U.S. patent number 10,385,088 [Application Number 15/026,836] was granted by the patent office on 2019-08-20 for polynucleotide molecules and uses thereof.
This patent grant is currently assigned to ModernaTX, Inc.. The grantee listed for this patent is ModernaTX, Inc.. Invention is credited to Andrew W. Fraley, Atanu Roy, Matthew Stanton.
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United States Patent |
10,385,088 |
Fraley , et al. |
August 20, 2019 |
Polynucleotide molecules and uses thereof
Abstract
The present disclosure provides alternative sugar moieties and
polynucleotides comprising such sugar moieties, and methods of use
thereof.
Inventors: |
Fraley; Andrew W. (Arlington,
MA), Roy; Atanu (Stoneham, MA), Stanton; Matthew
(Marlton, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
ModernaTX, Inc. (Cambridge,
MA)
|
Family
ID: |
52779294 |
Appl.
No.: |
15/026,836 |
Filed: |
October 2, 2014 |
PCT
Filed: |
October 02, 2014 |
PCT No.: |
PCT/US2014/058891 |
371(c)(1),(2),(4) Date: |
April 01, 2016 |
PCT
Pub. No.: |
WO2015/051169 |
PCT
Pub. Date: |
April 09, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160237108 A1 |
Aug 18, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61885979 |
Oct 2, 2013 |
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61896478 |
Oct 28, 2013 |
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61915907 |
Dec 13, 2013 |
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62036944 |
Aug 13, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H
19/167 (20130101); C07K 14/525 (20130101); C07H
19/20 (20130101); C07H 21/02 (20130101); C07K
14/565 (20130101); C07H 19/067 (20130101); C07H
19/10 (20130101); C07K 14/535 (20130101) |
Current International
Class: |
C07H
19/10 (20060101); C07H 19/20 (20060101); C07K
14/525 (20060101); C07K 14/535 (20060101); C07H
21/02 (20060101); C07H 19/167 (20060101); C07H
19/067 (20060101); C07K 14/565 (20060101) |
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|
Primary Examiner: Crane; Lawrence E
Attorney, Agent or Firm: Clark & Elbing LLP
Claims
The invention claimed is:
1. An mRNA 30 to 5000 nucleotides in length encoding a polypeptide,
the mRNA comprising at least one backbone moiety having the
structure: ##STR00057## wherein B is a nucleobase; or a salt
thereof.
2. The mRNA of claim 1, wherein B has the structure:
##STR00058##
3. The mRNA of claim 1, wherein B has the structure:
##STR00059##
4. The mRNA of claim 1, wherein B has the structure:
##STR00060##
5. The mRNA of claim 1, wherein B has the structure:
##STR00061##
6. The mRNA of claim 1, wherein B has the structure:
##STR00062##
7. The mRNA of claim 1, wherein B has the structure:
##STR00063##
8. The mRNA of claim 1, wherein B has the structure:
##STR00064##
9. The mRNA of claim 1, wherein B has the structure:
##STR00065##
10. The mRNA of claim 1, further comprising: (a) a 5' untranslated
region; (b) a 3' untranslated region; and (c) a 5' cap
structure.
11. The mRNA of claim 10, wherein the 5' cap structure has the
structure: ##STR00066## or a salt thereof.
12. The mRNA of claim 10, further comprising a poly-A tail 100 to
250 nucleotides in length at the 3'-terminus of the 3'-untranslated
region.
Description
REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable
form. The computer readable form is incorporated herein by
reference.
BACKGROUND
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.
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).
There is a need in the art for biological modalities to address the
modulation of intracellular translation of polynucleotides. The
present invention solves this problem by providing new mRNA
molecules incorporating chemical alternatives which impart
properties which are advantageous to therapeutic development.
SUMMARY OF THE INVENTION
The present disclosure provides nucleosides, nucleotides, and
polynucleotides having an alternative nucleobase, sugar, or
backbone and polynucleotides containing the same.
The present invention provides polynucleotides which may be
isolated and/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
alternative sugar moiety as compared to ribose. 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. Such polynucleotides may also be codon optimized.
In a first aspect, the invention features a compound of Formula
I:
##STR00001##
wherein the dotted line represents an optional double bond;
B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, O, or NR.sup.7;
R.sup.1 is hydrogen or fluorine;
R.sup.2 is hydrogen, fluorine, cyano, azido, or optionally
substituted C.sub.1-C.sub.6 alkyl;
R.sup.3 and R.sup.4 are independently hydrogen, optionally
substituted hydroxyl, or fluorine;
R.sup.5 and R.sup.6 are independently hydrogen or optionally
substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 and R.sup.6 are
combined to form an optionally substituted C.sub.3-C.sub.6
cycloalkyl, provided that one of R.sup.5 and R.sup.6 is absent when
the dotted line is a double bond;
R.sup.7 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl,
or optionally substituted amino;
each Y.sup.2 is independently hydroxyl or optionally substituted
C.sub.1-C.sub.6 heteroalkyl;
each Y.sup.3 is independently absent, O, or S;
each Y.sup.5 is independently O, NH, or CR.sup.8R.sup.9;
each Y.sup.6 is O or S;
each Y.sup.7 is O or NH; and
each R.sup.8 and R.sup.9 is independently hydrogen, fluorine, or
optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 and
R.sup.9 are combined to form an optionally substituted
C.sub.3-C.sub.6 cycloalkyl, provided that one of R.sup.8 and
R.sup.9 is absent when the dotted line is a double bond;
wherein if n is 0, X is O, R.sup.1, R.sup.2, R.sup.4, R.sup.5, and
R.sup.6 are hydrogen, and Y.sup.5 is O, then at least one of
Y.sup.1 and Y.sup.4 is optionally substituted amino, and, if m is
0, n is 1, Y.sup.1 is optionally substituted amino, Y.sup.2 is
optionally substituted C.sub.1-C.sub.6 heteroalkyl, Y.sup.3 is
absent, Y.sup.7 is O, X is O, and R.sup.1, R.sup.2, R.sup.4,
R.sup.5, and R.sup.6 are hydrogen, then Y.sup.4 is optionally
substituted amino; or a salt thereof.
In another aspect, the invention features a compound of Formula
II:
##STR00002##
wherein B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, or O;
R.sup.1 and R.sup.2 are independently hydrogen or fluorine;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl
(e.g., dimethoxytrityl), or optionally substituted amino;
Y.sup.2 is hydroxyl or optionally substituted C.sub.1-C.sub.6
heteroalkyl (e.g., optionally substituted C.sub.1-C.sub.6 alkoxy
such as .beta.-cyanoethoxy);
Y.sup.3 is absent or O;
wherein if n is 0, X is O, R.sup.1 and R.sup.2 are hydrogen, then
at least one of Y.sup.1 and Y.sup.4 is not hydroxyl or protected
hydroxyl, and, if m is 0, n is 1, Y.sup.1 is optionally substituted
amino, Y.sup.2 is optionally substituted C.sub.1-C.sub.6
heteroalkyl, Y.sup.3 is absent, X is O, and R.sup.1 and R.sup.2 are
hydrogen, then Y.sup.4 is not hydroxyl or protected hydroxyl;
or a salt thereof.
In some embodiments, the compound has the structure:
##STR00003## ##STR00004##
wherein m' is an integer from 0 to 2.
In certain embodiments of sugar A, B is uracil. In other
embodiments of sugar A, B is pseudouracil. In other embodiments of
sugar A, B is 1-methylpseudouracil. In other embodiments of sugar
A, B is 5-methoxyuracil. In other embodiments of sugar A, B is
cytosine. In other embodiments of sugar A, B is 5-methylcytosine.
In other embodiments of sugar A, B is guanine. In other embodiments
of sugar A, B is adenine.
In certain embodiments of sugar B, B is uracil. In other
embodiments of sugar B, B is pseudouracil. In other embodiments of
sugar B, B is 1-methylpseudouracil. In other embodiments of sugar
B, B is 5-methoxyuracil. In other embodiments of sugar B, B is
cytosine. In other embodiments of sugar B, B is 5-methylcytosine.
In other embodiments of sugar B, B is guanine. In other embodiments
of sugar B, B is adenine.
In certain embodiments of sugar C, B is uracil. In other
embodiments of sugar C, B is pseudouracil. In other embodiments of
sugar C, B is 1-methylpseudouracil. In other embodiments of sugar
C, B is 5-methoxyuracil. In other embodiments of sugar C, B is
cytosine. In other embodiments of sugar C, B is 5-methylcytosine.
In other embodiments of sugar C, B is guanine. In other embodiments
of sugar C, B is adenine.
In certain embodiments of sugar D, B is uracil. In other
embodiments of sugar D, B is pseudouracil. In other embodiments of
sugar D, B is 1-methylpseudouracil. In other embodiments of sugar
D, B is 5-methoxyuracil. In other embodiments of sugar D, B is
cytosine. In other embodiments of sugar D, B is 5-methylcytosine.
In other embodiments of sugar D, B is guanine. In other embodiments
of sugar D, B is adenine.
In certain embodiments of sugar E, B is uracil. In other
embodiments of sugar E, B is pseudouracil. In other embodiments of
sugar E, B is 1-methylpseudouracil. In other embodiments of sugar
E, B is 5-methoxyuracil. In other embodiments of sugar E, B is
cytosine. In other embodiments of sugar E, B is 5-methylcytosine.
In other embodiments of sugar E, B is guanine. In other embodiments
of sugar E, B is adenine.
In certain embodiments of sugar F, B is uracil. In other
embodiments of sugar F, B is pseudouracil. In other embodiments of
sugar F, B is 1-methylpseudouracil. In other embodiments of sugar
F, B is 5-methoxyuracil. In other embodiments of sugar F, B is
cytosine. In other embodiments of sugar F, B is 5-methylcytosine.
In other embodiments of sugar F, B is guanine. In other embodiments
of sugar F, B is adenine.
In certain embodiments of sugar G, B is uracil. In other
embodiments of sugar G, B is pseudouracil. In other embodiments of
sugar G, B is 1-methylpseudouracil. In other embodiments of sugar
G, B is 5-methoxyuracil. In other embodiments of sugar G, B is
cytosine. In other embodiments of sugar G, B is 5-methylcytosine.
In other embodiments of sugar G, B is guanine. In other embodiments
of sugar G, B is adenine.
In certain embodiments of sugar H, B is uracil. In other
embodiments of sugar H, B is pseudouracil. In other embodiments of
sugar H, B is 1-methylpseudouracil. In other embodiments of sugar
H, B is 5-methoxyuracil. In other embodiments of sugar H, B is
cytosine. In other embodiments of sugar H, B is 5-methylcytosine.
In other embodiments of sugar H, B is guanine. In other embodiments
of sugar H, B is adenine.
In certain embodiments of sugar I, B is uracil. In other
embodiments of sugar I, B is pseudouracil. In other embodiments of
sugar I, B is 1-methylpseudouracil. In other embodiments of sugar
I, B is 5-methoxyuracil. In other embodiments of sugar I, B is
cytosine. In other embodiments of sugar I, B is 5-methylcytosine.
In other embodiments of sugar I, B is guanine. In other embodiments
of sugar I, B is adenine.
In certain embodiments of sugar J, B is uracil. In other
embodiments of sugar J, B is pseudouracil. In other embodiments of
sugar J, B is 1-methylpseudouracil. In other embodiments of sugar
J, B is 5-methoxyuracil. In other embodiments of sugar J, B is
cytosine. In other embodiments of sugar J, B is 5-methylcytosine.
In other embodiments of sugar J, B is guanine. In other embodiments
of sugar J, B is adenine.
In certain embodiments of sugar K, B is uracil. In other
embodiments of sugar K, B is pseudouracil. In other embodiments of
sugar K, B is 1-methylpseudouracil. In other embodiments of sugar
K, B is 5-methoxyuracil. In other embodiments of sugar K, B is
cytosine. In other embodiments of sugar K, B is 5-methylcytosine.
In other embodiments of sugar K, B is guanine. In other embodiments
of sugar K, B is adenine.
In certain embodiments of sugar L, B is uracil. In other
embodiments of sugar L, B is pseudouracil. In other embodiments of
sugar L, B is 1-methylpseudouracil. In other embodiments of sugar
L, B is 5-methoxyuracil. In other embodiments of sugar L, B is
cytosine. In other embodiments of sugar L, B is 5-methylcytosine.
In other embodiments of sugar L, B is guanine. In other embodiments
of sugar L, B is adenine.
In certain embodiments of sugar M, B is uracil. In other
embodiments of sugar M, B is pseudouracil. In other embodiments of
sugar M, B is 1-methylpseudouracil. In other embodiments of sugar
M, B is 5-methoxyuracil. In other embodiments of sugar M, B is
cytosine. In other embodiments of sugar M, B is 5-methylcytosine.
In other embodiments of sugar M, B is guanine. In other embodiments
of sugar M, B is adenine.
In other embodiments, the compound has the structure:
##STR00005##
In some embodiments, the compound has the structure:
##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010##
wherein m' is an integer from 0 to 2.
In certain embodiments, the compound has the structure:
##STR00011## ##STR00012## ##STR00013##
In another aspect, the invention features a compound of Formula
IA:
##STR00014##
wherein the dotted line represents an optional double bond;
B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, O, or NR.sup.7;
R.sup.1 is hydrogen or fluorine;
R.sup.2 is hydrogen, fluorine, cyano, azido, or optionally
substituted C.sub.1-C.sub.6 alkyl;
R.sup.3 and R.sup.4 are independently hydrogen, optionally
substituted hydroxyl, or fluorine;
R.sup.5 and R.sup.6 are independently hydrogen or optionally
substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 and R.sup.6 are
combined to form an optionally substituted C.sub.3-C.sub.6
cycloalkyl, provided that one of R.sup.5 and R.sup.6 is absent when
the dotted line is a double bond;
R.sup.7 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl,
or optionally substituted amino;
each Y.sup.2 is independently hydroxyl or optionally substituted
C.sub.1-C.sub.6 heteroalkyl;
each Y.sup.3 is independently absent, O, or S;
each Y.sup.5 is independently O, NH, or CR.sup.8R.sup.9;
each Y.sup.6 is O or S;
each Y.sup.7 is O or NH and
each R.sup.8 and R.sup.9 is independently hydrogen, fluorine, or
optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 and
R.sup.9 are combined to form an optionally substituted
C.sub.3-C.sub.6 cycloalkyl, provided that one of R.sup.8 and
R.sup.9 is absent when the dotted line is a double bond;
or a salt thereof.
In some embodiments, if n is 0, X is O, R.sup.1, R.sup.2, R.sup.4,
R.sup.5, and R.sup.6 are hydrogen, and Y.sup.5 is O, then at least
one of Y.sup.1 and Y.sup.4 is optionally substituted amino, and, if
m is 0, n is 1, Y.sup.1 is optionally substituted amino, Y.sup.2 is
optionally substituted C.sub.1-C.sub.6 heteroalkyl, Y.sup.3 is
absent, Y.sup.7 is O, X is O, R.sup.1, R.sup.2, R.sup.4, R.sup.5,
and R.sup.6 are hydrogen, and R.sup.3 is hydroxyl, then Y.sup.4 is
optionally substituted amino.
In another aspect, the invention features a compound of Formula
IIA:
##STR00015##
wherein B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, or O;
R.sup.1 and R.sup.2 are independently hydrogen or fluorine;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl
(e.g., dimethoxytrityl), or optionally substituted amino;
Y.sup.2 is hydroxyl or optionally substituted C.sub.1-C.sub.6
heteroalkyl (e.g., optionally substituted C.sub.1-C.sub.6 alkoxy
such as .beta.-cyanoethoxy);
Y.sup.3 is absent or O; or a salt thereof.
In certain embodiments, if n is 0, X is O, R.sup.1 and R.sup.2 are
hydrogen, then at least one of Y.sup.1 and Y.sup.4 is optionally
substituted amino, or, if m is 0, n is 1, Y.sup.1 is optionally
substituted amino, Y.sup.2 is optionally substituted
C.sub.1-C.sub.6 heteroalkyl, Y.sup.3 is absent, X is O, and R.sup.1
and R.sup.2 are hydrogen, then Y.sup.4 is optionally substituted
amino.
In some embodiments, the compound has the structure:
##STR00016## ##STR00017##
wherein m' is an integer from 0 to 2.
In certain embodiments of sugar A', B is uracil. In other
embodiments of sugar A, B is pseudouracil. In other embodiments of
sugar A', B is 1-methylpseudouracil. In other embodiments of sugar
A', B is 5-methoxyuracil. In other embodiments of sugar A', B is
cytosine. In other embodiments of sugar A', B is 5-methylcytosine.
In other embodiments of sugar A', B is guanine. In other
embodiments of sugar A', B is adenine.
In certain embodiments of sugar B', B is uracil. In other
embodiments of sugar B', B is pseudouracil. In other embodiments of
sugar B', B is 1-methylpseudouracil. In other embodiments of sugar
B', B is 5-methoxyuracil. In other embodiments of sugar B', B is
cytosine. In other embodiments of sugar B', B is 5-methylcytosine.
In other embodiments of sugar B', B is guanine. In other
embodiments of sugar B', B is adenine.
In certain embodiments of sugar C', B is uracil. In other
embodiments of sugar C', B is pseudouracil. In other embodiments of
sugar C', B is 1-methylpseudouracil. In other embodiments of sugar
C', B is 5-methoxyuracil. In other embodiments of sugar C', B is
cytosine. In other embodiments of sugar C', B is 5-methylcytosine.
In other embodiments of sugar C', B is guanine. In other
embodiments of sugar C', B is adenine.
In certain embodiments of sugar D', B is uracil. In other
embodiments of sugar D', B is pseudouracil. In other embodiments of
sugar D', B is 1-methylpseudouracil. In other embodiments of sugar
D', B is 5-methoxyuracil. In other embodiments of sugar D', B is
cytosine. In other embodiments of sugar D', B is 5-methylcytosine.
In other embodiments of sugar D', B is guanine. In other
embodiments of sugar D', B is adenine.
In certain embodiments of sugar E', B is uracil. In other
embodiments of sugar E', B is pseudouracil. In other embodiments of
sugar E', B is 1-methylpseudouracil. In other embodiments of sugar
E', B is 5-methoxyuracil. In other embodiments of sugar E', B is
cytosine. In other embodiments of sugar E', B is 5-methylcytosine.
In other embodiments of sugar E', B is guanine. In other
embodiments of sugar E', B is adenine.
In certain embodiments of sugar F', B is uracil. In other
embodiments of sugar F', B is pseudouracil. In other embodiments of
sugar F', B is 1-methylpseudouracil. In other embodiments of sugar
F', B is 5-methoxyuracil. In other embodiments of sugar F', B is
cytosine. In other embodiments of sugar F', B is 5-methylcytosine.
In other embodiments of sugar F', B is guanine. In other
embodiments of sugar F', B is adenine.
In certain embodiments of sugar G', B is uracil. In other
embodiments of sugar G', B is pseudouracil. In other embodiments of
sugar G', B is 1-methylpseudouracil. In other embodiments of sugar
G', B is 5-methoxyuracil. In other embodiments of sugar G', B is
cytosine. In other embodiments of sugar G', B is 5-methylcytosine.
In other embodiments of sugar G', B is guanine. In other
embodiments of sugar G', B is adenine.
In certain embodiments of sugar H', B is uracil. In other
embodiments of sugar H', B is pseudouracil. In other embodiments of
sugar H', B is 1-methylpseudouracil. In other embodiments of sugar
H', B is 5-methoxyuracil. In other embodiments of sugar H', B is
cytosine. In other embodiments of sugar H', B is 5-methylcytosine.
In other embodiments of sugar H', B is guanine. In other
embodiments of sugar H', B is adenine.
In certain embodiments of sugar I', B is uracil. In other
embodiments of sugar I', B is pseudouracil. In other embodiments of
sugar I', B is 1-methylpseudouracil. In other embodiments of sugar
I', B is 5-methoxyuracil. In other embodiments of sugar I', B is
cytosine. In other embodiments of sugar I', B is 5-methylcytosine.
In other embodiments of sugar I', B is guanine. In other
embodiments of sugar I', B is adenine.
In certain embodiments of sugar J', B is uracil. In other
embodiments of sugar J', B is pseudouracil. In other embodiments of
sugar J', B is 1-methylpseudouracil. In other embodiments of sugar
J', B is 5-methoxyuracil. In other embodiments of sugar J', B is
cytosine. In other embodiments of sugar J', B is 5-methylcytosine.
In other embodiments of sugar J', B is guanine. In other
embodiments of sugar J', B is adenine.
In certain embodiments of sugar K', B is uracil. In other
embodiments of sugar K', B is pseudouracil. In other embodiments of
sugar K', B is 1-methylpseudouracil. In other embodiments of sugar
K', B is 5-methoxyuracil. In other embodiments of sugar K', B is
cytosine. In other embodiments of sugar K', B is 5-methylcytosine.
In other embodiments of sugar K', B is guanine. In other
embodiments of sugar K', B is adenine.
In certain embodiments of sugar L', B is uracil. In other
embodiments of sugar L', B is pseudouracil. In other embodiments of
sugar L', B is 1-methylpseudouracil. In other embodiments of sugar
L', B is 5-methoxyuracil. In other embodiments of sugar L', B is
cytosine. In other embodiments of sugar L', B is 5-methylcytosine.
In other embodiments of sugar L', B is guanine. In other
embodiments of sugar L', B is adenine.
In certain embodiments of sugar M', B is uracil. In other
embodiments of sugar M', B is pseudouracil. In other embodiments of
sugar M', B is 1-methylpseudouracil. In other embodiments of sugar
M', B is 5-methoxyuracil. In other embodiments of sugar M', B is
cytosine. In other embodiments of sugar M', B is 5-methylcytosine.
In other embodiments of sugar M', B is guanine. In other
embodiments of sugar M', B is adenine.
In some embodiments, the compound has the structure:
##STR00018##
In certain embodiments of compounds of Formula I, Formula II,
Formula IA, or Formula IIA, the nucleobase is uracil, pseudouracil,
1-methylpseudouracil, 5-methoxyuracil, cytosine, 5-methylcytosine,
guanine, or adenine. Such nucleobases may also be protected with
protecting groups as is known in the art. In other embodiments, the
compound of Formula I, Formula II, Formula IA, or Formula IIA is a
5' mono-, di-, or triphosphate, and n is 0. In other embodiments,
the compound of Formula I, Formula II, Formula IA, or Formula IIA
is a 3' phosphoramidite, i.e., n is 1, Y.sup.3 is absent, Y.sup.2
is optionally substituted C.sub.1-C.sub.6 heteroalkyl (e.g.,
.beta.-cyanoethoxy), Y.sup.1 is dialkyl substituted amino, (e.g.,
diisopropylamino), and m is 0.
