hz), 3.82-3.88 (m, 2H), 3.66-3.67 (m, 1H), 3.57-3.61 (m, 1H),
3.40-3.47 (m, 1H), 3.09 (s, 3H); MS: 281 (M+Na).sup.+) (FIGS. 6A
and 6B).
[0872] Alternative routes could be applied to obtain compound 4.
For example, pseudouridine could be reacted with an O-protecting
group (e.g., as described herein, such as TMS) and reacted with an
N-protecting group (e.g., as described herein, such as acetyl at
N1). Then, N3 of the nucleobase could be reacted with an alkylating
agent (e.g., dimethylamine/dimethoxymethyl) to provide compound 4
having N- and O-protecting groups. Finally, the resultant compound
would be deprotected (e.g., under basic conditions, such as
NH.sub.3/MeOH) to provide compound 4.
Example 12. Synthesis of N--Ac, 5-Ac--OCH.sub.2-cytidine (Compound
5)
##STR00171##
[0874] Uridine 5-a was protected to obtain isopropylidene compound
5-b, which was reacted with (CHCO).sub.n. Acetic acid with catalyst
amount of TFA was employed to obtain the desired selectively
acylated compound 5-f (30% yield). Further tritylation of the 5'-OH
group resulted in the desired orthogonally protected compound
5-g.
[0875] Compound 5-g was treated with POCl.sub.3 and triazole to
provide compound 5-h together with de-acylated compound 5-i.
Acetylation of these two compounds provided di-acylated, fully
protected compound 5-j. Deprotection of compound 5-j with acetic
acid under heating condition resulted in three products, one of
which was compound 5.
[0876] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
[0877] Alternative routes could be applied to obtain compound 5,
such as by beginning with cytidine as the starting material. In
such methods, the 5-position could be reacted with a halogen or a
halogenation agent (e.g., any described herein, such as
I.sub.2/meta-chloroperoxybenzoic acid), which can be displaced with
an alkylating agent. Further, such methods could include the use of
one or more N- or O-protecting groups (e.g., any described herein,
such as silylation or acetylation) to protect the amino group of
cytidine and/or hydroxyl groups of the sugar moiety.
Example 13. Synthesis of 5-TBDMS-OCH.sub.2-cytidine (Compound
6)
##STR00172##
[0879] A 5-hydroxyuracil compound `-b was glycosylated to obtain
compound 6`-d (28% yield), which was silylated to provide compound
6'-e. Activation of the protected uridine provided the desired
compound 6 after further amination and deprotection (800 mg of the
final compound). Molecular formula: C16H29N3O6Si; molecular weight:
387.50 g/mol; appearance: white solid; storage conditions: store at
25.degree. C.; HPLC: purity, 97.57%; .sup.1H NMR (CDCl.sub.3):
.delta. 7.81 (s, 1H), 7.40 (bs, 1H), 6.49 (bs, 1H), 5.79 (d, 1H,
J=2.4 Hz), 5.3-5.32 (m, 1H), 5.00-5.07 (m, 2H), 4.30-4.45 (m, 2H),
3.90-3.94 (m, 2H), 3.80-3.83 (m, 1H), 3.50-3.70 (m, 2H), 0.87 (s,
9H), 0.05 (S, 6H); MS: 388 (M+H).sup.+, 410 (M+Na).sup.+) (FIGS.
7A-7C).
[0880] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 14. Synthesis of 5-trifluoromethyl Cytidine (Compound
7)
##STR00173##
[0882] Compound 7-A was glycosylated to provide compound 7-B, which
was treated with 2,4,6-triisopropylbenzene sulfonyl chloride
(TPSCl) to activate the carbonyl group and to promote reductive
amination. Deprotection provided compound 7. Alternative activating
agents could be used instead of TPSCl, such as
2,4,6-trimethylbenzene sulfonyl chloride.
[0883] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 15. Synthesis of 5-trifluoromethyl Uridine (Compound 8)
##STR00174##
[0885] 5-Trifluoromethyluracil 8-A was glycosylated with
tetra-O-acetyl ribose, and the desired triprotected
5-trifluoromethyluridine 8-B was obtained in good yield. Further
deprotection gave desired compound 8, which was characterized with
NMR, MS and HPLC results. MS: 313 (M+H).sup.+, 335 (M+Na).sup.+;
HPLC: purity, 98.87%, ((FIGS. 8A-8C).
[0886] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 16. Synthesis of 5-(methoxycarbonyl)methyl Uridine
(Compound 9)
##STR00175##
[0888] Uridine 9-a was protected to provide compound 9-b (98%
yield). This compound was brominated with excess bromine in the
presence of acetic anhydride and acetic acid. The 5-bromo analog
9-c was obtained (60% yield) and further benzoylated to provide
desired compound 9-d (64% yield). 5-Bromo compound 9-d was
condensed with dimethyl malonate under basic condition to give the
arylated malonate and the fully protected diester 9-e (50% yield).
After de-carboxylation and deprotection, compound 9 was obtained
verified by NMR (FIG. 9).
[0889] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 17. Synthesis of 5-(methoxycarbonyl)methyl-2'-O-methyl
Uridine (2-OMe-MCM5U) (Compound 10)
##STR00176##
[0891] Similar strategy to the synthesis of compound 9 above,
2'-O-methyluridine 10-a was acylated and brominated to obtain
compound 10-c. Further benzoylation provided 5-bromo analog 10-d,
which was condensed with dimethyl malonate provide the desired
product 10-e (45% yield). Decarboxylation and deprotection provided
compound 10.
[0892] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 18. Synthesis of
5-trifluoroacetyl-aminomethyl-2-thiouridine (Compound 11)
##STR00177##
[0894] Glycosylation of 2-thiouracil 11-a provided compound 11-c,
which can be deprotected with any useful deprotection reagent. In
particular, LiOH provided desired product 11-d (80-90% yield).
Isopropylidene protection provided compound 11-e (90% yield).
Further 5-hydroxylmethylation provided compound 11-f Chlorination,
azidation, and further reduction provided methylamine compound
11-i, which was acetylated to provided compound 11.
[0895] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 19. Synthesis of 5-methylaminomethyl-2-uridine (Compound
12)
##STR00178##
[0897] Compound 12 can be obtained by any useful method (e.g., see
schemes (i) and (ii) above). For example, protected uracil can be
glycosylated and subsequently aminated to provide compound 12.
Additional protecting, deprotecting, and activating steps can be
conducted as needed. To obtain the corresponding NTP, a
triphosphate reaction can be conducted (e.g., any described
herein). Optionally, the NTP can be purified (e.g., using a
Sephadex DEAE-A25 column), lyophilized, or evaporated (e.g., from
EtOH).
Example 20. Synthesis of 5-TFA-methylaminomethyl-2-uridine
(Compound 13)
##STR00179##
[0899] Uridine 13-a was protected with isopropylidene to provide
compound 13-b and then 5-hydroxymethylated to provide compound
13-c. Chlorination and subsequent amination provided compound 13-e,
which can be protected to provided 13-f. Subsequent deprotection
provided compound 13.
[0900] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 21. Synthesis of 5-carboxymethylaminomethyl Uridine
(Compound 14)
##STR00180##
[0902] Uridine 14-a was protected with isopropylidene to provide
compound 14-b and then 5-aminoalkylated with the Mannich reaction
to provide compound 14-c. Methylation provided quaternary amine
14-d. Subsequent amination and deprotection steps can be used to
provide compound 14. To obtain the corresponding NTP, a
triphosphate reaction can be conducted (e.g., any described
herein). Optionally, the NTP can be purified (e.g., using a
Sephadex DEAE-A25 column), lyophilized, or evaporated (e.g., from
EtOH).
Example 22. Alternative synthesis of 5-methylaminomethyl-2-uridine
(Compound 12) and 5-carboxymethylaminomethyl-2-uridine (Compound
14)
##STR00181##
[0904] In addition to those strategies provided above for compounds
12 and 14, the following strategy can also be implemented.
5-Methyluridine A can be silylated to provide compound B. After
radical monobromination, the resultant intermediate bromide C can
be used for the preparation of compound 12 and compound 14 analogs.
Subsequent alkylamination of bromide compound C could provide
compounds D and E, which can be deprotected to provide compounds 14
and 12, respectively. To obtain the corresponding NTP, a
triphosphate reaction can be conducted (e.g., any described
herein). Optionally, the NTP can be purified (e.g., using a
Sephadex DEAE-A25 column), lyophilized, or evaporated (e.g., from
EtOH).
Example 23. Synthesis of dimethyl-pseudouridine (Compound 15) and
dimethyl-pseudo-UTP (NTP of Said Compound)
##STR00182##
[0906] Nucleosides can be phosphorylated by any useful method. For
example, as shown above, nucleosides can be reacted with phosphorus
oxychloride and subsequently treated with a monophosphate
intermediate with bis(tributylammonium)pyrophosphate (TBAPP) to
give the triphosphate.
Example 24. Synthesis of 2'-C-methyl Adenosine (Compound 16) and
2'-C-methyl ATP (NTP of Said Compound)
##STR00183##
[0908] About 5 g of compound 16-2 was prepared from 5 g of compound
16-1 via a Dess-Martin periodane reaction. Compound 16-2 was
reacted with MeMgI/TiCl4/-78.degree. C. to provide compound 16-3,
and crude compound 16-3 (6 g) was directly reacted with
benzylchloride to prepare compound 16-4. Reaction with the
nucleobase and deprotection provided compound 16 (0.56 g).
Example 25. Synthesis of 2'-C-methyl-cytidine Isomers (Compound 17
and Compound 18) and 2'-C-methyl UTP (NTP of Said Compounds)
##STR00184##
[0910] About 17.4 g of compound 17-3 was prepared from 20 g of
compound 17-1. Then, 2'-oxidation and alkylation with MeMgI
provided 300 mg of compound 17-5a and 80 mg of compound 17-5b.
About 9 g of compound 17-5a (about 90% pure) and 2.1 g of compound
17-5b (pure) were prepared from 17.4 g of compound 17-3 in 2
batches. N- and O-deprotection provided compounds 17 and 18.
Example 26. Synthesis of 2'-C-methyl Guanosine (Compound 19) and
2'-C-methyl GTP (NTP of Said Compound)
##STR00185##
[0912] 2'-Oxidation of protected ribose 19-1 and subsequent
alkylation with MeMgCl provided compound 19-3. The resultant
compound was further protected to provided compound 19-4, and 1.56
g of compound 19-5a was prepared from 3.1 g of compound 19-4.
Subsequent oxidation and deprotection provided compound 19 (about
90% pure, 50 mg).
[0913] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 27. Synthesis of 2'-C-methyl Uridine (compound 20) and
2'-C-methyl UTP (NTP of Said Compound)
##STR00186##
[0915] 2'-Oxidation of protected ribose 20-1 and subsequent
alkylation with MeMgCl provided compound 20-3. The resultant
compound was further protected to provide compound 20-4. Reaction
with uracil and deprotection provided pure compound 20 (50 mg).
[0916] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 28. Synthesis of (S)-2'-C-methyl Adenosine (Compound 21)
and (S)-2'-C-methyl ATP (NTP of Said Compound)
##STR00187##
[0918] Compound 21-1 (5 g) was protected to form compound 21-2a,
and chromium oxidation provided compound 21-3a. Alkylation via
route [i] (5 eq. MeMgI in ether at -50.degree. C.) provided
compound 21-4. Optionally, yield could be improved via route [ii]
by protecting the amino group to provide compound 21-3b and then
alkylating at the 2'-C position to provide compound 21-4a. Compound
21-3a was alkylated to provide crude compound 21-4 (3 g, 20% of
compound 3a in this crude product), where the product can be
optionally purified. Deprotection of compound 21-4 afforded
compound 21 (50% yield).
[0919] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 29. Synthesis of (S)-2'-C-methyl Guanosine (compound 22)
and (S)-2'-methyl GTP (NTP of Said Compound)
##STR00188## ##STR00189##
[0921] About 30 g of compound 22-1 was silylated to provide
compound 22-2 in three steps.
[0922] Further protection provided compound 22-3, and Dess-Martin
periodane oxidation provided compound 22-4 (1.6 g) in two batches.
2'-C alkylation (5 eq. MeMgI in ether, -50.degree. C. to RT)
provided compound 22-5, and further deprotection steps provided
compound 22.
[0923] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 30. Synthesis of (S)-2'-C-methyl Uridine (Compound 23) and
of (S)-2'-C-methyl UTP (NTP of Said Compound)
##STR00190##
[0925] Uridine 23-1 (2.0 g) was protected with TIPDSCl.sub.2
(1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane) to provide compound
23-2. Oxidation provided compound 23-3, and 2'-C alkylation
provided compound 23-4, which can be optionally purified with
Prep-HPLC prior to the next step. Then, deprotection provided
desired compound 23.
[0926] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 31. Synthesis of 4'-C-methyl Adenosine (Compound 24) and
4'-C-methyl ATP (NTP of Said Compound)
##STR00191## ##STR00192## ##STR00193##
[0928] 1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose 24-1 was
converted via sequential oxidation, reduction, and protection steps
to provide compound 24-4. The first oxidation step to provide
compound 24-2 can be implemented with any useful reagents, such as
0.75 eq. pyridinium dichromate (PDC) with 1 eq. Ac.sub.2O or 1.2
eq. of Dess-Martin periodane. Subsequent deprotection, formylation,
and reduction provided compound 24-7, which was followed with
protection and deoxygenation steps to provide compound 24-10. About
0.4 g of compound 24-14 was prepared from 1 g of compound 24-10 via
sequential protection and deprotection steps. Addition of
N6-benzoyladenine and subsequent deprotection provided compound
24.
[0929] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 32. Synthesis of 4'-C-methyl Cytidine (Compound 25) and
4'-C-methyl CTP (NTP of Said Compound)
##STR00194## ##STR00195## ##STR00196##
[0931] Similar to the strategy provided above for compound 24,
compound 25-14 was produced with compound 25-1. Addition of
cytidine and subsequent deprotection provided compound 25.
[0932] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 33. Synthesis of 4'-C-methyl Guanosine (Compound 26) and
4'-C-methyl GTP (NTP of Said Compound)
##STR00197## ##STR00198## ##STR00199##
[0934] Similar to the strategy provided above for compound 24,
compound 26-14 was produced with compound 26-1. Addition of
2-amino-6-chloropurine, subsequent oxidation, and then deprotection
provided compound 26.
[0935] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 34. Synthesis of 4'-C-methyl Uridine (Compound 27) and
4'-C-methyl UTP (NTP of Said Compound)
##STR00200## ##STR00201## ##STR00202##
[0937] Similar to the strategy provided above for compound 24,
compound 27-14 was produced with compound 27-1. Addition of uracil
and subsequent deprotection provided compound 27.
[0938] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 35. Synthesis of 2'-O,4'-C-methylene Adenosine (Compound
28) and 2'-O,4'-C-Methylene ATP (NTP of Said Compound)
##STR00203## ##STR00204## ##STR00205##
[0940] Similar to the strategy provided above for compound 24,
compound 28-7 was produced with compound 28-1. Subsequent
mesylation, deprotection, and acetylation provided compound 28-10,
which was followed by addition of N6-benzoyladenine and subsequent
internal cyclization. Various protection and deprotection steps
provided compound 28.
Example 36. Synthesis of 5-methyl-2'-O,4'-C-methylene Cytidine
(Compound 29) and 5-methyl-2'-O,4'-C-methylene CTP (NTP of Said
Compound)
##STR00206## ##STR00207##
[0942] Aldofuranose compound 29-1 was reacted via various
protection steps, and then 5-methyluracil was added to provide
compound 29-5. Subsequent internal cyclization, deprotection,
protection, and amination steps provided compound 29.
Example 37. Synthesis of 2'-O,4'-C-methylene Guanosine (Compound
30) and 2'-O,4'-C-Methylene GTP (NTP of Said Compound)
##STR00208## ##STR00209##
[0944] Similar to the strategy provided above for compound 29,
aldofuranose compound 30-1 was reacted via various protection
steps, and then 2-amino-6-chloropurine was added to provide
compound 30-5. Subsequent internal cyclization, amination, and
deprotection steps provided compound 30.
[0945] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 38. Synthesis of 2'-O,4'-C-methylene Uridine (Compound 31)
and 2'-O,4'-C-Methylene UTP (NTP of Said Compound)
##STR00210## ##STR00211## ##STR00212##
[0947] Similar to the strategy provided above for compound 24,
compound 31-7 was produced with compound 31-1. Subsequent
mesylation, deprotection, and acetylation provided compound 30-10.
Addition of uracil and subsequent internal cyclization provided
compound 31-12, and various protection and deprotection steps
provided compound 31. A subsequent triphosphate reaction (e.g., as
described herein) provided the NTP of compound 31, which can be
optionally purified (e.g., with HPLC).
Example 39. Synthesis of 2'-chloro Adenosine (Compound 32) and
2'-chloro ATP (NTP of Said Compound)
##STR00213## ##STR00214##
[0949] Arabinoadenosine 32-1 was protected via steps 1 and 2 and
then chlorinated to provide compound 32-4. Subsequent deprotection
provided compound 32, and the triphosphate reaction provided the
NTP of compound 32.
Example 40. Synthesis of 2'-iodo Adenosine (Compound 33) and
2'-iodo ATP (NTP of Said Compound)
##STR00215## ##STR00216##
[0951] Arabinoadenosine 33-1 was protected via steps 1 and 2 and
then iodinated to provide compound 33-4. Subsequent deprotection
provided compound 33, and the triphosphate reaction in DMF provided
the NTP of compound 33.
Example 41. Synthesis of 2'-bromo Cytidine (Compound 34) and
2'-bromo CTP (NTP of Said Compound)
##STR00217##
[0953] Arabinocytidine 34-1 was protected under various conditions
and then brominated to provide compound 34-4. Optionally, the
reaction can provide compound 34-4 via compound 34-3a under any
useful protection reactions, such as (i) 1.5 eq. Et.sub.3N, 1 eq.
DMAP, 1.2 eq. TfCl, in DCM (10 mL); (ii) 3 eq. DMAP, 1.2 eq. TfCl
in DCM (15 mL); or (iii) 15 eq. DMAP, 1.5 eq. Tf.sub.2O, in DCM (15
mL) at -10.degree. C. to 0.degree. C. for 2 hour. In particular, 55
mg of compound 34-3a was obtained from reaction condition (iii).
Subsequent deprotection provided compound 34, and the triphosphate
reaction in DMF provided the NTP of compound 34. Crude product 34
could be optionally purified prior to phosphorylation.
Example 42. Synthesis of 2'-chloro Guanosine (Compound 35) and
2'-chloro GTP (NTP of Said Compound)
##STR00218## ##STR00219##
[0955] Guanosine 35-1 was protected under various conditions and
then acetylated to provide compound 35-4. The reaction from
compound 35-2 to compound 35-3 was conducted with 2 eq. DMAP, 2 eq.
Et.sub.3N, 3 eq. Tf.sub.2O in 1,2-dichloroethane (10 mL) at
40.degree. C. for 4 hours. About 55 mg of compound 35-3 was
obtained after the purification.
[0956] Desired compound 35 can be obtained by any useful method.
For example, as shown above, compound 35-4 can be treated with
subsequent protection, chlorination, and deprotection steps to
provide compound 35. To obtain the corresponding NTP, a
triphosphate reaction can be conducted (e.g., any described
herein). Optionally, the NTP can be purified (e.g., using a
Sephadex DEAE-A25 column), lyophilized, or evaporated (e.g., from
EtOH).
Example 43. Synthesis of 2'-iodo Uridine (Compound 36) and 2'-iodo
UTP (NTP of Said Compound)
##STR00220##
[0958] O.sup.2,2'-Cyclouridine 36-1 was protected to provide
compound 36-2. Subsequent iodination, optionally mediated with
selenium, provided compound 36. A triphosphate reaction was
conducted to provide the NTP of compound 36. Optionally, the NTP
can be purified (e.g., using a Sephadex DEAE-A25 column),
lyophilized, or evaporated (e.g., from EtOH).
Example 44. Synthesis of 2'-O,4'-C-methylene Adenosine (Compound
37) and 2'-O,4'-C-Methylene ATP (NTP of Said Compound)
##STR00221## ##STR00222##
[0960] Similar to the strategy provided above for compound 24,
compound 37-7 was produced with compound 37-1. Subsequent
mesylation, deprotection, and acetylation provided compound 37-10.
Addition of uracil and subsequent internal cyclization provided
compound 37-12. Various protection and deprotection steps provided
compound 37.
[0961] To obtain the corresponding NTP, a triphosphate reaction can
be conducted (e.g., any described herein). Optionally, the NTP can
be purified (e.g., using a Sephadex DEAE-A25 column), lyophilized,
or evaporated (e.g., from EtOH).
Example 45. Synthesis of Cyclopentene Diol Cytidine (Compound 38)
and Cyclopentene Diol CTP (NTP of Said Compound)
##STR00223## ##STR00224## ##STR00225##
[0963] D-ribose was protected and then allylated to provide
compound 38-4, which was subsequently cyclized and reduced to
provide compound 38-7. Olefin metathesis and subsequent oxidation
provided compound 38-9, and further reduction reactions and
addition of N-benzoyluracil provided compound 38-14. Additional
deprotection and protection reactions provided compound 38, and
triphosphate reaction (e.g., with any useful reaction condition,
such as those described herein or in U.S. Pat. No. 7,893,227,
incorporated herein by reference) provided the NTP of compound
38.
Example 46. Synthesis of 2'-methyl Uridine (Compound 39) and
2'-methyl UTP (NTP of Said Compound)
##STR00226## ##STR00227##
[0965] Uridine 39-1 was protected and then oxidized with 2 eq. of
Dess-Martin periodane to provide compound 39-3. Subsequent Wittig
reaction, hydrogenation, and deprotection steps provided compound
39.
Example 47. Synthesis of 2'-methyl Cytidine (Compound 40) and
2'-methyl CTP (NTP of Said Compound)
##STR00228## ##STR00229##
[0967] Cytidine 40-1 was protected and then oxidized to provide
compound 40-3. Subsequent Wittig reaction, hydrogenation, and
deprotection steps provided compound 40.