In other embodiments, the compound is a 5' mono-, di-, or
triphosphate of any of the nucleosides provided herein.
In another aspect, the invention features a polynucleotide
comprising at least one backbone moiety of Formula III:
##STR00019##
wherein the dotted line represents an optional double bond;
B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, O, or NR.sup.7;
R.sup.1 is hydrogen or fluorine;
R.sup.2 is hydrogen, fluorine, cyano, azido, or optionally
substituted C.sub.1-C.sub.6 alkyl;
R.sup.3 and R.sup.4 are independently hydrogen, optionally
substituted hydroxyl, or fluorine;
R.sup.5 and R.sup.6 are independently hydrogen or optionally
substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 and R.sup.6 are
combined to form an optionally substituted C.sub.3-C.sub.6
cycloalkyl, provided that one of R.sup.5 and R.sup.6 is absent when
the dotted line is a double bond;
R.sup.7 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl;
each Y.sup.2 is independently hydroxyl or optionally substituted
C.sub.1-C.sub.6 heteroalkyl;
each Y.sup.3 is independently absent, O, or S;
each Y.sup.5 is independently O, NH, or CR.sup.8R.sup.9;
each Y.sup.6 is independently O or S;
each Y.sup.7 is independently O or NH; and
each R.sup.8 and R.sup.9 is independently hydrogen, fluorine, or
optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 and
R.sup.9 are combined to form an optionally substituted
C.sub.3-C.sub.6 cycloalkyl, provided that one of R.sup.8 and
R.sup.9 is absent when the dotted line is a double bond;
wherein if X is O and R.sup.1, R.sup.2, R.sup.4, R.sup.5, and
R.sup.6 are hydrogen, then at least one of Y.sup.5 and Y.sup.7 is
NH or Y.sup.5 is CR.sup.8R.sup.9;
provided that m and n are both 1 when the backbone moiety is not a
3' or 5' terminal moiety;
or a salt thereof.
In another aspect, the invention features a polynucleotide
comprising at least one backbone moiety of Formula IV:
##STR00020##
wherein B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, or O;
R.sup.1 and R.sup.2 are independently hydrogen or fluorine;
Y.sup.2 is hydroxyl or optionally substituted C.sub.1-C.sub.6
heteroalkyl;
Y.sup.3 is absent, O, or S;
wherein if X is O, then at least one of R.sup.1 and R.sup.2 is not
hydrogen;
provided that m and n are both 1 when the backbone moiety is not a
3' or 5' terminal moiety;
or a salt thereof.
In certain embodiments, the backbone moiety comprises:
##STR00021## ##STR00022##
In certain embodiments of backbone moiety A, B is uracil. In other
embodiments of backbone moiety A, B is pseudouracil. In other
embodiments of backbone moiety A, B is 1-methylpseudouracil. In
other embodiments of backbone moiety A, B is 5-methoxyuracil. In
other embodiments of backbone moiety A, B is cytosine. In other
embodiments of backbone moiety A, B is 5-methylcytosine. In other
embodiments of backbone moiety A, B is guanine. In other
embodiments of backbone moiety A, B is adenine.
In certain embodiments of backbone moiety B, B is uracil. In other
embodiments of backbone moiety B, B is pseudouracil. In other
embodiments of backbone moiety B, B is 1-methylpseudouracil. In
other embodiments of backbone moiety B, B is 5-methoxyuracil. In
other embodiments of backbone moiety B, B is cytosine. In other
embodiments of backbone moiety B, B is 5-methylcytosine. In other
embodiments of backbone moiety B, B is guanine. In other
embodiments of backbone moiety B, B is adenine.
In certain embodiments of backbone moiety C, B is uracil. In other
embodiments of backbone moiety C, B is pseudouracil. In other
embodiments of backbone moiety C, B is 1-methylpseudouracil. In
other embodiments of backbone moiety C, B is 5-methoxyuracil. In
other embodiments of backbone moiety C, B is cytosine. In other
embodiments of backbone moiety C, B is 5-methylcytosine. In other
embodiments of backbone moiety C, B is guanine. In other
embodiments of backbone moiety C, B is adenine.
In certain embodiments of backbone moiety D, B is uracil. In other
embodiments of backbone moiety D, B is pseudouracil. In other
embodiments of backbone moiety D, B is 1-methylpseudouracil. In
other embodiments of backbone moiety D, B is 5-methoxyuracil. In
other embodiments of backbone moiety D, B is cytosine. In other
embodiments of backbone moiety D, B is 5-methylcytosine. In other
embodiments of backbone moiety D, B is guanine. In other
embodiments of backbone moiety D, B is adenine.
In certain embodiments of backbone moiety E, B is uracil. In other
embodiments of backbone moiety E, B is pseudouracil. In other
embodiments of backbone moiety E, B is 1-methylpseudouracil. In
other embodiments of backbone moiety E, B is 5-methoxyuracil. In
other embodiments of backbone moiety E, B is cytosine. In other
embodiments of backbone moiety E, B is 5-methylcytosine. In other
embodiments of backbone moiety E, B is guanine. In other
embodiments of backbone moiety E, B is adenine.
In certain embodiments of backbone moiety F, B is uracil. In other
embodiments of backbone moiety F, B is pseudouracil. In other
embodiments of backbone moiety F, B is 1-methylpseudouracil. In
other embodiments of backbone moiety F, B is 5-methoxyuracil. In
other embodiments of backbone moiety F, B is cytosine. In other
embodiments of backbone moiety F, B is 5-methylcytosine. In other
embodiments of backbone moiety F, B is guanine. In other
embodiments of backbone moiety F, B is adenine.
In certain embodiments of backbone moiety G, B is uracil. In other
embodiments of backbone moiety G, B is pseudouracil. In other
embodiments of backbone moiety G, B is 1-methylpseudouracil. In
other embodiments of backbone moiety G, B is 5-methoxyuracil. In
other embodiments of backbone moiety G, B is cytosine. In other
embodiments of backbone moiety G, B is 5-methylcytosine. In other
embodiments of backbone moiety G, B is guanine. In other
embodiments of backbone moiety G, B is adenine.
In certain embodiments of backbone moiety H, B is uracil. In other
embodiments of backbone moiety H, B is pseudouracil. In other
embodiments of backbone moiety H, B is 1-methylpseudouracil. In
other embodiments of backbone moiety H, B is 5-methoxyuracil. In
other embodiments of backbone moiety H, B is cytosine. In other
embodiments of backbone moiety H, B is 5-methylcytosine. In other
embodiments of backbone moiety H, B is guanine. In other
embodiments of backbone moiety H, B is adenine.
In certain embodiments of backbone moiety I, B is uracil. In other
embodiments of backbone moiety I, B is pseudouracil. In other
embodiments of backbone moiety I, B is 1-methylpseudouracil. In
other embodiments of backbone moiety I, B is 5-methoxyuracil. In
other embodiments of backbone moiety I, B is cytosine. In other
embodiments of backbone moiety I, B is 5-methylcytosine. In other
embodiments of backbone moiety I, B is guanine. In other
embodiments of backbone moiety I, B is adenine.
In certain embodiments of backbone moiety J, B is uracil. In other
embodiments of backbone moiety J, B is pseudouracil. In other
embodiments of backbone moiety J, B is 1-methylpseudouracil. In
other embodiments of backbone moiety J, B is 5-methoxyuracil. In
other embodiments of backbone moiety J, B is cytosine. In other
embodiments of backbone moiety J, B is 5-methylcytosine. In other
embodiments of backbone moiety J, B is guanine. In other
embodiments of backbone moiety J, B is adenine.
In certain embodiments of backbone moiety K, B is uracil. In other
embodiments of backbone moiety K, B is pseudouracil. In other
embodiments of backbone moiety K, B is 1-methylpseudouracil. In
other embodiments of backbone moiety K, B is 5-methoxyuracil. In
other embodiments of backbone moiety K, B is cytosine. In other
embodiments of backbone moiety K, B is 5-methylcytosine. In other
embodiments of backbone moiety K, B is guanine. In other
embodiments of backbone moiety K, B is adenine.
In certain embodiments of backbone moiety L, B is uracil. In other
embodiments of backbone moiety L, B is pseudouracil. In other
embodiments of backbone moiety L, B is 1-methylpseudouracil. In
other embodiments of backbone moiety L, B is 5-methoxyuracil. In
other embodiments of backbone moiety L, B is cytosine. In other
embodiments of backbone moiety L, B is 5-methylcytosine. In other
embodiments of backbone moiety L, B is guanine. In other
embodiments of backbone moiety L, B is adenine.
In certain embodiments of backbone moiety M, B is uracil. In other
embodiments of backbone moiety M, B is pseudouracil. In other
embodiments of backbone moiety M, B is 1-methylpseudouracil. In
other embodiments of backbone moiety M, B is 5-methoxyuracil. In
other embodiments of backbone moiety M, B is cytosine. In other
embodiments of backbone moiety M, B is 5-methylcytosine. In other
embodiments of backbone moiety M, B is guanine. In other
embodiments of backbone moiety M, B is adenine.
In some embodiments, the backbone moiety comprises:
##STR00023##
In another aspect, the invention features a polynucleotide
comprising at least one backbone moiety of Formula IIIA:
##STR00024##
wherein the dotted line represents an optional double bond;
B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, O, or NR.sup.7;
R.sup.1 is hydrogen or fluorine;
R.sup.2 is hydrogen, fluorine, cyano, azido, or optionally
substituted C.sub.1-C.sub.6 alkyl;
R.sup.3 and R.sup.4 are independently hydrogen, optionally
substituted hydroxyl, or fluorine;
R.sup.5 and R.sup.6 are independently hydrogen or optionally
substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 and R.sup.6 are
combined to form an optionally substituted C.sub.3-C.sub.6
cycloalkyl, provided that one of R.sup.5 and R.sup.6 is absent when
the dotted line is a double bond;
R.sup.7 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl;
each Y.sup.2 is independently hydroxyl or optionally substituted
C.sub.1-C.sub.6 heteroalkyl
each Y.sup.3 is independently absent, O, or S;
each Y.sup.5 is independently O, NH, or CR.sup.8R.sup.9;
each Y.sup.6 is independently O or S;
each Y.sup.7 is independently O or NH; and
each R.sup.8 and R.sup.9 is independently hydrogen, fluorine, or
optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 and
R.sup.9 are combined to form an optionally substituted
C.sub.3-C.sub.6 cycloalkyl, provided that one of R.sup.8 and
R.sup.9 is absent when the dotted line is a double bond;
provided that m and n are both 1 when the backbone moiety is not a
3' or 5' terminal moiety;
or a salt thereof.
In another aspect, the invention features a polynucleotide
comprising at least one backbone moiety of Formula IVA:
##STR00025##
wherein B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, or O;
R.sup.1 and R.sup.2 are independently hydrogen or fluorine;
Y.sup.2 is hydroxyl or optionally substituted C.sub.1-C.sub.6
heteroalkyl;
Y.sup.3 is absent, O, or S;
provided that m and n are both 1 when the backbone moiety is not a
3' or 5' terminal moiety;
or a salt thereof.
In some embodiments, the backbone moiety comprises:
##STR00026## ##STR00027##
In certain embodiments of backbone moiety A', B is uracil. In other
embodiments of backbone moiety A, B is pseudouracil. In other
embodiments of backbone moiety A', B is 1-methylpseudouracil. In
other embodiments of backbone moiety A', B is 5-methoxyuracil. In
other embodiments of backbone moiety A', B is cytosine. In other
embodiments of backbone moiety A', B is 5-methylcytosine. In other
embodiments of backbone moiety A', B is guanine. In other
embodiments of backbone moiety A', B is adenine.
In certain embodiments of backbone moiety B', B is uracil. In other
embodiments of backbone moiety B', B is pseudouracil. In other
embodiments of backbone moiety B', B is 1-methylpseudouracil. In
other embodiments of backbone moiety B', B is 5-methoxyuracil. In
other embodiments of backbone moiety B', B is cytosine. In other
embodiments of backbone moiety B', B is 5-methylcytosine. In other
embodiments of backbone moiety B', B is guanine. In other
embodiments of backbone moiety B', B is adenine.
In certain embodiments of backbone moiety C', B is uracil. In other
embodiments of backbone moiety C', B is pseudouracil. In other
embodiments of backbone moiety C', B is 1-methylpseudouracil. In
other embodiments of backbone moiety C', B is 5-methoxyuracil. In
other embodiments of backbone moiety C', B is cytosine. In other
embodiments of backbone moiety C', B is 5-methylcytosine. In other
embodiments of backbone moiety C', B is guanine. In other
embodiments of backbone moiety C', B is adenine.
In certain embodiments of backbone moiety D', B is uracil. In other
embodiments of backbone moiety D', B is pseudouracil. In other
embodiments of backbone moiety D', B is 1-methylpseudouracil. In
other embodiments of backbone moiety D', B is 5-methoxyuracil. In
other embodiments of backbone moiety D', B is cytosine. In other
embodiments of backbone moiety D', B is 5-methylcytosine. In other
embodiments of backbone moiety D', B is guanine. In other
embodiments of backbone moiety D', B is adenine.
In certain embodiments of backbone moiety E', B is uracil. In other
embodiments of backbone moiety E', B is pseudouracil. In other
embodiments of backbone moiety E', B is 1-methylpseudouracil. In
other embodiments of backbone moiety E', B is 5-methoxyuracil. In
other embodiments of backbone moiety E', B is cytosine. In other
embodiments of backbone moiety E', B is 5-methylcytosine. In other
embodiments of backbone moiety E', B is guanine. In other
embodiments of backbone moiety E', B is adenine.
In certain embodiments of backbone moiety F', B is uracil. In other
embodiments of backbone moiety F', B is pseudouracil. In other
embodiments of backbone moiety F', B is 1-methylpseudouracil. In
other embodiments of backbone moiety F', B is 5-methoxyuracil. In
other embodiments of backbone moiety F', B is cytosine. In other
embodiments of backbone moiety F', B is 5-methylcytosine. In other
embodiments of backbone moiety F', B is guanine. In other
embodiments of backbone moiety F', B is adenine.
In certain embodiments of backbone moiety G', B is uracil. In other
embodiments of backbone moiety G', B is pseudouracil. In other
embodiments of backbone moiety G', B is 1-methylpseudouracil. In
other embodiments of backbone moiety G', B is 5-methoxyuracil. In
other embodiments of backbone moiety G', B is cytosine. In other
embodiments of backbone moiety G', B is 5-methylcytosine. In other
embodiments of backbone moiety G', B is guanine. In other
embodiments of backbone moiety G', B is adenine.
In certain embodiments of backbone moiety H', B is uracil. In other
embodiments of backbone moiety H', B is pseudouracil. In other
embodiments of backbone moiety H', B is 1-methylpseudouracil. In
other embodiments of backbone moiety H', B is 5-methoxyuracil. In
other embodiments of backbone moiety H', B is cytosine. In other
embodiments of backbone moiety H', B is 5-methylcytosine. In other
embodiments of backbone moiety H', B is guanine. In other
embodiments of backbone moiety H', B is adenine.
In certain embodiments of backbone moiety I', B is uracil. In other
embodiments of backbone moiety I', B is pseudouracil. In other
embodiments of backbone moiety I', B is 1-methylpseudouracil. In
other embodiments of backbone moiety I', B is 5-methoxyuracil. In
other embodiments of backbone moiety I', B is cytosine. In other
embodiments of backbone moiety I', B is 5-methylcytosine. In other
embodiments of backbone moiety I', B is guanine. In other
embodiments of backbone moiety I', B is adenine.
In certain embodiments of backbone moiety J', B is uracil. In other
embodiments of backbone moiety J', B is pseudouracil. In other
embodiments of backbone moiety J', B is 1-methylpseudouracil. In
other embodiments of backbone moiety J', B is 5-methoxyuracil. In
other embodiments of backbone moiety J', B is cytosine. In other
embodiments of backbone moiety J', B is 5-methylcytosine. In other
embodiments of backbone moiety J', B is guanine. In other
embodiments of backbone moiety J', B is adenine.
In certain embodiments of backbone moiety K', B is uracil. In other
embodiments of backbone moiety K', B is pseudouracil. In other
embodiments of backbone moiety K', B is 1-methylpseudouracil. In
other embodiments of backbone moiety K', B is 5-methoxyuracil. In
other embodiments of backbone moiety K', B is cytosine. In other
embodiments of backbone moiety K', B is 5-methylcytosine. In other
embodiments of backbone moiety K', B is guanine. In other
embodiments of backbone moiety K', B is adenine.
In certain embodiments of backbone moiety L', B is uracil. In other
embodiments of backbone moiety L', B is pseudouracil. In other
embodiments of backbone moiety L', B is 1-methylpseudouracil. In
other embodiments of backbone moiety L', B is 5-methoxyuracil. In
other embodiments of backbone moiety L', B is cytosine. In other
embodiments of backbone moiety L', B is 5-methylcytosine. In other
embodiments of backbone moiety L', B is guanine. In other
embodiments of backbone moiety L', B is adenine.
In certain embodiments of backbone moiety M', B is uracil. In other
embodiments of backbone moiety M', B is pseudouracil. In other
embodiments of backbone moiety M', B is 1-methylpseudouracil. In
other embodiments of backbone moiety M', B is 5-methoxyuracil. In
other embodiments of backbone moiety M', B is cytosine. In other
embodiments of backbone moiety M', B is 5-methylcytosine. In other
embodiments of backbone moiety M', B is guanine. In other
embodiments of backbone moiety M', B is adenine.
In some embodiments, the backbone moiety comprises:
##STR00028##
In certain embodiments of the polynucleotides, the nucleobase is
uracil, pseudouracil, 1-methylpseudouracil, 5-methoxyuracil,
cytosine, 5-methylcytosine, guanine, or adenine.
In some embodiments, the backbone moiety comprises:
##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033##
In certain embodiments, the backbone moiety comprises:
##STR00034## ##STR00035## ##STR00036##
In some embodiments, the polynucleotide further includes:
(a) a 5' UTR comprising at least one Kozak sequence;
(b) a 3' UTR; and
(c) at least one 5' cap structure.
In other embodiments, the at least one 5' cap structure is Cap0,
Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, or 2-azido-guanosine.
In some embodiments, the polynucleotide further includes a poly-A
tail.
In certain embodiments, the polynucleotide encodes a protein of
interest.
In some embodiments, the polynucleotide is purified.
The present invention also provides for pharmaceutical compositions
comprising the 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.
Methods of using the polynucleotides 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.
Administration of the polynucleotides 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.
Detection of the polynucleotides of the invention 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,
broncheoalveolar 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.
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.
Chemical Terms
The names of nucleobases correspond to the name given to the base
when part of a nucleoside or nucleotide. For example
"pseudo-uracil" refers to the nucleobase of pseudouridine.
As used herein, the term "compound," is meant to include all
stereoisomers, geometric isomers, tautomers, and isotopes of the
structures depicted.
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.
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.
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.
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.
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.
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.
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.
Non-limiting examples of optionally substituted acyl groups
include, alkoxycarbonyl, alkoxycarbonylacyl, arylalkoxycarbonyl,
aryloyl, carbamoyl, carboxyaldehyde, (heterocyclyl) imino, and
(heterocyclyl)oyl:
The "alkoxycarbonyl" group, which 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.
The "alkoxycarbonylacyl" group, which 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 hydroxyl group) for
each group.
The "arylalkoxycarbonyl" group, which 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.
The "aryloyl" group, which 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.
The "carbamoyl" group, which 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.
The "carboxyaldehyde" group, which as used herein, represents an
acyl group having the structure --CHO.
The "(heterocyclyl) imino" group, which 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.
The "(heterocyclyl)oyl" group, which 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.
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) hydroxyl, 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)
hydroxyl; (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.
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.
Non-limiting examples of optionally substituted alkyl and alkylene
groups include acylaminoalkyl, acyloxyalkyl, alkoxyalkyl,
alkoxycarbonylalkyl, alkylsulfinyl, alkylsulfinylalkyl, aminoalkyl,
carbamoylalkyl, carboxyalkyl, carboxyaminoalkyl, haloalkyl,
hydroxyalkyl, perfluoroalkyl, and sulfoalkyl:
The "acylaminoalkyl" group, which 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 through an alkylene
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 acylaminoalkyl 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 alkylene 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.
The "acyloxyalkyl" group, which 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 alkylene 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 alkylene group is, independently, further substituted with 1,
2, 3, or 4 substituents as described herein.
The "alkoxyalkyl" group, which as used herein, 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.
The "alkoxycarbonylalkyl" group, which 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 hydroxyl group).
The "alkylsulfinylalkyl" group, which as used herein, represents an
alkyl group, as defined herein, substituted with 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.
The "aminoalkyl" group, which as used herein, represents an alkyl
group, as defined herein, substituted with 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).
The "carbamoylalkyl" group, which as used herein, represents an
alkyl group, as defined herein, substituted with a carbamoyl group,
as defined herein. The alkyl group can be further substituted with
1, 2, 3, or 4 substituent groups as described herein.
The "carboxyalkyl" group, which as used herein, represents an alkyl
group, as defined herein, substituted with 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.
The "carboxyaminoalkyl" group, which as used herein, represents an
aminoalkyl group, as defined herein, substituted with 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 O-protecting group).
The "haloalkyl" group, which as used herein, represents an alkyl
group, as defined herein, substituted with 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.
The "hydroxyalkyl" group, which as used herein, represents an alkyl
group, as defined herein, substituted with one to three hydroxyl
groups, with the proviso that no more than one hydroxyl 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.
The "perfluoroalkyl" group, which 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.
The "sulfoalkyl" group, which as used herein, represents an alkyl
group, as defined herein, substituted with 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).
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.
Non-limiting examples of optionally substituted alkenyl groups
include, alkoxycarbonylalkenyl, aminoalkenyl, and
hydroxyalkenyl:
The "alkoxycarbonylalkenyl" group, which 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 hydroxyl group).
The "aminoalkenyl" group, which as used herein, represents an
alkenyl group, as defined herein, substituted with 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).
The "hydroxyalkenyl" group, which as used herein, represents an
alkenyl group, as defined herein, substituted with one to three
hydroxyl groups, with the proviso that no more than one hydroxyl
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.
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.
Non-limiting examples of optionally substituted alkynyl groups
include alkoxycarbonylalkynyl, aminoalkynyl, and
hydroxyalkynyl:
The "alkoxycarbonylalkynyl" group, which 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 hydroxyl group).
The "aminoalkynyl" group, which as used herein, represents an
alkynyl group, as defined herein, substituted with 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).
The "hydroxyalkynyl" group, which as used herein, represents an
alkynyl group, as defined herein, substituted with one to three
hydroxyl groups, with the proviso that no more than one hydroxyl
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.
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.
Non-limiting examples of optionally substituted amino groups
include acylamino and carbamyl:
The "acylamino" group, which 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.
The "carbamyl" group, which 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.
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) hydroxyl; (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)
hydroxyl; (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.
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) hydroxyl; (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.
The "arylalkyl" group, which 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 arylalkyl 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.
The term "azido" represents an --N.sub.3 group, which can also be
represented as --N.dbd.N.dbd.N.
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.
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.
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.
The term "carbonyl," as used herein, represents a C(O) group, which
can also be represented as C.dbd.O.
The term "carboxy," as used herein, means --CO.sub.2H.
The term "cyano," as used herein, represents an --CN group.
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) hydroxyl; (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)
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.
The "cycloalkylalkyl" group, which as used herein, 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.
The term "diastereomer," as used herein means stereoisomers that
are not mirror images of one another and are non-superimposable on
one another.
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%.
The term "halo," as used herein, represents a halogen selected from
bromine, chlorine, iodine, or fluorine.
The term "heteroalkyl," as used herein, refers to an alkyl 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 heteroalkyl group can be further substituted
with 1, 2, 3, or 4 substituent groups as described herein for alkyl
groups. The terms "heteroalkenyl" and heteroalkynyl," as used
herein refer to alkenyl and alkynyl groups, as defined herein,
respectively, in which one or two of the constituent carbon atoms
have each been replaced by nitrogen, oxygen, or sulfur. In some
embodiments, the heteroalkenyl and heteroalkynyl groups can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkyl groups.
Non-limiting examples of optionally substituted heteroalkyl,
heteroalkenyl, and heteroalkynyl groups include acyloxy,
alkenyloxy, alkoxy, alkoxyalkoxy, alkoxycarbonylalkoxy, alkynyloxy,
aminoalkoxy, arylalkoxy, carboxyalkoxy, cycloalkoxy, haloalkoxy,
(heterocyclyl)oxy, perfluoroalkoxy, thioalkoxy, and
thioheterocyclylalkyl:
The "acyloxy" group, which 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.
The "alkenyloxy" group, which as used here, 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 hydroxyl group).
The "alkoxy" group, which as used herein, 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., hydroxyl or
alkoxy).
The "alkoxyalkoxy" group, which as used herein, 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.
The "alkoxycarbonylalkoxy" group, which 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 hydroxyl group).
The "alkynyloxy" group, which as used herein, 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 hydroxyl group).
The "aminoalkoxy" group, which as used herein, represents an alkoxy
group, as defined herein, substituted with 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).
The "arylalkoxy" group, which 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.
The "aryloxy" group, which as used herein, 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.
The "carboxyalkoxy" group, which as used herein, represents an
alkoxy group, as defined herein, substituted with 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.
The "cycloalkoxy" group, which as used herein, 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.
The "haloalkoxy" group, which as used herein, represents an alkoxy
group, as defined herein, substituted with 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.
The "(heterocyclyl)oxy" group, which 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.
The "perfluoroalkoxy" group, which 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.
The "alkylsulfinyl" group, which 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.
The "thioarylalkyl" group, which as used herein, represents a
chemical substituent of formula --SR, where R is an arylalkyl
group. In some embodiments, the arylalkyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein.
The "thioalkoxy" group 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.
The "thioheterocyclylalkyl" group, which as used herein, represents
a chemical substituent of formula --SR, where R is an
heterocyclylalkyl group. In some embodiments, the heterocyclylalkyl
group can be further substituted with 1, 2, 3, or 4 substituent
groups as described herein.
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.
The term "heteroarylalkyl" refers to a heteroaryl group, as defined
herein, attached to the parent molecular group through an alkylene
group, as defined herein. Exemplary unsubstituted heteroarylalkyl
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.
Heteroarylalkyl groups are a subset of heterocyclylalkyl
groups.
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
##STR00037## where
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) hydroxyl; (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) 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.
The "heterocyclylalkyl" group, which as used herein, represents a
heterocyclyl group, as defined herein, attached to the parent
molecular group through an alkylene group, as defined herein.
Exemplary unsubstituted heterocyclylalkyl 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.
The term "hydrocarbon," as used herein, represents a group
consisting only of carbon and hydrogen atoms.
The term "hydroxyl," as used herein, represents an --OH group. In
some embodiments, the hydroxyl group can be substituted with a
substituent group (e.g., optionally substituted alkyl or an
O-protecting group).
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.
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.
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).
The term "nitro," as used herein, represents an --NO.sub.2
group.
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-methylphenoxycarbonyl,
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.
The term "oxo" as used herein, represents .dbd.O.
The prefix "perfluoro," as used herein, represents anyl group, as
defined herein, where each hydrogen radical bound to the alkyl
group has been replaced by a fluoride radical. For example,
perfluoroalkyl groups are exemplified by trifluoromethyl,
pentafluoroethyl, and the like.
The term "protected hydroxyl," as used herein, refers to an oxygen
atom bound to an O-protecting group.
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.
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.
The term "sulfonyl," as used herein, represents an --S(O).sub.2--
group.
The term "thiol," as used herein represents an --SH group.
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.
Other features and advantages of the present disclosure will be
apparent from the following detailed description and figures, and
from the claims.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure provides, alternative sugar moieties and
polynucleotides including these alternatives that may exhibit
improved therapeutic properties including, but not limited to, a
reduced innate immune response when introduced into a population of
cells.
As there remains a need in the art for therapeutic modalities to
address the myriad barriers surrounding the efficacious modulation
of intracellular translation and processing of polynucleotides
encoding polypeptides or fragments thereof, certain mRNA sequences
containing alternative sugar moieties may have the potential as
therapeutics with benefits beyond just evading, avoiding or
diminishing the immune response.
The present invention addresses this need by providing
polynucleotides which encode a polypeptide of interest (e.g.,
unnatural mRNA) and which have structural and/or chemical features
that preferably avoid one or more of the problems in the art, for
example, features which are useful for optimizing
polynucleotide-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.
Polypeptides of interest, according to the present invention may be
selected from any of those disclosed in US 2013/0259924, US
2013/0259923, WO 2013/151663, WO 2013/151669, WO 2013/151670, WO
2013/151664, WO 2013/151665, WO 2013/151736, U.S. Provisional
Patent Application No. 61/618,862, U.S. Provisional Patent
Application No. 61/681,645, U.S. Provisional Patent Application No.
61/618,873, U.S. Provisional Patent Application No. 61/681,650,
U.S. Provisional Patent Application No. 61/618,878, U.S.
Provisional Patent Application No. 61/681,654, U.S. Provisional
Patent Application No. 61/618,885, U.S. Provisional Patent
Application No. 61/681,658, U.S. Provisional Patent Application No.
61/618,911 s, U.S. Provisional Patent Application No. 61/681,667,
U.S. Provisional Patent Application No. 61/618,922, U.S.
Provisional Patent Application No. 61/681,675, U.S. Provisional
Patent Application No. 61/618,935, U.S. Provisional Patent
Application No. 61/681,687, U.S. Provisional Patent Application No.
61/618,945, U.S. Provisional Patent Application No. 61/681,696,
U.S. Provisional Patent Application No. 61/618,953, and U.S.
Provisional Patent Application No. 61/681,704, the contents of
which are incorporated herein by reference in their entirety.
Provided herein, in part, are polynucleotides encoding polypeptides
of interest which contain one or more of alternative sugar moieties
of the nucleotide compared to the natural counterpart, 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.
The alternative sugar moieties of the invention, including the
combination of alternatives taught herein may have superior
properties making them more suitable as therapeutic modalities.
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 mRNA containing alternative
nucleotides. To improve protein production, one may consider the
nature of the alternative nucleoside, nucleotide, or
polynucleotide, or combination of alternative groups, the percent
incorporation of the alternatives and survey more than one cytokine
or metric to determine the efficacy and risk profile of a
particular unnatural mRNA.
In one aspect of the invention, methods of determining the
effectiveness of an mRNA containing alternative sugar moieties as
compared to natural mRNA involves the measure and analysis of one
or more cytokines whose expression is triggered by the
administration of the exogenous polynucleotide of the invention.
These values are compared to administration of a natural
polynucleotide or to a standard metric such as cytokine response,
PolyIC, R-848 or other standard known in the art.
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 unnatural polynucleotide. Such ratios are referred
to herein as the Protein: Cytokine Ratio or "PC" Ratio. The higher
the PC ratio, the more efficacious the unnatural polynucleotide
(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. Alternative polynucleotides having higher PC
Ratios than an alternative polynucleotide of a different or natural
construct are preferred.
The PC ratio may be further qualified by the percentage of
alternative sugar moieties present in the polynucleotide. For
example, normalized to a 100% alternative polynucleotide, the
protein production as a function of cytokine (or risk) or cytokine
profile can be determined.
In one embodiment, the present invention provides a method for
determining, across chemistries, cytokines or percentage of
alternative nucleotides, the relative efficacy of any particular
polynucleotide by comparing the PC Ratio of the alternative
polynucleotide to the natural counterpart.
In another embodiment, the mRNA of the invention are substantially
non toxic and non mutagenic.
In one embodiment, the alternative sugar moieties and
polynucleotides can disrupt interactions, which may cause innate
immune responses. Further, these alternative sugar moieties and
polynucleotides can be used to deliver a payload, e.g., detectable
or therapeutic agent, to a biological target. For example, the
polynucleotides 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
extracellularly or intracellularly, as well as in assays such as
cell free assays.
In another aspect, the present disclosure provides alternative
sugar moieties that may reduce the cellular innate immune response,
as compared to the cellular innate immune induced by a
corresponding natural polynucleotide.
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.
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 polynucleotide made by the
methods described herein, wherein the secreted protein is active
upon a second population of human cells.
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.
In certain embodiments, provided herein are combination
therapeutics containing one or more alternative polynucleotides
containing translatable regions that preferably encode for a
protein or proteins that boost a mammalian subject's immunity along
with a protein that induces antibody dependent cellular
toxicity.
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.
Alternative Nucleotides, Nucleosides and Polynucleotides of the
Invention
Herein, in a sugar moiety or a polynucleotide (such as the
polynucleotides of the invention, e.g., mRNA molecule), the term
"alternative" refers to a compound differing chemically with
respect to ribose. Generally, herein, this term is 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.
The alternatives may be various. In some embodiments, where the
polynucleotide is an mRNA, the coding region, the flanking regions
and/or the terminal regions may contain one, two, or more
(optionally different) alternative sugar moieties. In some
embodiments, an alternative polynucleotide introduced to a cell may
exhibit reduced degradation in the cell, as compared to a natural
polynucleotide.
As described herein, the polynucleotides of the invention
preferably 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.
In certain embodiments, it may desirable for an alternative
polynucleotide molecule introduced into the cell to be degraded
intracellularly. For example, degradation of an alternative
polynucleotide molecule may be preferable if precise timing of
protein production is desired. Thus, in some embodiments, the
invention provides an alternative polynucleotide molecule
containing a degradation domain, which is capable of being acted on
in a directed manner within a cell.
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 alternative nucleoside or
nucleotides (i.e., unnatural mRNA molecules). Details for these
polynucleotides follow.
Polynucleotides
In some embodiments, the polynucleotides of the invention include 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.
Sugar Alternatives
The alternative nucleosides and nucleotides (e.g., building block
molecules), which may be incorporated into a polynucleotide (e.g.,
RNA or mRNA, as described herein), can include an alternative
sugar.
Generally, RNA includes the sugar group ribose, which is a
5-membered ring having an oxygen. Exemplary alternative sugar
moieties include compounds having the structure of Formula I:
##STR00038##
wherein the dotted line represents an optional double bond;
B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, O, or NR.sup.7;
R.sup.1 is hydrogen or fluorine;
R.sup.2 is hydrogen, fluorine, cyano, azido, or optionally
substituted C.sub.1-C.sub.6 alkyl;
R.sup.3 and R.sup.4 are independently hydrogen, optionally
substituted hydroxyl, or fluorine;
R.sup.5 and R.sup.6 are independently hydrogen or optionally
substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 and R.sup.6 are
combined to form an optionally substituted C.sub.3-C.sub.6
cycloalkyl, provided that one of R.sup.5 and R.sup.6 is absent when
the dotted line is a double bond;
R.sup.7 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl,
or optionally substituted amino;
each Y.sup.2 is independently hydroxyl or optionally substituted
C.sub.1-C.sub.6 heteroalkyl;
each Y.sup.3 is independently absent, O, or S;
each Y.sup.5 is independently O, NH, or CR.sup.8R.sup.9;
each Y.sup.6 is O or S;
each Y.sup.7 is O or NH; and
each R.sup.8 and R.sup.9 is independently hydrogen, fluorine, or
optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 and
R.sup.9 are combined to form an optionally substituted
C.sub.3-C.sub.6 cycloalkyl, provided that one of R.sup.8 and
R.sup.9 is absent when the dotted line is a double bond;
wherein if n is 0, X is O, R.sup.1, R.sup.2, R.sup.4, R.sup.5, and
R.sup.6 are hydrogen, and Y.sup.5 is O, then at least one of
Y.sup.1 and Y.sup.4 is optionally substituted amino, and, if m is
0, n is 1, Y.sup.1 is optionally substituted amino, Y.sup.2 is
optionally substituted C.sub.1-C.sub.6 heteroalkyl, Y.sup.3 is
absent, Y.sup.7 is O, X is O, and R.sup.1, R.sup.2, R.sup.4,
R.sup.5, and R.sup.6 are hydrogen, then Y.sup.4 is optionally
substituted amino;
or a salt thereof.
Additional alternative sugar moieties include compounds having the
structure of Formula II:
##STR00039##
wherein B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, or O;
R.sup.1 and R.sup.2 are independently hydrogen or fluorine;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl,
or optionally substituted amino;
Y.sup.2 is hydroxyl or optionally substituted C.sub.1-C.sub.6
heteroalkyl (e.g., .beta.-cyanoethyl);
Y.sup.3 is absent or O;
wherein if n is 0, X is O, R.sup.1 and R.sup.2 are hydrogen, then
at least one of Y.sup.1 and Y.sup.4 is not hydroxyl or protected
hydroxy, and, if m is 0, n is 1, Y.sup.1 is optionally substituted
amino, Y.sup.2 is optionally substituted C.sub.1-C.sub.6
heteroalkyl, Y.sup.3 is absent, X is O, and R.sup.1 and R.sup.2 are
hydrogen, then Y.sup.4 is not hydroxyl or protected hydroxyl;
or a salt thereof.
Additional alternative sugar moieties include compounds having the
structure of Formula IA:
##STR00040##
wherein the dotted line represents an optional double bond;
B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, O, or NR.sup.7;
R.sup.1 is hydrogen or fluorine;
R.sup.2 is hydrogen, fluorine, cyano, azido, or optionally
substituted C.sub.1-C.sub.6 alkyl;
R.sup.3 and R.sup.4 are independently hydrogen, optionally
substituted hydroxyl, or fluorine;
R.sup.5 and R.sup.6 are independently hydrogen or optionally
substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 and R.sup.6 are
combined to form an optionally substituted C.sub.3-C.sub.6
cycloalkyl, provided that one of R.sup.5 and R.sup.6 is absent when
the dotted line is a double bond;
R.sup.7 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl,
or optionally substituted amino;
each Y.sup.2 is independently hydroxyl or optionally substituted
C.sub.1-C.sub.6 heteroalkyl;
each Y.sup.3 is independently absent, O, or S;
each Y.sup.5 is independently O, NH, or CR.sup.8R.sup.9;
each Y.sup.6 is O or S;
each Y.sup.7 is O or NH and
each R.sup.8 and R.sup.9 is independently hydrogen, fluorine, or
optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 and
R.sup.9 are combined to form an optionally substituted
C.sub.3-C.sub.6 cycloalkyl, provided that one of R.sup.8 and
R.sup.9 is absent when the dotted line is a double bond;
or a salt thereof.
In some embodiments, if n is 0, X is O, R.sup.1, R.sup.2, R.sup.4,
R.sup.5, and R.sup.6 are hydrogen, and Y.sup.5 is O, then at least
one of Y.sup.1 and Y.sup.4 is optionally substituted amino, and, if
m is 0, n is 1, Y.sup.1 is optionally substituted amino, Y.sup.2 is
optionally substituted C.sub.1-C.sub.6 heteroalkyl, Y.sup.3 is
absent, Y.sup.7 is O, X is O, R.sup.1, R.sup.2, R.sup.4, R.sup.5,
and R.sup.6 are hydrogen, and R.sup.3 is hydroxyl, then Y.sup.4 is
optionally substituted amino.
Additional alternative sugar moieties include compounds having the
structure of Formula IIA:
##STR00041##
wherein B is a nucleobase;
m and n are independently an integer from 0 to 3;
X is S, CH.sub.2, SO.sub.2, or O;
R.sup.1 and R.sup.2 are independently hydrogen or fluorine;
Y.sup.1 and Y.sup.4 are independently hydroxyl, protected hydroxyl
(e.g., dimethoxytrityl), or optionally substituted amino
Y.sup.2 is hydroxyl or optionally substituted C.sub.1-C.sub.6
heteroalkyl (e.g., optionally substituted C.sub.1-C.sub.6 alkoxy
such as .beta.-cyanoethoxy);
Y.sup.3 is absent or O; or a salt thereof.
In certain embodiments, if n is 0, X is O, R.sup.1 and R.sup.2 are
hydrogen, then at least one of Y.sup.1 and Y.sup.4 is optionally
substituted amino, or, if m is 0, n is 1, Y.sup.1 is optionally
substituted amino, Y.sup.2 is optionally substituted
C.sub.1-C.sub.6 heteroalkyl, Y.sup.3 is absent, X is O, and R.sup.1
and R.sup.2 are hydrogen, then Y.sup.4 is optionally substituted
amino.
Synthesis of Polynucleotide Molecules
The polynucleotide molecules for use in accordance with the
invention may be prepared according to any useful technique, as
described herein. The alternative sugar moieties 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.
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.
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.
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.
Resolution of racemic mixtures of unnatural polynucleotides (e.g.,
polynucleotides or unnatural 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.
Different sugar alternatives may exist at various positions in the
polynucleotide. One of ordinary skill in the art will appreciate
that the alternative sugar moieties may be located at any
position(s) of a polynucleotide such that the function of the
polynucleotide is not substantially decreased. A polynucleotide may
also include a 5' or 3' terminal alternative. The polynucleotide
may contain from about 1% to about 100% alternative sugar moieties
(either in relation to overall sugar content, or in relation to one
or more types of sugar) 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%).
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
alternatives. In such embodiments, sugar alternatives may also be
present in the translatable region. Also provided are
polynucleotides containing a Kozak sequence.
Alternative Polynucleotides
The present disclosure provides polynucleotides, including RNAs
such as mRNAs that contain one or more alternative sugar moieties
(termed "alternative polynucleotides") as described herein, which
may 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 alternative polynucleotides may
enhance the efficiency of protein production, intracellular
retention of polynucleotides, and viability of contacted cells, as
well as possess reduced immunogenicity, these polynucleotides
having these properties are also termed "enhanced polynucleotides"
herein.
The term "polynucleotide," in its broadest sense, includes any
compound that an oligonucleotide chain of two or more nucleotides.
Exemplary polynucleotides 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.
Provided are alternative polynucleotides containing a translatable
region and one, two, or more than two different sugar alternatives.
In some embodiments, the alternative polynucleotide exhibits
reduced degradation in a cell into which the polynucleotide is
introduced, relative to a corresponding natural polynucleotide.
Exemplary polynucleotides include ribonucleic acids (RNAs) and
deoxyribonucleic acids (DNAs), or a hybrid thereof. In preferred
embodiments, the alternative polynucleotide includes messenger RNAs
(mRNAs). As described herein, the polynucleotides of the present
disclosure preferably do not substantially induce an innate immune
response of a cell into which the mRNA is introduced.
In certain embodiments, it is desirable to intracellularly degrade
an alternative polynucleotide introduced into the cell, for example
if precise timing of protein production is desired. Thus, the
present disclosure provides an alternative polynucleotide
containing a degradation domain, which is capable of being acted on
in a directed manner within a cell.
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 sugar alternatives.
In such embodiments, sugar alternatives may also be present in the
translatable region. Also provided are polynucleotides containing a
Kozak sequence.
Further, provided are polynucleotides 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 polynucleotides 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).
Major Groove Interacting Partners
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 polynucleotide. As such, RNA ligands
comprising alternative sugar or polynucleotides 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.
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
The term "innate immune response" includes a cellular response to
exogenous single stranded polynucleotides, 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 polynucleotides, the present disclosure provides
alternative polynucleotides such as mRNAs that may 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 natural polynucleotide.
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
unnatural 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 natural polynucleotide.
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
alternative polynucleotides.
In some embodiments, the alternative polynucleotides, including
mRNA molecules preferably do 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 may be a
novel feature of the unnatural polynucleotides of the present
invention.
The present disclosure provides for the repeated introduction
(e.g., transfection) of alternative polynucleotides 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
alternative polynucleotides is repeated a number of times
sufficient such that a predetermined efficiency of protein
translation in the cell population is achieved. Given the
preferable reduced cytotoxicity of the target cell population
provided by the polynucleotide alternatives, such repeated
transfections may be achievable in a diverse array of cell types in
vitro and/or in vivo.
Polypeptide Variants
Provided are polynucleotides 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).
In some embodiments, the polypeptide variant preferably has the
same or a similar activity as the reference polypeptide.
Alternatively, the variant may have 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.
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
Also provided are polynucleotide libraries containing alternative
nucleosides, wherein the polynucleotides individually contain a
first polynucleotide 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.
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-Polynucleotide Complexes
Proper protein translation involves the physical aggregation of a
number of polypeptides and polynucleotides associated with the
mRNA. Provided by the present disclosure are protein-polynucleotide
complexes, containing a translatable mRNA having one or more
alternative sugars (e.g., at least two different alternative
sugars) 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 Alternative Polynucleotides
As described herein, provided are mRNAs having sequences that are
substantially not translatable. Such mRNA is may be effective as a
vaccine when administered to a mammalian subject.
Also provided are alternative polynucleotides that contain one or
more noncoding regions. Such alternative polynucleotides are
generally not translated, but may be 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 alternative
polynucleotide may contain a small nucleolar RNA (sno-RNA), micro
RNA (miRNA), small interfering RNA (sRNA) or Piwi-interacting RNA
(piRNA).