Example 48. Synthesis of N-acetyl Cytidine (Compound 41) and
N-acetyl CTP (NTP of Said Compound)
##STR00230##
[0969] A solution of N-acetyl-cytidine (compound 41) (103.0 mg,
0.36 mmol) was added to proton sponge (115.72 mg, 0.54 mmol, 1.50
equiv) in 1.0 mL trimethylphosphate (TMP) and 1.0 mL of anhydrous
tetrahydrofuran (THF). The solution was stirred for 10 minutes at
0.degree. C. Phosphorous oxychloride (POCl.sub.3) (67.2 ul, 0.72
mmol, 2.0 eqiv.) was added dropwise to the solution before being
kept stirring for 2 hours under N.sub.2 atmosphere. After 2 hours
the solution was reacted with a mixture of bistributylammonium
pyrophosphate (TBAPP or (n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7)
(1.28 g, 2.34 mmol, 6.5 eqiv.) and tributylamine (350.0 ul, 1.45
mmol, 4.0 equiv.) in 2.5 ml of dimethylformamide. After
approximately 15 minutes, the reaction was quenched with 24.0 ml of
0.2M triethylammonium bicarbonate (TEAB) and the clear solution was
stirred at room temperature for an hour. The reaction mixture was
lyophilized overnight and the crude reaction mixture was purified
by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 16.81-17.80 min). Fractions containing the desired
compound were pooled and lyophilized to produce the NTP of compound
41. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 49. Synthesis of 5-methoxy Uridine (Compound 42) and
5-methoxy UTP (NTP of Said Compound)
##STR00231##
[0971] A solution of 5-methoxy uridine (compound 42) (69.0 mg, 0.25
mmol, plus heat to make it soluble) was added to proton sponge
(80.36 mg, 0.375 mmol, 1.50 equiv.) in 0.7 mL trimethylphosphate
(TMP) and was stirred for 10 minutes at 0.degree. C. Phosphorous
oxychloride (POCl.sub.3) (46.7 ul, 0.50 mmol, 2.0 equiv.) was added
dropwise to the solution before being kept stirring for 2 hours
under N.sub.2 atmosphere. After 2 hours the solution was reacted
with a mixture of bistributylammonium pyrophosphate (TBAPP or
(n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7) (894.60 mg, 1.63 mmol,
6.50 equiv.) and tributylamine (243.0 ul, 1.00 mmol, 4.0 equiv.) in
2.0 ml of dimethylformamide. After approximately 15 minutes, the
reaction was quenched with 17.0 ml of 0.2M triethylammonium
bicarbonate (TEAB) and the clear solution was stirred at room
temperature for an hour. The reaction mixture was lyophilized
overnight and the crude reaction mixture was purified by HPLC
(Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 16.57-17.51 min). Fractions containing the desired
compound were pooled and lyophilized to produce the NTP of compound
42. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 50. Synthesis of 5-formyl Cytidine (Compound 43) and
5-formyl CTP (NTP of Said Compound)
##STR00232##
[0973] A solution of 5-formyl cytidine (compound 43)) (48.4 mg,
0.18 mmol, plus heat to make it soluble) was added to proton sponge
(57.86 mg, 0.27 mmol, 1.50 equiv.) in 0.7 mL trimethylphosphate
(TMP) and was stirred for 10 minutes at 0.degree. C. Phosphorous
oxychloride (POCl.sub.3) (33.6 ul, 0.36 mmol, 2.0 equiv.) was added
dropwise to the solution before being kept stirring for 2 hours
under N.sub.2 atmosphere. After 2 hours the solution was reacted
with a mixture of bistributylammonium pyrophosphate (TBAPP or
(n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7) (642.0 mg, 1.17 mmol,
6.50 equiv.) and tributylamine (175.0 ul, 0.72 mmol, 4.0 equiv.) in
1.7 ml of dimethylformamide. After approximately 15 minutes, the
reaction was quenched with 12.0 ml of 0.2M triethylammonium
bicarbonate (TEAB) and the clear solution was stirred at room
temperature for an hour. The reaction mixture was lyophilized
overnight and the crude reaction mixture was purified by HPLC
(Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 17.04-17.87 min). Fractions containing the desired
compound were pooled and lyophilized to provide the NTP of compound
43. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 51. Synthesis of 3-methyl Uridine (Compound 44) and
3-methyl UTP (NTP of Said Compound)
##STR00233##
[0975] A solution of 3-methyl uridine (compound 44) (45.80 mg, 0.18
mmol) was added to proton sponge (57.86 mg, 0.27 mmol, 1.50 equiv.)
in 0.5 mL trimethylphosphate (TMP) and was stirred for 10 minutes
at 0.degree. C. Phosphorous oxychloride (POCl.sub.3) (33.6 ul, 0.36
mmol, 2.0 equiv.) was added dropwise to the solution before being
kept stirring for 2 hours under N.sub.2 atmosphere. After 2 hours
the solution was reacted with a mixture of bistributylammonium
pyrophosphate (TBAPP or (n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7)
(652.0 mg, 1.19 mmol, 6.60 equiv.) and tributylamine (175.0 ul,
0.72 mmol, 4.0 equiv.) in 1.3 ml of dimethylformamide. After
approximately 15 minutes, the reaction was quenched with 12.0 ml of
0.2M triethylammonium bicarbonate (TEAB) and the clear solution was
stirred at room temperature for an hour. The reaction mixture was
lyophilized overnight and the crude reaction mixture was purified
by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 18.52-19.57 min). Fractions containing the desired
compound were pooled and lyophilized to provide the NTP of compound
44. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 52. Synthesis of N1-methyl Pseudouridine (Compound 45) and
N1-methyl PseudoUTP (NTP of Said Compound)
##STR00234##
[0977] A solution of N1-methyl pseudouridine (compound 45) (96.6
mg, 0.374 mmol, plus heat to make it soluble) was added to proton
sponge (120.0 mg, 0.56 mmol, 1.50 equiv.) in 0.8 mL
trimethylphosphate (TMP) and was stirred for 10 minutes at
0.degree. C. Phosphorous oxychloride (POCl.sub.3) (70.0 ul, 0.75
mmol, 2.0 equiv.) was added dropwise to the solution before being
kept stirring for 2 hours under N.sub.2 atmosphere. After 2 hours
the solution was reacted with a mixture of bistributylammonium
pyrophosphate (TBAPP or (n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7)
(1.36 g, 2.47 mmol, 6.60 equiv.) and tributylamine (362.0 ul, 1.5
mmol, 4.0 equiv.) in 2.5 ml of dimethylformamide. After
approximately 15 minutes, the reaction was quenched with 17.0 ml of
0.2M triethylammonium bicarbonate (TEAB) and the clear solution was
stirred at room temperature for an hour. The reaction mixture was
lyophilized overnight and the crude reaction mixture was purified
by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 15.91-17.01 min). Fractions containing the desired
compound were pooled and lyophilized was subjected to a
triphosphorylation reaction to provide the NTP of compound 45. The
triphosphorylation reactions were carried out in a two-neck flask
flame-dried under N.sub.2 atmosphere. Nucleosides and the protein
sponge were dried over P.sub.2O.sub.5 under vacuum overnight prior
to use. The formation of monophosphates was monitored by LCMS.
Example 53. Synthesis of 5-methoxycarbonylethenyl Uridine (Compound
46) and 5-methoxycarbonylethenyl UTP (NTP of Said Compound)
##STR00235##
[0979] A solution of 5-methoxycarbonylethenyl uridine (compound 46)
(102.0 mg, 0.31 mmol) was added to proton sponge (99.65 mg, 0.46
mmol, 1.50 equiv.) in 0.8 mL trimethylphosphate (TMP) and was
stirred for 10 minutes at 0.degree. C. Phosphorous oxychloride
(POCl.sub.3) (57.8 ul, 0.62 mmol, 2.0 equiv) was added dropwise to
the solution before being kept stirring for 2 hours under N.sub.2
atmosphere. After 2 hours the solution was reacted with a mixture
of bistributylammonium pyrophosphate (TBAPP or
(n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7) (1.12 g, 2.05 mol, 6.60
equiv.) and tributylamine (300.0 ul, 1.24 mmol, 4.0 equiv.) in 2.5
ml of dimethylformamide. After approximately 15 minutes, the
reaction was quenched with 20.0 ml of 0.2M triethylammonium
bicarbonate (TEAB) and the clear solution was stirred at room
temperature for an hour. The reaction mixture was lyophilized
overnight and the crude reaction mixture was purified by HPLC
(Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 21.56-23.21 min). Fractions containing the desired
compound were pooled and lyophilized to provide the NTP of compound
46. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 54. Synthesis of 5-aminopropenyl Uridine (Compound 47) and
5-aminopropenyl UTP (NTP of Said Compound)
##STR00236##
[0981] 5-Aminopropenyl uridine 47 was protected and a solution of
protected compound 47 (86.0 mg, 0.22 mmol) was added to proton
sponge (70.7 mg, 0.33 mmol, 1.50 equiv.) in 0.7 mL
trimethylphosphate (TMP) and was stirred for 10 minutes at
0.degree. C. Phosphorous oxychloride (POCl.sub.3) (41.1 ul, 0.44
mmol, 2.0 equiv.) was added dropwise to the solution before being
kept stirring for 2 hours under N.sub.2 atmosphere. After 2 hours
the solution was reacted with a mixture of bistributylammonium
pyrophosphate (TBAPP or (n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7)
(784.6 mg, 1.43 mmol, 6.50 equiv.) and tributylamine (213.0 ul,
0.88 mmol, 4.0 equiv.) in 1.6 ml of dimethylformamide. After
approximately 15 minutes, the reaction was quenched with 15.0 ml of
0.2M triethylammonium bicarbonate (TEAB) and the clear solution was
stirred at room temperature for an hour. 18.0 ml of concentrated
ammonium hydroxide was added to the reaction mixture to remove the
trifluoroacetyl group. It was then stored stirring overnight. The
reaction mixture was lyophilized overnight and the crude reaction
mixture was purified by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18
preparative column, 250.times.21.20 mm, 10.0 micron; gradient: 100%
A for 3.0 min, then 1% B/min, A=100 mM TEAB buffer, B=ACN; flow
rate: 10.0 mL/min; retention time: 16.14-17.02 min). Fractions
containing the desired compound were pooled and lyophilized to
provide the NTP of compound 47. The triphosphorylation reactions
were carried out in a two-neck flask flame-dried under N.sub.2
atmosphere. Nucleosides and the protein sponge were dried over
P.sub.2O.sub.5 under vacuum overnight prior to use. The formation
of monophosphates was monitored by LCMS.
Example 55. Synthesis of N-PEG Adenosine (Compound 48) and N-PEG
ATP (NTP of Said Compound)
##STR00237##
[0983] N-PEG adenosine 48 was protected and a solution of the
protected compound 48 (100.0 mg, 0.15 mmol) was added to proton
sponge (49.3 mg, 0.23 mmol, 1.50 equiv.) in 0.65 mL
trimethylphosphate (TMP) and was stirred for 10 minutes at
0.degree. C. Phosphorous oxychloride (POCl.sub.3) (28.0 ul, 0.3
mmol, 2.0 equiv.) was added dropwise to the solution before being
kept stirring for 2 hours under N.sub.2 atmosphere. After 2 hours
the solution was reacted with a mixture of bistributylammonium
pyrophosphate (TBAPP or (n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7)
(537.7 mg, 0.98 mmol, 6.50 equiv.) and tributylamine (146.0 ul, 0.6
mmol, 4.0 equiv.) in 1.2 ml of dimethylformamide. After
approximately 15 minutes, the reaction was quenched with 10.0 ml of
0.2M triethylammonium bicarbonate (TEAB) and the clear solution was
stirred at room temperature for an hour. 18.0 ml of concentrated
ammonium hydroxide was added to the reaction mixture to remove the
trifluoroacetyl group. It was then stored stirring overnight. The
reaction mixture was lyophilized overnight and the crude reaction
mixture was purified by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18
preparative column, 250.times.21.20 mm, 10.0 micron; gradient: 100%
A for 3.0 min, then 1% B/min, A=100 mM TEAB buffer, B=ACN; flow
rate: 10.0 mL/min; retention time: 24.5-25.5 min). Fractions
containing the desired compound were pooled and lyophilized to
provide the NTP of compound 48. The triphosphorylation reactions
were carried out in a two-neck flask flame-dried under N.sub.2
atmosphere. Nucleosides and the protein sponge were dried over
P.sub.2O.sub.5 under vacuum overnight prior to use. The formation
of monophosphates was monitored by LCMS.
Example 56. Synthesis of N-methyl Adenosine (Compound 49) and
N-methyl ATP (NTP of Said Compound)
##STR00238##
[0985] A solution of N-methyl adenosine (compound 49) (70.0 mg,
0.25 mmol) was added to proton sponge (79.29 mg, 0.37 mmol, 1.50
equiv.) in 0.7 mL trimethylphosphate (TMP) and was stirred for 10
minutes at 0.degree. C. Phosphorous oxychloride (POCl.sub.3) (46.66
ul, 0.50 mmol, 2.0 equiv.) was added dropwise to the solution
before being kept stirring for 2 hours under N.sub.2 atmosphere.
After 2 hours the solution was reacted with a mixture of
bistributylammonium pyrophosphate (TBAPP or
(n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7) (888.85 mg, 1.62 mmol,
6.50 equiv.) and tributylamine (241.0 ul, 1.0 mmol, 4.0 equiv.) in
1.3 ml of dimethylformamide. After approximately 15 minutes, the
reaction was quenched with 16.0 ml of 0.2 M triethylammonium
bicarbonate (TEAB) and the clear solution was stirred at room
temperature for an hour. The reaction mixture was lyophilized
overnight and the crude reaction mixture was purified by HPLC
(Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 19.62-20.14 min). Fractions containing the desired
compound were pooled and lyophilized to provide the NTP of compound
49. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 57. Synthesis of N,N-dimethyl Guanosine (Compound 50) and
N,N-dimethyl GTP (NTP of Said Compound)
##STR00239##
[0987] A solution of N,N-dimethyl guanosine (compound 50) (65.8 mg,
0.21 mmol) was added to proton sponge (68.58 mg, 0.32 mmol, 1.50
equiv) in 0.7 mL trimethylphosphate (TMP) and was stirred for 10
minutes at 0.degree. C. Phosphorous oxychloride (POCl.sub.3) (39.20
ul, 0.42 mmol, 2.0 equiv.) was added dropwise to the solution
before being kept stirring for 2 hours under N.sub.2 atmosphere.
After 2 hours the solution was reacted with a mixture of
bistributylammonium pyrophosphate (TBAPP or
(n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7) (751.67 mg, 1.37 mmol,
6.50 equiv.) and tributylamine (204.0 ul, 0.84 mmol, 4.0 equiv.) in
1.5 ml of dimethylformamide. After approximately 15 minutes, the
reaction was quenched with 14.0 ml of 0.2 M triethylammonium
bicarbonate (TEAB) and the clear solution was stirred at room
temperature for an hour. The reaction mixture was lyophilized
overnight and the crude reaction mixture was purified by HPLC
(Shimadzu, Kyoto Japan, Phenomenex C18 preparative column,
250.times.21.20 mm, 10.0 micron; gradient: 100% A for 3.0 min, then
1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;
retention time: 19.27-19.95 min). Fractions containing the desired
compound were pooled and lyophilized to provide the NTP of compound
50. The triphosphorylation reactions were carried out in a two-neck
flask flame-dried under N.sub.2 atmosphere. Nucleosides and the
protein sponge were dried over P.sub.2O.sub.5 under vacuum
overnight prior to use. The formation of monophosphates was
monitored by LCMS.
Example 58. General Methods for Triphosphate Synthesis of NTPS
##STR00240##
[0989] The nucleoside i can be phosphorylated by any useful method
to provide a triphosphate compound ii. For example, the nucleoside
can be added to proton sponge and trimethylphosphate (TMP) and
cooled (e.g., to -40.degree. C.). Phosphorous oxychloride
(POCl.sub.3) can be added dropwise before reacting with
bistributylammonium pyrophosphate (TBAPP or
(n-Bu.sub.3NH).sub.2H.sub.2P.sub.2O.sub.7) and tributylamine. The
reaction can then be quickly quenched with triethylammonium
bicarbonate (TEAB). Exemplary conditions are provided in U.S. Pat.
No. 7,893,227, which is incorporated herein by reference.
[0990] After the phosphorylation reaction, the reaction mixture can
be optionally lyophilized, purified (e.g., by ion-exchange
chromatography and/or HPLC), or converted to a sodium salt (e.g.,
by dissolving in MeOH and adding sodium perchlorate in
acetone).
Example 59: PCR for cDNA Production
[0991] PCR procedures for the preparation of cDNA are performed
using 2.times.KAPA HIFI.TM. HotStart ReadyMix by Kapa Biosystems
(Woburn, Mass.). This system includes 2.times.KAPA ReadyMix 12.5
.mu.l; Forward Primer (10 uM) 0.75 .mu.l; Reverse Primer (10 uM)
0.75 .mu.l; Template cDNA 100 ng; and dH.sub.2O diluted to 25.0
.mu.l. The reaction conditions are at 95.degree. C. for 5 min. and
25 cycles of 98.degree. C. for 20 sec, then 58.degree. C. for 15
sec, then 72.degree. C. for 45 sec, then 72.degree. C. for 5 min.
then 4.degree. C. to termination.
[0992] The reverse primer of the instant invention incorporates a
poly-T.sub.120 for a poly-A.sub.120 in the mRNA. Other reverse
primers with longer or shorter poly-T tracts can be used to adjust
the length of the poly-A tail in the mRNA.
[0993] The reaction is cleaned up using Invitrogen's PURELINK.TM.
PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions
(up to 5 .mu.g). Larger reactions will require a cleanup using a
product with a larger capacity. Following the cleanup, the cDNA is
quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the cDNA is the expected size. The cDNA
is then submitted for sequencing analysis before proceeding to the
in vitro transcription reaction.
Example 60. In Vitro Transcription (IVT)
[0994] The in vitro transcription reaction generates mRNA
containing modified nucleotides or modified RNA. The input
nucleotide triphosphate (NTP) mix is made in-house using natural
and un-natural NTPs.
[0995] A typical in vitro transcription reaction includes the
following:
TABLE-US-00004 Template cDNA 1.0 .mu.g 10x transcription buffer
(400 mM Tris-HCl pH 2.0 .mu.l 8.0, 190 mM MgCl2, 50 mM DTT, 10 mM
Spermidine) Custom NTPs (25 mM each 7.2 .mu.l RNase Inhibitor 20 U
T7 RNA polymerase 3000 U dH.sub.20 .sub.Up to 20.0 .mu.l
[0996] Incubation at 37.degree. C. for 3 hr-5 hrs.
[0997] The crude IVT mix may be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase is then used to
digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA is purified using Ambion's MEGACLEAR.TM.
Kit (Austin, Tex.) following the manufacturer's instructions. This
kit can purify up to 500 .mu.g of RNA. Following the cleanup, the
RNA is quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred.
[0998] The T7 RNA polymerase may be selected from, T7 RNA
polymerase, T3 RNA polymerase and mutant polymerases such as, but
not limited to, the novel polymerases able to incorporate modified
NTPs as well as those polymerases described by Liu (Esvelt et al.
(Nature (2011) 472(7344):499-503 and U.S. Publication No.
20110177495) which recognize alternate promoters, Ellington
(Chelliserrykattil and Ellington, Nature Biotechnology (2004)
22(9):1155-1160) describing a T7 RNA polymerase variant to
transcribe 2'-O-methyl RNA and Sousa (Padilla and Sousa, Nucleic
Acids Research (2002) 30(24): e128) describing a T7 RNA polymerase
double mutant; herein incorporated by reference in their
entireties.
Example 61. Enzymatic Capping of mRNA
[0999] Capping of the mRNA is performed as follows where the
mixture includes: IVT RNA 60 .mu.g-180 .mu.g and dH.sub.20 up to 72
.mu.l. The mixture is incubated at 65.degree. C. for 5 minutes to
denature RNA, and then is transferred immediately to ice.
[1000] The protocol then involves the mixing of 10.times. Capping
Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl.sub.2)
(10.0 .mu.l); 20 mM GTP (5.0 .mu.l); 20 mM S-Adenosyl Methionine
(2.5 .mu.l); RNase Inhibitor (100 U); 2'-O-Methyltransferase
(400U); Vaccinia capping enzyme (Guanylyl transferase) (40 U);
dH.sub.2O (Up to 28 .mu.l); and incubation at 37.degree. C. for 30
minutes for 60 .mu.g RNA or up to 2 hours for 180 .mu.g of RNA.
[1001] The mRNA is then purified using Ambion's MEGACLEAR.TM. Kit
(Austin, Tex.) following the manufacturer's instructions. Following
the cleanup, the RNA is quantified using the NANODROP.TM.
(ThermoFisher, Waltham, Mass.) and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred. The RNA product may also be
sequenced by running a reverse-transcription-PCR to generate the
cDNA for sequencing.
Example 62. PolyA Tailing Reaction
[1002] Without a poly-T in the cDNA, a poly-A tailing reaction must
be performed before cleaning the final product. This is done by
mixing Capped IVT RNA (100 .mu.l); RNase Inhibitor (20 U);
10.times. Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100
mM MgCl.sub.2)(12.0 .mu.l); 20 mM ATP (6.0 .mu.l); Poly-A
Polymerase (20 U); dH.sub.2O up to 123.5 .mu.l and incubation at
37.degree. C. for 30 min. If the poly-A tail is already in the
transcript, then the tailing reaction may be skipped and proceed
directly to cleanup with Ambion's MEGACLEAR.TM. kit (Austin, Tex.)
(up to 500 .mu.g). Poly-A Polymerase is preferably a recombinant
enzyme expressed in yeast.
[1003] For studies performed and described herein, the poly-A tail
is encoded in the IVT template to comprise 160 nucleotides in
length. However, it should be understood that the processivity or
integrity of the poly-A tailing reaction may not always result in
exactly 160 nucleotides. Hence poly-A tails of approximately 160
nucleotides, e.g, about 150-165, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164 or 165 are within the scope of the invention.
Example 63. Method of Screening for Protein Expression
A. Electrospray Ionization
[1004] A biological sample which may contain proteins encoded by
modified RNA administered to the subject is prepared and analyzed
according to the manufacturer protocol for electrospray ionization
(ESI) using 1, 2, 3 or 4 mass analyzers. A biologic sample may also
be analyzed using a tandem ESI mass spectrometry system.
[1005] Patterns of protein fragments, or whole proteins, are
compared to known controls for a given protein and identity is
determined by comparison.