Synthesis of Alternative Polynucleotides
Polynucleotides 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).
Different sugar alternatives and/or backbone structures may exist
at various positions in the polynucleotide. One of ordinary skill
in the art will appreciate that the sugar alternative(s) may be
located at any position(s) of a polynucleotide such that the
function of the polynucleotide is not substantially decreased. The
5' or 3' terminus may also include an alternative. The
polynucleotides may contain at a minimum one and at maximum 100%
alternative sugar, or any intervening percentage, such as at least
5% alternative sugars, at least 10% alternative sugars, at least
25% alternative sugars, at least 50% alternative sugars, at least
80% alternative sugars, or at least 90% alternative sugars.
Generally, the shortest length of an unnatural 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.
Examples of dipeptides that the alternative polynucleotide
sequences can encode for include, but are not limited to, carnosine
and anserine.
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.
For example, the alternative polynucleotides described herein can
be prepared using methods that are known to those skilled in the
art of polynucleotide synthesis.
5' Capping
The 5' cap structure of an mRNA is involved in nuclear export,
increasing mRNA stability and binds the mRNA Cap Binding Protein
(CBP), which is responsible for mRNA stability in the cell and
translation competency through the association of CBP with poly(A)
binding protein to form the mature cyclic mRNA species. The cap
further assists the removal of 5' proximal introns removal during
mRNA splicing.
Endogenous mRNA molecules may be 5'-end capped generating a
5'-ppp-5'-triphosphate linkage between a terminal guanosine cap
residue and the 5'-terminal transcribed sense nucleotide of the
mRNA. This 5'-guanylate cap may then be methylated to generate an
N7-methyl-guanylate residue. The ribose sugars of the terminal
and/or anteterminal transcribed nucleotides of the 5' end of the
mRNA may optionally also be 2'-O-methylated. 5'-decapping through
hydrolysis and cleavage of the guanylate cap structure may target a
nucleic acid molecule, such as an mRNA molecule, for
degradation.
Modifications to the nucleic acids of the present invention may
generate a non-hydrolyzable cap structure preventing decapping and
thus increasing mRNA half-life. Because cap structure hydrolysis
requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified
nucleotides may be used during the capping reaction. For example, a
Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.)
may be used with .alpha.-thio-guanosine nucleotides according to
the manufacturer's instructions to create a phosphorothioate
linkage in the 5'-ppp-5' cap. Additional modified guanosine
nucleotides may be used such as .alpha.-methyl-phosphonate and
seleno-phosphate nucleotides.
Additional modifications include, but are not limited to,
2'-O-methylation of the ribose sugars of 5'-terminal and/or
5'-anteterminal nucleotides of the mRNA (as mentioned above) on the
2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap
structures can be used to generate the 5'-cap of a nucleic acid
molecule, such as an mRNA molecule.
5' Cap structures include those described in International Patent
Publication Nos. WO2008127688, WO 2008016473, and WO 2011015347,
each of which is incorporated herein by reference in its
entirety.
Cap analogs, which herein are also referred to as synthetic cap
analogs, chemical caps, chemical cap analogs, or structural or
functional cap analogs, differ from natural (i.e. endogenous,
wild-type or physiological) 5'-caps in their chemical structure,
while retaining cap function. Cap analogs may be chemically (i.e.
non-enzymatically) or enzymatically synthesized and/linked to a
nucleic acid molecule.
For example, the Anti-Reverse Cap Analog (ARCA) cap contains two
guanines linked by a 5'-5'-triphosphate group, wherein one guanine
contains an N7 methyl group as well as a 3'-O-methyl group (i.e.,
N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine
(m.sup.7G-3'mppp-G; which may equivalently be designated 3'
O-Me-m7G(5)ppp(5')G). The 3'-O atom of the other, unmodified,
guanine becomes linked to the 5'-terminal nucleotide of the capped
nucleic acid molecule (e.g. an mRNA or mmRNA). The N7- and
3'-O-methylated guanine provides the terminal moiety of the capped
nucleic acid molecule (e.g. mRNA or mmRNA).
Another exemplary cap is mCAP, which is similar to ARCA but has a
2'-O-methyl group on guanosine (i.e.,
N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine,
m.sup.7Gm-ppp-G).
In one embodiment, the cap is a dinucleotide cap analog. As a
non-limiting example, the dinucleotide cap analog may be modified
at different phosphate positions with a boranophosphate group or a
phophoroselenoate group such as the dinucleotide cap analogs
described in U.S. Pat. No. 8,519,110, the contents of which are
herein incorporated by reference in its entirety.
In another embodiment, the cap analog is a
N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap
analog known in the art and/or described herein. Non-limiting
examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide
form of a cap analog include a
N7-(4-chlorophenoxyethyl)-G(5)ppp(5')G and a
N7-(4-chlorophenoxyethyl)-m.sup.3'-OG(5)ppp(5')G cap analog (See
e.g., the various cap analogs and the methods of synthesizing cap
analogs described in Kore et al. Bioorganic & Medicinal
Chemistry 2013 21:4570-4574; the contents of which are herein
incorporated by reference in its entirety). In another embodiment,
a cap analog of the present invention is a
4-chloro/bromophenoxyethyl analog.
While cap analogs allow for the concomitant capping of a nucleic
acid molecule in an in vitro transcription reaction, up to 20% of
transcripts remain uncapped. This, as well as the structural
differences of a cap analog from endogenous 5'-cap structures of
nucleic acids produced by the endogenous, cellular transcription
machinery, may lead to reduced translational competency and reduced
cellular stability.
Modified nucleic acids of the invention may also be capped
post-transcriptionally, using enzymes, in order to generate more
authentic 5'-cap structures. As used herein, the phrase "more
authentic" refers to a feature that closely mirrors or mimics,
either structurally or functionally, an endogenous or wild type
feature. That is, a "more authentic" feature is better
representative of an endogenous, wild-type, natural or
physiological cellular function and/or structure as compared to
synthetic features or analogs, etc., of the prior art, or which
outperforms the corresponding endogenous, wild-type, natural or
physiological feature in one or more respects. Non-limiting
examples of more authentic 5'-cap structures of the present
invention are those which, among other things, have enhanced
binding of cap binding proteins, increased half life, reduced
susceptibility to 5' endonucleases and/or reduced 5' decapping, as
compared to synthetic 5'-cap structures known in the art (or to a
wild-type, natural or physiological 5'-cap structure). For example,
recombinant Vaccinia Virus Capping Enzyme and recombinant
2'-O-methyltransferase enzyme can create a canonical
5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an
mRNA and a guanine cap nucleotide wherein the cap guanine contains
an N7 methylation and the 5'-terminal nucleotide of the mRNA
contains a 2'-O-methyl. Such a structure is termed the Cap1
structure. This cap results in a higher translational-competency
and cellular stability and a reduced activation of cellular
pro-inflammatory cytokines, as compared, e.g., to other 5'cap
analog structures known in the art. Cap structures include
7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NImpNp (cap 1),
7mG(5')-ppp(5')NImpN2mp (cap 2) and
m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).
Because the modified nucleic acids may be capped
post-transcriptionally, and because this process is more efficient,
nearly 100% of the modified nucleic acids may be capped. This is in
contrast to .about.80% when a cap analog is linked to an mRNA in
the course of an in vitro transcription reaction.
According to the present invention, 5' terminal caps may include
endogenous caps or cap analogs. According to the present invention,
a 5' terminal cap may comprise a guanine analog. Useful guanine
analogs include inosine, N1-methyl-guanosine, 2'fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, and 2-azido-guanosine.
In one embodiment, the nucleic acids described herein may contain a
modified 5'-cap. A modification on the 5'-cap may increase the
stability of mRNA, increase the half-life of the mRNA, and could
increase the mRNA translational efficiency. The modified 5'-cap may
include, but is not limited to, one or more of the following
modifications: modification at the 2' and/or 3' position of a
capped guanosine triphosphate (GTP), a replacement of the sugar
ring oxygen (that produced the carbocyclic ring) with a methylene
moiety (CH.sub.2), a modification at the triphosphate bridge moiety
of the cap structure, or a modification at the nucleobase (G)
moiety.
The 5'-cap structure that may be modified includes, but is not
limited to, the caps described herein such as Cap0 having the
substrate structure for cap dependent translation of:
##STR00042## or Cap1 having the substrate structure for cap
dependent translation of:
##STR00043##
As a non-limiting example, the modified 5'-cap may have the
substrate structure for cap dependent translation of:
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
where R.sub.1 and R.sub.2 are defined in Table 1:
TABLE-US-00001 TABLE 1 R.sub.1 and R.sub.2 for CAP-022 to CAP096
Cap Structure Number R.sub.1 R.sub.2 CAP-022 C.sub.2H.sub.5 (Ethyl)
H CAP-023 H C.sub.2H.sub.5 (Ethyl) CAP-024 C.sub.2H.sub.5 (Ethyl)
C.sub.2H.sub.5 (Ethyl) CAP-025 C.sub.3H.sub.7 (Propyl) H CAP-026 H
C.sub.3H.sub.7 (Propyl) CAP-027 C.sub.3H.sub.7 (Propyl)
C.sub.3H.sub.7 (Propyl) CAP-028 C.sub.4H.sub.9 (Butyl) H CAP-029 H
C.sub.4H.sub.9 (Butyl) CAP-030 C.sub.4H.sub.9 (Butyl)
C.sub.4H.sub.9 (Butyl) CAP-031 C.sub.5H.sub.11 (Pentyl) H CAP-032 H
C.sub.5H.sub.11 (Pentyl) CAP-033 C.sub.5H.sub.11 (Pentyl)
C.sub.5H.sub.11 (Pentyl) CAP-034 H.sub.2C--C.ident.CH (Propargyl) H
CAP-035 H H.sub.2C--C.ident.CH (Propargyl) CAP-036
H.sub.2C--C.ident.CH (Propargyl) H.sub.2C--C.ident.CH (Propargyl)
CAP-037 CH.sub.2CH.dbd.CH.sub.2 (Allyl) H CAP-038 H
CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-039 CH.sub.2CH.dbd.CH.sub.2
(Allyl) CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-040 CH.sub.2OCH.sub.3
(MOM) H CAP-041 H CH.sub.2OCH.sub.3 (MOM) CAP-042 CH.sub.2OCH.sub.3
(MOM) CH.sub.2OCH.sub.3 (MOM) CAP-043
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) H CAP-044 H
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-045
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM)
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-046 CH.sub.2SCH.sub.3
(MTM) H CAP-047 H CH.sub.2SCH.sub.3 (MTM) CAP-048 CH.sub.2SCH.sub.3
(MTM) CH.sub.2SCH.sub.3 (MTM) CAP-049 CH.sub.2C.sub.6H.sub.5
(Benzyl) H CAP-050 H CH.sub.2C.sub.6H.sub.5 (Benzyl) CAP-051
CH.sub.2C.sub.6H.sub.5 (Benzyl) CH.sub.2C.sub.6H.sub.5 (Benzyl)
CAP-052 CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) H CAP-053 H
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-054
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM)
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-055
CH.sub.2C.sub.6H.sub.4--OMe (p- H Methoxybenzyl) CAP-056 H
CH.sub.2C.sub.6H.sub.4--OMe (p- Methoxybenzyl) CAP-057
CH.sub.2C.sub.6H.sub.4--OMe (p- CH.sub.2C.sub.6H.sub.4--OMe (p-
Methoxybenzyl) Methoxybenzyl) CAP-058
CH.sub.2C.sub.6H.sub.4--NO.sub.2 H (p-Nitrobenzyl) CAP-059 H
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) CAP-060
CH.sub.2C.sub.6H.sub.4--NO.sub.2 CH.sub.2C.sub.6H.sub.4--NO.sub.2
(p-Nitrobenzyl) (p-Nitrobenzyl) CAP-061 CH.sub.2C.sub.6H.sub.4--X
(p-Halobenzyl) H where X = F, Cl, Br or I CAP-062 H
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X = F, Cl, Br or I
CAP-063 CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl)
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X = F, Cl, Br or I
where X = F, Cl, Br or I CAP-064 CH.sub.2C.sub.6H.sub.4--N.sub.3 H
(p-Azidobenzyl) CAP-065 H CH.sub.2C.sub.6H.sub.4--N.sub.3
(p-Azidobenzyl) CAP-066 CH.sub.2C.sub.6H.sub.4--N.sub.3
CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl) (p-Azidobenzyl)
CAP-067 CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- H
Trifluoromethylbenzyl) CAP-068 H CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- Trifluoromethylbenzyl) CAP-069 CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- Trifluoromethylbenzyl)
Trifluoromethylbenzyl) CAP-070 CH.sub.2C.sub.6H.sub.4--OCF.sub.3
(p- H Trifluoromethoxylbenzyl) CAP-071 H
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
CAP-072 CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p-
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
Trifluoromethoxylbenzyl) CAP-073
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4- H
bis(Trifluoromethyl)benzyl] CAP-074 H
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] CAP-075
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-076
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t- H Butyldiphenylsilyl)
CAP-077 H Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t-
Butyldiphenylsilyl) CAP-078 Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9
(t- Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t- Butyldiphenylsilyl)
Butyldiphenylsilyl) CAP-079 CH.sub.2CH.sub.2CH.dbd.CH.sub.2 H
(Homoallyl) CAP-080 H CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl)
CAP-081 CH.sub.2CH.sub.2CH.dbd.CH.sub.2
CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl) (Homoallyl) CAP-082
P(O)(OH).sub.2 (MP) H CAP-083 H P(O)(OH).sub.2 (MP) CAP-084
P(O)(OH).sub.2 (MP) P(O)(OH).sub.2 (MP) CAP-085 P(S)(OH).sub.2
(Thio-MP) H CAP-086 H P(S)(OH).sub.2 (Thio-MP) CAP-087
P(S)(OH).sub.2 (Thio-MP) P(S)(OH).sub.2 (Thio-MP) CAP-088
P(O)(CH.sub.3)(OH) H (Methylphophonate) CAP-089 H
P(O)(CH.sub.3)(OH) (Methylphophonate) CAP-090 P(O)(CH.sub.3)(OH)
P(O)(CH.sub.3)(OH) (Methylphophonate) (Methylphophonate) CAP-091
PN(.sup.IPr).sub.2(OCH.sub.2CH.sub.2CN) H (Phosporamidite) CAP-092
H PN(.sup.IPr).sub.2(OCH.sub.2CH.sub.2CN) (Phosporamidite) CAP-093
PN(.sup.IPr).sub.2(OCH.sub.2CH.sub.2CN) PN(.sup.IPr).sub.2(OCH.sub-
.2CH.sub.2CN) (Phosporamidite) (Phosporamidite) CAP-094
SO.sub.2CH.sub.3 H (Methanesulfonic acid) CAP-095 H
SO.sub.2CH.sub.3 (Methanesulfonic acid) CAP-096 SO.sub.2CH.sub.3
SO.sub.2CH.sub.3 (Methanesulfonic acid) (Methanesulfonic acid)
or
##STR00049## where R.sub.1 and R.sub.2 are defined in Table 5:
TABLE-US-00002 TABLE 2 R.sub.1 and R.sub.2 for CAP-097 to CAP111
Cap Structure Number R.sub.1 R.sub.2 CAP-097 NH.sub.2 (amino) H
CAP-098 H NH.sub.2 (amino) CAP-099 NH.sub.2 (amino) NH.sub.2
(amino) CAP-100 N.sub.3 (Azido) H CAP-101 H N.sub.3 (Azido) CAP-102
N.sub.3 (Azido) N.sub.3 (Azido) CAP-103 X (Halo: F, Cl, Br, I) H
CAP-104 H X (Halo: F, Cl, Br, I) CAP-105 X (Halo: F, Cl, Br, I) X
(Halo: F, Cl, Br, I) CAP-106 SH (Thiol) H CAP-107 H SH (Thiol)
CAP-108 SH (Thiol) SH (Thiol) CAP-109 SCH.sub.3 (Thiomethyl) H
CAP-110 H SCH.sub.3 (Thiomethyl) CAP-111 SCH.sub.3 (Thiomethyl)
SCH.sub.3 (Thiomethyl)
In Table 1, "MOM" stands for methoxymethyl, "MEM" stands for
methoxyethoxymethyl, "MTM" stands for methylthiomethyl, "BOM"
stands for benzyloxymethyl and "MP" stands for monophosphonate. In
Table 1 and 2, "F" stands for fluorine, "Cl" stands for chlorine,
"Br" stands for bromine and "I" stands for iodine.
In a non-limiting example, the modified 5'cap may have the
substrate structure for vaccinia mRNA capping enzyme of:
##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054##
##STR00055## where R.sub.1 and R.sub.2 are defined in Table 3:
TABLE-US-00003 TABLE 3 R.sub.1 and R.sub.2 for CAP-136 to CAP-210
Cap Structure Number R.sub.1 R.sub.2 CAP-136 C.sub.2H.sub.5 (Ethyl)
H CAP-137 H C.sub.2H.sub.5 (Ethyl) CAP-138 C.sub.2H.sub.5 (Ethyl)
C.sub.2H.sub.5 (Ethyl) CAP-139 C.sub.3H.sub.7 (Propyl) H CAP-140 H
C.sub.3H.sub.7 (Propyl) CAP-141 C.sub.3H.sub.7 (Propyl)
C.sub.3H.sub.7 (Propyl) CAP-142 C.sub.4H.sub.9 (Butyl) H CAP-143 H
C.sub.4H.sub.9 (Butyl) CAP-144 C.sub.4H.sub.9 (Butyl)
C.sub.4H.sub.9 (Butyl) CAP-145 C.sub.5H.sub.11 (Pentyl) H CAP-146 H
C.sub.5H.sub.11 (Pentyl) CAP-147 C.sub.5H.sub.11 (Pentyl)
C.sub.5H.sub.11 (Pentyl) CAP-148 H.sub.2C--C.ident.CH (Propargyl) H
CAP-149 H H.sub.2C--C.ident.CH (Propargyl) CAP-150
H.sub.2C--C.ident.CH (Propargyl) H.sub.2C--C.ident.CH (Propargyl)
CAP-151 CH.sub.2CH.dbd.CH.sub.2 (Allyl) H CAP-152 H
CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-153 CH.sub.2CH.dbd.CH.sub.2
(Allyl) CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-154 CH.sub.2OCH.sub.3
(MOM) H CAP-155 H CH.sub.2OCH.sub.3 (MOM) CAP-156 CH.sub.2OCH.sub.3
(MOM) CH.sub.2OCH.sub.3 (MOM) CAP-157
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) H CAP-158 H
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-159
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM)
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-160 CH.sub.2SCH.sub.3
(MTM) H CAP-161 H CH.sub.2SCH.sub.3 (MTM) CAP-162 CH.sub.2SCH.sub.3
(MTM) CH.sub.2SCH.sub.3 (MTM) CAP-163 CH.sub.2C.sub.6H.sub.5
(Benzyl) H CAP-164 H CH.sub.2C.sub.6H.sub.5 (Benzyl) CAP-165
CH.sub.2C.sub.6H.sub.5 (Benzyl) CH.sub.2C.sub.6H.sub.5 (Benzyl)
CAP-166 CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) H CAP-167 H
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-168
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM)
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-169
CH.sub.2C.sub.6H.sub.4--OMe (p- H Methoxybenzyl) CAP-170 H
CH.sub.2C.sub.6H.sub.4--OMe (p- Methoxybenzyl) CAP-171
CH.sub.2C.sub.6H.sub.4--OMe (p- CH.sub.2C.sub.6H.sub.4--OMe (p-
Methoxybenzyl) Methoxybenzyl) CAP-172
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p- H Nitrobenzyl) CAP-173 H
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p- Nitrobenzyl) CAP-174
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p- Nitrobenzyl) Nitrobenzyl)
CAP-175 CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) H where X = F, Cl,
Br or I CAP-176 H CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X
= F, Cl, Br or I CAP-177 CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl)
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X = F, Cl, Br or I
where X = F, Cl, Br or I CAP-178 CH.sub.2C.sub.6H.sub.4--N.sub.3 H
(p-Azidobenzyl) CAP-179 H CH.sub.2C.sub.6H.sub.4--N.sub.3
(p-Azidobenzyl) CAP-180 CH.sub.2C.sub.6H.sub.4--N.sub.3
CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl) (p-Azidobenzyl)
CAP-181 CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- H
Trifluoromethylbenzyl) CAP-182 H CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- Trifluoromethylbenzyl) CAP-183 CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- Trifluoromethylbenzyl)
Trifluoromethylbenzyl) CAP-184 CH.sub.2C.sub.6H.sub.4--OCF.sub.3
(p- H Trifluoromethoxylbenzyl) CAP-185 H
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
CAP-186 CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p-
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
Trifluoromethoxylbenzyl) CAP-187
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4- H
bis(Trifluoromethyl)benzyl] CAP-188 H
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] CAP-189
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-190
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t- H Butyldiphenylsilyl)
CAP-191 H Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9
(t-Butyldiphenylsilyl) CAP-192
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t-
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 Butyldiphenylsilyl)
(t-Butyldiphenylsilyl) CAP-193 CH.sub.2CH.sub.2CH.dbd.CH.sub.2 H
(Homoallyl) CAP-194 H CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl)
CAP-195 CH.sub.2CH.sub.2CH.dbd.CH.sub.2
CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl) (Homoallyl) CAP-196
P(O)(OH).sub.2 (MP) H CAP-197 H P(O)(OH).sub.2 (MP) CAP-198
P(O)(OH).sub.2 (MP) P(O)(OH).sub.2 (MP) CAP-199 P(S)(OH).sub.2
(Thio-MP) H CAP-200 H P(S)(OH).sub.2 (Thio-MP) CAP-201
P(S)(OH).sub.2 (Thio-MP) P(S)(OH).sub.2 (Thio-MP) CAP-202
P(O)(CH.sub.3)(OH) H (Methylphophonate) CAP-203 H
P(O)(CH.sub.3)(OH) (Methylphophonate) CAP-204 P(O)(CH.sub.3)(OH)
P(O)(CH.sub.3)(OH) (Methylphophonate) (Methylphophonate) CAP-205
PN(.sup.IPr).sub.2(OCH.sub.2CH.sub.2CN) H (Phosporamidite) CAP-206
H PN(.sup.IPr).sub.2(OCH.sub.2CH.sub.2CN) (Phosporamidite) CAP-207
PN(.sup.IPr).sub.2(OCH.sub.2CH.sub.2CN) PN(.sup.IPr).sub.2(OCH.sub-
.2CH.sub.2CN) (Phosporamidite) (Phosporamidite) CAP-208
SO.sub.2CH.sub.3 H (Methanesulfonic acid) CAP-209 H
SO.sub.2CH.sub.3 (Methanesulfonic acid) CAP-210 SO.sub.2CH.sub.3
SO.sub.2CH.sub.3 (Methanesulfonic acid) (Methanesulfonic acid)
or
##STR00056## where R.sub.1 and R.sub.2 are defined in Table 4:
TABLE-US-00004 TABLE 4 R.sub.1 and R.sub.2 for CAP-211 to 225 Cap
Structure Number R.sub.1 R.sub.2 CAP-211 NH.sub.2 (amino) H CAP-212
H NH.sub.2 (amino) CAP-213 NH.sub.2 (amino) NH.sub.2 (amino)
CAP-214 N.sub.3 (Azido) H CAP-215 H N.sub.3 (Azido) CAP-216 N.sub.3
(Azido) N.sub.3 (Azido) CAP-217 X (Halo: F, Cl, Br, I) H CAP-218 H
X (Halo: F, Cl, Br, I) CAP-219 X (Halo: F, Cl, Br, I) X (Halo: F,
Cl, Br, I) CAP-220 SH (Thiol) H CAP-221 H SH (Thiol) CAP-222 SH
(Thiol) SH (Thiol) CAP-223 SCH.sub.3 (Thiomethyl) H CAP-224 H
SCH.sub.3 (Thiomethyl) CAP-225 SCH.sub.3 (Thiomethyl) SCH.sub.3
(Thiomethyl)
In Table 3, "MOM" stands for methoxymethyl, "MEM" stands for
methoxyethoxymethyl, "MTM" stands for methylthiomethyl, "BOM"
stands for benzyloxymethyl and "MP" stands for monophosphonate. In
Table 3 and 4, "F" stands for fluorine, "Cl" stands for chlorine,
"Br" stands for bromine and "I" stands for iodine.
In another non-limiting example, of the modified capping structure
substrates CAP-112-CAP-225 could be added in the presence of
vaccinia capping enzyme with a component to create enzymatic
activity such as, but not limited to, S-adenosylmethionine
(AdoMet), to form a modified cap for mRNA.
In one embodiment, the replacement of the sugar ring oxygen (that
produced the carbocyclic ring) with a methylene moiety (CH.sub.2)
could create greater stability to the C--N bond against
phosphorylases as the C--N bond is resistant to acid or enzymatic
hydrolysis. The methylene moiety may also increase the stability of
the triphosphate bridge moiety and thus increasing the stability of
the mRNA. As a non-limiting example, the cap substrate structure
for cap dependent translation may have the structure such as, but
not limited to, CAP-014 and CAP-015 and/or the cap substrate
structure for vaccinia mRNA capping enzyme such as, but not limited
to, CAP-123 and CAP-124. In another example, CAP-112-CAP-122 and/or
CAP-125-CAP-225, can be modified by replacing the sugar ring oxygen
(that produced the carbocyclic ring) with a methylene moiety
(CH.sub.2).
In another embodiment, the triphophosphate bridge may be modified
by the replacement of at least one oxygen with sulfur (thio), a
borane (BH.sub.3) moiety, a methyl group, an ethyl group, a methoxy
group and/or combinations thereof. This modification could increase
the stability of the mRNA towards decapping enzymes. As a
non-limiting example, the cap substrate structure for cap dependent
translation may have the structure such as, but not limited to,
CAP-016-CAP-021 and/or the cap substrate structure for vaccinia
mRNA capping enzyme such as, but not limited to, CAP-125-CAP-130.
In another example, CAP-003-CAP-015, CAP-022-CAP-124 and/or
CAP-131-CAP-225, can be modified on the triphosphate bridge by
replacing at least one of the triphosphate bridge oxygens with
sulfur (thio), a borane (BH.sub.3) moiety, a methyl group, an ethyl
group, a methoxy group and/or combinations thereof.
In one embodiment, CAP-001-134 and/or CAP-136-CAP-225 may be
modified to be a thioguanosine analog similar to CAP-135. The
thioguanosine analog may comprise additional modifications such as,
but not limited to, a modification at the triphosphate moiety
(e.g., thio, BH.sub.3, CH.sub.3, C.sub.2H.sub.5, OCH.sub.3, S and S
with OCH.sub.3), a modification at the 2' and/or 3' positions of
6-thio guanosine as described herein and/or a replacement of the
sugar ring oxygen (that produced the carbocyclic ring) as described
herein.
In one embodiment, CAP-001-121 and/or CAP-123-CAP-225 may be
modified to be a modified 5'cap similar to CAP-122. The modified
5'cap may comprise additional modifications such as, but not
limited to, a modification at the triphosphate moiety (e.g., thio,
BH.sub.3, CH.sub.3, C.sub.2H.sub.5, OCH.sub.3, S and S with
OCH.sub.3), a modification at the 2' and/or 3' positions of 6-thio
guanosine as described herein and/or a replacement of the sugar
ring oxygen (that produced the carbocyclic ring) as described
herein.
In one embodiment, the 5'cap modification may be the attachment of
biotin or conjugation at the 2' or 3' position of a GTP.
In another embodiment, the 5' cap modification may include a
CF.sub.2 modified triphosphate moiety.
In another embodiment, the triphosphate bridge of any of the cap
structures described herein may be replaced with a tetraphosphate
or pentaphosphate bridge. Examples of tetraphosphate and
pentaphosphate containing bridges and other cap modifications are
described in Jemielity, J. et al. RNA 2003 9:1108-1122;
Grudzien-Nogalska, E. et al. Methods Mol. Biol. 2013 969:55-72; and
Grudzien, E. et al. RNA, 2004 10:1479-1487, each of which is
incorporated herein by reference in its entirety.
Terminal Architecture Modifications: Stem Loop
In one embodiment, the nucleic acids of the present invention may
include a stem loop such as, but not limited to, a histone stem
loop. The stem loop may be a nucleotide sequence that is about 25
or about 26 nucleotides in length such as, but not limited to, SEQ
ID NOs: 7-17 as described in International Patent Publication No.
WO2013103659, incorporated herein by reference in its entirety. The
histone stem loop may be located 3' relative to the coding region
(e.g., at the 3' terminus of the coding region). As a non-limiting
example, the stem loop may be located at the 3' end of a nucleic
acid described herein.
In one embodiment, the stem loop may be located in the second
terminal region. As a non-limiting example, the stem loop may be
located within an untranslated region (e.g., 3'UTR) in the second
terminal region.
In one embodiment, the nucleic acid such as, but not limited to
mRNA, which comprises the histone stem loop may be stabilized by
the addition of at least one chain terminating nucleoside. Not
wishing to be bound by theory, the addition of at least one chain
terminating nucleoside may slow the degradation of a nucleic acid
and thus can increase the half-life of the nucleic acid.
In one embodiment, the chain terminating nucleoside may be, but is
not limited to, those described in International Patent Publication
No. WO2013103659, incorporated herein by reference in its entirety.
In another embodiment, the chain terminating nucleosides which may
be used with the present invention includes, but is not limited to,
3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine,
3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such
as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine,
2',3'-dideoxycytosine, 2',3'-dideoxyguanosine,
2',3'-dideoxythymine, a 2'-deoxynucleoside, or a
--O-methylnucleoside.
In another embodiment, the nucleic acid such as, but not limited to
mRNA, which comprises the histone stem loop may be stabilized by a
modification to the 3'region of the nucleic acid that can prevent
and/or inhibit the addition of oligio(U) (see e.g., International
Patent Publication No. WO2013103659, incorporated herein by
reference in its entirety).
In yet another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprises the histone stem loop may be
stabilized by the addition of an oligonucleotide that terminates in
a 3'-deoxynucleoside, 2',3'-dideoxynucleoside
3'-0-methylnucleosides, 3'-0-ethylnucleosides, 3'-arabinosides, and
other modified nucleosides known in the art and/or described
herein.
In one embodiment, the nucleic acids of the present invention may
include a histone stem loop, a polyA tail sequence and/or a 5'cap
structure. The histone stem loop may be before and/or after the
polyA tail sequence. The nucleic acids comprising the histone stem
loop and a polyA tail sequence may include a chain terminating
nucleoside described herein.
In another embodiment, the nucleic acids of the present invention
may include a histone stem loop and a 5'cap structure. The 5'cap
structure may include, but is not limited to, those described
herein and/or known in the art.
In one embodiment, the conserved stem loop region may comprise a
miR sequence described herein. As a non-limiting example, the stem
loop region may comprise the seed sequence of a miR sequence
described herein. In another non-limiting example, the stem loop
region may comprise a miR-122 seed sequence.
In another embodiment, the conserved stem loop region may comprise
a miR sequence described herein and may also include a TEE
sequence.
In one embodiment, the incorporation of a miR sequence and/or a TEE
sequence changes the shape of the stem loop region which may
increase and/or decrease translation. (see e.g, Kedde et al. A
Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221
and miR-22 accessibility. Nature Cell Biology. 2010, herein
incorporated by reference in its entirety).
In one embodiment, the modified nucleic acids described herein may
comprise at least one histone stem-loop and a polyA sequence or
polyadenylation signal. Non-limiting examples of nucleic acid
sequences encoding for at least one histone stem-loop and a polyA
sequence or a polyadenylation signal are described in International
Patent Publication No. WO2013120497, WO2013120629, WO2013120500,
WO2013120627, WO2013120498, WO2013120626, WO2013120499 and
WO2013120628, the contents of each of which are incorporated herein
by reference in their entirety. In one embodiment, the nucleic acid
encoding for a histone stem loop and a polyA sequence or a
polyadenylation signal may code for a pathogen antigen or fragment
thereof such as the nucleic acid sequences described in
International Patent Publication No WO2013120499 and WO2013120628,
the contents of both of which are incorporated herein by reference
in their entirety. In another embodiment, the nucleic acid encoding
for a histone stem loop and a polyA sequence or a polyadenylation
signal may code for a therapeutic protein such as the nucleic acid
sequences described in International Patent Publication No
WO2013120497 and WO2013120629, the contents of both of which are
incorporated herein by reference in their entirety. In one
embodiment, the nucleic acid encoding for a histone stem loop and a
polyA sequence or a polyadenylation signal may code for a tumor
antigen or fragment thereof such as the nucleic acid sequences
described in International Patent Publication No WO2013120500 and
WO2013120627, the contents of both of which are incorporated herein
by reference in their entirety. In another embodiment, the nucleic
acid encoding for a histone stem loop and a polyA sequence or a
polyadenylation signal may code for a allergenic antigen or an
autoimmune self-antigen such as the nucleic acid sequences
described in International Patent Publication No WO2013120498 and
WO2013120626, the contents of both of which are incorporated herein
by reference in their entirety.
Terminal Architecture Modifications: 3'UTR and Triple Helices
In one embodiment, nucleic acids of the present invention may
include a triple helix on the 3' end of the modified nucleic acid,
enhanced modified RNA or ribonucleic acid. The 3' end of the
nucleic acids of the present invention may include a triple helix
alone or in combination with a Poly-A tail.
In one embodiment, the nucleic acid of the present invention may
comprise at least a first and a second U-rich region, a conserved
stem loop region between the first and second region and an A-rich
region. The first and second U-rich region and the A-rich region
may associate to form a triple helix on the 3' end of the nucleic
acid. This triple helix may stabilize the nucleic acid, enhance the
translational efficiency of the nucleic acid and/or protect the 3'
end from degradation. Exemplary triple helices include, but are not
limited to, the triple helix sequence of metastasis-associated lung
adenocarcinoma transcript 1 (MALAT1), MEN-.beta. and polyadenylated
nuclear (PAN) RNA (See Wilusz et al., Genes & Development 2012
26:2392-2407; herein incorporated by reference in its entirety). In
one embodiment, the 3' end of the modified nucleic acids, enhanced
modified RNA or ribonucleic acids of the present invention
comprises a first U-rich region comprising TTTTTCTTTT (SEQ ID NO:
1), a second U-rich region comprising TTTTGCTTTTT (SEQ ID NO: 2) or
TTTTGCTTTT (SEQ ID NO: 3), an A-rich region comprising AAAAAGCAAAA
(SEQ ID NO: 4). In another embodiment, the 3' end of the nucleic
acids of the present invention comprises a triple helix formation
structure comprising a first U-rich region, a conserved region, a
second U-rich region and an A-rich region.
In one embodiment, the triple helix may be formed from the cleavage
of a MALAT1 sequence prior to the cloverleaf structure. While not
meaning to be bound by theory, MALAT1 is a long non-coding RNA
which, when cleaved, forms a triple helix and a tRNA-like
cloverleaf structure. The MALAT1 transcript then localizes to
nuclear speckles and the tRNA-like cloverleaf localizes to the
cytoplasm (Wilusz et al. Cell 2008 135(5): 919-932; incorporated
herein by reference in its entirety).
As a non-limiting example, the terminal end of the nucleic acid of
the present invention comprising the MALAT1 sequence can then form
a triple helix structure, after RNaseP cleavage from the cloverleaf
structure, which stabilizes the nucleic acid (Peart et al. Non-mRNA
3' end formation: how the other half lives; WIREs RNA 2013;
incorporated herein by reference in its entirety).
In one embodiment, the nucleic acids or mRNA described herein
comprise a MALAT1 sequence. In another embodiment, the nucleic
acids or mRNA may be polyadenylated. In yet another embodiment, the
nucleic acids or mRNA is not polyadenylated but has an increased
resistance to degradation compared to unmodified nucleic acids or
mRNA.
In one embodiment, the nucleic acids of the present invention may
comprise a MALAT1 sequence in the second flanking region (e.g., the
3'UTR). As a non-limiting example, the MALAT1 sequence may be human
or mouse.
In another embodiment, the cloverleaf structure of the MALAT1
sequence may also undergo processing by RNaseZ and CCA adding
enzyme to form a tRNA-like structure called mascRNA
(MALAT1-associated small cytoplasmic RNA). As a non-limiting
example, the mascRNA may encode a protein or a fragment thereof
and/or may comprise a microRNA sequence. The mascRNA may comprise
at least one chemical modification described herein.
Terminal Architecture Modifications: Poly-A Tails
During RNA processing, a long chain of adenine nucleotides (poly-A
tail) is normally added to a messenger RNA (mRNA) molecules to
increase the stability of the molecule. Immediately after
transcription, the 3' end of the transcript is cleaved to free a 3'
hydroxyl. Then poly-A polymerase adds a chain of adenine
nucleotides to the RNA. The process, called polyadenylation, adds a
poly-A tail that is between 100 and 250 residues long.
Methods for the stabilization of RNA by incorporation of
chain-terminating nucleosides at the 3'-terminus include those
described in International Patent Publication No. WO2013103659,
incorporated herein in its entirety.
Unique poly-A tail lengths may provide certain advantages to the
modified RNAs of the present invention.
Generally, the length of a poly-A tail of the present invention is
greater than 30 nucleotides in length. In another embodiment, the
poly-A tail 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 1700 nucleotides. In another
embodiment, the length is at least 1800 nucleotides. In another
embodiment, the length is at least 1900 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 some embodiments, the nucleic acid or mRNA includes from about
30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100,
from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000,
from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to
100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to
1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from
50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000,
from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100
to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500,
from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000
to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to
3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to
3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to
3,000).
In one embodiment, the poly-A tail may be 80 nucleotides, 120
nucleotides, 160 nucleotides in length on a modified RNA molecule
described herein.
In another embodiment, the poly-A tail may be 20, 40, 80, 100, 120,
140 or 160 nucleotides in length on a modified RNA molecule
described herein.
In one embodiment, the poly-A tail is designed relative to the
length of the overall modified RNA molecule. This design may be
based on the length of the coding region of the modified RNA, the
length of a particular feature or region of the modified RNA (such
as the mRNA), or based on the length of the ultimate product
expressed from the modified RNA. When relative to any additional
feature of the modified RNA (e.g., other than the mRNA portion
which includes the poly-A tail) the poly-A tail may be 10, 20, 30,
40, 50, 60, 70, 80, 90 or 100% greater in length than the
additional feature. The poly-A tail may also be designed as a
fraction of the modified RNA to which it belongs. In this context,
the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or
more of the total length of the construct or the total length of
the construct minus the poly-A tail.
In one embodiment, engineered binding sites and/or the conjugation
of nucleic acids or mRNA for Poly-A binding protein may be used to
enhance expression. The engineered binding sites may be sensor
sequences which can operate as binding sites for ligands of the
local microenvironment of the nucleic acids and/or mRNA. As a
non-limiting example, the nucleic acids and/or mRNA may comprise at
least one engineered binding site to alter the binding affinity of
Poly-A binding protein (PABP) and analogs thereof. The
incorporation of at least one engineered binding site may increase
the binding affinity of the PABP and analogs thereof.
Additionally, multiple distinct nucleic acids or mRNA may be linked
together to the PABP (Poly-A binding protein) through the 3'-end
using modified nucleotides at the 3'-terminus of the poly-A tail.
Transfection experiments can be conducted in relevant cell lines at
and protein production can be assayed by ELISA at 12 hr, 24 hr, 48
hr, 72 hr and day 7 post-transfection. As a non-limiting example,
the transfection experiments may be used to evaluate the effect on
PABP or analogs thereof binding affinity as a result of the
addition of at least one engineered binding site.
In one embodiment, a polyA tail may be used to modulate translation
initiation. While not wishing to be bound by theory, the polyA til
recruits PABP which in turn can interact with translation
initiation complex and thus may be essential for protein
synthesis.
In another embodiment, a polyA tail may also be used in the present
invention to protect against 3'-5' exonuclease digestion.
In one embodiment, the nucleic acids or mRNA of the present
invention are designed to include a polyA-G Quartet. The G-quartet
is a cyclic hydrogen bonded array of four guanine nucleotides that
can be formed by G-rich sequences in both DNA and RNA. In this
embodiment, the G-quartet is incorporated at the end of the poly-A
tail. The resultant nucleic acid or mRNA may be assayed for
stability, protein production and other parameters including
half-life at various time points. It has been discovered that the
polyA-G quartet results in protein production equivalent to at
least 75% of that seen using a poly-A tail of 120 nucleotides
alone.
In one embodiment, the nucleic acids or mRNA of the present
invention may comprise a polyA tail and may be stabilized by the
addition of a chain terminating nucleoside. The nucleic acids
and/or mRNA with a polyA tail may further comprise a 5'cap
structure.
In another embodiment, the nucleic acids or mRNA of the present
invention may comprise a polyA-G Quartet. The nucleic acids and/or
mRNA with a polyA-G Quartet may further comprise a 5'cap
structure.
In one embodiment, the chain terminating nucleoside which may be
used to stabilize the nucleic acid or mRNA comprising a polyA tail
or polyA-G Quartet may be, but is not limited to, those described
in International Patent Publication No. WO2013103659, incorporated
herein by reference in its entirety. In another embodiment, the
chain terminating nucleosides which may be used with the present
invention includes, but is not limited to, 3'-deoxyadenosine
(cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine,
3'-deoxythymine, 2',3'-dideoxynucleosides, such as
2',3'-dideoxyadenosine, 2',3'-dideoxyuridine,
2',3'-dideoxycytosine, 2',3'-dideoxyguanosine,
2',3'-dideoxythymine, a 2'-deoxynucleoside, or a
--O-methylnucleoside.
In another embodiment, the nucleic acid such as, but not limited to
mRNA, which comprise a polyA tail or a polyA-G Quartet may be
stabilized by a modification to the 3'region of the nucleic acid
that can prevent and/or inhibit the addition of oligio(U) (see
e.g., International Patent Publication No. WO2013103659,
incorporated herein by reference in its entirety).
In yet another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprise a polyA tail or a polyA-G Quartet
may be stabilized by the addition of an oligonucleotide that
terminates in a 3'-deoxynucleoside, 2',3'-dideoxynucleoside
3'-0-methylnucleosides, 3'-0-ethylnucleosides, 3'-arabinosides, and
other modified nucleosides known in the art and/or described
herein.
5'UTR, 3'UTR and Translation Enhancer Elements (TEEs)
In one embodiment, the 5'UTR of the polynucleotides, primary
constructs, modified nucleic acids and/or mmRNA may include at
least one translational enhancer polynucleotide, translation
enhancer element, translational enhancer elements (collectively
referred to as "TEE"s). As a non-limiting example, the TEE may be
located between the transcription promoter and the start codon. The
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA with at least one TEE in the 5'UTR may include a cap at the
5'UTR. Further, at least one TEE may be located in the 5'UTR of
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA undergoing cap-dependent or cap-independent translation.
The term "translational enhancer element" or "translation enhancer
element" (herein collectively referred to as "TEE") refers to
sequences that increase the amount of polypeptide or protein
produced from an mRNA.
In one aspect, TEEs are conserved elements in the UTR which can
promote translational activity of a nucleic acid such as, but not
limited to, cap-dependent or cap-independent translation. The
conservation of these sequences has been previously shown by Panek
et al (Nucleic Acids Research, 2013, 1-10; incorporated herein by
reference in its entirety) across 14 species including humans.
In one non-limiting example, the TEEs known may be in the 5'-leader
of the Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad.
Sci. USA 101:9590-9594, 2004, incorporated herein by reference in
their entirety).
In another non-limiting example, TEEs are disclosed as SEQ ID NOs:
1-35 in US Patent Publication No. US20090226470, SEQ ID NOs: 1-35
in US Patent Publication US20130177581, SEQ ID NOs: 1-35 in
International Patent Publication No. WO2009075886, SEQ ID NOs: 1-5,
and 7-645 in International Patent Publication No. WO2012009644, SEQ
ID NO: 1 in International Patent Publication No. WO1999024595, SEQ
ID NO: 1 in U.S. Pat. No. 6,310,197, and SEQ ID NO: 1 in U.S. Pat.
No. 6,849,405, each of which is incorporated herein by reference in
its entirety.
In yet another non-limiting example, the TEE may be an internal
ribosome entry site (IRES), HCV-IRES or an IRES element such as,
but not limited to, those described in U.S. Pat. No. 7,468,275, US
Patent Publication Nos. US20070048776 and US20110124100 and
International Patent Publication Nos. WO2007025008 and
WO2001055369, each of which is incorporated herein by reference in
its entirety. The IRES elements may include, but are not limited
to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described
by Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004)
and Zhou et al. (PNAS 102:6273-6278, 2005) and in US Patent
Publication Nos. US20070048776 and US20110124100 and International
Patent Publication No. WO2007025008, each of which is incorporated
herein by reference in its entirety.