B. Matrix-Assisted Laser Desorption/Ionization
[1006] A biological sample which may contain proteins encoded by
modified RNA administered to the subject is prepared and analyzed
according to the manufacturer protocol for matrix-assisted laser
desorption/ionization (MALDI).
[1007] Patterns of protein fragments, or whole proteins, are
compared to known controls for a given protein and identity is
determined by comparison.
C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry
[1008] A biological sample, which may contain proteins encoded by
modified RNA, may be treated with a trypsin enzyme to digest the
proteins contained within. The resulting peptides are analyzed by
liquid chromatography-mass spectrometry-mass spectrometry
(LC/MS/MS). The peptides are fragmented in the mass spectrometer to
yield diagnostic patterns that can be matched to protein sequence
databases via computer algorithms. The digested sample may be
diluted to achieve 1 ng or less starting material for a given
protein. Biological samples containing a simple buffer background
(e.g. water or volatile salts) are amenable to direct in-solution
digest; more complex backgrounds (e.g. detergent, non-volatile
salts, glycerol) require an additional clean-up step to facilitate
the sample analysis.
[1009] Patterns of protein fragments, or whole proteins, are
compared to known controls for a given protein and identity is
determined by comparison.
Example 64. Cytokine Study: PBMC
A. PBMC Isolation and Culture
[1010] 50 mL of human blood from two donors was received from
Research Blood Components (lots KP30928 and KP30931) in sodium
heparin tubes. For each donor, the blood was pooled and diluted to
70 mL with DPBS (SAFC Bioscience 59331C, lot 071M8408) and split
evenly between two 50 mL conical tubes. 10 mL of Ficoll Paque (GE
Healthcare 17-5442-03, lot 10074400) was gently dispensed below the
blood layer. The tubes were centrifuged at 2000 rpm for 30 minutes
with low acceleration and braking. The tubes were removed and the
buffy coat PBMC layers were gently transferred to a fresh 50 mL
conical and washed with DPBS. The tubes were centrifuged at 1450
rpm for 10 minutes.
[1011] The supernatant was aspirated and the PBMC pellets were
resuspended and washed in 50 mL of DPBS. The tubes were centrifuged
at 1250 rpm for 10 minutes. This wash step was repeated, and the
PBMC pellets were resuspended in 19 mL of Optimem I (Gibco 11058,
lot 1072088) and counted. The cell suspensions were adjusted to a
concentration of 3.0.times.10{circumflex over ( )}6 cells/mL live
cells.
[1012] These cells were then plated on five 96 well tissue culture
treated round bottom plates (Costar 3799) per donor at 50 uL per
well. Within 30 minutes, transfection mixtures were added to each
well at a volume of 50 uL per well. After 4 hours post
transfection, the media was supplemented with 10 uL of Fetal Bovine
Serum (Gibco 10082, lot 1012368)
B. Transfection Preparation
[1013] Modified mRNA encoding human G-CSF (mRNA sequence shown in
SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shown
in sequence; 5'cap, Cap1) (containing either (1) natural NTPs, (2)
100% substitution with 5-methyl cytidine and pseudouridine, or (3)
100% substitution with 5-methyl cytidine and N1-methyl
pseudouridine; mRNA encoding luciferase (IVT cDNA sequence shown in
SEQ ID NO: 2; mRNA sequence shown in SEQ ID NO: 3, polyA tail of
approximately 160 nucleotides not shown in sequence, 5'cap, Cap1,
fully modified with 5-methylcytosine at each cytosine and
pseudouridine replacement at each uridine site) (containing either
(1) natural NTPs or (2) 100% substitution with 5-methyl cytidine
and pseudouridine) and TLR agonist R848 (Invivogen tlrl-r848) were
diluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.
[1014] Separately, 110 uL of Lipofectamine 2000 (Invitrogen
11668-027, lot 1070962) was diluted with 6.76 mL Optimem I. In a 96
well plate nine aliquots of 135 uL of each mRNA, positive control
(R-848) or negative control (Optimem I) was added to 135 uL of the
diluted Lipofectamine 2000. The plate containing the material to be
transfected was incubated for 20 minutes. The transfection mixtures
were then transferred to each of the human PBMC plates at 50 uL per
well. The plates were then incubated at 37.degree. C. At 2, 4, 8,
20, and 44 hours each plate was removed from the incubator, and the
supernatants were frozen.
[1015] After the last plate was removed, the supernatants were
assayed using a human G-CSF ELISA kit (Invitrogen KHC2032) and
human IFN-alpha ELISA kit (Thermo Scientific 41105-2). Each
condition was done in duplicate.
C. Protein and Innate Immune Response Analysis
[1016] The ability of unmodified and modified mRNA to produce the
encoded protein was assessed (G-CSF production) over time as was
the ability of the mRNA to trigger innate immune recognition as
measured by interferon-alpha production. Use of in vitro PBMC
cultures is an accepted way to measure the immunostimulatory
potential of oligonucleotides (Robbins et al., Oligonucleotides
2009 19:89-102).
[1017] Results were interpolated against the standard curve of each
ELISA plate using a four parameter logistic curve fit. Shown in
Tables 4 and 5 are the average from 3 separate PBMC donors of the
G-CSF, interferon-alpha (IFN-alpha) and tumor necrosis factor alpha
(TNF-alpha) production over time as measured by specific ELISA.
[1018] In the G-CSF ELISA, background signal from the Lipofectamine
2000 (LF2000) untreated condition was subtracted at each time
point. The data demonstrated specific production of human G-CSF
protein by human peripheral blood mononuclear is seen with G-CSF
mRNA containing natural NTPs, 100% substitution with 5-methyl
cytidine and pseudouridine, or 100% substitution with 5-methyl
cytidine and N1-methyl pseudouridine. Production of G-CSF was
significantly increased through the use of 5-methyl cytidine and
N1-methyl pseudouridine modified mRNA relative to 5-methyl cytidine
and pseudouridine modified mRNA.
[1019] With regards to innate immune recognition, while both
modified mRNA chemistries largely prevented IFN-alpha and TNF-alpha
production relative to positive controls (R848, p(I)p(C)),
significant differences did exist between the chemistries. 5-methyl
cytidine and pseudouridine modified mRNA resulted in low but
detectable levels of IFN-alpha and TNF-alpha production, while
5-methyl cytidine and N1-methyl pseudouridine modified mRNA
resulted in no detectable IFN-alpha and TNF-alpha production.
[1020] Consequently, it has been determined that, in addition to
the need to review more than one cytokine marker of the activation
of the innate immune response, it has surprisingly been found that
combinations of modifications provide differing levels of cellular
response (protein production and immune activation). The
modification, N1-methyl pseudouridine, in this study has been shown
to convey added protection over the standard combination of
5-methylcytidine/pseudouridine explored by others resulting in
twice as much protein and almost 150 fold reduction in immune
activation (TNF-alpha).
[1021] Given that PBMC contain a large array of innate immune RNA
recognition sensors and are also capable of protein translation, it
offers a useful system to test the interdependency of these two
pathways. It is known that mRNA translation can be negatively
affected by activation of such innate immune pathways (Kariko et
al. Immunity (2005) 23:165-175; Warren et al. Cell Stem Cell (2010)
7:618-630). Using PBMC as an in vitro assay system it is possible
to establish a correlation between translation (in this case G-CSF
protein production) and cytokine production (in this case
exemplified by IFN-alpha and TNF-alpha protein production). Better
protein production is correlated with lower induction of innate
immune activation pathway, and new chemistries can be judged
favorably based on this ratio (Table 6).
[1022] In this study, the PC Ratio for the two chemical
modifications, pseudouridine and N1-methyl pseudouridine, both with
5-methy cytosine was 4742/141=34 as compared to 9944/1=9944 for the
cytokine IFN-alpha. For the cytokine, TNF-alpha, the two
chemistries had PC Ratios of 153 and 1243, respectively suggesting
that for either cytokine, the N1-methylpseudouridine is the
superior modification. In Tables 4 and 5, "NT" means not
tested.
TABLE-US-00005 TABLE 4 G-CSF G-CSF: 3 Donor Average (pg/ml) G-CSF
4742 5-methyl cytosine/ pseudouridine G-CSF 9944 5-methylcytosine/
N1-methylpseudouridine Luciferase 18 LF2000 16
TABLE-US-00006 TABLE 5 IFN-alpha and TNF-alpha IFN-alpha: 3 Donor
TNF-alpha: 3 Donor Average (pg/ml) Average (pg/ml) G-CSF 141 31
5-methyl cytosine/ pseudouridine G-CSF 1 8 5-methylcytosine/
N1-methylpseudouridine P(I)P(C) 1104 NT R-848 NT 1477 LF2000 17
25
TABLE-US-00007 TABLE 6 G-CSF to Cytokine Ratios G-CSF/IFN-alpha
(ratio) G-CSF/TNF-alpha (ratio) 5-methyl- 5-methyl- 5-methyl
cytosine/ 5-methyl cytosine/ cytosine/ N1-methyl- cytosine/
N1-methyl- pseudouridine pseudouridine pseudouridine pseudouridine
PC 34 9944 153 1243 Ratio
Example 65. Chemical Modification Ranges of Modified mRNA
[1023] Modified nucleosides such as, but not limited to, the
chemical modifications 5-methylcytosine and pseudouridine have been
shown to lower the innate immune response and increase expression
of RNA in mammalian cells. Surprisingly and not previously known,
the effects manifested by these chemical modifications can be
titrated when the amount of chemical modification of a particular
nucleotide is less than 100%. Previously, it was believed that the
benefit of chemical modification could be derived using less than
complete replacement of a modified nucleoside and published reports
suggest no loss of benefit until the level of substitution with a
modified nucleoside is less than 50% (Kariko et al., Immunity
(2005) 23:165-175).
[1024] However, it has now been shown that the benefits of chemical
modification are directly correlated with the degree of chemical
modification and must be considered in view of more than a single
measure of immune response. Such benefits include enhanced protein
production or mRNA translation and reduced or avoidance of
stimulating the innate immune response as measured by cytokine
profiles and metrics of immune response triggers.
[1025] Enhanced mRNA translation and reduced or lack of innate
immune stimulation are seen with 100% substitution with a modified
nucleoside. Lesser percentages of substitution result in less mRNA
translation and more innate immune stimulation, with unmodified
mRNA showing the lowest translation and the highest innate immune
stimulation.
In Vitro PBMC Studies: Percent Modification
[1026] 480 ng of G-CSF mRNA modified with 5-methylcytosine (5mC)
and pseudouridine (pseudoU) or unmodified G-CSF mRNA was
transfected with 0.4 uL of Lipofectamine 2000 into peripheral blood
mononuclear cells (PBMC) from three normal blood donors (D1, D2,
and D3). The G-CSF mRNA (SEQ ID NO: 1; polyA tail of approximately
160 nucleotides not shown in sequence; 5'cap, Cap1) was completely
modified with 5mC and pseudo (100% modification), not modified with
5mC and pseudo (0% modification) or was partially modified with 5mC
and pseudoU so the mRNA would contain 75% modification, 50%
modification or 25% modification. A control sample of Luciferase
(mRNA sequence shown in SEQ ID NO: 3; polyA tail of approximately
160 nucleotides not shown in sequence; 5'cap, Cap1; fully modified
5meC and pseudoU) was also analyzed for G-CSF expression. For
TNF-alpha and IFN-alpha control samples of Lipofectamine2000, LPS,
R-848, Luciferase (mRNA sequence shown in SEQ ID NO: 3; polyA tail
of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1; fully modified 5mC and pseudo), and P(I)P(C) were also
analyzed. The supernatant was harvested and run by ELISA 22 hours
after transfection to determine the protein expression. The
expression of G-CSF is shown in Table 7 and the expression of
IFN-alpha and TNF-alpha is shown in Table 8. The expression of
IFN-alpha and TNF-alpha may be a secondary effect from the
transfection of the G-CSF mRNA. Tables 7, 8 and FIG. 10 show that
the amount of chemical modification of G-CSF, interferon alpha
(IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) is
titratable when the mRNA is not fully modified and the titratable
trend is not the same for each target.
[1027] As mentioned above, using PBMC as an in vitro assay system
it is possible to establish a correlation between translation (in
this case G-CSF protein production) and cytokine production (in
this case exemplified by IFN-alpha protein production). Better
protein production is correlated with lower induction of innate
immune activation pathway, and the percentage modification of a
chemistry can be judged favorably based on this ratio (Table 9). As
calculated from Tables 7 and 8 and shown in Table 9, full
modification with 5-methylcytidine and pseudouridine shows a much
better ratio of protein/cytokine production than without any
modification (natural G-CSF mRNA) (100-fold for IFN-alpha and
27-fold for TNF-alpha). Partial modification shows a linear
relationship with increasingly less modification resulting in a
lower protein/cytokine ratio.
TABLE-US-00008 TABLE 7 G-CSF Expression G-CSF Expression (pg/ml) D1
D2 D3 100% modification 1968.9 2595.6 2835.7 75% modification 566.7
631.4 659.5 50% modification 188.9 187.2 191.9 25% modification
139.3 126.9 102.0 0% modification 194.8 182.0 183.3 Luciferase 90.2
0.0 22.1
TABLE-US-00009 TABLE 8 IFN-alpha and TNF-alpha Expression IFN-alpha
Expression (pg/ml) TNF-alpha Expression (pg/ml) D1 D2 D3 D1 D2 D3
100% modification 336.5 78.0 46.4 115.0 15.0 11.1 75% modification
339.6 107.6 160.9 107.4 21.7 11.8 50% modification 478.9 261.1
389.7 49.6 24.1 10.4 25% modification 564.3 400.4 670.7 85.6 26.6
19.8 0% modification 1421.6 810.5 1260.5 154.6 96.8 45.9 LPS 0.0
0.6 0.0 0.0 12.6 4.3 R-848 0.5 3.0 14.1 655.2 989.9 420.4 P(I)P(C)
130.8 297.1 585.2 765.8 2362.7 1874.4 Lipid only 1952.2 866.6 855.8
248.5 82.0 60.7
TABLE-US-00010 TABLE 9 PC Ratio and Effect of Percentage of
Modification Average Average Average G-CSF/ G-CSF/ % G-CSF IFN-a
TNF-a IFN-alpha TNF-alpha Modification (pg/ml) (pg/ml) (pg/ml) (PC
ratio) (PC ratio) 100 2466 153 47 16 52 75 619 202 47 3.1 13 50 189
376 28 0.5 6.8 25 122 545 44 0.2 2.8 0 186 1164 99 0.16 1.9
Example 66. Modified RNA Transfected in PBMC
[1028] 500 ng of G-CSF mRNA modified with 5-methylcytosine (5mC)
and pseudouridine (pseudoU) or unmodified G-CSF mRNA was
transfected with 0.4 uL of Lipofectamine 2000 into peripheral blood
mononuclear cells (PBMC) from three normal blood donors (D1, D2,
and D3). The G-CSF mRNA (SEQ ID NO: 1; polyA tail of approximately
160 nucleotides not shown in sequence; 5'cap, Cap1) was completely
modified with 5mC and pseudo (100% modification), not modified with
5mC and pseudo (0% modification) or was partially modified with 5mC
and pseudoU so the mRNA would contain 50% modification, 25%
modification, 10% modification, %5 modification, 1% modification or
0.1% modification. A control sample of mCherry (mRNA sequence shown
in SEQ ID NO: 6; polyA tail of approximately 160 nucleotides not
shown in sequence; 5'cap, Cap1; fully modified 5meC and
pseudouridine) and G-CSF fully modified with 5-methylcytosine and
pseudouridine (Control G-CSF) was also analyzed for G-CSF
expression. For tumor necrosis factor-alpha (TNF-alpha) and
interferon-alpha (IFN-alpha) control samples of Lipofectamine2000,
LPS, R-848, Luciferase (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1; fully modified 5mC and pseudo), and P(I)P(C) were also
analyzed. The supernatant was harvested 6 hours and 18 hours after
transfection and run by ELISA to determine the protein expression.
The expression of G-CSF, IFN-alpha, and TNF-alpha for Donor 1 is
shown in Table 10, Donor 2 is shown in Table 11 and Donor 3 is
shown in Table 12.
[1029] Full 100% modification with 5-methylcytidine and
pseudouridine resulted in the most protein translation (G-CSF) and
the least amount of cytokine produced across all three human PBMC
donors. Decreasing amounts of modification results in more cytokine
production (IFN-alpha and TNF-alpha), thus further highlighting the
importance of fully modification to reduce cytokines and to improve
protein translation (as evidenced here by G-CSF production).
TABLE-US-00011 TABLE 10 Donor 1 G-CSF (pg/mL) IFN-alpha (pg/mL)
TNF-alpha (pg/mL) 6 hours 18 hours 6 hours 18 hours 6 hours 18
hours 100% Mod 1815 2224 1 13 0 0 75% Mod 591 614 0 89 0 0 50% Mod
172 147 0 193 0 0 25% Mod 111 92 2 219 0 0 10% Mod 138 138 7 536 18
0 1% Mod 199 214 9 660 18 3 0.1% Mod 222 208 10 597 0 6 0% Mod 273
299 10 501 10 0 Control G-CSF 957 1274 3 123 18633 1620 mCherry 0 0
0 10 0 0 Untreated N/A N/A 0 0 1 1
TABLE-US-00012 TABLE 11 Donor 2 G-CSF (pg/mL) IFN-alpha (pg/mL)
TNF-alpha (pg/mL) 6 hours 18 hours 6 hours 18 hours 6 hours 18
hours 100% Mod 2184 2432 0 7 0 11 75% Mod 935 958 3 130 0 0 50% Mod
192 253 2 625 7 23 25% Mod 153 158 7 464 6 6 10% Mod 203 223 25 700
22 39 1% Mod 288 275 27 962 51 66 0.1% Mod 318 288 33 635 28 5 0%
Mod 389 413 26 748 1 253 Control G-CSF 1461 1634 1 59 481 814
mCherry 0 7 0 1 0 0 Untreated N/A N/A 1 0 0 0
TABLE-US-00013 TABLE 12 Donor 3 G-CSF (pg/mL) IFN-alpha (pg/mL)
TNF-alpha (pg/mL) 6 hours 18 hours 6 hours 18 hours 6 hours 18
hours 100% Mod 6086 7549 7 658 11 11 75% Mod 2479 2378 23 752 4 35
50% Mod 667 774 24 896 22 18 25% Mod 480 541 57 1557 43 115 10% Mod
838 956 159 2755 144 123 1% Mod 1108 1197 235 3415 88 270 0.1% Mod
1338 1177 191 2873 37 363 0% Mod 1463 1666 215 3793 74 429 Control
G-CSF 3272 3603 16 1557 731 9066 mCherry 0 0 2 645 0 0 Untreated
N/A N/A 1 1 0 8
Example 67. Microames Reverse Mutation Screen of Modifications
Background and Methods
[1030] The microames screen is a version of the full Ames
preincubation assay. It detects both frameshift and base-pair
substitution mutations using four Salmonella tester strains (TA97a,
TA98, TA100 and TA1535) and one Escherichia coli strain (WP2 uvrA
pKM101). Strains TA97a and TA98 detect frameshift mutations, and
TA100, TA1535 and WP2 uvrA pKM101 detect base-pair substitution
mutations. This scaled-down Ames test uses minimal compound, is
conducted with and without metabolic activation (S9 fraction), and
uses multiwell plates. This teste is a microbial assay to detect
the mutagenic potential of test compounds.
[1031] The microAmes screen for 5-Methylcytidine, Pseudouridine or
N'-methylpseudouridine test article was tested in duplicate with
strains TA97a, TA98, TA100, TA1535 and WP2 uvrA pKM101 in the
presence and absence of a metabolic activation system (AROCLOR.TM.
1254 induced rat liver S9 microsomal fraction) at 0.25, 2.5, 12.5,
25, 75, and 250 ug/well. Positive control compounds were used at 4
different concentrations to ensure the assay system was sensitive
to known mutagenic compounds. DMSO was used as the vehicle control.
Positive and vehicle controls yielded the expected results,
demonstrating that the microAmes screen is sufficiently sensitive
to detect mutagens.
Results
[1032] For 5-methylcytosine, precipitates were not observed with
any tester strain either with or without metabolic activation.
Cytotoxicity (reduction in the background lawn and/or number of
revertants) was not observed in any strain either with or without
metabolic activation. There was no increase in the number of
revertant colonies as compared with the vehicle control in any
strain with or without metabolic activation. Therefore,
5-Methylcytidine was not mutagenic up to 250 ug/well in strains
TA97a, TA98, TA100, TA1535 and WP2 uvrA pKM101 with or without
metabolic activation under the conditions of the microAmes
screen.
[1033] Precipitates were not observed with any tester strain either
with or without metabolic activation for pseudouridine.
Cytotoxicity (reduction in the number of revertants) was observed
with strain TA100 without metabolic activation. Cytotoxicity
(reduction in the background lawn and/or number of revertants) was
not observed in any other strain either with or without metabolic
activation. There was no increase in the number of revertant
colonies as compared with the vehicle control in any strain with or
without metabolic activation. Therefore, pseudouridine was not
mutagenic up to 75 ug/well in strain TA100 without metabolic
activation and up to 250 .mu.g/well in strains TA97a, TA98, TA1535
and WP2 uvrA pKM101 with or without metabolic activation and strain
TA100 without metabolic activation under the conditions of this
microAmes screen.
[1034] For the modification, N1-methylpseudouridine precipitates
were not observed with any tester strain either with or without
metabolic activation. Cytotoxicity (reduction in the background
lawn and/or number of revertants) was not observed in any strain
either with or without metabolic activation. There was no increase
in the number of revertant colonies as compared with the vehicle
control in any strain with or without metabolic activation.
N1-methylpseudouridine was not mutagenic up to 250 .mu.g/well in
strains TA97a, TA98, TA100, TA1535 and WP2 uvrA pKM101 with or
without metabolic activation under the conditions of this microAmes
screen. N1-methylpseudouridine was found less mutagenic than
pseudouridine.
[1035] The comparison in this microAMES test of 5 methyl cytidine,
pseudouridine, and N1-methylpseudouridine reveal them to be
generally non-mutagenic. Of particular note, however, was the
difference between pseudouridine and N1-methylpseudouridine, where
pseudouridine did show a cytotoxic response in one bacterial strain
where N1-methylpseudouridine did not. These microAMES tests are
routinely used as part of the pre-clinical assessment of compound
safety and highlight an important difference between
N1-methylpseudouridine and pseudouridine.