"Translational enhancer polynucleotides" or "translation enhancer
polynucleotide sequences" are polynucleotides which include one or
more of the specific TEE exemplified herein and/or disclosed in the
art (see e.g., U.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273,
7,183,395, US20090226470, US20070048776, US20110124100,
US20090093049, US20130177581, WO2009075886, WO2007025008,
WO2012009644, WO2001055371 WO1999024595, and EP2610341A1 and
EP2610340A1; each of which is incorporated herein by reference in
its entirety) or their variants, homologs or functional
derivatives. One or multiple copies of a specific TEE can be
present in the polynucleotides, primary constructs, modified
nucleic acids and/or mmRNA. The TEEs in the translational enhancer
polynucleotides can be organized in one or more sequence segments.
A sequence segment can harbor one or more of the specific TEEs
exemplified herein, with each TEE being present in one or more
copies. When multiple sequence segments are present in a
translational enhancer polynucleotide, they can be homogenous or
heterogeneous. Thus, the multiple sequence segments in a
translational enhancer polynucleotide can harbor identical or
different types of the specific TEEs exemplified herein, identical
or different number of copies of each of the specific TEEs, and/or
identical or different organization of the TEEs within each
sequence segment.
In one embodiment, the polynucleotides, primary constructs,
modified nucleic acids and/or mmRNA may include at least one TEE
that is described in International Patent Publication No.
WO1999024595, WO2012009644, WO2009075886, WO2007025008,
WO1999024595, European Patent Publication No. EP2610341A1 and
EP2610340A1, U.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273,
7,183,395, US Patent Publication No. US20090226470, US20110124100,
US20070048776, US20090093049, and US20130177581 each of which is
incorporated herein by reference in its entirety. The TEE may be
located in the 5'UTR of the polynucleotides, primary constructs,
modified nucleic acids and/or mmRNA.
In another embodiment, the polynucleotides, primary constructs,
modified nucleic acids and/or mmRNA may include at least one TEE
that has at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95% or at least 99% identity with the TEEs described in US
Patent Publication Nos. US20090226470, US20070048776, US20130177581
and US20110124100, International Patent Publication No.
WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European
Patent Publication No. EP2610341A1 and EP2610340A1, U.S. Pat. Nos.
6,310,197, 6,849,405, 7,456,273, 7,183,395, each of which is
incorporated herein by reference in its entirety.
In one embodiment, the 5'UTR of the polynucleotides, primary
constructs, modified nucleic acids and/or mmRNA may include at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18 at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, at least 25, at least 30, at
least 35, at least 40, at least 45, at least 50, at least 55 or
more than 60 TEE sequences. The TEE sequences in the 5'UTR of the
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may be the same or different TEE
sequences. The TEE sequences may be in a pattern such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than three times. In these patterns, each letter, A, B, or
C represent a different TEE sequence at the nucleotide level.
In one embodiment, the 5'UTR may include a spacer to separate two
TEE sequences. As a non-limiting example, the spacer may be a 15
nucleotide spacer and/or other spacers known in the art. As another
non-limiting example, the 5'UTR may include a TEE sequence-spacer
module repeated at least once, at least twice, at least 3 times, at
least 4 times, at least 5 times, at least 6 times, at least 7
times, at least 8 times and at least 9 times or more than 9 times
in the 5'UTR.
In another embodiment, the spacer separating two TEE sequences may
include other sequences known in the art which may regulate the
translation of the polynucleotides, primary constructs, modified
nucleic acids and/or mmRNA of the present invention such as, but
not limited to, miR sequences described herein (e.g., miR binding
sites and miR seeds). As a non-limiting example, each spacer used
to separate two TEE sequences may include a different miR sequence
or component of a miR sequence (e.g., miR seed sequence).
In one embodiment, the TEE in the 5'UTR of the polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA of the
present invention may include at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 99% or more than 99% of
the TEE sequences disclosed in US Patent Publication Nos.
US20090226470, US20070048776, US20130177581 and US20110124100,
International Patent Publication No. WO1999024595, WO2012009644,
WO2009075886 and WO2007025008, European Patent Publication No.
EP2610341A1 and EP2610340A1, U.S. Pat. Nos. 6,310,197, 6,849,405,
7,456,273, and U.S. Pat. No. 7,183,395 each of which is
incorporated herein by reference in its entirety. In another
embodiment, the TEE in the 5'UTR of the polynucleotides, primary
constructs, modified nucleic acids and/or mmRNA of the present
invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide
fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a
5-10 nucleotide fragment of the TEE sequences disclosed in US
Patent Publication Nos. US20090226470, US20070048776, US20130177581
and US20110124100, International Patent Publication No.
WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European
Patent Publication No. EP2610341A1 and EP2610340A1, U.S. Pat. Nos.
6,310,197, 6,849,405, 7,456,273, and U.S. Pat. No. 7,183,395; each
of which is incorporated herein by reference in its entirety.
In one embodiment, the TEE in the 5'UTR of the polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA of the
present invention may include at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 99% or more than 99% of
the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad.
Sci. USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278,
2005), in Supplemental Table 1 and in Supplemental Table 2
disclosed by Wellensiek et al (Genome-wide profiling of human
cap-independent translation-enhancing elements, Nature Methods,
2013; DOI:10.1038/NMETH.2522); each of which is herein incorporated
by reference in its entirety. In another embodiment, the TEE in the
5'UTR of the polynucleotides, primary constructs, modified nucleic
acids and/or mmRNA of the present invention may include a 5-30
nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide
fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of
the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad.
Sci. USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278,
2005), in Supplemental Table 1 and in Supplemental Table 2
disclosed by Wellensiek et al (Genome-wide profiling of human
cap-independent translation-enhancing elements, Nature Methods,
2013; DOI:10.1038/NMETH.2522); each of which is incorporated herein
by reference in its entirety.
In one embodiment, the TEE used in the 5'UTR of the
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention is an IRES sequence such as, but not
limited to, those described in U.S. Pat. No. 7,468,275 and
International Patent Publication No. WO2001055369, each of which is
incorporated herein by reference in its entirety.
In one embodiment, the TEEs used in the 5'UTR of the
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may be identified by the methods
described in US Patent Publication No. US20070048776 and
US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2012009644, each of which is incorporated herein
by reference in its entirety.
In another embodiment, the TEEs used in the 5'UTR of the
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may be a transcription regulatory
element described in U.S. Pat. Nos. 7,456,273 and 7,183,395, US
Patent Publication No. US20090093049, and International Publication
No. WO2001055371, each of which is incorporated herein by reference
in its entirety. The transcription regulatory elements may be
identified by methods known in the art, such as, but not limited
to, the methods described in U.S. Pat. Nos. 7,456,273 and
7,183,395, US Patent Publication No. US20090093049, and
International Publication No. WO2001055371, each of which is
incorporated herein by reference in its entirety.
In yet another embodiment, the TEE used in the 5'UTR of the
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention is an oligonucleotide or portion
thereof as described in U.S. Pat. Nos. 7,456,273 and 7,183,395, US
Patent Publication No. US20090093049, and International Publication
No. WO2001055371, each of which is incorporated herein by reference
in its entirety.
The 5' UTR comprising at least one TEE described herein may be
incorporated in a monocistronic sequence such as, but not limited
to, a vector system or a nucleic acid vector. As a non-limiting
example, the vector systems and nucleic acid vectors may include
those described in U.S. Pat. Nos. 7,456,273 and 7,183,395, US
Patent Publication No. US20070048776, US20090093049 and
US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2001055371, each of which is incorporated herein
by reference in its entirety.
In one embodiment, the TEEs described herein may be located in the
5'UTR and/or the 3'UTR of the polynucleotides, primary constructs,
modified nucleic acids and/or mmRNA. The TEEs located in the 3'UTR
may be the same and/or different than the TEEs located in and/or
described for incorporation in the 5'UTR.
In one embodiment, the 3'UTR of the polynucleotides, primary
constructs, modified nucleic acids and/or mmRNA may include at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18 at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, at least 25, at least 30, at
least 35, at least 40, at least 45, at least 50, at least 55 or
more than 60 TEE sequences. The TEE sequences in the 3'UTR of the
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may be the same or different TEE
sequences. The TEE sequences may be in a pattern such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than three times. In these patterns, each letter, A, B, or
C represent a different TEE sequence at the nucleotide level.
In one embodiment, the 3'UTR may include a spacer to separate two
TEE sequences. As a non-limiting example, the spacer may be a 15
nucleotide spacer and/or other spacers known in the art. As another
non-limiting example, the 3'UTR may include a TEE sequence-spacer
module repeated at least once, at least twice, at least 3 times, at
least 4 times, at least 5 times, at least 6 times, at least 7
times, at least 8 times and at least 9 times or more than 9 times
in the 3'UTR.
In another embodiment, the spacer separating two TEE sequences may
include other sequences known in the art which may regulate the
translation of the polynucleotides, primary constructs, modified
nucleic acids and/or mmRNA of the present invention such as, but
not limited to, miR sequences described herein (e.g., miR binding
sites and miR seeds). As a non-limiting example, each spacer used
to separate two TEE sequences may include a different miR sequence
or component of a miR sequence (e.g., miR seed sequence).
In one embodiment, the incorporation of a miR sequence and/or a TEE
sequence changes the shape of the stem loop region which may
increase and/or decrease translation. (see e.g, Kedde et al. A
Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221
and miR-22 accessibility. Nature Cell Biology. 2010, herein
incorporated by reference in its entirety).
Heterologous 5'UTRs
A 5' UTR may be provided as a flanking region to the modified
nucleic acids (mRNA), enhanced modified RNA or ribonucleic acids of
the invention. 5'UTR may be homologous or heterologous to the
coding region found in the modified nucleic acids (mRNA), enhanced
modified RNA or ribonucleic acids of the invention. Multiple 5'
UTRs may be included in the flanking region and may be the same or
of different sequences. Any portion of the flanking regions,
including none, may be codon optimized and any may independently
contain one or more different structural or chemical modifications,
before and/or after codon optimization.
Shown in Lengthy Table 21 in U.S. Provisional Application No.
61/775,509, filed Mar. 9, 2013, entitled Heterologous Untranslated
Regions for mRNA and in Lengthy Table 21 and in Table 22 in U.S.
Provisional Application No. 61/829,372, filed May 31, 2013,
entitled Heterologous Untranslated Regions for mRNA, the contents
of each of which are incorporated herein by reference in their
entirety, is a listing of the start and stop site of the modified
nucleic acids (mRNA), enhanced modified RNA or ribonucleic acids of
the invention. In Table 21 each 5'UTR (5'UTR-005 to 5'UTR 68511) is
identified by its start and stop site relative to its native or
wild type (homologous) transcript (ENST; the identifier used in the
ENSEMBL database).
To alter one or more properties of the polynucleotides, primary
constructs or mmRNA of the invention, 5'UTRs which are heterologous
to the coding region of the modified nucleic acids (mRNA), enhanced
modified RNA or ribonucleic acids of the invention are engineered
into compounds of the invention. The modified nucleic acids (mRNA),
enhanced modified RNA or ribonucleic acids are then administered to
cells, tissue or organisms and outcomes such as protein level,
localization and/or half-life are measured to evaluate the
beneficial effects the heterologous 5'UTR may have on the modified
nucleic acids (mRNA), enhanced modified RNA or ribonucleic acids of
the invention. Variants of the 5' UTRs may be utilized wherein one
or more nucleotides are added or removed to the termini, including
A, T, C or G. 5'UTRs may also be codon-optimized or modified in any
manner described herein.
Incorporating microRNA Binding Sites
In one embodiment, modified nucleic acids (mRNA), enhanced modified
RNA or ribonucleic acids of the invention would not only encode a
polypeptide but also a sensor sequence. Sensor sequences include,
for example, microRNA binding sites, transcription factor binding
sites, structured mRNA sequences and/or motifs, artificial binding
sites engineered to act as pseudo-receptors for endogenous nucleic
acid binding molecules. Non-limiting examples, of polynucleotides
comprising at least one sensor sequence are described in co-pending
and co-owned U.S. Provisional Patent Application No. U.S.
61/753,661, filed Jan. 17, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironments, U.S. Provisional Patent Application No. U.S.
61/754,159, filed Jan. 18, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironments, U.S. Provisional Patent Application No. U.S.
61/781,097, filed Mar. 14, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironments, U.S. Provisional Patent Application No. U.S.
61/829,334, filed May 31, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironments, U.S. Provisional Patent Application No. U.S.
61/839,893, filed Jun. 27, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironments, U.S. Provisional Patent Application No. U.S.
61/842,733, filed Jul. 3, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironment, and US Provisional Patent Application No. U.S.
61/857,304, filed Jul. 23, 2013, entitled Signal-Sensor
Polynucleotide for the Alteration of Cellular Phenotypes and
Microenvironment, the contents of each of which are incorporated
herein by reference in their entirety.
In one embodiment, microRNA (miRNA) profiling of the target cells
or tissues is conducted to determine the presence or absence of
miRNA in the cells or tissues.
microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that
bind to the 3'UTR of nucleic acid molecules and down-regulate gene
expression either by reducing nucleic acid molecule stability or by
inhibiting translation. The modified nucleic acids (mRNA), enhanced
modified RNA or ribonucleic acids of the invention may comprise one
or more microRNA target sequences, microRNA sequences, or microRNA
seeds. Such sequences may correspond to any known microRNA such as
those taught in US Publication US2005/0261218 and US Publication
US200510059005, the contents of which are incorporated herein by
reference in their entirety.
A microRNA sequence comprises a "seed" region, i.e., a sequence in
the region of positions 2-8 of the mature microRNA, which sequence
has perfect Watson-Crick complementarity to the miRNA target
sequence. A microRNA seed may comprise positions 2-8 or 2-7 of the
mature microRNA. In some embodiments, a microRNA seed may comprise
7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA),
wherein the seed-complementary site in the corresponding miRNA
target is flanked by an adenine (A) opposed to microRNA position 1.
In some embodiments, a microRNA seed may comprise 6 nucleotides
(e.g., nucleotides 2-7 of the mature microRNA), wherein the
seed-complementary site in the corresponding miRNA target is
flanked by an adenine (A) opposed to microRNA position 1. See for
example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L
P, Bartel D P; Mol Cell. 2007 July 6; 27(1):91-105. The bases of
the microRNA seed have complete complementarity with the target
sequence. By engineering microRNA target sequences into the 3'UTR
of nucleic acids or mRNA of the invention one can target the
molecule for degradation or reduced translation, provided the
microRNA in question is available. This process will reduce the
hazard of off target effects upon nucleic acid molecule delivery.
Identification of microRNA, microRNA target regions, and their
expression patterns and role in biology have been reported (Bonauer
et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr
Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012
26:404-413 (2011 Dec. 20. doi: 10.1038/Ieu.2011.356); Bartel Cell
2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner
and Naldini, Tissue Antigens. 2012 80:393-403 and all references
therein; each of which is incorporated herein by reference in its
entirety).
For example, if the mRNA is not intended to be delivered to the
liver but ends up there, then miR-122, a microRNA abundant in
liver, can inhibit the expression of the gene of interest if one or
multiple target sites of miR-122 are engineered into the 3'UTR of
the modified nucleic acids, enhanced modified RNA or ribonucleic
acids. Introduction of one or multiple binding sites for different
microRNA can be engineered to further decrease the longevity,
stability, and protein translation of a modified nucleic acids,
enhanced modified RNA or ribonucleic acids. As used herein, the
term "microRNA site" refers to a microRNA target site or a microRNA
recognition site, or any nucleotide sequence to which a microRNA
binds or associates. It should be understood that "binding" may
follow traditional Watson-Crick hybridization rules or may reflect
any stable association of the microRNA with the target sequence at
or adjacent to the microRNA site.
Conversely, for the purposes of the modified nucleic acids,
enhanced modified RNA or ribonucleic acids of the present
invention, microRNA binding sites can be engineered out of (i.e.
removed from) sequences in which they naturally occur in order to
increase protein expression in specific tissues. For example,
miR-122 binding sites may be removed to improve protein expression
in the liver.
In one embodiment, the modified nucleic acids, enhanced modified
RNA or ribonucleic acids of the present invention may include at
least one miRNA-binding site in the 3'UTR in order to direct
cytotoxic or cytoprotective mRNA therapeutics to specific cells
such as, but not limited to, normal and/or cancerous cells (e.g.,
HEP3B or SNU449).
In another embodiment, the modified nucleic acids, enhanced
modified RNA or ribonucleic acids of the present invention may
include three miRNA-binding sites in the 3'UTR in order to direct
cytotoxic or cytoprotective mRNA therapeutics to specific cells
such as, but not limited to, normal and/or cancerous cells (e.g.,
HEP3B or SNU449).
Regulation of expression in multiple tissues can be accomplished
through introduction or removal or one or several microRNA binding
sites. The decision of removal or insertion of microRNA binding
sites, or any combination, is dependent on microRNA expression
patterns and their profilings in diseases.
Examples of tissues where microRNA are known to regulate mRNA, and
thereby protein expression, include, but are not limited to, liver
(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells
(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p,
miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7,
miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194,
miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
Specifically, microRNAs are known to be differentially expressed in
immune cells (also called hematopoietic cells), such as antigen
presenting cells (APCs) (e.g. dendritic cells and macrophages),
macrophages, monocytes, B lymphocytes, T lymphocytes, granuocytes,
natural killer cells, etc. Immune cell specific microRNAs are
involved in immunogenicity, autoimmunity, the immune-response to
infection, inflammation, as well as unwanted immune response after
gene therapy and tissue/organ transplantation. Immune cells
specific microRNAs also regulate many aspects of development,
proliferation, differentiation and apoptosis of hematopoietic cells
(immune cells). For example, miR-142 and miR-146 are exclusively
expressed in the immune cells, particularly abundant in myeloid
dendritic cells. It was demonstrated in the art that the immune
response to exogenous nucleic acid molecules was shut-off by adding
miR-142 binding sites to the 3'UTR of the delivered gene construct,
enabling more stable gene transfer in tissues and cells. miR-142
efficiently degrades the exogenous mRNA in antigen presenting cells
and suppresses cytotoxic elimination of transduced cells (Annoni A
et al., blood, 2009, 114, 5152-5161; Brown B D, et al., Nat med.
2006, 12(5), 585-591; Brown B D, et al., blood, 2007, 110(13):
4144-4152, each of which is incorporated herein by reference in its
entirety).
An antigen-mediated immune response can refer to an immune response
triggered by foreign antigens, which, when entering an organism,
are processed by the antigen presenting cells and displayed on the
surface of the antigen presenting cells. T cells can recognize the
presented antigen and induce a cytotoxic elimination of cells that
express the antigen.
Introducing the miR-142 binding site into the 3'-UTR of a
polypeptide of the present invention can selectively repress the
gene expression in the antigen presenting cells through miR-142
mediated mRNA degradation, limiting antigen presentation in APCs
(e.g. dendritic cells) and thereby preventing antigen-mediated
immune response after the delivery of the polynucleotides. The
polynucleotides are therefore stably expressed in target tissues or
cells without triggering cytotoxic elimination.
In one embodiment, microRNAs binding sites that are known to be
expressed in immune cells, in particular, the antigen presenting
cells, can be engineered into the polynucleotide to suppress the
expression of the sensor-signal polynucleotide in APCs through
microRNA mediated RNA degradation, subduing the antigen-mediated
immune response, while the expression of the polynucleotide is
maintained in non-immune cells where the immune cell specific
microRNAs are not expressed. For example, to prevent the
immunogenic reaction caused by a liver specific protein expression,
the miR-122 binding site can be removed and the miR-142 (and/or
mirR-146) binding sites can be engineered into the 3-UTR of the
polynucleotide.
To further drive the selective degradation and suppression of mRNA
in APCs and macrophage, the polynucleotide may include another
negative regulatory element in the 3-UTR, either alone or in
combination with mir-142 and/or mir-146 binding sites. As a
non-limiting example, one regulatory element is the Constitutive
Decay Elements (CDEs).
Immune cells specific microRNAs include, but are not limited to,
hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c,
hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p,
hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184,
hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p,
miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p,
miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p,
miR-143-5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p,
miR-147a, miR-147b, miR-148a-5p, miR-148a-3p, miR-150-3p,
miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a-3p,
miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p, miR-16-2-3p,
miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p, miR-181a-2-3p,
miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p, miR-21-5p,
miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,
miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,
miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p,
miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p,
miR-29b-1-5p, miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p,
miR-30e-5p, miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p,
miR-345-5p, miR-346, miR-34a-3p, miR-34a-5p, miR-363-3p,
miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p,
miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j,
miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p,
miR-99a-5p, miR-99b-3p and miR-99b-5p. microRNAs that are enriched
in specific types of immune cells are listed in Table 13.
Furthermore, novel miroRNAs are discovered in the immune cells in
the art through micro-array hybridization and microtome analysis
(Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC
Genomics, 2010, 11,288, the content of each of which is
incorporated herein by reference in its entirety.)
MicroRNAs that are known to be expressed in the liver include, but
are not limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p,
miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p,
miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p,
miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557,
miR-581, miR-939-3p, miR-939-5p. MicroRNA binding sites from any
liver specific microRNA can be introduced to or removed from the
polynucleotides to regulate the expression of the polynucleotides
in the liver. Liver specific microRNAs binding sites can be
engineered alone or further in combination with immune cells (e.g.
APCs) microRNA binding sites in order to prevent immune reaction
against protein expression in the liver.
MicroRNAs that are known to be expressed in the lung include, but
are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p,
miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p,
miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p,
miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p,
miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p,
miR-337-5p, miR-381-3p, miR-381-5p. MicroRNA binding sites from any
lung specific microRNA can be introduced to or removed from the
polynucleotide to regulate the expression of the polynucleotide in
the lung. Lung specific microRNAs binding sites can be engineered
alone or further in combination with immune cells (e.g. APCs)
microRNA binding sites in order to prevent an immune reaction
against protein expression in the lung.
MicroRNAs that are known to be expressed in the heart include, but
are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p,
miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210,
miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p,
miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p and
miR-92b-5p. MicroRNA binding sites from any heart specific microRNA
can be introduced to or removed from the polynucleotides to
regulate the expression of the polynucleotides in the heart. Heart
specific microRNAs binding sites can be engineered alone or further
in combination with immune cells (e.g. APCs) microRNA binding sites
to prevent an immune reaction against protein expression in the
heart.