Example 68. Toxicity of Nucleoside Triphosphates (NTPs)
[1036] The cytotoxicity of natural and modified nucleoside
triphosphates (NTPs) alone or in combination with other bases, was
analyzed in human embryonic kidney 293 (HEK293) cells in the
absence of transfection reagent. HEK293 cells were seeded on
96-well plates at a density of 30,000 cells per well having 0.75 ul
of RNAiMAX.TM. (Invitrogen, Carlsbad, Calif.) per well at a total
well volume of 100 ul. 10 ul of the NTPs outlined in Table 12 were
combined with 10 ul of lipid dilution and incubated for 30 minutes
to form a complex before 80 ul of the HEK293 cell suspension was
added to the NTP complex.
[1037] Natural and modified NTPs were transfected at a
concentration of 2.1 nM, 21 nM, 210 nM, 2.1 um, 21 uM, 210 um or
2.1 mM. NTPs in combination were transfected at a total
concentration of NTPs of 8.4 nM, 84 nM, 840 nM, 8.4 uM, 84 uM, 840
uM and 8.4 mM. As a control modified G-CSF mRNA (SEQ ID NO: 1;
polyA tail of approximately 160 nucleotides not shown in sequence;
5'cap, Cap1; fully modified 5-methylcytosine and pseudouridine) was
transfected in HEK293 cells at a concentration of 8.4 nM. The
cytotoxicity of the NTPs and the modified G-CSF mRNA was assayed at
4, 24, 48 and 72 hours post addition to the HEK293 cells using a
CYTO TOX-GLO.TM. assay from Promega (Madison, Wis.) following the
manufacturer protocol except pippeting was used for lysing the
cells instead of shaking the plates.
[1038] Table 13 and 14 show the percent of viable cells for each of
the NTPs, NTP combinations and controls tested. There was no
toxicity seen with the individual NTPs as compared to the untreated
cells. These data demonstrate that introduction of individual NTPs,
including 5-methylcytidine, pseudouridine, and
N1-methylpseudouridine, into mammalian cells is not toxic at doses
1,000,000 times an effective dose when introduced as a modified
mRNA.
TABLE-US-00014 TABLE 13 Cytotoxicity of Individual NTPs Individual
NTP Cytotoxicity Dose Time 2.1 mM 210 uM 21 uM 2.1 uM 210 nM 21 nM
2.1 nM Adenine 4 hr 90.03 85.97 91.20 90.23 90.36 93.21 93.48 24 hr
88.42 87.31 86.86 86.81 86.94 87.19 86.44 48 hr 93.71 90.55 89.94
89.80 89.17 91.13 92.12 72 hr 97.49 94.81 93.83 94.58 92.22 93.88
95.74 Cytosine 4 hr 90.51 89.88 91.41 90.49 88.95 93.11 93.34 24 hr
86.92 86.33 85.72 86.70 86.12 86.16 85.78 48 hr 94.23 87.81 87.28
87.73 85.36 88.95 88.99 72 hr 97.15 92.34 92.22 88.93 88.22 91.80
94.22 Guanine 4 hr 90.96 90.14 91.36 90.60 90.00 92.84 93.33 24 hr
86.37 85.86 85.93 86.13 86.35 85.50 85.41 48 hr 93.83 87.05 88.18
87.89 85.31 87.92 89.57 72 hr 97.04 91.41 92.39 92.30 92.19 92.55
93.72 Uracil 4 hr 90.97 89.60 91.95 90.90 91.05 92.90 93.15 24 hr
87.68 86.48 85.89 86.75 86.52 87.23 87.63 48 hr 94.39 88.98 89.11
89.44 88.33 88.89 91.28 72 hr 96.82 93.45 93.63 94.60 94.50 94.53
95.51 Pseudouridine 4 hr 92.09 92.37 91.35 92.02 92.84 91.96 92.26
24 hr 88.38 86.68 86.05 86.75 85.91 87.59 87.31 48 hr 88.62 87.79
87.73 87.66 87.82 89.03 91.99 72 hr 96.87 89.82 94.23 93.54 92.37
94.26 94.25 5-methyl 4 hr 92.01 91.54 91.16 91.31 92.31 91.40 92.23
cytosine 24 hr 87.97 85.76 84.72 85.14 84.71 86.37 86.35 48 hr
87.29 85.94 85.74 86.18 86.44 87.10 88.18 72 hr 96.08 88.10 92.26
90.92 89.97 92.10 91.93 N1-methyl 4 hr 92.45 91.43 91.48 90.41
92.15 91.44 91.89 pseudouridine 24 hr 88.92 86.48 85.17 85.72 85.89
86.85 87.79 48 hr 89.84 86.02 87.52 85.85 87.38 86.72 87.81 72 hr
96.80 93.03 93.83 92.25 92.40 92.84 92.98 Untreated 4 hr 92.77 --
-- -- -- -- -- 24 hr 87.52 -- -- -- -- -- -- 48 hr 92.95 -- -- --
-- -- -- 72 hr 96.97 -- -- -- -- -- --
TABLE-US-00015 TABLE 14 Cytotoxicity of NTPs in Combination NTP
Combination Cytotoxicity Dose Time 8.4 mM 840 uM 84 uM 8.4 uM 840
nM 84 nM 8.4 nM Pseudouridine/ 4 hr 92.27 92.04 91.47 90.86 90.87
91.10 91.50 5-methylcytosine/ 24 hr 88.51 86.90 86.43 88.15 88.46
86.28 87.51 Adenine/Guanine 48 hr 88.30 87.36 88.58 88.13 87.39
88.72 90.55 72 hr 96.53 94.42 94.31 94.53 94.38 94.36 93.65
N1-methyl 4 hr 92.31 91.71 91.36 91.15 91.30 90.86 91.38
pseudouridine/ 24 hr 88.19 87.07 86.46 87.70 88.13 85.30 87.21
5-methylcytosine/ 48 hr 87.17 86.53 87.51 85.85 84.69 87.73 86.79
Adenine/Guanine 72 hr 96.40 94.88 94.40 93.65 94.82 92.72 93.10
G-CSF 4 hr na na na na na na 92.63 modified 24 hr na na na na na na
87.53 mRNA 48 hr na na na na na na 91.70 72 hr na na na na na na
96.36
Example 69. Innate Immune Response Study in BJ Fibroblasts
[1039] Human primary foreskin fibroblasts (BJ fibroblasts) were
obtained from American Type Culture Collection (ATCC) (catalog
#CRL-2522) and grown in Eagle's Minimum Essential Medium (ATCC,
catalog #30-2003) supplemented with 10% fetal bovine serum at
37.degree. C., under 5% CO.sub.2. BJ fibroblasts were seeded on a
24-well plate at a density of 300,000 cells per well in 0.5 ml of
culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shown
in SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not
shown in sequence; 5'cap, Cap1) fully modified with
5-methylcytosine and pseudouridine (Gen1) or fully modified with
5-methylcytosine and N1-methylpseudouridine (Gen2) having Cap0,
Cap1 or no cap was transfected using Lipofectamine 2000
(Invitrogen, catalog #11668-019), following manufacturer's
protocol. Control samples of poly I:C (PIC), Lipofectamine 2000
(Lipo), natural luciferase mRNA (mRNA sequence shown in SEQ ID NO:
3; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1) and natural G-CSF mRNA were also
transfected. The cells were harvested after 18 hours, the total RNA
was isolated and DNASE.RTM. treated using the RNeasy micro kit
(catalog #74004) following the manufacturer's protocol. 100 ng of
total RNA was used for cDNA synthesis using High Capacity cDNA
Reverse Transcription kit (catalog #4368814) following the
manufacturer's protocol. The cDNA was then analyzed for the
expression of innate immune response genes by quantitative real
time PCR using SybrGreen in a Biorad CFX 384 instrument following
manufacturer's protocol. Table 15 shows the expression level of
innate immune response transcripts relative to house-keeping gene
HPRT (hypoxanthine phosphoribosytransferase) and is expressed as
fold-induction relative to HPRT. In the table, the panel of
standard metrics includes: RIG-I is retinoic acid inducible gene 1,
IL6 is interleukin-6, OAS-1 is oligoadenylate synthetase 1, IFNb is
interferon-beta, AIM2 is absent in melanoma-2, IFIT-1 is
interferon-induced protein with tetratricopeptide repeats 1, PKR is
protein kinase R, TNFa is tumor necrosis factor alpha and IFNa is
interferon alpha.
TABLE-US-00016 TABLE 15 Innate Immune Response Transcript Levels
Formulation RIG-I IL6 OAS-1 IFNb AIM2 IFIT-1 PKR TNFa IFNa Natural
71.5 20.6 20.778 11.404 0.251 151.218 16.001 0.526 0.067 Luciferase
Natural G-CSF 73.3 47.1 19.359 13.615 0.264 142.011 11.667 1.185
0.153 G-CSF Gen1-UC 0.81 0.22 0.080 0.009 0.008 2.220 1.592 0.090
0.027 G-CSF Gen1-Cap0 0.54 0.26 0.042 0.005 0.008 1.314 1.568 0.088
0.038 G-CSF Gen1-Cap1 0.58 0.30 0.035 0.007 0.006 1.510 1.371 0.090
0.040 G-CSF Gen2-UC 0.21 0.20 0.002 0.007 0.007 0.603 0.969 0.129
0.005 G-CSF Gen2-Cap0 0.23 0.21 0.002 0.0014 0.007 0.648 1.547
0.121 0.035 G-CSF Gen2-Cap1 0.27 0.26 0.011 0.004 0.005 0.678 1.557
0.099 0.037 Lipo 0.27 0.53 0.001 0 0.007 0.954 1.536 0.158
0.064
Example 70. In Vivo Detection of Innate Immune Response
[1040] In an effort to distinguish the importance of different
chemical modification of mRNA on in vivo protein production and
cytokine response in vivo, female BALB/C mice (n=5) are injected
intramuscularly with G-CSF mRNA (GCSF mRNA unmod) (mRNA sequence
shown in SEQ ID NO: 1; polyA tail of approximately 160 nucleotides
not shown in sequence;) with a 5'cap of Cap1, G-CSF mRNA fully
modified with 5-methylcytosine and pseudouridine (GCSF mRNA
5mc/pU), G-CSF mRNA fully modified with 5-methylcytosine and
N1-methylpseudouridine with (GCSF mRNA 5mc/N1pU) or without a 5'
cap (GCSF mRNA 5mc/N1 pU no cap) or a control of either R848 or 5%
sucrose as described in Table 16.
TABLE-US-00017 TABLE 16 Dosing Chart Dose Dose Formulation Route
(ug/mouse) (ul) G-CSF mRNA I.M. 200 50 unmod G-CSF mRNA I.M. 200 50
5mc/pU G-CSF mRNA I.M. 200 50 5mc/N1pU G-CSF mRNA I.M. 200 50
5mc/N1pU no cap R848 I.M. 75 50 5% sucrose I.M. -- 50 Untreated
I.M. -- --
[1041] Blood is collected at 8 hours after dosing. Using ELISA the
protein levels of G-CSF, TNF-alpha and IFN-alpha is determined by
ELISA. 8 hours after dosing, muscle is collected from the injection
site and quantitative real time polymerase chain reaction (QPCR) is
used to determine the mRNA levels of RIG-I, PKR, AIM-2, IFIT-1,
OAS-2, MDA-5, IFN-beta, TNF-alpha, IL-6, G-CSF, CD45 in the
muscle.
Example 71. In Vivo Detection of Innate Immune Response Study
[1042] Female BALB/C mice (n=5) were injected intramuscularly with
G-CSF mRNA (GCSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 1;
polyA tail of approximately 160 nucleotides not shown in sequence;)
with a 5'cap of Cap1, G-CSF mRNA fully modified with
5-methylcytosine and pseudouridine (GCSF mRNA 5mc/pU), G-CSF mRNA
fully modified with 5-methylcytosine and N1-methylpseudouridine
with (GCSF mRNA 5mc/N1pU) or without a 5' cap (GCSF mRNA 5mc/N1 pU
no cap) or a control of either R848 or 5% sucrose as described in
Table 17. Blood is collected at 8 hours after dosing and using
ELISA the protein levels of G-CSF and interferon-alpha (IFN-alpha)
is determined by ELISA and are shown in Table 17.
[1043] As shown in Table 17, unmodified, 5mc/pU, and 5mc/N1pU
modified G-CSF mRNA resulted in human G-CSF expression in mouse
serum. The uncapped 5mC/N1pU modified G-CSF mRNA showed no human
G-CSF expression in serum, highlighting the importance of having a
5' cap structure for protein translation.
[1044] As expected, no human G-CSF protein was expressed in the
R848, 5% sucrose only, and untreated groups. Importantly,
significant differences were seen in cytokine production as
measured by mouse IFN-alpha in the serum. As expected, unmodified
G-CSF mRNA demonstrated a robust cytokine response in vivo (greater
than the R848 positive control). The 5mc/pU modified G-CSF mRNA did
show a low but detectable cytokine response in vivo, while the
5mc/N1pU modified mRNA showed no detectable IFN-alpha in the serum
(and same as vehicle or untreated animals).
[1045] Also, the response of 5mc/N1pU modified mRNA was the same
regardless of whether it was capped or not. These in vivo results
reinforce the conclusion that 1) that unmodified mRNA produce a
robust innate immune response, 2) that this is reduced, but not
abolished, through 100% incorporation of 5mc/pU modification, and
3) that incorporation of 5mc/N1pU modifications results in no
detectable cytokine response.
[1046] Lastly, given that these injections are in 5% sucrose (which
has no effect by itself), these result should accurately reflect
the immunostimulatory potential of these modifications.
[1047] From the data it is evident that N1pU modified molecules
produce more protein while concomitantly having little or no effect
on IFN-alpha expression. It is also evident that capping is
required for protein production for this chemical modification. The
Protein: Cytokine Ratio of 748 as compared to the PC Ratio for the
unmodified mRNA (PC=9) means that this chemical modification is far
superior as related to the effects or biological implications
associated with IFN-alpha.
TABLE-US-00018 TABLE 17 Human G-CSF and Mouse IFN-alpha in serum
G-CSF IFN-alpha Dose Dose protein expression PC Formulation Route
(ug/mouse) (ul) (pg/ml) (pg/ml) Ratio GCSF mRNA unmod I.M. 200 50
605.6 67.01 9 GCSF mRNA 5mc/pU I.M. 200 50 356.5 8.87 40 GCSF
mRNA5mc/N1pU I.M. 200 50 748.1 0 748 GCSF mRNA5mc/N1pU no cap I.M.
200 50 6.5 0 6.5 R848 I.M. 75 50 3.4 40.97 .08 5% sucrose I.M. --
50 0 1.49 0 Untreated I.M. -- -- 0 0 0
Example 72: In Vivo Delivery Using Lipoplexes
A. Human G-CSF Modified RNA
[1048] A formulation containing 100 .mu.g of one of two versions of
modified human G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1;
polyA tail of approximately 160 nucleotides not shown in sequence;
5' cap, Cap1) (G-CSF fully modified with 5-methylcytosine and
pseudouridine (G-CSF) or G-CSF fully modified with 5-methylcytosine
and N1-methyl-pseudouridine (G-CSF-N1) lipoplexed with 30% by
volume of RNAIMAX.TM. and delivered in 150 uL intramuscularly
(I.M.) and in 225 uL intravenously (I.V.) to C57/BL6 mice.
[1049] Three control groups were administered either 100 .mu.g of
modified luciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 2;
mRNA sequence shown in SEQ ID NO: 3, polyA tail of approximately
160 nucleotides not shown in sequence, 5'cap, Cap1, fully modified
with 5-methylcytosine at each cytosine and pseudouridine
replacement at each uridine site) intramuscularly (Luc-unsp I.M.)
or 150 .mu.g of modified luciferase mRNA intravenously (Luc-unsp
I.V.) or 150 uL of the formulation buffer intramuscularly (Buffer
I.M.). 6 hours after administration of a formulation, serum was
collected to measure the amount of human G-CSF protein in the mouse
serum by human G-CSF ELISA and the results are shown in Table
18.
[1050] These results demonstrate that both
5-methylcytosine/pseudouridine and
5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA
can result in specific human G-CSF protein expression in serum when
delivered via I.V. or I.M. route of administration in a lipoplex
formulation.
TABLE-US-00019 TABLE 18 Human G-CSF in Serum (I.M. and I.V.
Injection Route) Formulation Route G-CSF (pg/ml) G-CSF I.M. 85.6
G-CSF-N1 I.M. 40.1 G-CSF I.V. 31.0 G-CSF-N1 I.V. 6.1 Luc-unsp I.M.
0.0 Luc-unsp I.V. 0.0 Buffer I.M. 0.0
B. Human G-CSF Modified RNA Comparison
[1051] A formulation containing 100 .mu.g of either modified human
G-CSF mRNA lipoplexed with 30% by volume of RNAIMAX.TM. with a
5-methylcytosine (5mc) and a pseudouridine (.psi.) modification
(G-CSF-Gen1-Lipoplex), modified human G-CSF mRNA with a 5mc and
.psi. modification in saline (G-CSF-Gen1-Saline), modified human
G-CSF mRNA with a N1-5-methylcytosine (N1-5mc) and a .psi.
modification lipoplexed with 30% by volume of RNAIMAX.TM.
(G-CSF-Gen2-Lipoplex), modified human G-CSF mRNA with a N1-5mc and
.psi. modification in saline (G-CSF-Gen2-Saline), modified
luciferase with a 5mc and .psi. modification lipoplexed with 30% by
volume of RNAIMAX.TM. (Luc-Lipoplex), or luciferase mRNA fully
modified with 5mc and .psi. modifications in saline (Luc-Saline)
was delivered intramuscularly (I.M.) or subcutaneously (S.C.) and a
control group for each method of administration was giving a dose
of 80 uL of the formulation buffer (F. Buffer) to C57/BL6 mice. 13
hours post injection serum and tissue from the site of injection
were collected from each mouse and analyzed by G-CSF ELISA to
compare human G-CSF protein levels. The results of the human G-CSF
protein in mouse serum from the intramuscular administration and
the subcutaneous administration results are shown in Table 19.
[1052] These results demonstrate that
5-methylcytosine/pseudouridine and
5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA
can result in specific human G-CSF protein expression in serum when
delivered via I.M. or S.C. route of administration whether in a
saline formulation or in a lipoplex formulation. As shown in Table
19, 5-methylcytosine/N1-methylpseudouridine modified human G-CSF
mRNA generally demonstrates increased human G-CSF protein
production relative to 5-methylcytosine/pseudouridine modified
human G-CSF mRNA.
TABLE-US-00020 TABLE 19 Human G-CSF Protein in Mouse Serum G-CSF
(pg/ml) Formulation I.M. Injection Route S.C. Injenction Route
G-CSF-Gen1-Lipoplex 13.988 42.855 GCSF-Gen1-saline 9.375 4.614
GCSF-Gen2-lipoplex 75.572 32.107 GCSF-Gen2-saline 20.190 45.024 Luc
lipoplex 0 3.754 Luc saline 0.0748 0 F. Buffer 4.977 2.156
Example 73. Multi-Site Administration: Intramuscular and
Subcutaneous
[1053] Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO:
1; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1) modified as either Gen1 or Gen2
(5-methylcytosine (5mc) and a pseudouridine (.psi.) modification,
G-CSF-Gen1; or N1-5-methylcytosine (N1-5mc) and a .psi.
modification, G-CSF-Gen2) and formulated in saline were delivered
to mice via intramuscular (IM) or subcutaneous (SC) injection.
Injection of four doses or 2.times.50 ug (two sites) daily for
three days (24 hrs interval) was performed. The fourth dose was
administered 6 hrs before blood collection and CBC analysis.
Controls included Luciferase (cDNA sequence for IVT shown in SEQ ID
NO: 2; mRNA sequence shown in SEQ ID NO: 3, polyA tail of
approximately 160 nucleotides not shown in sequence, 5'cap, Cap1,
fully modified with 5-methylcytosine at each cytosine and
pseudouridine replacement at each uridine site) or the formulation
buffer (F.Buffer). The mice were bled at 72 hours after the first
mRNA injection (6 hours after the last mRNA dose) to determine the
effect of mRNA-encoded human G-CSF on the neutrophil count. The
dosing regimen is shown in Table 20 as are the resulting neutrophil
counts (thousands/uL). In Table 20, an asterisks (*) indicate
statistical significance at p<0.05.
[1054] For intramuscular administration, the data reveal a four
fold increase in neutrophil count above control at day 3 for the
Gen1 G-CSF mRNA and a two fold increase for the Gen2 G-CSF mRNA.
For subcutaneous administration, the data reveal a two fold
increase in neutrophil count above control at day 3 for the Gen2
G-CSF mRNA.
[1055] These data demonstrate that both
5-methylcytidine/pseudouridine and
5-methylcytidine/N1-methylpseudouridine-modified mRNA can be
biologically active, as evidenced by specific increases in blood
neutrophil counts.
TABLE-US-00021 TABLE 20 Dosing Regimen Dose Dose Vol. Dosing
Neutrophil Gr. Treatment Route N= (.mu.g/mouse) (.mu.l/mouse)
Vehicle Thous/uL 1 G-CSF (Gen1) I.M 5 2 .times. 50 ug (four doses)
50 F. buffer 840* 2 G-CSF (Gen1) S.C 5 2 .times. 50 ug (four doses)
50 F. buffer 430 3 G-CSF (Gen2) I.M 5 2 .times. 50 ug (four doses)
50 F. buffer 746* 4 G-CSF (Gen2) S.C 5 2 .times. 50 ug (four doses)
50 F. buffer 683 5 Luc (Gen1) I.M. 5 2 .times. 50 ug (four doses)
50 F. buffer 201 6 Luc (Gen1) S.C. 5 2 .times. 50 ug (four doses)
50 F. buffer 307 7 Luc (Gen2) I.M 5 2 .times. 50 ug (four doses) 50
F. buffer 336 8 Luc (Gen2) S.C 5 2 .times. 50 ug (four doses) 50 F.
buffer 357 9 F. Buffer I.M 4 0 (four doses) 50 F. buffer 245 10 F.
Buffer S.C. 4 0 (four doses) 50 F. buffer 509 11 Untreated -- 4 --
312
Example 74. Intravenous Administration
[1056] Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO:
1; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1) modified with 5-methylcytosine (5mc) and a
pseudouridine (.psi.) modification (Gen1); or having no
modifications and formulated in 10% lipoplex (RNAIMAX.TM.) were
delivered to mice at a dose of 50 ug RNA and in a volume of 100 ul
via intravenous (IV) injection at days 0, 2 and 4. Neutrophils were
measured at days 1, 5 and 8. Controls included non-specific
mammalian RNA or the formulation buffer alone (F.Buffer). The mice
were bled at days 1, 5 and 8 to determine the effect of
mRNA-encoded human G-CSF to increase neutrophil count. The dosing
regimen is shown in Table 21 as are the resulting neutrophil counts
(thousands/uL; K/uL).
[1057] For intravenous administration, the data reveal a four to
five fold increase in neutrophil count above control at day 5 with
G-CSF modified mRNA but not with unmodified G-CSF mRNA or
non-specific controls. Blood count returned to baseline four days
after the final injection. No other changes in leukocyte
populations were observed.
[1058] In Table 21, an asterisk (*) indicates statistical
significance at p<0.001 compared to buffer.
[1059] These data demonstrate that lipoplex-formulated
5-methylcytidine/pseudouridine-modified mRNA can be biologically
active, when delivered through an I.V. route of administration as
evidenced by specific increases in blood neutrophil counts. No
other cell subsets were significantly altered. Unmodified G-CSF
mRNA similarly administered showed no pharmacologic effect on
neutrophil counts.
TABLE-US-00022 TABLE 21 Dosing Regimen Dose Vol. Dosing Neutrophil
Gr. Treatment N (.mu.l/mouse) Vehicle K/uL 1 G-CSF (Gen1) Day 1 5
100 10% lipoplex 2.91 2 G-CSF (Gen1) Day 5 5 100 10% lipoplex 5.32*
3 G-CSF (Gen1) Day 8 5 100 10% lipoplex 2.06 4 G-CSF (no
modification) 5 100 10% lipoplex 1.88 Day 1 5 G-CSF (no
modification) 5 100 10% lipoplex 1.95 Day 5 6 G-CSF (no
modification) 5 100 10% lipoplex 2.09 Day 8 7 RNA control Day 1 5
100 10% lipoplex 2.90 8 RNA control Day 5 5 100 10% lipoplex 1.68 9
RNA control Day 8 4 100 10% lipoplex 1.72 10 F. Buffer Day 1 4 100
10% lipoplex 2.51 11 F. Buffer Day 5 4 100 10% lipoplex 1.31 12 F.
Buffer Day 8 4 100 10% lipoplex 1.92
Example 75: Routes of Administration
[1060] Studies were performed to investigate split dosing using
different routes of administration. Studies utilizing multiple
subcutaneous or intramuscular injection sites at one time point
were designed and performed to investigate ways to increase
modified mRNA drug exposure and improve protein production. In
addition to detection of the expressed protein product, an
assessment of the physiological function of proteins was also
determined through analyzing samples from the animal tested.
[1061] Surprisingly, it has been determined that split dosing of
modified mRNA produces greater protein production and phenotypic
responses than those produced by single unit dosing or multi-dosing
schemes.
[1062] The design of a split dose experiment involved using human
erythropoietin (EPO) modified mRNA (mRNA sequence shown in SEQ ID
NO: 5; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1) or luciferase modified mRNA (mRNA sequence
shown in SEQ ID NO: 3; polyA tail of approximately 160 nucleotides
not shown in sequence; 5'cap, Cap1) administered in buffer alone or
formulated with 30% lipoplex (RNAIMAX.TM.). The dosing vehicle
(buffer) consisted of 150 mM NaCl, 2 mM CaCl.sub.2, 2 mM
Na.sup.+-phosphate (1.4 mM monobasic sodium phosphate; 0.6 mM
dibasic sodium phosphate), and 0.5 mM EDTA, pH 6.5. The pH was
adjusted using sodium hydroxide and the final solution was filter
sterilized. The mRNA was modified with 5methylC (5meC) at each
cytosine and pseudouridine replacement at each uridine site.
[1063] 4 mice per group were dosed intramuscularly (I.M.),
intravenously (I.V.) or subcutaneously (S.C.) by the dosing chart
outlined in Table 22. Serum was collected 13 hours post injection
from all mice, tissue was collected from the site of injection from
the intramuscular and subcutaneous group and the spleen, liver and
kidneys were collected from the intravenous group. The results from
the intramuscular group and the subcutaneous group results are
shown in Table 23.
TABLE-US-00023 TABLE 22 Dosing Chart Group Treatment Route Dose of
modified mRNA Total Dose Dosing Vehicle 1 Lipoplex-human EPO I.M. 4
.times. 100 ug + 30% Lipoplex 4 .times. 70 ul Lipoplex modified
mRNA 2 Lipoplex-human EPO I.M. 4 .times. 100 ug 4 .times. 70 ul
Buffer modified mRNA 3 Lipoplex-human EPO S.C. 4 .times. 100 ug +
30% Lipoplex 4 .times. 70 ul Lipoplex modified mRNA 4
Lipoplex-human EPO S.C. 4 .times. 100 ug 4 .times. 70 ul Buffer
modified mRNA 5 Lipoplex-human EPO I.V. 200 ug + 30% Lipoplex 140
ul Lipoplex modified mRNA 6 Lipoplexed-Luciferase I.M. 100 ug + 30%
Lipoplex 4 .times. 70 ul Lipoplex modified mRNA 7
Lipoplexed-Luciferase I.M. 100 ug 4 .times. 70 ul Buffer modified
mRNA 8 Lipoplexed-Luciferase S.C. 100 ug + 30% Lipoplex 4 .times.
70 ul Lipoplex modified mRNA 9 Lipoplexed-Luciferase S.C. 100 ug 4
.times. 70 ul Buffer modified mRNA 10 Lipoplexed-human EPO I.V. 200
ug + 30% Lipoplex 140 ul Lipoplex modified mRNA 11 Formulation
Buffer I.M. 4.times. multi dosing 4 .times. 70 ul Buffer
TABLE-US-00024 TABLE 23 Human EPO Protein in Mouse Serum (I.M.
Injection Route) EPO (pg/ml) Formulation I.M. Injection Route S.C.
Injection Route Epo-Lipoplex 67.1 2.2 Luc-Lipoplex 0 0 Epo-Saline
100.9 11.4 Luc-Saline 0 0 Formulation Buffer 0 0
Example 76: In Vivo Delivery Using Varying Lipid Ratios
[1064] Modified mRNA was delivered to C57/BL6 mice to evaluate
varying lipid ratios and the resulting protein expression.
Formulations of 100 .mu.g modified human EPO mRNA (mRNA sequence
shown in SEQ ID NO: 5; polyA tail of approximately 160 nucleotides
not shown in sequence; 5'cap, Cap1; fully modified with
5-methylcytosine and pseudouridine) lipoplexed with 10%, 30% or 50%
RNAIMAX.TM., 100 .mu.g modified luciferase mRNA (IVT cDNA sequence
shown in SEQ ID NO: 2; mRNA sequence shown in SEQ ID NO: 3, polyA
tail of approximately 160 nucleotides not shown in sequence, 5'cap,
Cap1, fully modified with 5-methylcytosine at each cytosine and
pseudouridine replacement at each uridine site) lipoplexed with
10%, 30% or 50% RNAIMAX.TM. or a formulation buffer were
administered intramuscularly to mice in a single 70 .mu.l dose.
Serum was collected 13 hours post injection to undergo a human EPO
ELISA to determine the human EPO protein level in each mouse. The
results of the human EPO ELISA, shown in Table 24, show that
modified human EPO expressed in the muscle is secreted into the
serum for each of the different percentage of RNAIMAX.TM..
TABLE-US-00025 TABLE 24 Human EPO Protein in Mouse Serum (IM
Injection Route) Formulation EPO (pg/ml) Epo + 10% RNAiMAX 11.4 Luc
+ 10% RNAiMAX 0 Epo + 30% RNAiMAX 27.1 Luc + 30% RNAiMAX 0 Epo +
50% RNAiMAX 19.7 Luc + 50% RNAiMAX 0 F. Buffer 0
Example 77: In Vivo Delivery of Modified RNA in Rats
[1065] Protein production of modified mRNA was evaluated by
delivering modified G-CSF mRNA or modified Factor IX mRNA to female
Sprague Dawley rats (n=6). Rats were injected with 400 ug in 100 ul
of G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail of
approximately 160 nucleotides not shown in sequence; 5'cap, Cap1)
fully modified with 5-methylcytosine and pseudouridine (G-CSF
Gen1), G-CSF mRNA fully modified with 5-methylcytosine and
N1-methylpseudouridine (G-CSF Gen2) or Factor IX mRNA (mRNA
sequence shown in SEQ ID NO: 6; polyA tail of approximately 160
nucleotides not shown in sequence; 5'cap, Cap1) fully modified with
5-methylcytosine and pseudouridine (Factor IX Gen1) reconstituted
from the lyophilized form in 5% sucrose. Blood was collected 8
hours after injection and the G-CSF protein level in serum was
measured by ELISA. Table 25 shows the G-CSF protein levels in serum
after 8 hours.
[1066] These results demonstrate that both G-CSF Gen 1 and G-CSF
Gen 2 modified mRNA can produce human G-CSF protein in a rat
following a single intramuscular injection, and that human G-CSF
protein production is improved when using Gen 2 chemistry over Gen
1 chemistry.
TABLE-US-00026 TABLE 25 G-CSF Protein in Rat Serum (I.M. Injection
Route) Formulation G-CSF protein (pg/ml) G-CSF Gen1 19.37 G-CSF
Gen2 64.72 Factor IX Gen 1 2.25
Example 78. Chemical Modification: In Vitro Studies
A. In Vitro Screening in PBMC
[1067] 500 ng of G-CSF (mRNA sequence shown in SEQ ID NO: 1; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) mRNA fully modified with the chemical modification outlined
Tables 26 and 27 was transfected with 0.4 uL Lipofectamine 2000
into peripheral blood mononuclear cells (PBMC) from three normal
blood donors. Control samples of LPS, R848, P(I)P(C) and mCherry
(mRNA sequence shown in SEQ ID NO: 4; polyA tail of approximately
160 nucleotides not shown in sequence, 5'cap, Cap1; fully modified
with 5-methylcytosine and pseudouridine) were also analyzed. The
supernatant was harvested and stored frozen until analyzed by ELISA
to determine the G-CSF protein expression, and the induction of the
cytokines interferon-alpha (IFN-.alpha.) and tumor necrosis factor
alpha (TNF-.alpha.). The protein expression of G-CSF is shown in
Table 26, the expression of IFN-.alpha. and TNF-.alpha. is shown in
Table 27.
[1068] The data in Table 26 demonstrates that many, but not all,
chemical modifications can be used to productively produce human
G-CSF in PBMC. Of note, 100% N1-methylpseudouridine substitution
demonstrates the highest level of human G-CSF production (almost
10-fold higher than pseudouridine itself). When
N1-methylpseudouridine is used in combination with 5-methylcytidine
a high level of human G-CSF protein is also produced (this is also
higher than when pseudouridine is used in combination with 5
methylcytidine).
[1069] Given the inverse relationship between protein production
and cytokine production in PBMC, a similar trend is also seen in
Table 27, where 100% substitution with N1-methylpseudouridine
results no cytokine induction (similar to transfection only
controls) and pseudouridine shows detectable cytokine induction
which is above background.
[1070] Other modifications such as N6-methyladenosine and
.alpha.-thiocytidine appear to increase cytokine stimulation.
TABLE-US-00027 TABLE 26 Chemical Modifications and G-CSF Protein
Expression G-CSF Protein Expression (pg/ml) Donor Donor Donor
Chemical Modifications 1 2 3 Pseudouridine 2477 1,909 1,498
5-methyluridine 318 359 345 N1-methylpseudouridine 21,495 16,550
12,441 2-thiouridine 932 1,000 600 4-thiouridine 5 391 218
5-methoxyuridine 2,964 1,832 1,800 5-methylcytosine and
pseudouridine (1.sup.st set) 2,632 1,955 1,373 5-methylcytosine and
N1-methyl- 10,232 7,245 6,214 pseudouridine (1.sup.st set)
2'Fluoroguanosine 59 186 177 2'Fluorouridine 118 209 191
5-methylcytosine and pseudouridine (2.sup.nd set) 1,682 1,382 1,036
5-methylcytosine and N1-methyl- 9,564 8,509 7,141 pseudouridine
(2.sup.nd set) 5-bromouridine 314 482 291
5-(2-carbomethoxyvinyl)uridine 77 286 177
5-[3(1-E-propenylamino)uridine 541 491 550 .alpha.-thiocytidine 105
264 245 5-methylcytosine and pseudouridine (3.sup.rd set) 1,595
1,432 955 N1-methyladenosine 182 177 191 N6-methyladenosine 100 168
200 5-methylcytidine 291 277 359 N4-acetylcytidine 50 136 36
5-formylcytidine 18 205 23 5-methylcytosine and pseudouridine
(4.sup.th set) 264 350 182 5-methylcytosine and N1-methyl- 9,505
6,927 5,405 pseudouridine (4.sup.th set) LPS 1,209 786 636 mCherry
5 168 164 R848 709 732 636 P(I)P(C) 5 186 182
TABLE-US-00028 TABLE 27 Chemical Modifications and Cytokine
Expression Chemical IFN-.alpha. Expression (pg/ml) TNF-.alpha.
Expression (pg/ml) Modifications Donor 1 Donor 2 Donor 3 Donor 1
Donor 2 Donor 3 Pseudouridine 120 77 171 36 81 126 5-methyluridine
245 135 334 94 100 157 N1-methyl- 26 75 138 101 106 134
pseudouridine 2-thiouridine 100 108 154 133 133 141 4-thiouridine
463 258 659 169 126 254 5-methoxyuridine 0 64 133 39 74 111
5-methylcytosine 88 94 148 64 89 121 and pseudouridine (1.sup.st
set) 5-methylcytosine 0 60 136 54 79 126 and N1-methyl-
pseudouridine (1.sup.st set) 2'Fluoroguanosine 107 97 194 91 94 141
2'Fluorouridine 158 103 178 164 121 156 5-methylcytosine 133 92 167
99 111 150 and pseudouridine (2.sup.nd set) 5-methylcytosine 0 66
140 54 97 149 and N1-methyl- pseudouridine (2.sup.nd set)
5-bromouridine 95 86 181 87 106 157 5-(2-carbometh- 0 61 130 40 81
116 oxyvinyl)uridine 5-[3(1-E- 0 58 132 71 90 119 propenylamino)
uridine .alpha.-thiocytidine 1,138 565 695 300 273 277
5-methylcytosine 88 75 150 84 89 130 and pseudouridine (3.sup.rd
set) N1-methyl- 322 255 377 256 157 294 adenosine N6-methyl- 1,935
1,065 1,492 1,080 630 857 adenosine 5-methylcytidine 643 359 529
176 136 193 N4-acetylcytidine 789 593 431 263 67 207
5-formylcytidine 180 93 88 136 30 40 5-methylcytosine 131 28 18 53
24 29 and pseudouridine (4.sup.th set) 5-methylcytosine 0 0 0 36 14
13 and N1-methyl- pseudouridine (4.sup.th set) LPS 0 67 146 7,004
3,974 4,020 mCherry 100 75 143 67 100 133 R848 674 619 562 11,179
8,546 9,907 P(I)P(C) 470 117 362 249 177 197
B. In Vitro Screening in HeLa Cells
[1071] The day before transfection, 20,000 HeLa cells (ATCC no.
CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA
solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a
total volume of 100 ul EMEM medium (supplemented with 10% FCS and
1.times. Glutamax) per well in a 96-well cell culture plate
(Corning, Manassas, Va.). The cells were grown at 37.degree.G in 5%
CO.sub.2 atmosphere overnight. Next day, 83 ng of Luciferase
modified RNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail of
approximately 160 nucleotides not shown in sequence; 5'cap, Cap1)
with the chemical modification described in Table 28, were diluted
in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,
N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)
was used as transfection reagent and 0.2 ul were diluted in 10 ul
final volume of OPTI-MEM. After 5 minutes of incubation at room
temperature, both solutions were combined and incubated an
additional 15 minute at room temperature. Then the 20 ul combined
solution was added to the 100 ul cell culture medium containing the
HeLa cells and incubated at room temperature.
[1072] After 18 to 22 hours of incubation cells expressing
luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega,
Madison, Wis.) according to manufacturer instructions. Aliquots of
the lysates were transferred to white opaque polystyrene 96-well
plates (Corning, Manassas, Va.) and combined with 100 ul complete
luciferase assay solution (Promega, Madison, Wis.). The lysate
volumes were adjusted or diluted until no more than 2 mio relative
light units (RLU) per well were detected for the strongest signal
producing samples and the RLUs for each chemistry tested are shown
in Table 28. The plate reader was a BioTek Synergy H1 (BioTek,
Winooski, Vt.). The background signal of the plates without reagent
was about 200 relative light units per well.
[1073] These results demonstrate that many, but not all, chemical
modifications can be used to productively produce human G-CSF in
HeLa cells. Of note, 100% N1-methylpseudouridine substitution
demonstrates the highest level of human G-CSF production.
TABLE-US-00029 TABLE 28 Relative Light Units of Luciferase Chemical
Modification RLU N6-methyladenosine (m6a) 534 5-methylcytidine
(m5c) 138,428 N4-acetylcytidine (ac4c) 235,412 5-formylcytidine
(f5c) 436 5-methylcytosine/pseudouridine, test A1 48,659
5-methylcytosine/N1-methylpseudouridine, test A1 190,924
Pseudouridine 655,632 1-methylpseudouridine (m1u) 1,517,998
2-thiouridine (s2u) 3387 5-methoxyuridine (mo5u) 253,719
5-methylcytosine/pseudouridine, test B1 317,744
5-methylcytosine/N1-methylpseudouridine, test B1 265,871
5-Bromo-uridine 43,276 5 (2 carbovinyl) uridine 531 5 (3-1E
propenyl Amino) uridine 446 5-methylcytosine/pseudouridine, test A2
295,824 5-methylcytosine/N1-methylpseudouridine, test A2 233,921
5-methyluridine 50,932 .alpha.-Thio-cytidine 26,358
5-methylcytosine/pseudouridine, test B2 481,477
5-methylcytosine/N1-methylpseudouridine, test B2 271,989
5-methylcytosine/pseudouridine, test A3 438,831
5-methylcytosine/N1-methylpseudouridine, test A3 277,499 Unmodified
Luciferase 234,802
C. In Vitro Screening in Rabbit Reticulocyte Lysates
[1074] Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) was modified with the chemical modification listed in Table
29 and were diluted in sterile nuclease-free water to a final
amount of 250 ng in 10 ul. The diluted luciferase was added to 40
ul of freshly prepared Rabbit Reticulocyte Lysate and the in vitro
translation reaction was done in a standard 1.5 mL polypropylene
reaction tube (Thermo Fisher Scientific, Waltham, Mass.) at
30.degree. C. in a dry heating block. The translation assay was
done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit
(Promega, Madison, Wis.) according to the manufacturer's
instructions. The reaction buffer was supplemented with a
one-to-one blend of provided amino acid stock solutions devoid of
either Leucine or Methionine resulting in a reaction mix containing
sufficient amounts of both amino acids to allow effective in vitro
translation.
[1075] After 60 minutes of incubation, the reaction was stopped by
placing the reaction tubes on ice. Aliquots of the in vitro
translation reaction containing luciferase modified RNA were
transferred to white opaque polystyrene 96-well plates (Corning,
Manassas, Va.) and combined with 100 ul complete luciferase assay
solution (Promega, Madison, Wis.). The volumes of the in vitro
translation reactions were adjusted or diluted until no more than 2
mio relative light units (RLUs) per well were detected for the
strongest signal producing samples and the RLUs for each chemistry
tested are shown in Table 29. The plate reader was a BioTek Synergy
H1 (BioTek, Winooski, Vt.). The background signal of the plates
without reagent was about 200 relative light units per well.
[1076] These cell-free translation results very nicely correlate
with the protein production results in HeLa, with the same
modifications generally working or not working in both systems. One
notable exception is 5-formylcytidine modified luciferase mRNA
which worked in the cell-free translation system, but not in the
HeLa cell-based transfection system. A similar difference between
the two assays was also seen with 5-formylcytidine modified G-CSF
mRNA.
TABLE-US-00030 TABLE 29 Relative Light Units of Luciferase Chemical
Modification RLU N6-methyladenosine (m6a) 398 5-methylcytidine
(m5c) 152,989 N4-acetylcytidine (ac4c) 60,879 5-formylcytidine
(f5c) 55,208 5-methylcytosine/pseudouridine, test A1 349,398
5-methylcytosine/N1-methylpseudouridine, test A1 205,465
Pseudouridine 587,795 1-methylpseudouridine (m1u) 589,758
2-thiouridine (s2u) 708 5-methoxyuridine (mo5u) 288,647
5-methylcytosine/pseudouridine, test B1 454,662
5-methylcytosine/N1-methylpseudouridine, test B1 223,732
5-Bromo-uridine 221,879 5 (2 carbovinyl) uridine 225 5 (3-1E
propenyl Amino) uridine 211 5-methylcytosine/pseudouridine, test A2
558,779 5-methylcytosine/N1-methylpseudouridine, test A2 333,082
5-methyluridine 214,680 .alpha.-Thio-cytidine 123,878
5-methylcytosine/pseudouridine, test B2 487,805
5-methylcytosine/N1-methylpseudouridine, test B2 154,096
5-methylcytosine/pseudouridine, test A3 413,535
5-methylcytosine/N1-methylpseudouridine, test A3 292,954 Unmodified
Luciferase 225,986
Example 79. Chemical Modification: In Vivo Studies
[1077] A. In Vivo Screening of G-CSF Modified mRNA
[1078] Balb-C mice (n=4) are intramuscularly injected in each leg
with modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1;
polyA tail of approximately 160 nucleotides not shown in sequence;
5'cap, Cap1), fully modified with the chemical modifications
outlined in Table 30, is formulated in 1.times.PBS. A control of
luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 3;
polyA tail of approximately 160 nucleotides not shown in sequence;
5'cap, Cap1; fully modified with pseudouridine and
5-methylcytosine) and a control of PBS are also tested. After 8
hours serum is collected to determine G-CSF protein levels cytokine
levels by ELISA.