MicroRNAs that are known to be expressed in the nervous system
include, but are not limited to, miR-124-5p, miR-125a-3p,
miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p,
miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p,
miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p,
miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,
miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b,
miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p,
miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p,
miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329,
miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383,
miR-410, miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483,
miR-510, miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571,
miR-7-1-3p, miR-7-2-3p, miR-7-5p, miR-802, miR-922, miR-9-3p and
miR-9-5p. MicroRNAs enriched in the nervous system further include
those specifically expressed in neurons, including, but not limited
to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p,
miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e,
miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328,
miR-922 and those specifically expressed in glial cells, including,
but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,
miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,
miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, miR-657. MicroRNA
binding sites from any CNS specific microRNA can be introduced to
or removed from the polynucleotides to regulate the expression of
the polynucleotide in the nervous system. Nervous system specific
microRNAs binding sites can be engineered alone or further in
combination with immune cells (e.g. APCs) microRNA binding sites in
order to prevent immune reaction against protein expression in the
nervous system.
MicroRNAs that are known to be expressed in the pancreas include,
but are not limited to, miR-105-3p, miR-105-5p, miR-184,
miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p,
miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p,
miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p
and miR-944. MicroRNA binding sites from any pancreas specific
microRNA can be introduced to or removed from the polynucleotide to
regulate the expression of the polynucleotide in the pancreas.
Pancreas specific microRNAs binding sites can be engineered alone
or further in combination with immune cells (e.g. APCs) microRNA
binding sites in order to prevent an immune reaction against
protein expression in the pancreas.
MicroRNAs that are known to be expressed in the kidney further
include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p,
miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p,
miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p,
miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p,
miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p, miR-324-3p,
miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p and miR-562.
MicroRNA binding sites from any kidney specific microRNA can be
introduced to or removed from the polynucleotide to regulate the
expression of the polynucleotide in the kidney. Kidney specific
microRNAs binding sites can be engineered alone or further in
combination with immune cells (e.g. APCs) microRNA binding sites to
prevent an immune reaction against protein expression in the
kidney.
MicroRNAs that are known to be expressed in the muscle further
include, but are not limited to, let-7g-3p, let-7g-5p, miR-1,
miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p,
miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a,
miR-208b, miR-25-3p and miR-25-5p. MicroRNA binding sites from any
muscle specific microRNA can be introduced to or removed from the
polynucleotide to regulate the expression of the polynucleotide in
the muscle. Muscle specific microRNAs binding sites can be
engineered alone or further in combination with immune cells (e.g.
APCs) microRNA binding sites to prevent an immune reaction against
protein expression in the muscle.
MicroRNAs are differentially expressed in different types of cells,
such as endothelial cells, epithelial cells and adipocytes. For
example, microRNAs that are expressed in endothelial cells include,
but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p,
miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p,
miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p,
miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p,
miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p,
miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p,
miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p,
miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p,
miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p and
miR-92b-5p. Many novel microRNAs are discovered in endothelial
cells from deep-sequencing analysis (Voellenkle C et al., RNA,
2012, 18, 472-484, herein incorporated by reference in its
entirety) microRNA binding sites from any endothelial cell specific
microRNA can be introduced to or removed from the polynucleotide to
modulate the expression of the polynucleotide in the endothelial
cells in various conditions.
For further example, microRNAs that are expressed in epithelial
cells include, but are not limited to, let-7b-3p, let-7b-5p,
miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p,
miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b,
miR-494, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a,
miR-449b-3p, miR-449b-5p specific in respiratory ciliated
epithelial cells; let-7 family, miR-133a, miR-133b, miR-126
specific in lung epithelial cells; miR-382-3p, miR-382-5p specific
in renal epithelial cells and miR-762 specific in corneal
epithelial cells. MicroRNA binding sites from any epithelial cell
specific MicroRNA can be introduced to or removed from the
polynucleotide to modulate the expression of the polynucleotide in
the epithelial cells in various conditions.
In addition, a large group of microRNAs are enriched in embryonic
stem cells, controlling stem cell self-renewal as well as the
development and/or differentiation of various cell lineages, such
as neural cells, cardiac, hematopoietic cells, skin cells,
osteogenic cells and muscle cells (Kuppusamy K T et al., Curr. Mol
Med, 2013, 13(5), 757-764; Vidigal J A and Ventura A, Semin Cancer
Biol. 2012, 22(5-6), 428-436; Goff L A et al., PLoS One, 2009,
4:e7192; Morin R D et al., Genome Res, 2008, 18, 610-621; Yoo J K
et al., Stem Cells Dev. 2012, 21(11), 2049-2057, each of which is
herein incorporated by reference in its entirety). MicroRNAs
abundant in embryonic stem cells include, but are not limited to,
let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,
miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,
miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,
miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p,
miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p,
miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e,
miR-367-3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371,
miR-373, miR-380-5p, miR-423-3p, miR-423-5p, miR-486-5p,
miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR-548g-5p,
miR-548i, miR-548k, miR-548l, miR-548m, miR-548n, miR-548o-3p,
miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR-664b-3p,
miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p, miR-885-5p,
miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p, miR-99b-3p and
miR-99b-5p. Many predicted novel microRNAs are discovered by deep
sequencing in human embryonic stem cells (Morin R D et al., Genome
Res, 2008, 18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;
Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each
of which is incorporated herein by references in its entirety).
In one embodiment, the binding sites of embryonic stem cell
specific microRNAs can be included in or removed from the 3-UTR of
the polynucleotide to modulate the development and/or
differentiation of embryonic stem cells, to inhibit the senescence
of stem cells in a degenerative condition (e.g. degenerative
diseases), or to stimulate the senescence and apoptosis of stem
cells in a disease condition (e.g. cancer stem cells).
Many microRNA expression studies are conducted in the art to
profile the differential expression of microRNAs in various cancer
cells/tissues and other diseases. Some microRNAs are abnormally
over-expressed in certain cancer cells and others are
under-expressed. For example, microRNAs are differentially
expressed in cancer cells (WO2008/154098, US2013/0059015,
US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224);
pancreatic cancers and diseases (US2009/0131348, US2011/0171646,
US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation
(U.S. Pat. No. 8,415,096); prostate cancer (US2013/0053264);
hepatocellular carcinoma (WO2012/151212, US2012/0329672,
WO2008/054828, U.S. Pat. No. 8,252,538); lung cancer cells
(WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357);
cutaneous T cell lymphoma (WO2013/011378); colorectal cancer cells
(WO2011/0281756, WO2011/076142); cancer positive lymph nodes
(WO2009/100430, US2009/0263803); nasopharyngeal carcinoma
(EP2112235); chronic obstructive pulmonary disease (US2012/0264626,
US2013/0053263); thyroid cancer (WO2013/066678); ovarian cancer
cells (US2012/0309645, WO2011/095623); breast cancer cells
(WO2008/154098, WO2007/081740, US2012/0214699), leukemia and
lymphoma (WO2008/073915, US2009/0092974, US2012/0316081,
US2012/0283310, WO2010/018563, the content of each of which is
incorporated herein by reference in its entirety.)
As a non-limiting example, microRNA sites that are over-expressed
in certain cancer and/or tumor cells can be removed from the 3-UTR
of the polynucleotide encoding the polypeptide of interest,
restoring the expression suppressed by the over-expressed microRNAs
in cancer cells, thus ameliorating the corresponsive biological
function, for instance, transcription stimulation and/or
repression, cell cycle arrest, apoptosis and cell death. Normal
cells and tissues, wherein microRNAs expression is not
up-regulated, will remain unaffected.
MicroRNA can also regulate complex biological processes such as
angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011
18:171-176). In the modified nucleic acids, enhanced modified RNA
or ribonucleic acids of the invention, binding sites for microRNAs
that are involved in such processes may be removed or introduced,
in order to tailor the expression of the modified nucleic acids,
enhanced modified RNA or ribonucleic acids expression to
biologically relevant cell types or to the context of relevant
biological processes. In this context, the mRNA are defined as
auxotrophic mRNA.
MicroRNA gene regulation may be influenced by the sequence
surrounding the microRNA such as, but not limited to, the species
of the surrounding sequence, the type of sequence (e.g.,
heterologous, homologous and artificial), regulatory elements in
the surrounding sequence and/or structural elements in the
surrounding sequence. The microRNA may be influenced by the 5'UTR
and/or the 3'UTR. As a non-limiting example, a non-human 3'UTR may
increase the regulatory effect of the microRNA sequence on the
expression of a polypeptide of interest compared to a human 3'UTR
of the same sequence type.
In one embodiment, other regulatory elements and/or structural
elements of the 5'-UTR can influence microRNA mediated gene
regulation. One example of a regulatory element and/or structural
element is a structured IRES (Internal Ribosome Entry Site) in the
5'UTR, which is necessary for the binding of translational
elongation factors to initiate protein translation. EIF4A2 binding
to this secondarily structured element in the 5'UTR is necessary
for microRNA mediated gene expression (Meijer H A et al., Science,
2013, 340, 82-85, herein incorporated by reference in its
entirety). The modified nucleic acids, enhanced modified RNA or
ribonucleic acids of the invention can further be modified to
include this structured 5'-UTR in order to enhance microRNA
mediated gene regulation.
At least one microRNA site can be engineered into the 3' UTR of the
modified nucleic acids, enhanced modified RNA or ribonucleic acids
of the present invention. In this context, at least two, at least
three, at least four, at least five, at least six, at least seven,
at least eight, at least nine, at least ten or more microRNA sites
may be engineered into the 3' UTR of the ribonucleic acids of the
present invention. In one embodiment, the microRNA sites
incorporated into the modified nucleic acids, enhanced modified RNA
or ribonucleic acids may be the same or may be different microRNA
sites. In another embodiment, the microRNA sites incorporated into
the modified nucleic acids, enhanced modified RNA or ribonucleic
acids may target the same or different tissues in the body. As a
non-limiting example, through the introduction of tissue-,
cell-type-, or disease-specific microRNA binding sites in the 3'
UTR of a modified nucleic acid mRNA, the degree of expression in
specific cell types (e.g. hepatocytes, myeloid cells, endothelial
cells, cancer cells, etc.) can be reduced.
In one embodiment, a microRNA site can be engineered near the 5'
terminus of the 3'UTR, about halfway between the 5' terminus and
3'terminus of the 3'UTR and/or near the 3'terminus of the 3'UTR. As
a non-limiting example, a microRNA site may be engineered near the
5' terminus of the 3'UTR and about halfway between the 5' terminus
and 3'terminus of the 3'UTR. As another non-limiting example, a
microRNA site may be engineered near the 3'terminus of the 3'UTR
and about halfway between the 5' terminus and 3'terminus of the
3'UTR. As yet another non-limiting example, a microRNA site may be
engineered near the 5' terminus of the 3'UTR and near the 3'
terminus of the 3'UTR.
In another embodiment, a 3'UTR can comprise 4 microRNA sites. The
microRNA sites may be complete microRNA binding sites, microRNA
seed sequences and/or microRNA binding site sequences without the
seed sequence.
In one embodiment, a nucleic acid of the invention may be
engineered to include at least one microRNA in order to dampen the
antigen presentation by antigen presenting cells. The microRNA may
be the complete microRNA sequence, the microRNA seed sequence, the
microRNA sequence without the seed or a combination thereof. As a
non-limiting example, the microRNA incorporated into the nucleic
acid may be specific to the hematopoietic system. As another
non-limiting example, the microRNA incorporated into the nucleic
acid of the invention to dampen antigen presentation is
miR-142-3p.
In one embodiment, a nucleic acid may be engineered to include
microRNA sites which are expressed in different tissues of a
subject. As a non-limiting example, a modified nucleic acid,
enhanced modified RNA or ribonucleic acid of the present invention
may be engineered to include miR-192 and miR-122 to regulate
expression of the modified nucleic acid, enhanced modified RNA or
ribonucleic acid in the liver and kidneys of a subject. In another
embodiment, a modified nucleic acid, enhanced modified RNA or
ribonucleic acid may be engineered to include more than one
microRNA sites for the same tissue. For example, a modified nucleic
acid, enhanced modified RNA or ribonucleic acid of the present
invention may be engineered to include miR-17-92 and miR-126 to
regulate expression of the modified nucleic acid, enhanced modified
RNA or ribonucleic acid in endothelial cells of a subject.
In one embodiment, the therapeutic window and or differential
expression associated with the target polypeptide encoded by the
modified nucleic acid, enhanced modified RNA or ribonucleic acid
encoding a signal (also referred to herein as a polynucleotide) of
the invention may be altered. For example, polynucleotides may be
designed whereby a death signal is more highly expressed in cancer
cells (or a survival signal in a normal cell) by virtue of the
miRNA signature of those cells. Where a cancer cell expresses a
lower level of a particular miRNA, the polynucleotide encoding the
binding site for that miRNA (or miRNAs) would be more highly
expressed. Hence, the target polypeptide encoded by the
polynucleotide is selected as a protein which triggers or induces
cell death. Neighboring noncancer cells, harboring a higher
expression of the same miRNA would be less affected by the encoded
death signal as the polynucleotide would be expressed at a lower
level due to the effects of the miRNA binding to the binding site
or "sensor" encoded in the 3'UTR. Conversely, cell survival or
cytoprotective signals may be delivered to tissues containing
cancer and non-cancerous cells where a miRNA has a higher
expression in the cancer cells--the result being a lower survival
signal to the cancer cell and a larger survival signature to the
normal cell. Multiple polynucleotides may be designed and
administered having different signals according to the previous
paradigm.
In one embodiment, the expression of a nucleic acid may be
controlled by incorporating at least one sensor sequence in the
nucleic acid and formulating the nucleic acid. As a non-limiting
example, a nucleic acid may be targeted to an orthotopic tumor by
having a nucleic acid incorporating a miR-122 binding site and
formulated in a lipid nanoparticle comprising the cationic lipid
DLin-KC2-DMA.
According to the present invention, the polynucleotides may be
modified as to avoid the deficiencies of other polypeptide-encoding
molecules of the art. Hence, in this embodiment the polynucleotides
are referred to as modified polynucleotides.
Through an understanding of the expression patterns of microRNA in
different cell types, modified nucleic acids, enhanced modified RNA
or ribonucleic acids such as polynucleotides can be engineered for
more targeted expression in specific cell types or only under
specific biological conditions. Through introduction of
tissue-specific microRNA binding sites, modified nucleic acids,
enhanced modified RNA or ribonucleic acids, could be designed that
would be optimal for protein expression in a tissue or in the
context of a biological condition.
Transfection experiments can be conducted in relevant cell lines,
using engineered modified nucleic acids, enhanced modified RNA or
ribonucleic acids and protein production can be assayed at various
time points post-transfection. For example, cells can be
transfected with different microRNA binding site-engineering
nucleic acids or mRNA and by using an ELISA kit to the relevant
protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr,
72 hr and 7 days post-transfection. In vivo experiments can also be
conducted using microRNA-binding site-engineered molecules to
examine changes in tissue-specific expression of formulated
modified nucleic acids, enhanced modified RNA or ribonucleic
acids.
Non-limiting examples of cell lines which may be useful in these
investigations include those from ATCC (Manassas, Va.) including
MRC-5, A549, T84, NCI-H2126 [H2126], NCI-H1688 [H1688], WI-38,
WI-38 VA-13 subline 2RA, WI-26 VA4, C3A [HepG2/C3A, derivative of
Hep G2 (ATCC HB-8065)], THLE-3, H69AR, NCI-H292 [H292], CFPAC-1,
NTERA-2 cl.D1 [NT2/D1], DMS 79, DMS 53, DMS 153, DMS 114,
MSTO-211H, SW 1573 [SW-1573, SW1573], SW 1271 [SW-1271, SW1271],
SHP-77, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387, SNU-423, NL20,
NL20-TA [NL20T-A], THLE-2, HBE135-E6E7, HCC827, HCC4006, NCI-H23
[H23], NCI-H1299, NCI-H187 [H187], NCI-H358 [H-358, H358], NCI-H378
[H378], NCI-H522 [H522], NCI-H526 [H526], NCI-H727 [H727], NCI-H810
[H810], NCI-H889 [H889], NCI-H1155 [H1155], NCI-H1404 [H1404],
NCI-N87 [N87], NCI-H196 [H196], NCI-H211 [H211], NCI-H220 [H220],
NCI-H250 [H250], NCI-H524 [H524], NCI-H647 [H647], NCI-H650 [H650],
NCI-H711 [H711], NCI-H719 [H719], NCI-H740 [H740], NCI-H748 [H748],
NCI-H774 [H774], NCI-H838 [H838], NCI-H841 [H841], NCI-H847 [H847],
NCI-H865 [H865], NCI-H920 [H920], NCI-H1048 [H1048], NCI-H1092
[H1092], NCI-H1105 [H1105], NCI-H1184 [H1184], NCI-H1238 [H1238],
NCI-H1341 [H1341], NCI-H1385 [H1385], NCI-H1417 [H1417], NCI-H1435
[H1435], NCI-H1436 [H1436], NCI-H1437 [H1437], NCI-H1522 [H1522],
NCI-H1563 [H1563], NCI-H1568 [H1568], NCI-H1573 [H1573], NCI-H1581
[H1581], NCI-H1618 [H1618], NCI-H1623 [H1623], NCI-H1650 [H-1650,
H1650], NCI-H1651 [H1651], NCI-H1666 [H-1666, H1666], NCI-H1672
[H1672], NCI-H1693 [H1693], NCI-H1694 [H1694], NCI-H1703 [H1703],
NCI-H1734 [H-1734, H1734], NCI-H1755 [H1755], NCI-H1755 [H1755],
NCI-H1770 [H1770], NCI-H1793 [H1793], NCI-H1836 [H1836], NCI-H1838
[H1838], NCI-H1869 [H1869], NCI-H1876 [H1876], NCI-H1882 [H1882],
NCI-H1915 [H1915], NCI-H1930 [H1930], NCI-H1944 [H1944], NCI-H1975
[H-1975, H1975], NCI-H1993 [H1993], NCI-H2023 [H2023], NCI-H2029
[H2029], NCI-H2030 [H2030], NCI-H2066 [H2066], NCI-H2073 [H2073],
NCI-H2081 [H2081], NCI-H2085 [H2085], NCI-H2087 [H2087], NCI-H2106
[H2106], NCI-H2110 [H2110], NCI-H2135 [H2135], NCI-H2141 [H2141],
NCI-H2171 [H2171], NCI-H2172 [H2172], NCI-H2195 [H2195], NCI-H2196
[H2196], NCI-H2198 [H2198], NCI-H2227 [H2227], NCI-H2228 [H2228],
NCI-H2286 [H2286], NCI-H2291 [H2291], NCI-H2330 [H2330], NCI-H2342
[H2342], NCI-H2347 [H2347], NCI-H2405 [H2405], NCI-H2444 [H2444],
UMC-11, NCI-H64 [H64], NCI-H735 [H735], NCI-H735 [H735], NCI-H1963
[H1963], NCI-H2107 [H2107], NCI-H2108 [H2108], NCI-H2122 [H2122],
Hs 573.T, Hs 573.Lu, PLC/PRF/5, BEAS-2B, Hep G2, Tera-1, Tera-2,
NCI-H69 [H69], NCI-H128 [H128], ChaGo-K-1, NCI-H446 [H446],
NCI-H209 [H209], NCI-H146 [H146], NCI-H441 [H441], NCI-H82 [H82],
NCI-H460 [H460], NCI-H596 [H596], NCI-H676B [H676B], NCI-H345
[H345], NCI-H820 [H820], NCI-H520 [H520], NCI-H661 [H661],
NCI-H510A [H510A, NCI-H510], SK-HEP-1, A-427, Calu-1, Calu-3,
Calu-6, SK-LU-1, SK-MES-1, SW 900 [SW-900, SW900], Malme-3M, and
Capan-1.
In some embodiments, modified messenger RNA can be designed to
incorporate microRNA binding region sites that either have 100%
identity to known seed sequences or have less than 100% identity to
seed sequences. The seed sequence can be partially mutated to
decrease microRNA binding affinity and as such result in reduced
downmodulation of that mRNA transcript. In essence, the degree of
match or mis-match between the target mRNA and the microRNA seed
can act as a rheostat to more finely tune the ability of the
microRNA to modulate protein expression. In addition, mutation in
the non-seed region of a microRNA binding site may also impact the
ability of a microRNA to modulate protein expression.
In one embodiment, a miR sequence may be incorporated into the loop
of a stem loop.
In another embodiment, a miR seed sequence may be incorporated in
the loop of a stem loop and a miR binding site may be incorporated
into the 5' or 3' stem of the stem loop.
In one embodiment, a TEE may be incorporated on the 5'end of the
stem of a stem loop and a miR seed may be incorporated into the
stem of the stem loop. In another embodiment, a TEE may be
incorporated on the 5'end of the stem of a stem loop, a miR seed
may be incorporated into the stem of the stem loop and a miR
binding site may be incorporated into the 3'end of the stem or the
sequence after the stem loop. The miR seed and the miR binding site
may be for the same and/or different miR sequences.
In one embodiment, the incorporation of a miR sequence and/or a TEE
sequence changes the shape of the stem loop region which may
increase and/or decrease translation. (see e.g, Kedde et al. A
Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221
and miR-22 accessibility. Nature Cell Biology. 2010, incorporated
herein by reference in its entirety).
In one embodiment, the incorporation of a miR sequence and/or a TEE
sequence changes the shape of the stem loop region which may
increase and/or decrease translation. (see e.g, Kedde et al. A
Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221
and miR-22 accessibility. Nature Cell Biology. 2010, incorporated
herein by reference in its entirety).
In one embodiment, the 5'UTR may comprise at least one microRNA
sequence. The microRNA sequence may be, but is not limited to, a 19
or 22 nucleotide sequence and/or a microRNA sequence without the
seed.
In one embodiment the microRNA sequence in the 5'UTR may be used to
stabilize the nucleic acid and/or mRNA described herein.
In another embodiment, a microRNA sequence in the 5'UTR may be used
to decrease the accessibility of the site of translation initiation
such as, but not limited to a start codon. Matsuda et al (PLoS One.
2010 11(5):e15057; incorporated herein by reference in its
entirety) used antisense locked nucleic acid (LNA) oligonucleotides
and exon-junction complexes (EJCs) around a start codon (-4 to +37
where the A of the AUG codons is +1) in order to decrease the
accessibility to the first start codon (AUG). Matsuda showed that
altering the sequence around the start codon with an LNA or EJC the
efficiency, length and structural stability of the nucleic acid or
mRNA is affected. The nucleic acids or mRNA of the present
invention may comprise a microRNA sequence, instead of the LNA or
EJC sequence described by Matsuda et al, near the site of
translation initiation in order to decrease the accessibility to
the site of translation initiation. The site of translation
initiation may be prior to, after or within the microRNA sequence.