TABLE-US-00031 TABLE 30 G-CSF mRNA Chemical Modifications G-CSF
Pseudouridine G-CSF 5-methyluridine G-CSF 2-thiouridine G-CSF
4-thiouridine G-CSF 5-methoxyuridine G-CSF 2'-fluorouridine G-CSF
5-bromouridine G-CSF 5-[3(1-E-propenylamino)uridine) G-CSF
alpha-thio-cytidine G-CSF 5-methylcytidine G-CSF N4-acetylcytidine
G-CSF Pseudouridine and 5-methylcytosine G-CSF
N1-methyl-pseudouridine and 5-methylcytosine Luciferase
Pseudouridine and 5-methylcytosine PBS None
B. In Vivo Screening of Luciferase Modified mRNA
[1079] Balb-C mice (n=4) were subcutaneously injected with 200 ul
containing 42 to 103 ug of modified luciferase mRNA (mRNA sequence
shown in SEQ ID NO: 3; polyA tail of approximately 160 nucleotides
not shown in sequence; 5'cap, Cap1), fully modified with the
chemical modifications outlined in Table 31, was formulated in
1.times.PBS. A control of PBS was also tested. The dosages of the
modified luciferase mRNA is also outlined in Table 31. 8 hours
after dosing the mice were imaged to determine luciferase
expression. Twenty minutes prior to imaging, mice were injected
intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals
were then anesthetized and images were acquired with an IVIS Lumina
II imaging system (Perkin Elmer). Bioluminescence was measured as
total flux (photons/second) of the entire mouse.
[1080] As demonstrated in Table 31, all luciferase mRNA modified
chemistries demonstrated in vivo activity, with the exception of
2'-fluorouridine. In addition 1-methylpseudouridine modified mRNA
demonstrated very high expression of luciferase (5-fold greater
expression than pseudouridine containing mRNA).
TABLE-US-00032 TABLE 31 Luciferase Screening Luciferase Dose Dose
expression (ug) of volume (photon/ mRNA Chemical Modifications mRNA
(ml) second) Luciferase 5-methylcytidine 83 0.72 1.94E+07
Luciferase N4-acetylcytidine 76 0.72 1.11E07 Luciferase
Pseudouridine 95 1.20 1.36E+07 Luciferase 1-methylpseudouridine 103
0.72 7.40E+07 Luciferase 5-methoxyuridine 95 1.22 3.32+07
Luciferase 5-methyluridine 94 0.86 7.42E+06 Luciferase
5-bromouridine 89 1.49 3.75E+07 Luciferase 2'-fluoroguanosine 42
0.72 5.88E+05 Luciferase 2'-fluorocytidine 47 0.72 4.21E+05
Luciferase 2'-flurorouridine 59 0.72 3.47E+05 PBS None -- 0.72
3.16E+05
Example 80. In Vivo Screening of Combination Luciferase Modified
mRNA
[1081] Balb-C mice (n=4) were subcutaneously injected with 200 ul
of 100 ug of modified luciferase mRNA (mRNA sequence shown in SEQ
ID NO: 3; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1), fully modified with the chemical
modifications outlined in Table 32, was formulated in 1.times.PBS.
A control of PBS was also tested. The dosages of the modified
luciferase mRNA is also outlined in Table 29. 8 hours after dosing
the mice were imaged to determine luciferase expression. Twenty
minutes prior to imaging, mice were injected intraperitoneally with
a D-luciferin solution at 150 mg/kg. Animals were then anesthetized
and images were acquired with an IVIS Lumina II imaging system
(Perkin Elmer). Bioluminescence was measured as total flux
(photons/second) of the entire mouse.
[1082] As demonstrated in Table 32, all luciferase mRNA modified
chemistries (in combination) demonstrated in vivo activity. In
addition the presence of N1-methylpseudouridine in the modified
mRNA (with N4-acetylcytidine or 5 methylcytidine) demonstrated
higher expression than when the same combinations where tested
using with pseudouridine. Taken together, these data demonstrate
that N1-methylpseudouridine containing luciferase mRNA results in
improved protein expression in vivo whether used alone (Table 31)
or when used in combination with other modified nulceotides (Table
32).
TABLE-US-00033 TABLE 32 Luciferase Screening Combinations
Luciferase expression (photon/ mRNA Chemical Modifications second)
Luciferase N4-acetylcytidine/pseudouridine 4.18E+06 Luciferase
N4-acetylcytidine/N1-methylpseudouridine 2.88E+07 Luciferase
5-methylcytidine/5-methoxyuridine 3.48E+07 Luciferase
5-methylcytidine/5-methyluridine 1.44E+07 Luciferase
5-methylcytidine/where 50% of the uridine 2.39E+06 is replaced with
2-thiouridine Luciferase 5-methylcytidine/pseudouridine 2.36E+07
Luciferase 5-methylcytidine/N1-methyl-pseudouridine 4.15E+07 PBS
None 3.59E+05
Example 81. Stability of Modified RNA
A. Storage of Modified RNA
[1083] Stability experiments were conducted to obtain a better
understanding of storage conditions to retain the integrity of
modified RNA. Unmodified G-CSF mRNA (mRNA sequence shown in SEQ ID
NO: 1; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1), G-CSF mRNA fully modified with
5-methylcytosine and pseudouridine and G-CSF mRNA fully modified
with 5-methylcytosine and pseudouridine lipoplexed with 0.75% by
volume of RNAIMAX.TM. was stored at 50.degree. C., 40.degree. C.,
37.degree. C., 25.degree. C., 4.degree. C. or -20.degree. C. After
the mRNA had been stored for 0 hours, 2 hours, 6 hours, 24 hours,
48 hours, 5 days and 14 days, the mRNA was analyzed by gel
electrophoresis using a Bio-Rad EXPERION.TM. system. The modified,
unmodified and lipoplexed G-CSF mRNA was also stored in
RNASTABLE.RTM. (Biomatrica, Inc. San Diego, Calif.) at 40.degree.
C. or water at -80.degree. C. or 40.degree. C. for 35 days before
being analyzed by gel electrophoresis.
[1084] All mRNA samples without stabilizer were stable after 2
weeks after storage at 4.degree. C. or -20.degree. C. Modified
G-CSF mRNA, with or without lipoplex, was more stable than
unmodified G-CSF when stored at 25.degree. C. (stable out to 5 days
versus 48 hours), 37.degree. C. (stable out to 24 hours versus 6
hours) and 50.degree. C. (stable out to 6 hours versus 2 hours).
Unmodified G-CSF mRNA, modified G-CSF mRNA with or without lipoplex
tolerated 12 freeze/thaw cycles.
[1085] mRNA samples stored in stabilizer at 40.degree. C. showed
similar stability to the mRNA samples stored in water at
-80.degree. C. after 35 days whereas the mRNA stored in water at
40.degree. C. showed heavy degradation after 18 days.
Example 82. Cell Viability in BJ Fibroblasts
[1086] Human primary foreskin fibroblasts (BJ fibroblasts) were
obtained from American Type Culture Collection (ATCC) (catalog
#CRL-2522) and grown in Eagle's Minimum Essential Medium (ATCC, cat
#30-2003) supplemented with 10% fetal bovine serum at 37.degree.
C., under 5% CO.sub.2. BJ fibroblasts were seeded on a 24-well
plate at a density of 130,000 cells per well in 0.5 ml of culture
medium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ
ID NO: 1; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1) fully modified with 5-methylcytosine and
pseudouridine (Gen1) or fully modified with 5-methylcytosine and
N1-methylpseudouridine (Gen2) was transfected using Lipofectamine
2000 (Invitrogen, cat #11668-019), following manufacturer's
protocol. Control samples of Lipofectamine 2000 (LF2000) and
unmodified G-CSF mRNA were also transfected. The modified mRNA or
control samples were transfected daily for 4 days. The viability of
the cells after transfection was evaluated 6 hours and 24 hours
after the first transfection (T1, 6 hours or T1, 24 hours), and 24
hours after the second (T2, 24 hours) and fourth transfection (T4,
24 hours).
[1087] To determine cell viability, the culture medium was
completely removed and the cells were washed once with 600 ul of
sterile PBS without Ca2+/Mg2+ (Gibco/Life Technologies, Manassas,
Va.) in order to rinse-off loosely attached cells. PBS was removed
and discarded. The cleaned fibroblasts in each well were treated
with 220 ul of a diluted CELL TITER GLO.RTM. (Promega, catalog
#G7570) stock solution (the CELL TITER GLO.RTM. stock solution was
further diluted 1:1 with an equal amount of sterile PBS). A sterile
pipet tip was used to scratch the cells off the plate and
accelerate the lysis process.
[1088] For two time intervals, T1, 24 hours and T2, 24 hours, an
alternative protocol was applied. Cells were washed with PBS, as
described above, and subsequently trypsinized with Trypsin/EDTA
solution (Gibco/Life Technologies, Manassas, Va.). Cells were
detached and collected in 500 ul of medium containing trypsin
inhibitor. Cells were harvested by centrifugation at 1200 rcf for 5
minutes. The cell pellet was resuspended in 500 ul PBS. This cell
suspension was kept on ice, and 100 ul of this was combined with
100 ul of undiluted Cell Titer Glo solution.
[1089] All of the CELL TITER GLO.RTM. lysates were then incubated
at room temperature for 20 minutes. 20 ul of the lysates were
transferred to a white opaque polystyrene 96-well plate (Corning,
Manassas, Va.) and combined with 100 ul diluted CELL TITER GLO.RTM.
solution. The plate reader used was from BioTek Synergy H1 (BioTek,
Winooski, Vt.) and the absolute values were normalized to signal of
the untreated BJ Fibroblasts to 100% cell vitality. The percent
viability for the BJ fibroblasts are shown in Table 33.
[1090] Importantly, all of these experiments are conducted in the
absence of any interferon or other cytokine inhibitors and thus
represent an accurate measure of the cytotoxicity of the different
mRNA.
[1091] These results demonstrate that repeated transfection of BJ
fibroblasts with unmodified mRNA results in loss of cell viability
that is apparent as early as 24 hrs after the first transfection
(T1, 24 hours) and continues to be apparent and more pronounced at
subsequent time points.
[1092] There is also a loss of viability with repeated transfection
of 5methylcytidine and pseudouridine modified mRNA that is apparent
24 hours after the fourth daily transfection (T4, 24 hours). No
loss of cell viability over the course of this experiment is seen
using 5methylcytidine and N1-methylpseudouridine modified mRNA.
These results demonstrate that 5methylcytidine and
N1-methylpseudouridine containing mRNA have improved cell viability
when analyzed under repeated transfection. The ability to
repeatedly administer modified mRNA is important in most
therapeutic applications, and as such the ability to do so without
cytotoxicity is also important. While not wishing to be bound by
theory, it is believed that response genes following a single
transfection may lead to a decrease in protein production, cytokine
induction, and eventually loss of cell viability. These results are
consistent with N1-methylpseudouridine-containing mRNA showing an
improved profile in this respect relative to both unmodified mRNA
and pseudouridine-modified mRNA.
TABLE-US-00034 TABLE 33 Percent Viability T1, 6 hours T1, 24 hours
T2, 24 hours T4, 24 hours Gen 1 G-CSF 81 108 91 65 Gen 2 G-CSF 99
102 128 87 Unmodified G-CSF 101 72 74 42 LF2000 99 80 114 106
Untreated 100 100 100 100
Example 83. Innate Immune Response in BJ Fibroblasts
[1093] Human primary foreskin fibroblasts (BJ fibroblasts) are
obtained from American Type Culture Collection (ATCC) (catalog
#CRL-2522) and grown in Eagle's Minimum Essential Medium (ATCC, cat
#30-2003) supplemented with 10% fetal bovine serum at 37.degree.
C., under 5% CO.sub.2. BJ fibroblasts are seeded on a 24-well plate
at a density of 130,000 cells per well in 0.5 ml of culture medium.
250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1;
polyA tail of approximately 160 nucleotides not shown in sequence;
5'cap, Cap1) fully modified with 5-methylcytosine and pseudouridine
(Gen1) or fully modified with 5-methylcytosine and
N1-methylpseudouridine (Gen2) is transfected using Lipofectamine
2000 (Invitrogen, cat #11668-019), following manufacturer's
protocol. Control samples of Lipofectamine 2000 and unmodified
G-CSF mRNA (natural G-CSF) are also transfected. The cells are
transfected for five consecutive days. The transfection complexes
are removed four hours after each round of transfection.
[1094] The culture supernatant is assayed for secreted GCSF
(R&D Systems, catalog #DCS50), tumor necrosis factor-alpha
(TNF-alpha) and interferon alpha (IFN-alpha) by ELISA every day
after transfection following manufacturer's protocols. The cells
are analyzed for viability using CELL TITER GLO.RTM. (Promega,
catalog #G7570) 6 hrs and 18 hrs after the first round of
transfection and every alternate day following that. At the same
time from the harvested cells, total RNA is isolated and treated
with DNASE.RTM. using the RNAEASY micro kit (catalog #74004)
following the manufacturer's protocol. 100 ng of total RNA is used
for cDNA synthesis using the High Capacity cDNA Reverse
Transcription kit (Applied Biosystems, cat #4368814) following the
manufacturer's protocol. The cDNA is then analyzed for the
expression of innate immune response genes by quantitative real
time PCR using SybrGreen in a Biorad CFX 384 instrument following
the manufacturer's protocol.
Example 84. In Vitro Transcription with Wild-Type T7 Polymerase
[1095] Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) were fully modified with different chemistries and chemistry
combinations listed in Tables 34-37 using wild-type T7 polymerase
as previously described.
[1096] The yield of the translation reactions was determined by
spectrophometric measurement (OD260) and the yield for Luciferase
is shown in Table 34 and G-CSF is shown in Table 36.
[1097] The luciferase and G-CSF modified mRNA were also subjected
to an enzymatic capping reaction and each modified mRNA capping
reaction was evaluated for yield by spectrophometic measurement
(OD260) and correct size assessed using bioanalyzer. The yield from
the capping reaction for luciferase is shown in Table 35 and G-CSF
is shown in Table 37.
TABLE-US-00035 TABLE 34 In vitro transcription chemistry for
Luciferase Chemical Modification Yield (mg) N6-methyladenosine 0.99
5-methylcytidine 1.29 N4-acetylcytidine 1.0 5-formylcytidine 0.55
Pseudouridine 2.0 N1-methylpseudouridine 1.43 2-thiouridine 1.56
5-methoxyuridine 2.35 5-methyluridine 1.01 .alpha.-Thio-cytidine
0.83 5-Br-uridine (5Bru) 1.96 5 (2 carbomethoxyvinyl) uridine 0.89
5 (3-1E propenyl Amino) uridine 2.01
N4-acetylcytidine/pseudouridine 1.34
N4-acetylcytidine/N1-methylpseudouridine 1.26
5-methylcytidine/5-methoxyuridine 1.38
5-methylcytidine/5-bromouridine 0.12
5-methylcytidine/5-methyluridine 2.97 5-methylcytidine/half of the
uridines are 1.59 modified with 2-thiouridine
5-methylcytidine/2-thiouridine 0.90 5-methylcytidine/pseudouridine
1.83 5-methylcytidine/N1 methyl pseudouridine 1.33
TABLE-US-00036 TABLE 35 Capping chemistry and yield for Luciferase
modified mRNA Chemical Modification Yield (mg) 5-methylcytidine
1.02 N4-acetylcytidine 0.93 5-formylcytidine 0.55 Pseudouridine
2.07 N1-methylpseudouridine 1.27 2-thiouridine 1.44
5-methoxyuridine 2 5-methyluridine 0.8 .alpha.-Thio-cytidine 0.74
5-Br-uridine (5Bru) 1.29 5 (2 carbomethoxyvinyl) uridine 0.54 5
(3-1E propenyl Amino) uridine 1.39 N4-acetylcytidine/pseudouridine
0.99 N4-acetylcytidine/N1-methylpseudouridine 1.08
5-methylcytidine/5-methoxyuridine 1.13
5-methylcytidine/5-methyluridine 1.08 5-methylcytidine/half of the
uridines are 1.2 modified with 2-thiouridine
5-methylcytidine/2-thiouridine 1.27 5-methylcytidine/pseudouridine
1.19 5-methylcytidine/N1 methyl pseudouridine 1.04
TABLE-US-00037 TABLE 36 In vitro transcription chemistry and yield
for G-CSF modified mRNA Chemical Modification Yield (mg)
N6-methyladenosine 1.57 5-methylcytidine 2.05 N4-acetylcytidine
3.13 5-formylcytidine 1.41 Pseudouridine 4.1 N1-methylpseudouridine
3.24 2-thiouridine 3.46 5-methoxyuridine 2.57 5-methyluridine 4.27
4-thiouridine 1.45 2'-F-uridine 0.96 .alpha.-Thio-cytidine 2.29
2'-F-guanosine 0.6 N-1-methyladenosine 0.63 5-Br-uridine (5Bru)
1.08 5 (2 carbomethoxyvinyl) uridine 1.8 5 (3-1E propenyl Amino)
uridine 2.09 N4-acetylcytidine/pseudouridine 1.72
N4-acetylcytidine/N1-methylpseudouridine 1.37
5-methylcytidine/5-methoxyuridine 1.85
5-methylcytidine/5-methyluridine 1.56 5-methylcytidine/half of the
uridines are 1.84 modified with 2-thiouridine
5-methylcytidine/2-thiouridine 2.53 5-methylcytidine/pseudouridine
0.63 N4-acetylcytidine/2-thiouridine 1.3
N4-acetylcytidine/5-bromouridine 1.37 5-methylcytidine/N1 methyl
pseudouridine 1.25 N4-acetylcytidine/pseudouridine 2.24
TABLE-US-00038 TABLE 37 Capping chemistry and yield for G-CSF
modified mRNA Chemical Modification Yield (mg) N6-methyladenosine
1.04 5-methylcytidine 1.08 N4-acetylcytidine 2.73 5-formylcytidine
0.95 Pseudouridine 3.88 N1-methylpseudouridine 2.58 2-thiouridine
2.57 5-methoxyuridine 2.05 5-methyluridine 3.56 4-thiouridine 0.91
2'-F-uridine 0.54 .alpha.-Thio-cytidine 1.79 2'-F-guanosine 0.14
5-Br-uridine (5Bru) 0.79 5 (2 carbomethoxyvinyl) uridine 1.28 5
(3-1E propenyl Amino) uridine 1.78 N4-acetylcytidine/pseudouridine
0.29 N4-acetylcytidine/N1-methylpseudouridine 0.33
5-methylcytidine/5-methoxyuridine 0.91
5-methylcytidine/5-methyluridine 0.61 5-methylcytidine/half of the
uridines are 1.24 modified with 2-thiouridine
5-methylcytidine/pseudouridine 1.08 N4-acetylcytidine/2-thiouridine
1.34 N4-acetylcytidine/5-bromouridine 1.22 5-methylcytidine/N1
methyl pseudouridine 1.56
Example 85. In Vitro Transcription with Mutant T7 Polymerase
[1098] Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) were fully modified with different chemistries and chemistry
combinations listed in Tables 38-41 using a mutant T7 polymerase
(Durascribe.RTM. T7 Transcription kit (Cat. No. DS010925)
(Epicentre.RTM., Madison, Wis.).
[1099] The yield of the translation reactions was determined by
spectrophometric measurement (OD260) and the yield for Luciferase
is shown in Table 38 and G-CSF is shown in Table 40.
[1100] The luciferase and G-CSF modified mRNA were also subjected
to an enzymatic capping reaction and each modified mRNA capping
reaction was evaluated for yield by spectrophometic measurement
(OD260) and correct size assessed using bioanalyzer. The yield from
the capping reaction for luciferase is shown in Table 39 and G-CSF
is shown in Table 41.
TABLE-US-00039 TABLE 38 In vitro transcription chemistry and yield
for Luciferase modified mRNA Chemical Modification Yield (ug)
2'Fluorocytosine 71.4 2'Fluorouridine 57.5
5-methylcytosine/pseudouridine, test A 26.4
5-methylcytosine/N1-methylpseudouridine, test A 73.3
N1-acetylcytidine/2-fluorouridine 202.2
5-methylcytidine/2-fluorouridine 131.9
2-fluorocytosine/pseudouridine 119.3
2-fluorocytosine/N1-methylpseudouridine 107.0
2-fluorocytosine/2-thiouridine 34.7 2-fluorocytosine/5-bromouridine
81.0 2-fluorocytosine/2-fluorouridine 80.4
2-fluoroguanine/5-methylcytosine 61.2
2-fluoroguanine/5-methylcytosine/pseudouridine 65.0
2-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 41.2
2-fluoroguanine/pseudouridine 79.1
2-fluoroguanine/N1-methylpseudouridine 74.6
5-methylcytidine/pseudouridine, test B 91.8
5-methylcytidine/N1-methylpseudouridine, test B 72.4
2'fluoroadenosine 190.98
TABLE-US-00040 TABLE 39 Capping chemistry and yield for Luciferase
modified mRNA Chemical Modification Yield (ug) 2'Fluorocytosine
19.2 2'Fluorouridine 16.7 5-methylcytosine/pseudouridine, test A
7.0 5-methylcytosine/N1-methylpseudouridine, test A 21.5
N1-acetylcytidine/2-fluorouridine 47.5
5-methylcytidine/2-fluorouridine 53.2
2-fluorocytosine/pseudouridine 58.4
2-fluorocytosine/N1-methylpseudouridine 26.2
2-fluorocytosine/2-thiouridine 12.9 2-fluorocytosine/5-bromouridine
26.5 2-fluorocytosine/2-fluorouridine 35.7
2-fluoroguanine/5-methylcytosine 24.7
2-fluoroguanine/5-methylcytosine/pseudouridine 32.3
2-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 31.3
2-fluoroguanine/pseudouridine 20.9
2-fluoroguanine/N1-methylpseudouridine 29.8
5-methylcytidine/pseudouridine, test B 58.2
5-methylcytidine/N1-methylpseudouridine, test B 44.4
TABLE-US-00041 TABLE 40 In vitro transcription chemistry and yield
for G-CSF modified mRNA Chemical Modification Yield (ug)
2'Fluorocytosine 56.5 2'Fluorouridine 79.4
5-methylcytosine/pseudouridine, test A 21.2
5-methylcytosine/N1-methylpseudouridine, test A 77.1
N1-acetylcytidine/2-fluorouridine 168.6
5-methylcytidine/2-fluorouridine 134.7
2-fluorocytosine/pseudouridine 97.8
2-fluorocytosine/N1-methylpseudouridine 103.1
2-fluorocytosine/2-thiouridine 58.8 2-fluorocytosine/5-bromouridine
88.8 2-fluorocytosine/2-fluorouridine 93.9
2-fluoroguanine/5-methylcytosine 97.3
2-fluoroguanine/5-methylcytosine/pseudouridine 96.0
2-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 82.0
2-fluoroguanine/pseudouridine 68.0
2-fluoroguanine/N1-methylpseudouridine 59.3
5-methylcytidine/pseudouridine, test B 58.7
5-methylcytidine/N1-methylpseudouridine, test B 78.0
TABLE-US-00042 TABLE 41 Capping chemistry and yield for G-CSF
modified mRNA Chemical Modification Yield (ug) 2'Fluorocytosine
16.9 2'Fluorouridine 17.0 5-methylcytosine/pseudouridine, test A
10.6 5-methylcytosine/N1-methylpseudouridine, test A 22.7
N1-acetylcytidine/2-fluorouridine 19.9
5-methylcytidine/2-fluorouridine 21.3
2-fluorocytosine/pseudouridine 65.2
2-fluorocytosine/N1-methylpseudouridine 58.9
2-fluorocytosine/2-thiouridine 41.2 2-fluorocytosine/5-bromouridine
35.8 2-fluorocytosine/2-fluorouridine 36.7
2-fluoroguanine/5-methylcytosine 36.6
2-fluoroguanine/5-methylcytosine/pseudouridine 37.3
2-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 30.7
2-fluoroguanine/pseudouridine 29.0
2-fluoroguanine/N1-methylpseudouridine 22.7
5-methylcytidine/pseudouridine, test B 60.4
5-methylcytidine/N1-methylpseudouridine, test B 33.0
Example 86. 2'O-methyl and 2'Fluoro Compounds
[1101] Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) were produced as fully modified versions with the chemistries
in Table 42 and transcribed using mutant T7 polymerase
(Durascribe.RTM. T7 Transcription kit (Cat. No. DS010925)
(Epicentre.RTM., Madison, Wis.). 2' fluoro-containing mRNA were
made using Durascribe T7, however, 2'Omethyl-containing mRNA could
not be transcribed using Durascribe T7.
[1102] Incorporation of 2'Omethyl modified mRNA might possibly be
accomplished using other mutant T7 polymerases (Nat Biotechnol.
(2004) 22:1155-1160; Nucleic Acids Res. (2002) 30:e138).
Alternatively, 2'OMe modifications could be introduced
post-transcriptionally using enzymatic means.
[1103] Introduction of modifications on the 2' group of the sugar
has many potential advantages. 2'OMe substitutions, like 2' fluoro
substitutions are known to protect against nucleases and also have
been shown to abolish innate immune recognition when incorporated
into other nucleic acids such as siRNA and anti-sense (incorporated
in its entirety, Crooke, ed. Antisense Drug Technology, 2.sup.nd
edition; Boca Raton: CRC press).
[1104] The 2'Fluoro-modified mRNA were then transfected into HeLa
cells to assess protein production in a cell context and the same
mRNA were also assessed in a cell-free rabbit reticulocyte system.
A control of unmodified luciferase (natural luciferase) was used
for both transcription experiments, a control of untreated and mock
transfected (Lipofectamine 2000 alone) were also analyzed for the
HeLa transfection and a control of no RNA was analyzed for the
rabbit reticulysates.
[1105] For the HeLa transfection experiments, the day before
transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.)
were harvested by treatment with Trypsin-EDTA solution
(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume
of 100 ul EMEM medium (supplemented with 10% FCS and 1.times.
Glutamax) per well in a 96-well cell culture plate (Corning,
Manassas, Va.). The cells were grown at 37.degree.G in 5% CO.sub.2
atmosphere overnight. Next day, 83 ng of the 2'fluoro-containing
luciferase modified RNA (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) with the chemical modification described in Table 42, were
diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand
Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island,
N.Y.) was used as transfection reagent and 0.2 ul were diluted in
10 ul final volume of OPTI-MEM. After 5 minutes of incubation at
room temperature, both solutions were combined and incubated an
additional 15 minute at room temperature. Then the 20 ul combined
solution was added to the 100 ul cell culture medium containing the
HeLa cells and incubated at room temperature. After 18 to 22 hours
of incubation cells expressing luciferase were lysed with 100 ul of
Passive Lysis Buffer (Promega, Madison, Wis.) according to
manufacturer instructions. Aliquots of the lysates were transferred
to white opaque polystyrene 96-well plates (Corning, Manassas, Va.)
and combined with 100 ul complete luciferase assay solution
(Promega, Madison, Wis.). The lysate volumes were adjusted or
diluted until no more than 2 mio relative light units (RLU) per
well were detected for the strongest signal producing samples and
the RLUs for each chemistry tested are shown in Table 42. The plate
reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The
background signal of the plates without reagent was about 200
relative light units per well.
[1106] For the rabbit reticulocyte lysate assay,
2'-fluoro-containing luciferase mRNA were diluted in sterile
nuclease-free water to a final amount of 250 ng in 10 ul and added
to 40 ul of freshly prepared Rabbit Reticulocyte Lysate and the in
vitro translation reaction was done in a standard 1.5 mL
polypropylene reaction tube (Thermo Fisher Scientific, Waltham,
Mass.) at 30.degree. C. in a dry heating block. The translation
assay was done with the Rabbit Reticulocyte Lysate
(nuclease-treated) kit (Promega, Madison, Wis.) according to the
manufacturer's instructions. The reaction buffer was supplemented
with a one-to-one blend of provided amino acid stock solutions
devoid of either Leucine or Methionine resulting in a reaction mix
containing sufficient amounts of both amino acids to allow
effective in vitro translation. After 60 minutes of incubation, the
reaction was stopped by placing the reaction tubes on ice.
[1107] Aliquots of the in vitro translation reaction containing
luciferase modified RNA were transferred to white opaque
polystyrene 96-well plates (Corning, Manassas, Va.) and combined
with 100 ul complete luciferase assay solution (Promega, Madison,
Wis.). The volumes of the in vitro translation reactions were
adjusted or diluted until no more than 2 mio relative light units
(RLUs) per well were detected for the strongest signal producing
samples and the RLUs for each chemistry tested are shown in Table
43. The plate reader was a BioTek Synergy H1 (BioTek, Winooski,
Vt.). The background signal of the plates without reagent was about
160 relative light units per well.
[1108] As can be seen in Table 42 and 43, multiple
2'Fluoro-containing compounds are active in vitro and produce
luciferase protein.
TABLE-US-00043 TABLE 42 HeLa Cells Chemical Concentration Volume
Yield Modification (ug/ml) (ul) (ug) RLU 2'Fluoroadenosine 381.96
500 190.98 388.5 2'Fluorocytosine 654.56 500 327.28 2420
2'Fluoroguanine 541.795 500 270.90 11,705.5 2'Flurorouridine
944.005 500 472.00 6767.5 Natural luciferase N/A N/A N/A 133,853.5
Mock N/A N/A N/A 340 Untreated N/A N/A N/A 238
TABLE-US-00044 TABLE 43 Rabbit Reticulysates Chemical Modification
RLU 2'Fluoroadenosine 162 2'Fluorocytosine 208 2'Fluoroguanine
371,509 2'Flurorouridine 258 Natural luciferase 2,159,968 No RNA
156
Example 87. Luciferase in HeLa Cells Using a Combination of
Modifications
[1109] To evaluate using of 2'fluoro-modified mRNA in combination
with other modification a series of mRNA were transcribed using
either wild-type T7 polymerase (non-fluoro-containing compounds) or
using mutant T7 polymerases (fluyoro-containing compounds) as
described in Example 86. All modified mRNA were tested by in vitro
transfection in HeLa cells.
[1110] The day before transfection, 20,000 HeLa cells (ATCC no.
CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA
solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a
total volume of 100 ul EMEM medium (supplemented with 10% FCS and
1.times. Glutamax) per well in a 96-well cell culture plate
(Corning, Manassas, Va.). The cells were grown at 37.degree.G in 5%
CO.sub.2 atmosphere overnight. Next day, 83 ng of Luciferase
modified RNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail of
approximately 160 nucleotides not shown in sequence; 5'cap, Cap1)
with the chemical modification described in Table 44, were diluted
in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,
N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)
was used as transfection reagent and 0.2 ul were diluted in 10 ul
final volume of OPTI-MEM. After 5 minutes of incubation at room
temperature, both solutions were combined and incubated an
additional 15 minute at room temperature. Then the 20 ul combined
solution was added to the 100 ul cell culture medium containing the
HeLa cells and incubated at room temperature.
[1111] After 18 to 22 hours of incubation cells expressing
luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega,
Madison, Wis.) according to manufacturer instructions. Aliquots of
the lysates were transferred to white opaque polystyrene 96-well
plates (Corning, Manassas, Va.) and combined with 100 ul complete
luciferase assay solution (Promega, Madison, Wis.). The lysate
volumes were adjusted or diluted until no more than 2 mio relative
light units (RLU) per well were detected for the strongest signal
producing samples and the RLUs for each chemistry tested are shown
in Table 44. The plate reader was a BioTek Synergy H1 (BioTek,
Winooski, Vt.). The background signal of the plates without reagent
was about 200 relative light units per well.
[1112] As evidenced in Table 44, most combinations of modifications
resulted in mRNA which produced functional luciferase protein,
including all the non-flouro containing compounds and many of the
combinations containing 2'fluro modifications.
TABLE-US-00045 TABLE 44 Luciferase Chemical Modification RLU
N4-acetylcytidine/pseudouridine 113,796
N4-acetylcytidine/N1-methylpseudouridine 316,326
5-methylcytidine/5-methoxyuridine 24,948
5-methylcytidine/5-methyluridine 43,675 5-methylcytidine/half of
the uridines modified with 50% 41,601 2-thiouridine
5-methylcytidine/2-thiouridine 1,102 5-methylcytidine/pseudouridine
51,035 5-methylcytidine/N1 methyl pseudouridine 152,151
N4-acetylcytidine/2'Fluorouridine triphosphate 288
5-methylcytidine/2'Fluorouridine triphosphate 269 2'Fluorocytosine
triphosphate/pseudouridine 260 2'Fluorocytosine
triphosphate/N1-methylpseudouridine 412 2'Fluorocytosine
triphosphate/2-thiouridine 427 2'Fluorocytosine
triphosphate/5-bromouridine 253 2'Fluorocytosine
triphosphate/2'Fluorouridine triphosphate 184 2'Fluoroguanine
triphosphate/5-methylcytidine 321 2'Fluoroguanine
triphosphate/5-methylcytidine/Pseudouridine 207
2'Fluoroguanine/5-methylcytidine/N1 methylpsuedouridine 235
2'Fluoroguanine/pseudouridine 218
2'Fluoroguanine/N1-methylpsuedouridine 247
5-methylcytidine/pseudouridine, test A 13,833
5-methylcytidine/N-methylpseudouridine, test A 598 2'Fluorocytosine
triphosphate 201 2'Fluorouridine triphosphate 305
5-methylcytidine/pseudouridine, test B 115,401
5-methylcytidine/N-methylpseudouridine, test B 21,034 Natural
luciferase 30,801 Untreated 344 Mock 262
Example 88. G-CSF In Vitro Transcription
[1113] To assess the activity of all our different chemical
modifications in the context of a second open reading frame, we
replicated experiments previously conducted using luciferase mRNA,
with human G-CSF mRNA. G-CSF mRNA (mRNA sequence shown in SEQ ID
NO: 1; polyA tail of approximately 160 nucleotides not shown in
sequence; 5'cap, Cap1) were fully modified with the chemistries in
Tables 45 and 46 using wild-type T7 polymerase (for all
non-fluoro-containing compounds) or mutant T7 polymerase (for all
fluoro-containing compounds). The mutant T7 polymerase was obtained
commercially (Durascribe.RTM. T7 Transcription kit (Cat. No.
DS010925) (Epicentre.RTM., Madison, Wis.).
[1114] The modified RNA in Tables 45 and 46 were transfected in
vitro in HeLa cells or added to rabbit reticulysates (250 ng of
modified mRNA) as indicated. A control of untreated, mock
transfected (transfection reagent alone), G-CSF fully modified with
5-methylcytosine and N1-methylpseudouridine or luciferase control
(mRNA sequence shown in SEQ ID NO: 3; polyA tail of approximately
160 nucleotides not shown in sequence; 5'cap, Cap1) fully modified
with 5-methylcytosine and N1-methylpseudouridine were also
analyzed. The expression of G-CSF protein was determined by ELISA
and the values are shown in Tables 45 and 46. In Table 45, "NT"
means not tested.
[1115] As shown in Table 45, many, but not all, chemical
modifications resulted in human G-CSF protein production. These
results from cell-based and cell-free translation systems correlate
very nicely with the same modifications generally working or not
working in both systems. One notable exception is 5-formylcytidine
modified G-CSF mRNA which worked in the cell-free translation
system, but not in the HeLa cell-based transfection system. A
similar difference between the two assays was also seen with
5-formylcytidine modified luciferase mRNA.
[1116] As demonstrated in Table 46, many, but not all, G-CSF mRNA
modified chemistries (when used in combination) demonstrated in
vivo activity. In addition the presence of N1-methylpseudouridine
in the modified mRNA (with N4-acetylcytidine or 5 methylcytidine)
demonstrated higher expression than when the same combinations
where tested using with pseudouridine. Taken together, these data
demonstrate that N1-methylpseudouridine containing G-CSF mRNA
results in improved protein expression in vitro.
TABLE-US-00046 TABLE 45 G-CSF Expression G-CSF protein G-CSF
(pg/ml) protein Rabbit (pg/ml) reticulysates Chemical Modification
HeLa cells cells Pseudouridine 1,150,909 147,875 5-methyluridine
347,045 147,250 2-thiouridine 417,273 18,375 N1-methylpseudouridine
NT 230,000 4-thiouridine 107,273 52,375 5-methoxyuridine 1,715,909
201,750 5-methylcytosine/pseudouridine, Test A 609,545 119,750
5-methylcytosine/N1-methylpseudouridine, 1,534,318 110,500 Test A
2'-Fluoro-guanosine 11,818 0 2'-Fluoro-uridine 60,455 0
5-methylcytosine/pseudouridine, Test B 358,182 57,875
5-methylcytosine/N1-methylpseudouridine, 1,568,636 76,750 Test B
5-Bromo-uridine 186,591 72,000 5-(2carbomethoxyvinyl) uridine 1,364
0 5-[3(1-E-propenylamino) uridine 27,955 32,625
.alpha.-thio-cytidine 120,455 42,625
5-methylcytosine/pseudouridine, Test C 882,500 49,250
N1-methyl-adenosine 4,773 0 N6-methyl-adenosine 1,591 0
5-methyl-cytidine 646,591 79,375 N4-acetylcytidine 39,545 8,000
5-formyl-cytidine 0 24,000 5-methylcytosine/pseudouridine, Test D
87,045 47,750 5-methylcytosine/N1-methylpseudouridine, 1,168,864
97,125 Test D Mock 909 682 Untreated 0 0
5-methylcytosine/N1-methylpseudouridine, 1,106,591 NT Control
Luciferase control NT 0
TABLE-US-00047 TABLE 46 Combination Chemistries in HeLa cells G-CSF
protein (pg/ml) Chemical Modification HeLa cells
N4-acetylcytidine/pseudouridine 537,273
N4-acetylcytidine/N1-methylpseudouridine 1,091,818
5-methylcytidine/5-methoxyuridine 516,136
5-methylcytidine/5-bromouridine 48,864
5-methylcytidine/5-methyluridine 207,500
5-methylcytidine/2-thiouridine 33,409
N4-acetylcytidine/5-bromouridine 211,591
N4-acetylcytidine/2-thiouridine 46,136
5-methylcytosine/pseudouridine 301,364
5-methylcytosine/N1-methylpseudouridine 1,017,727
N4-acetylcytidine/2'Fluorouridine triphosphate 62,273
5-methylcytidine/2'Fluorouridine triphosphate 49,318
2'Fluorocytosine triphosphate/pseudouridine 7,955 2'Fluorocytosine
triphosphate/N1-methylpseudouridine 1,364 2'Fluorocytosine
triphosphate/2-thiouridine 0 2'Fluorocytosine
triphosphate/5-bromouridine 1,818 2'Fluorocytosine
triphosphate/2'Fluorouridine triphosphate 909 2'Fluoroguanine
triphosphate/5-methylcytidine 0 2'Fluoroguanine
triphosphate/5-methylcytidine/ 0 pseudouridine 2'Fluoroguanine
triphosphate/5-methylcytidine/N1 1,818 methylpseudouridine
2'Fluoroguanine triphosphate/pseudouridine 1,136 2'Fluoroguanine
triphosphate/2'Fluorocytosine 0 triphosphate/N1-methylpseudouridine
5-methylcytidine/pseudouridine 617,727
5-methylcytidine/N1-methylpseudouridine 747,045
5-methylcytidine/pseudouridine 475,455
5-methylcytidine/N1-methylpseudouridine 689,091
5-methylcytosine/N1-methylpseudouridine, Control 1 848,409
5-methylcytosine/N1-methylpseudouridine, Control 2 581,818 Mock 682
Untreated 0 Luciferase 2'Fluorocytosine triphosphate 0 Luciferase
2'Fluorouridine triphosphate 0
Example 89. Screening of Chemistries
[1117] The tables listed in below (Tables 47-49) summarize much of
the in vitro and in vitro screening data with the different
compounds presented in the previous examples. A good correlation
exists between cell-based and cell-free translation assays. The
same chemistry substitutions generally show good concordance
whether tested in the context of luciferase or G-CSF mRNA. Lastly,
N1-methylpseudouridine containing mRNA show a very high level of
protein expression with little to no detectable cytokine
stimulation in vitro and in vivo, and is superior to mRNA
containing pseudouridine both in vitro and in vivo.
[1118] Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA
tail of approximately 160 nucleotides not shown in sequence; 5'cap,
Cap1) were modified with naturally and non-naturally occurring
chemistries described in Tables 47 and 48 or combination
chemistries described in Table 48 and tested using methods
described herein.
[1119] In Tables 47 and 48, "*" refers to in vitro transcription
reaction using a mutant T7 polymerase (Durascribe.RTM. T7
Transcription kit (Cat. No. DS010925) (Epicentre.RTM., Madison,
Wis.); "**" refers to the second result in vitro transcription
reaction using a mutant T7 polymerase (Durascribe.RTM. T7
Transcription kit (Cat. No. DS010925) (Epicentre.RTM., Madison,
Wis.); "***" refers to production seen in cell free translations
(rabbit reticulocyte lysates); the protein production of HeLa is
judged by "+," "+/-" and "-"; when referring to G-CSF PBMC "++++"
means greater than 6,000 pg/ml G-CSF, "+++" means greater than
3,000 pg/ml G-CSF, "++" means greater than 1,500 pg/ml G-CSF, "+"
means greater than 300 pg/ml G-CSF, "+/-" means 150-300 pg/ml G-CSF
and the background was about 110 pg/ml; when referring to cytokine
PBMC "++++" means greater than 1,000 pg/ml interferon-alpha
(IFN-alpha), "+++" means greater than 600 pg/ml IFN-alpha, "++"
means greater than 300 pg/ml IFN-alpha, "+" means greater than 100
pg/ml IFN-alpha, "-" means less than 100 pg/ml and the background
was about 70 pg/ml; and "NT" means not tested. In Table 48, the
protein production was evaluated using a mutant T7 polymerase
(Durascribe.RTM. T7 Transcription kit (Cat. No. DS010925)
(Epicentre.RTM., Madison, Wis.).
TABLE-US-00048 TABLE 47 Naturally Occurring Protein Protein Protein
Cytokines In Vivo In Vivo Common Name IVT IVT (Luc; (G-CSF; (G-CSF;
(G-CSF; Protein Protein (symbol) (Luc) (G-CSF) HeLa) HeLa) PBMC)
PBMC) (Luc) (G-CSF) 1-methyladenosine Fail Pass NT - +/- ++ NT NT
(m.sup.1A) N.sup.6-methyladenosine Pass Pass - - +/- ++++ NT NT
(m.sup.6A) 2'-O- Fail* Not Done NT NT NT NT NT NT methyladenosine
(Am) 5-methylcytidine Pass Pass + + + ++ + NT (m.sup.5C)
2'-O-methylcytidine Fail* Not Done NT NT NT NT NT NT (Cm)
2-thiocytidine (s.sup.2C) Fail Fail NT NT NT NT NT NT
N.sup.4-acetylcytidine Pass Pass + + +/- +++ + NT (ac.sup.4C)
5-formylcytidine Pass Pass -*** -*** - + NT NT (f.sup.5C) 2'-O-
Fail* Not Done NT NT NT NT NT NT methylguanosine (Gm) inosine (I)
Fail Fail NT NT NT NT NT NT pseudouridine (Y) Pass Pass + + ++ + +
NT 5-methyluridine Pass Pass + + +/- + NT NT (m.sup.5U)
2'-O-methyluridine Fail* Not Done NT NT NT NT NT NT (Um) 1- Pass
Pass + Not Done ++++ - + NT methylpseudouridine (m.sup.1Y)
2-thiouridine (s.sup.2U) Pass Pass - + + + NT NT 4-thiouridine
(s.sup.4U) Fail Pass + +/- ++ NT NT 5-methoxyuridine Pass Pass + +
++ - + NT (mo.sup.5U) 3-methyluridine Fail Fail NT NT NT NT NT NT
(m.sup.3U)
TABLE-US-00049 TABLE 48 Non-Naturally Occurring Protein Protein
Protein Cytokines In Vivo In Vivo IVT IVT (Luc; (G-CSF; (G-CSF;
(G-CSF; Protein Protein Common Name (Luc) (G-CSF) HeLa) HeLa) PBMC)
PBMC) (Luc) (G-CSF) 2'-F-ara-guanosine Fail Fail NT NT NT NT NT NT
2'-F-ara-adenosine Fail Fail NT NT NT NT NT NT 2'-F-ara-cytidine
Fail Fail NT NT NT NT NT NT 2'-F-ara-uridine Fail Fail NT NT NT NT
NT NT 2'-F-guanosine Fail/Pass** Pass/Fail** +** +/- - + + NT
2'-F-adenosine Fail/Pass** Fail/Fail** -** NT NT NT NT NT
2'-F-cytidine Fail/Pass** Fail/Pass** +** NT NT NT + NT
2'-F-uridine Fail/Pass** Pass/Pass** +** + +/- + - NT
2'-OH-ara-guanosine Fail Fail NT NT NT NT NT NT 2'-OH-ara-adenosine
Not Done Not Done NT NT NT NT NT NT 2'-OH-ara-cytidine Fail Fail NT
NT NT NT NT NT 2'-OH-ara-uridine Fail Fail NT NT NT NT NT NT
5-Br-Uridine Pass Pass + + + + + 5-(2- Pass Pass - - +/- -
carbomethoxyvinyl) Uridine 5-[3-(1-E- Pass Pass - + + -
Propenylamino) Uridine (aka Chem 5) N6-(19-Amino- Fail Fail NT NT
NT NT NT NT pentaoxanonadecyl) A 2-Dimethylamino Fail Fail NT NT NT
NT NT NT guanosine 6-Aza-cytidine Fail Fail NT NT NT NT NT NT
a-Thio-cytidine Pass Pass + + +/- +++ NT NT Pseudo-isocytidine NT
NT NT NT NT NT NT NT 5-Iodo-uridine NT NT NT NT NT NT NT NT
a-Thio-uridine NT NT NT NT NT NT NT NT 6-Aza-uridine NT NT NT NT NT
NT NT NT Deoxy-thymidine NT NT NT NT NT NT NT NT a-Thio guanosine
NT NT NT NT NT NT NT NT 8-Oxo-guanosine NT NT NT NT NT NT NT NT
O6-Methyl- NT NT NT NT NT NT NT NT guanosine 7-Deaza-guanosine NT
NT NT NT NT NT NT NT 6-Chloro-purine NT NT NT NT NT NT NT NT
a-Thio-adenosine NT NT NT NT NT NT NT NT 7-Deaza-adenosine NT NT NT
NT NT NT NT NT 5-iodo-cytidine NT NT NT NT NT NT NT NT
[1120] In Table 49, the protein production of HeLa is judged by
"+," "+/-" and "-"; when referring to G-CSF PBMC "++++" means
greater than 6,000 pg/ml G-CSF, "+++" means greater than 3,000
pg/ml G-CSF, "++" means greater than 1,500 pg/ml G-CSF, "+" means
greater than 300 pg/ml G-CSF, "+/-" means 150-300 pg/ml G-CSF and
the background was about 110 pg/ml; when referring to cytokine PBMC
"++++" means greater than 1,000 pg/ml interferon-alpha (IFN-alpha),
"+++" means greater than 600 pg/ml IFN-alpha, "++" means greater
than 300 pg/ml IFN-alpha, "+" means greater than 100 pg/ml
IFN-alpha, "-" means less than 100 pg/ml and the background was
about 70 pg/ml; "WT" refers to the wild type T7 polymerase, "MT"
refers to mutant T7 polymerase (Durascribe.RTM. T7 Transcription
kit (Cat. No. DS010925) (Epicentre.RTM., Madison, Wis.) and "NT"
means not tested.
TABLE-US-00050 TABLE 49 Combination Chemistry Protein Protein
Protein Cytokines In Vivo Cytidine Uridine IVT IVT (Luc; (G-CSF;
(G-CSF; (G-CSF; Protein analog analog Purine Luc (G-CSF) HeLa)
HeLa) PBMC) PBMC) (Luc) N4- pseudouridine A, G Pass Pass + + NT NT
+ acetylcytidine WT WT N4- N1- A, G Pass Pass + + NT NT +
acetylcytidine methylpseudouridine WT WT 5- 5- A, G Pass Pass + +
NT NT + methylcytidine methoxyuridine WT WT 5- 5- A, G Pass Pass
Not Done + NT NT methylcytidine bromouridine WT WT 5- 5- A, G Pass
Pass + + NT NT + methylcytidine methyluridine WT WT 5- 50% 2- A, G
Pass Pass + NT NT NT + methylcytidine thiouridine; WT WT 50%
uridine 5- 100% 2- A, G Pass Pass - + NT NT methylcytidine
thiouridine WT WT 5- pseudouridine A, G Pass Pass + + ++ + +
methylcytidine WT WT 5- N1- A, G Pass Pass + + ++++ - +
methylcytidine methylpseudouridine WT WT N4- 2-thiouridine A, G Not
Done Pass Not Done + NT NT NT acetylcytidine WT N4- 5- A, G Not
Done Pass Not Done + NT NT NT acetylcytidine bromouridine WT N4- 2
A, G Pass Pass - + NT NT NT acetylcytidine Fluorouridine
triphosphate 5- 2 A, G Pass Pass - + NT NT NT methylcytidine
Fluorouridine triphosphate 2 pseudouridine A, G Pass Pass - + NT NT
NT Fluorocytosine triphosphate 2 N1- A, G Pass Pass - +/- NT NT NT
Fluorocytosine methylpseudouridine triphosphate 2 2-thiouridine A,
G Pass Pass - - NT NT NT Fluorocytosine triphosphate 2 5- A, G Pass
Pass - +/- NT NT NT Fluorocytosine bromouridine triphosphate 2 2 A,
G Pass Pass - +/- NT NT NT Fluorocytosine Fluorouridine
triphosphate triphosphate 5- uridine A, 2 Pass Pass - - NT NT NT
methylcytidine Fluoro GTP 5- pseudouridine A, 2 Pass Pass - - NT NT
NT methylcytidine Fluoro GTP 5- N1- A, 2 Pass Pass - +/- NT NT NT
methylcytidine methylpseudouridine Fluoro GTP 2 pseudouridine A, 2
Pass Pass - +/- NT NT NT Fluorocytosine Fluoro triphosphate FTP 2
N1- Fluorocytosine methylpseudouridine A, 2 Pass Pass - - NT NT NT
triphosphate Fluoro GTP
Example 90. 2'Fluoro Chemistries in PBMC
[1121] The ability of G-CSF modified mRNA (mRNA sequence shown in
SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shown
in sequence; 5'cap, Cap1) to trigger innate an immune response was
determined by measuring interferon-alpha (IFN-alpha) and tumor
necrosis factor-alpha (TNF-alpha) production. Use of in vitro PBMC
cultures is an accepted way to measure the immunostimulatory
potential of oligonucleotides (Robbins et al., Oligonucleotides
2009 19:89-102) and transfection methods are described herein.
Shown in Table 50 are the average from 2 or 3 separate PBMC donors
of the interferon-alpha (IFN-alpha) and tumor necrosis factor alpha
(TNF-alpha) production over time as measured by specific ELISA.
Controls of R848, P(I)P(C), LPS and Lipofectamine 2000 (L2000) were
also analyzed.
[1122] With regards to innate immune recognition, while both
modified mRNA chemistries largely prevented IFN-alpha and TNF-alpha
production relative to positive controls (R848, P(I)P(C)), 2'fluoro
compounds reduce IFN-alpha and TNF-alpha production even lower than
other combinations and N4-acetylcytidine combinations raised the
cytokine profile.
TABLE-US-00051 TABLE 50 IFN-alpha and TNF-alpha IFN-alpha:
TNF-alpha: 3 Donor 2 Donor Average Average (pg/ml) (pg/ml) L2000 1
361 P(I)P(C) 482 544 R848 45 8,235 LPS 0 6,889
N4-acetylcytidine/pseudouridine 694 528
N4-acetylcytidine/N1-methylpseudouridine 307 283
5-methylcytidine/5-methoxyuridine 0 411
5-methylcytidine/5-bromouridine 0 270
5-methylcytidine/5-methyluridine 456 428
5-methylcytidine/2-thiouridine 274 277
N4-acetylcytidine/2-thiouridine 0 285
N4-acetylcytidine/5-bromouridine 44 403
5-methylcytidine/pseudouridine 73 332
5-methylcytidine/N1-methylpseudouridine 31 280
N4-acetylcytidine/2'fluorouridine triphosphate 35 32
5-methylcytodine/2'fluorouridine triphosphate 24 0 2'fluorocytidine
triphosphate/N1- 0 11 methylpseudouridine 2'fluorocytidine
triphosphate/2-thiouridine 0 0
2'fluorocytidine/triphosphate5-bromouridine 12 2 2'fluorocytidine
triphosphate/2'fluorouridine 11 0 triphosphate 2'fluorocytidine
triphosphate/5-methylcytidine 14 23 2'fluorocytidine
triphosphate/5- 6 21 methylcytidine/pseudouridine 2'fluorocytidine
triphosphate/5- 3 15 methylcytidine/N1-methylpseudouridine
2'fluorocytidine triphosphate/pseudouridine 0 4 2'fluorocytidine
triphosphate/N1- 6 20 methylpseudouridine
5-methylcytidine/pseudouridine 82 18
5-methylcytidien/N1-methylpseudouridine 35 3
OTHER EMBODIMENTS
[1123] It is to be understood that while the present disclosure has
been described in conjunction with the detailed description
thereof, the foregoing description is intended to illustrate and
not limit the scope of the present disclosure, which is defined by
the scope of the appended claims. Other aspects, advantages, and
modifications are within the scope of the following claims.
Sequence CWU 1
1
61758RNAHomo Sapiens 1gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug gccggucccg 60cgacccaaag ccccaugaaa cuuauggccc ugcaguugcu
gcuuuggcac ucggcccucu 120ggacagucca agaagcgacu ccucucggac
cugccucauc guugccgcag ucauuccuuu 180ugaagugucu ggagcaggug
cgaaagauuc agggcgaugg agccgcacuc caagagaagc 240ucugcgcgac
auacaaacuu ugccaucccg aggagcucgu acugcucggg cacagcuugg
300ggauucccug ggcuccucuc ucguccuguc cgucgcaggc uuugcaguug
gcagggugcc 360uuucccagcu ccacuccggu uuguucuugu aucagggacu
gcugcaagcc cuugagggaa 420ucucgccaga auugggcccg acgcuggaca
cguugcagcu cgacguggcg gauuucgcaa 480caaccaucug gcagcagaug
gaggaacugg ggauggcacc cgcgcugcag cccacgcagg 540gggcaaugcc
ggccuuugcg uccgcguuuc agcgcagggc ggguggaguc cucguagcga
600gccaccuuca aucauuuuug gaagucucgu accgggugcu gagacaucuu
gcgcagccgu 660gaagcgcugc cuucugcggg gcuugccuuc uggccaugcc
cuucuucucu cccuugcacc 720uguaccucuu ggucuuugaa uaaagccuga guaggaag
75821838DNAHomo Sapiens 2taatacgact cactataggg aaataagaga
gaaaagaaga gtaagaagaa atataagagc 60caccatggaa gatgcgaaga acatcaagaa
gggacctgcc ccgttttacc ctttggagga 120cggtacagca ggagaacagc
tccacaaggc gatgaaacgc tacgccctgg tccccggaac 180gattgcgttt
accgatgcac atattgaggt agacatcaca tacgcagaat acttcgaaat
240gtcggtgagg ctggcggaag cgatgaagag atatggtctt aacactaatc
accgcatcgt 300ggtgtgttcg gagaactcat tgcagttttt catgccggtc
cttggagcac ttttcatcgg 360ggtcgcagtc gcgccagcga acgacatcta
caatgagcgg gaactcttga atagcatggg 420aatctcccag ccgacggtcg
tgtttgtctc caaaaagggg ctgcagaaaa tcctcaacgt 480gcagaagaag
ctccccatta ttcaaaagat catcattatg gatagcaaga cagattacca
540agggttccag tcgatgtata cctttgtgac atcgcatttg ccgccagggt
ttaacgagta 600tgacttcgtc cccgagtcat ttgacagaga taaaaccatc
gcgctgatta tgaattcctc 660gggtagcacc ggtttgccaa agggggtggc
gttgccccac cgcactgctt gtgtgcggtt 720ctcgcacgct agggatccta
tctttggtaa tcagatcatt cccgacacag caatcctgtc 780cgtggtacct
tttcatcacg gttttggcat gttcacgact ctcggctatt tgatttgcgg
840tttcagggtc gtacttatgt atcggttcga ggaagaactg tttttgagat
ccttgcaaga 900ttacaagatc cagtcggccc tccttgtgcc aacgcttttc
tcattctttg cgaaatcgac 960acttattgat aagtatgacc tttccaatct
gcatgagatt gcctcagggg gagcgccgct 1020tagcaaggaa gtcggggagg
cagtggccaa gcgcttccac cttcccggaa ttcggcaggg 1080atacgggctc
acggagacaa catccgcgat ccttatcacg cccgagggtg acgataagcc
1140gggagccgtc ggaaaagtgg tccccttctt tgaagccaag gtcgtagacc
tcgacacggg 1200aaaaaccctc ggagtgaacc agaggggcga gctctgcgtg
agagggccga tgatcatgtc 1260aggttacgtg aataaccctg aagcgacgaa
tgcgctgatc gacaaggatg ggtggttgca 1320ttcgggagac attgcctatt
gggatgagga tgagcacttc tttatcgtag atcgacttaa 1380gagcttgatc
aaatacaaag gctatcaggt agcgcctgcc gagctcgagt caatcctgct
1440ccagcacccc aacattttcg acgccggagt ggccgggttg cccgatgacg
acgcgggtga 1500gctgccagcg gccgtggtag tcctcgaaca tgggaaaaca
atgaccgaaa aggagatcgt 1560ggactacgta gcatcacaag tgacgactgc
gaagaaactg aggggagggg tagtctttgt 1620ggacgaggtc ccgaaaggct
tgactgggaa gcttgacgct cgcaaaatcc gggaaatcct 1680gattaaggca
aagaaaggcg ggaaaatcgc tgtctgataa gctgccttct gcggggcttg
1740ccttctggcc atgcccttct tctctccctt gcacctgtac ctcttggtct
ttgaataaag 1800cctgagtagg aaggcggccg ctcgagcatg catctaga
183831796RNAHomo sapiens 3gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug gaagaugcga 60agaacaucaa gaagggaccu gccccguuuu
acccuuugga ggacgguaca gcaggagaac 120agcuccacaa ggcgaugaaa
cgcuacgccc ugguccccgg aacgauugcg uuuaccgaug 180cacauauuga
gguagacauc acauacgcag aauacuucga aaugucggug aggcuggcgg
240aagcgaugaa gagauauggu cuuaacacua aucaccgcau cguggugugu
ucggagaacu 300cauugcaguu uuucaugccg guccuuggag cacuuuucau
cggggucgca gucgcgccag 360cgaacgacau cuacaaugag cgggaacucu
ugaauagcau gggaaucucc cagccgacgg 420ucguguuugu cuccaaaaag
gggcugcaga aaauccucaa cgugcagaag aagcucccca 480uuauucaaaa
gaucaucauu auggauagca agacagauua ccaaggguuc cagucgaugu
540auaccuuugu gacaucgcau uugccgccag gguuuaacga guaugacuuc
guccccgagu 600cauuugacag agauaaaacc aucgcgcuga uuaugaauuc
cucggguagc accgguuugc 660caaagggggu ggcguugccc caccgcacug
cuugugugcg guucucgcac gcuagggauc 720cuaucuuugg uaaucagauc
auucccgaca cagcaauccu guccguggua ccuuuucauc 780acgguuuugg
cauguucacg acucucggcu auuugauuug cgguuucagg gucguacuua
840uguaucgguu cgaggaagaa cuguuuuuga gauccuugca agauuacaag
auccagucgg 900cccuccuugu gccaacgcuu uucucauucu uugcgaaauc
gacacuuauu gauaaguaug 960accuuuccaa ucugcaugag auugccucag
ggggagcgcc gcuuagcaag gaagucgggg 1020aggcaguggc caagcgcuuc
caccuucccg gaauucggca gggauacggg cucacggaga 1080caacauccgc
gauccuuauc acgcccgagg gugacgauaa gccgggagcc gucggaaaag
1140ugguccccuu cuuugaagcc aaggucguag accucgacac gggaaaaacc
cucggaguga 1200accagagggg cgagcucugc gugagagggc cgaugaucau
gucagguuac gugaauaacc 1260cugaagcgac gaaugcgcug aucgacaagg
augggugguu gcauucggga gacauugccu 1320auugggauga ggaugagcac
uucuuuaucg uagaucgacu uaagagcuug aucaaauaca 1380aaggcuauca
gguagcgccu gccgagcucg agucaauccu gcuccagcac cccaacauuu
1440ucgacgccgg aguggccggg uugcccgaug acgacgcggg ugagcugcca
gcggccgugg 1500uaguccucga acaugggaaa acaaugaccg aaaaggagau
cguggacuac guagcaucac 1560aagugacgac ugcgaagaaa cugaggggag
ggguagucuu uguggacgag gucccgaaag 1620gcuugacugg gaagcuugac
gcucgcaaaa uccgggaaau ccugauuaag gcaaagaaag 1680gcgggaaaau
cgcugucuga uaagcugccu ucugcggggc uugccuucug gccaugcccu
1740ucuucucucc cuugcaccug uaccucuugg ucuuugaaua aagccugagu aggaag
17964854RNAHomo sapiens 4gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug guauccaagg 60gggaggagga caacauggcg aucaucaagg
aguucaugcg auucaaggug cacauggaag 120guucggucaa cggacacgaa
uuugaaaucg aaggagaggg ugaaggaagg cccuaugaag 180ggacacagac
cgcgaaacuc aaggucacga aagggggacc acuuccuuuc gccugggaca
240uucuuucgcc ccaguuuaug uacgggucca aagcauaugu gaagcauccc
gccgauauuc 300cugacuaucu gaaacucagc uuucccgagg gauucaagug
ggagcggguc augaacuuug 360aggacggggg uguagucacc guaacccaag
acucaagccu ccaagacggc gaguucaucu 420acaaggucaa acugcggggg
acuaacuuuc cgucggaugg gccggugaug cagaagaaaa 480cgaugggaug
ggaagcguca ucggagagga uguacccaga agauggugca uugaaggggg
540agaucaagca gagacugaag uugaaagaug ggggacauua ugaugccgag
gugaaaacga 600cauacaaagc gaaaaagccg gugcagcuuc ccggagcgua
uaaugugaau aucaaguugg 660auauuacuuc acacaaugag gacuacacaa
uugucgaaca guacgaacgc gcugagggua 720gacacucgac gggaggcaug
gacgaguugu acaaaugaua agcugccuuc ugcggggcuu 780gccuucuggc
caugcccuuc uucucucccu ugcaccugua ccucuugguc uuugaauaaa
840gccugaguag gaag 8545725RNAHomo sapiens 5gggaaauaag agagaaaaga
agaguaagaa gaaauauaag agccaccaug ggagugcacg 60agugucccgc gugguugugg
uugcugcugu cgcucuugag ccucccacug ggacugccug 120ugcugggggc
accacccaga uugaucugcg acucacgggu acuugagagg uaccuucuug
180aagccaaaga agccgaaaac aucacaaccg gaugcgccga gcacugcucc
cucaaugaga 240acauuacugu accggauaca aaggucaauu ucuaugcaug
gaagagaaug gaaguaggac 300agcaggccgu cgaagugugg caggggcucg
cgcuuuuguc ggaggcggug uugcgggguc 360aggcccuccu cgucaacuca
ucacagccgu gggagccccu ccaacuucau gucgauaaag 420cggugucggg
gcuccgcagc uugacgacgu ugcuucgggc ucugggcgca caaaaggagg
480cuauuucgcc gccugacgcg gccuccgcgg caccccuccg aacgaucacc
gcggacacgu 540uuaggaagcu uuuuagagug uacagcaauu uccuccgcgg
aaagcugaaa uuguauacug 600gugaagcgug uaggacaggg gaucgcugau
aagcugccuu cugcggggcu ugccuucugg 660ccaugcccuu cuucucuccc
uugcaccugu accucuuggu cuuugaauaa agccugagua 720ggaag
72561536RNAHomo sapiens 6gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaau gcagcgcguc 60aacaugauua uggccgaauc gccgggacuc
aucacaaucu gccucuuggg uuaucucuug 120ucggcagaau guaccguguu
cuuggaucac gaaaacgcga acaaaauucu uaaucgcccg 180aagcgguaua
acuccgggaa acuugaggag uuugugcagg gcaaucuuga acgagagugc
240auggaggaga aaugcuccuu ugaggaggcg agggaagugu uugaaaacac
agagcgaaca 300acggaguuuu ggaagcaaua cguagauggg gaccagugug
agucgaaucc gugccucaau 360gggggaucau guaaagauga caucaauagc
uaugaaugcu ggugcccguu uggguuugaa 420gggaagaacu gugagcugga
ugugacgugc aacaucaaaa acggacgcug ugagcaguuu 480uguaagaacu
cggcugacaa uaagguagua ugcucgugca cagagggaua ccggcuggcg
540gagaaccaaa aaucgugcga gcccgcaguc ccguucccuu gugggagggu
gagcguguca 600cagacuagca aguugacgag agcggagacu guauuccccg
acguggacua cgucaacagc 660accgaagccg aaacaauccu cgauaacauc
acgcagagca cucaguccuu caaugacuuu 720acgagggucg uaggugguga
ggacgcgaaa cccggucagu uccccuggca ggugguauug 780aacggaaaag
ucgaugccuu uuguggaggu uccauuguca acgagaagug gauugucaca
840gcggcacacu gcguagaaac aggagugaaa aucacgguag uggcgggaga
gcauaacauu 900gaagagacag agcacacgga acaaaagcga aaugucauca
gaaucauucc acaccauaac 960uauaacgcgg caaucaauaa guacaaucac
gacaucgcac uuuuggagcu ugacgaaccu 1020uuggugcuua auucguacgu
caccccuauu uguauugccg acaaagagua uacaaacauc 1080uucuugaaau
ucggcuccgg guacguaucg ggcuggggca gaguguucca uaaggguaga
1140uccgcacugg uguugcaaua ccucagggug ccccucgugg aucgagccac
uugucugcgg 1200uccaccaaau ucacaaucua caacaauaug uucugugcgg
gauuccauga aggugggaga 1260gauagcugcc agggagacuc aggggguccc
cacgugacgg aagucgaggg gacgucauuu 1320cugacgggaa uuaucucaug
gggagaggaa ugugcgauga aggggaaaua uggcaucuac 1380acuaaagugu
cacgguaugu caauuggauc aaggaaaaga cgaaacucac gugaucagcc
1440agcgcugccu ucugcggggc uugccuucug gccaugcccu ucuucucucc
cuugcaccug 1500uaccucuugg ucuuugaaua aagccugagu aggaag 1536
* * * * *