As a non-limiting example, the site of translation initiation may
be located within a microRNA sequence such as a seed sequence or
binding site. As another non-limiting example, the site of
translation initiation may be located within a miR-122 sequence
such as the seed sequence or the mir-122 binding site.
In one embodiment, the nucleic acids or mRNA of the present
invention may include at least one microRNA in order to dampen the
antigen presentation by antigen presenting cells. The microRNA may
be the complete microRNA sequence, the microRNA seed sequence, the
microRNA sequence without the seed or a combination thereof. As a
non-limiting example, the microRNA incorporated into the nucleic
acids or mRNA of the present invention may be specific to the
hematopoietic system. As another non-limiting example, the microRNA
incorporated into the nucleic acids or mRNA of the present
invention to dampen antigen presentation is miR-142-3p.
In one embodiment, the nucleic acids or mRNA of the present
invention may include at least one microRNA in order to dampen
expression of the encoded polypeptide in a cell of interest. As a
non-limiting example, the nucleic acids or mRNA of the present
invention may include at least one miR-122 binding site in order to
dampen expression of an encoded polypeptide of interest in the
liver. As another non-limiting example, the nucleic acids or mRNA
of the present invention may include at least one miR-142-3p
binding site, miR-142-3p seed sequence, miR-142-3p binding site
without the seed, miR-142-5p binding site, miR-142-5p seed
sequence, miR-142-5p binding site without the seed, miR-146 binding
site, miR-146 seed sequence and/or miR-146 binding site without the
seed sequence.
In one embodiment, the nucleic acids or mRNA of the present
invention may comprise at least one microRNA binding site in the
3'UTR in order to selectively degrade mRNA therapeutics in the
immune cells to subdue unwanted immunogenic reactions caused by
therapeutic delivery. As a non-limiting example, the microRNA
binding site may be the modified nucleic acids more unstable in
antigen presenting cells. Non-limiting examples of these microRNA
include mir-142-5p, mir-142-3p, mir-146a-5p and mir-146-3p.
In one embodiment, the nucleic acids or mRNA of the present
invention comprises at least one microRNA sequence in a region of
the nucleic acid or mRNA which may interact with a RNA binding
protein.
RNA Motifs for RNA Binding Proteins (RBPs)
RNA binding proteins (RBPs) can regulate numerous aspects of co-
and post-transcription gene expression such as, but not limited to,
RNA splicing, localization, translation, turnover, polyadenylation,
capping, modification, export and localization. RNA-binding domains
(RBDs), such as, but not limited to, RNA recognition motif (RR) and
hnRNP K-homology (KH) domains, typically regulate the sequence
association between RBPs and their RNA targets (Ray et al. Nature
2013. 499:172-177; incorporated herein by reference in its
entirety). In one embodiment, the canonical RBDs can bind short RNA
sequences. In another embodiment, the canonical RBDs can recognize
structure RNAs.
In one embodiment, to increase the stability of the mRNA of
interest, an mRNA encoding HuR can be co-transfected or co-injected
along with the mRNA of interest into the cells or into the tissue.
These proteins can also be tethered to the mRNA of interest in
vitro and then administered to the cells together. Poly A tail
binding protein, PABP interacts with eukaryotic translation
initiation factor eIF4G to stimulate translational initiation.
Co-administration of mRNAs encoding these RBPs along with the mRNA
drug and/or tethering these proteins to the mRNA drug in vitro and
administering the protein-bound mRNA into the cells can increase
the translational efficiency of the mRNA. The same concept can be
extended to co-administration of mRNA along with mRNAs encoding
various translation factors and facilitators as well as with the
proteins themselves to influence RNA stability and/or translational
efficiency.
In one embodiment, the nucleic acids and/or mRNA may comprise at
least one RNA-binding motif such as, but not limited to a
RNA-binding domain (RBD).
In one embodiment, the RBD may be any of the RBDs, fragments or
variants thereof descried by Ray et al. (Nature 2013. 499:172-177;
incorporated herein by reference in its entirety).
In one embodiment, the nucleic acids or mRNA of the present
invention may comprise a sequence for at least one RNA-binding
domain (RBDs). When the nucleic acids or mRNA of the present
invention comprise more than one RBD, the RBDs do not need to be
from the same species or even the same structural class.
In one embodiment, at least one flanking region (e.g., the 5'UTR
and/or the 3'UTR) may comprise at least one RBD. In another
embodiment, the first flanking region and the second flanking
region may both comprise at least one RBD. The RBD may be the same
or each of the RBDs may have at least 60% sequence identity to the
other RBD. As a non-limiting example, at least on RBD may be
located before, after and/or within the 3'UTR of the nucleic acid
or mRNA of the present invention. As another non-limiting example,
at least one RBD may be located before or within the first 300
nucleosides of the 3'UTR.
In another embodiment, the nucleic acids and/or mRNA of the present
invention may comprise at least one RBD in the first region of
linked nucleosides. The RBD may be located before, after or within
a coding region (e.g., the ORF).
In yet another embodiment, the first region of linked nucleosides
and/or at least one flanking region may comprise at least on RBD.
As a non-limiting example, the first region of linked nucleosides
may comprise a RBD related to splicing factors and at least one
flanking region may comprise a RBD for stability and/or translation
factors.
In one embodiment, the nucleic acids and/or mRNA of the present
invention may comprise at least one RBD located in a coding and/or
non-coding region of the nucleic acids and/or mRNA.
In one embodiment, at least one RBD may be incorporated into at
least one flanking region to increase the stability of the nucleic
acid and/or mRNA of the present invention.
In one embodiment, a microRNA sequence in a RNA binding protein
motif may be used to decrease the accessibility of the site of
translation initiation such as, but not limited to a start codon.
The nucleic acids or mRNA of the present invention may comprise a
microRNA sequence, instead of the LNA or EJC sequence described by
Matsuda et al, near the site of translation initiation in order to
decrease the accessibility to the site of translation initiation.
The site of translation initiation may be prior to, after or within
the microRNA sequence. As a non-limiting example, the site of
translation initiation may be located within a microRNA sequence
such as a seed sequence or binding site. As another non-limiting
example, the site of translation initiation may be located within a
miR-122 sequence such as the seed sequence or the mir-122 binding
site.
In another embodiment, an antisense locked nucleic acid (LNA)
oligonucleotides and exon-junction complexes (EJCs) may be used in
the RNA binding protein motif. The LNA and EJCs may be used around
a start codon (-4 to +37 where the A of the AUG codons is +1) in
order to decrease the accessibility to the first start codon
(AUG).
Codon Optimization
The polynucleotides of the invention, their regions or parts or
subregions may be codon optimized. Codon optimization methods are
known in the art and may be useful in efforts to achieve one or
more of several goals. These goals include to match codon
frequencies in target and host organisms to ensure proper folding,
bias GC content to increase mRNA stability or reduce secondary
structures, minimize tandem repeat codons or base runs that may
impair gene construction or expression, customize transcriptional
and translational control regions, insert or remove protein
trafficking sequences, remove/add post translation modification
sites in encoded protein (e.g., glycosylation sites), add, remove
or shuffle protein domains, insert or delete restriction sites,
modify ribosome binding sites and mRNA degradation sites, to adjust
translational rates to allow the various domains of the protein to
fold properly, or to reduce or eliminate problem secondary
structures within the polynucleotide. Codon optimization tools,
algorithms and services are known in the art, non-limiting examples
include services from GeneArt (Life Technologies), DNA2.0 (Menlo
Park Calif.) and/or proprietary methods. In one embodiment, the ORF
sequence is optimized using optimization algorithms. Codon options
for each amino acid are given in Table 5.
TABLE-US-00005 TABLE 5 Codon Options Single Letter Amino Acid Code
Codon Options Isoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA,
CTG, TTA, TTG Valine V GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC
Methionine M ATG Cysteine C TGT, TGC Alanine A GCT, GCC, GCA, GCG
Glycine G GGT, GGC, GGA, GGG Proline P CCT, CCC, CCA, CCG Threonine
T ACT, ACC, ACA, ACG Serine S TCT, TCC, TCA, TCG, AGT, AGC Tyrosine
Y TAT, TAC Tryptophan W TGG Glutamine Q CAA, CAG Asparagine N AAT,
AAC Histidine H CAT, CAC Glutamic acid E GAA, GAG Aspartic acid D
GAT, GAC Lysine K AAA, AAG Arginine R CGT, CGC, CGA, CGG, AGA, AGG
Selenocysteine Sec UGA in mRNA in presence of Selenocystein
insertion element (SECIS) Stop codons Stop TAA, TAG, TGA
"Codon optimized" refers to the modification of a starting
nucleotide sequence by replacing at least one codon of the starting
nucleotide sequence with a codon that is more frequently used in
the group of abundant polypeptides of the host organism. Table 6
contains the codon usage frequency for humans (Codon usage
database:
[[www.]]kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606&aa=1&style=-
N).
Codon optimization may be used to increase the expression of
polypeptides by the replacement of at least one, at least two, at
least three, at least four, at least five, at least six, at least
seven, at least eight, at least nine, at least ten or at least 1%,
at least 2%, at least 4%, at least 6%, at least 8%, at least 10%,
at least 20%, at least 40%, at least 60%, at least 80%, at least
90% or at least 95%, or all codons of the starting nucleotide
sequence with more frequently or the most frequently used codons
for the respective amino acid as determined for the group of
abundant proteins.
In one embodiment of the invention, the modified nucleotide
sequences contain for each amino acid the most frequently used
codons of the abundant proteins of the respective host cell.
TABLE-US-00006 TABLE 6 Codon usage frequency table for humans.
Amino Amino Amino Amino Codon Acid % Codon Acid % Codon Acid %
Codon Acid % UUU F 46 UCU S 19 UAU Y 44 UGU C 46 UUC F 54 UCC S 22
UAC Y 56 UGC C 54 UUA L 8 UCA S 15 UAA * 30 UGA * 47 UUG L 13 UCG S
5 UAG * 24 UGG W 100 CUU L 13 CCU P 29 CAU H 42 CGU R 8 CUC L 20
CCC P 32 CAC H 58 CGC R 18 CUA L 7 CCA P 28 CAA Q 27 CGA R 11 CUG L
40 CCG P 11 CAG Q 73 CGG R 20 AUU I 36 ACU T 25 AAU N 47 AGU S 15
AUC I 47 ACC T 36 AAC N 53 AGC S 24 AUA I 17 ACA T 28 AAA K 43 AGA
R 21 AUG M 100 ACG T 11 AAG K 57 AGG R 21 GUU V 18 GCU A 27 GAU D
46 GGU G 16 GUC V 24 GCC A 40 GAC D 54 GGC G 34 GUA V 12 GCA A 23
GAA E 42 GGA G 25 GUG V 46 GCG A 11 GAG E 58 GGG G 25
In one embodiment, after a nucleotide sequence has been codon
optimized it may be further evaluated for regions containing
restriction sites. At least one nucleotide within the restriction
site regions may be replaced with another nucleotide in order to
remove the restriction site from the sequence but the replacement
of nucleotides does alter the amino acid sequence which is encoded
by the codon optimized nucleotide sequence.
Features, which may be considered beneficial in some embodiments of
the present invention, may be encoded by regions of the
polynucleotide and such regions may be upstream (5') or downstream
(3') to a region which encodes a polypeptide. These regions may be
incorporated into the polynucleotide before and/or after codon
optimization of the protein encoding region or open reading frame
(ORF). It is not required that a polynucleotide contain both a 5'
and 3' flanking region. Examples of such features include, but are
not limited to, untranslated regions (UTRs), Kozak sequences, an
oligo(dT) sequence, and detectable tags and may include multiple
cloning sites which may have XbaI recognition.
In some embodiments, a 5' UTR and/or a 3' UTR region may be
provided as flanking regions. Multiple 5' or 3' UTRs may be
included in the flanking regions and may be the same or of
different sequences. Any portion of the flanking regions, including
none, may be codon optimized and any may independently contain one
or more different structural or chemical modifications, before
and/or after codon optimization.
After optimization (if desired), the polynucleotides components are
reconstituted and transformed into a vector such as, but not
limited to, plasmids, viruses, cosmids, and artificial chromosomes.
For example, the optimized polynucleotide may be reconstituted and
transformed into chemically competent E. coli, yeast, neurospora,
maize, drosophila, etc. where high copy plasmid-like or chromosome
structures occur by methods described herein.
Uses of Alternative Polynucleotides
Therapeutic Agents
The alternative polynucleotides described herein can be used as
therapeutic agents. For example, an alternative polynucleotide
described herein can be administered to an animal or subject,
wherein the alternative polynucleotide 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 alternative polynucleotides, cells
containing alternative polynucleotides or polypeptides translated
from the alternative polynucleotides, polypeptides translated from
alternative polynucleotides, cells contacted with cells containing
alternative polynucleotides or polypeptides translated from the
alternative polynucleotides, tissues containing cells containing
alternative polynucleotides and organs containing tissues
containing cells containing alternative polynucleotides.
Provided are methods of inducing translation of a synthetic or
recombinant polynucleotide to produce a polypeptide in a cell
population using the alternative polynucleotides 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 polynucleotide that has at least one
nucleoside alternative, and a translatable region encoding the
polypeptide. The population is contacted under conditions such that
the polynucleotide is localized into one or more cells of the cell
population and the recombinant polypeptide is translated in the
cell from the polynucleotide.
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 polynucleotide
(e.g., size, and extent of alternative 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 natural
polynucleotide. Increased efficiency may be demonstrated by
increased cell transfection (i.e., the percentage of cells
transfected with the polynucleotide), increased protein translation
from the polynucleotide, decreased polynucleotide degradation (as
demonstrated, e.g., by increased duration of protein translation
from an alternative polynucleotide), or reduced innate immune
response of the host cell or improve therapeutic utility.
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 polynucleotide that has at least one
alternative nucleoside and a translatable region encoding the
polypeptide is administered to the subject using the delivery
methods described herein. The polynucleotide is provided in an
amount and under other conditions such that the polynucleotide is
localized into a cell or cells of the subject and the recombinant
polypeptide is translated in the cell from the polynucleotide. The
cell in which the polynucleotide is localized, or the tissue in
which the cell is present, may be targeted with one or more than
one rounds of polynucleotide administration.
Other aspects of the present disclosure relate to transplantation
of cells containing alternative polynucleotides 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 alternative polynucleotides are formulated
for administration intramuscularly, transarterially,
intraperitoneally, intravenously, intranasally, subcutaneously,
endoscopically, transdermally, or intrathecally. In some
embodiments, the composition is formulated for extended
release.
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.
In certain embodiments, the administered alternative polynucleotide
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.
In other embodiments, the administered alternative polynucleotide
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 alternative polynucleotide
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
polynucleotide, a carbohydrate, or a small molecule toxin.
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.
As described herein, a useful feature of the alternative
polynucleotides of the present disclosure is the capacity to
reduce, evade, avoid or eliminate the innate immune response of a
cell to an exogenous polynucleotide. 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 polynucleotide including a
translatable region and at least one alternative nucleoside, and
the level of the innate immune response of the cell to the first
exogenous polynucleotide is determined. Subsequently, the cell is
contacted with a second composition, which includes a second dose
of the first exogenous polynucleotide, the second dose containing a
lesser amount of the first exogenous polynucleotide as compared to
the first dose. Alternatively, the cell is contacted with a first
dose of a second exogenous polynucleotide. The second exogenous
polynucleotide may contain one or more alternative nucleosides,
which may be the same or different from the first exogenous
polynucleotide or, alternatively, the second exogenous
polynucleotide may not contain alternative 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
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 unnatural 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 unnatural 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
polynucleotide or cell-based therapeutics containing the
alternative polynucleotides provided herein, wherein the
alternative polynucleotides encode for a protein that replaces the
protein activity missing from the target cells of the subject.
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 polynucleotide or cell-based therapeutics containing
the alternative polynucleotides provided herein, wherein the
alternative polynucleotides encode for a protein that antagonizes
or otherwise overcomes the aberrant protein activity present in the
cell of the subject.
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.
Thus, provided are methods of treating cystic fibrosis in a
mammalian subject by contacting a cell of the subject with an
alternative polynucleotide 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.
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 an unnatural 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 Polynucleotide Delivery
Methods of the present disclosure enhance polynucleotide 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 polynucleotide having
at least one nucleoside alternative and, optionally, a translatable
region. The composition also generally contains a transfection
reagent or other compound that increases the efficiency of enhanced
polynucleotide uptake into the host cells. The enhanced
polynucleotide exhibits enhanced retention in the cell population,
relative to a corresponding natural polynucleotide. The retention
of the enhanced polynucleotide is greater than the retention of the
corresponding polynucleotide. 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 natural polynucleotide. Such
retention advantage may be achieved by one round of transfection
with the enhanced polynucleotide, or may be obtained following
repeated rounds of transfection.
In some embodiments, the enhanced polynucleotide is delivered to a
target cell population with one or more additional polynucleotides.
Such delivery may be at the same time, or the enhanced
polynucleotide is delivered prior to delivery of the one or more
additional polynucleotides. The additional one or more
polynucleotides may be alternative polynucleotides or natural
polynucleotides. It is understood that the initial presence of the
enhanced polynucleotides 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 natural polynucleotides. In this regard, the enhanced
polynucleotide may not itself contain a translatable region, if the
protein desired to be present in the target cell population is
translated from the natural polynucleotides.
Targeting Moieties
In embodiments of the present disclosure, alternative
polynucleotides 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, alternative
polynucleotides can be employed to direct the synthesis and
extracellular localization of lipids, carbohydrates, or other
biological moieties.
Permanent Gene Expression Silencing
Methods of the present disclosure include a method for
epigenetically silencing gene expression in a mammalian subject,
comprising a polynucleotide 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
The alternative nucleosides, alternative nucleotides, and
alternative polynucleotides 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.
For example, the alternative nucleosides, alternative nucleotides,
and alternative polynucleotides 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 alternative polynucleotide 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 an alternative
polynucleotide in reversible drug delivery into cells.
The alternative nucleosides, alternative nucleotides, and
alternative polynucleotides 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.
In addition, the alternative nucleosides, alternative nucleotides,
and alternative nucleic acids described herein can be used to
deliver therapeutic agents to cells or tissues, e.g., in living
animals. For example, the alternative nucleosides, alternative
nucleotides, and alternative nucleic acids described herein can be
used to deliver highly polar chemotherapeutics agents to kill
cancer cells. The alternative 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.
In another example, the alternative nucleosides, alternative
nucleotides, and alternative 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 alternative nucleosides, alternative
nucleotides, and alternative 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
The present disclosure provides proteins generated from unnatural
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 an alternative nucleic acids 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.RTM.), 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.
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.
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..
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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).
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.
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.
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
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.
In one aspect, the disclosure provides kits for protein production,
comprising a first isolated nucleic acid comprising a translatable
region and an alternative nucleic acid, 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.
In one aspect, the disclosure provides kits for protein production,
comprising: a first isolated alternative 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.
In one aspect, the disclosure provides kits for protein production,
comprising a first isolated nucleic acid comprising a translatable
region and an alternative sugar, wherein the nucleic acid exhibits
reduced degradation by a cellular nuclease, and packaging and
instructions.
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 alternative sugars, wherein the
nucleic acid exhibits reduced degradation by a cellular nuclease,
and packaging and instructions.
In one aspect, the disclosure provides kits for protein production,
comprising a first isolated nucleic acid comprising a translatable
region and at least one alternative sugar, wherein the nucleic acid
exhibits reduced degradation by a cellular nuclease; a second
nucleic acid comprising an inhibitory nucleic acid; and packaging
and instructions.
In another aspect, the disclosure provides compositions for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a sugar alternative, 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
About: As used herein, the term "about" means+/-10% of the recited
value.
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.
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.
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.
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).
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.
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.
Biodegradable: As used herein, the term "biodegradable" means
capable of being broken down into innocuous products by the action
of living things.
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.
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.
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.
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.
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.
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.
Delivery: As used herein, "delivery" refers to the act or manner of
delivering a compound, substance, entity, moiety, cargo or
payload.
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.
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.
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.
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.
Distal: As used herein, the term "distal" means situated away from
the center or away from a point or region of interest.
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.
Encoded protein cleavage signal: As used herein, "encoded protein
cleavage signal" refers to the nucleotide sequence which encodes a
protein cleavage signal.
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.
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.
Feature: As used herein, a "feature" refers to a characteristic, a
property, or a distinctive element.
Formulation: As used herein, a "formulation" includes at least a
polynucleotide and a delivery agent.
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.
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.
Homology: As used herein, the term "homology" 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. 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.
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 polynucleotide 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)).
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.
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).
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).
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.
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 an alternative 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 polynucleotide 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.
Naturally occurring: As used herein, "naturally occurring" means
existing in nature without artificial aid.
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.
Off-target: As used herein, "off target" refers to any unintended
effect on any one or more target, gene, or cellular transcript.
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.
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.
Paratope: As used herein, a "paratope" refers to the
antigen-binding site of an antibody.
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.
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.
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.
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.
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.
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.
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."
Physicochemical: As used herein, "physicochemical" means of or
relating to a physical and/or chemical property.
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.
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.
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.
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.
Protein cleavage signal: As used herein "protein cleavage signal"
refers to at least one amino acid that flags or marks a polypeptide
for cleavage.
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.
Proximal: As used herein, the term "proximal" means situated nearer
to the center or to a point or region of interest.
Purified: As used herein, "purify," "purified," "purification"
means to make substantially pure or clear from unwanted components,
material defilement, admixture or imperfection.
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 polynucleotide molecule.
Signal Sequences: As used herein, the phrase "signal sequences"
refers to a sequence which can direct the transport or localization
of a protein.
Significant or Significantly: As used herein, the terms
"significant" or "significantly" are used synonymously with the
term "substantially."
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.
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.
Split dose: As used herein, a "split dose" is the division of
single unit dose or total daily dose into two or more doses.
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.
Stabilized: As used herein, the term "stabilize", "stabilized,"
"stabilized region" means to make or become stable.
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.
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.
Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
Substantially simultaneously: As used herein and as it relates to
plurality of doses, the term means within 2 seconds.
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.
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 polynucleotide 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.
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.
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.
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.
Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., polynucleotide, 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.
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.
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.
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.
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.
Equivalents and Scope
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.
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.
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.
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.
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 polynucleotide 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.
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
Example 1: PCR for cDNA Production
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 ReadyMix12.5 .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.
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.
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 2: In Vitro Transcription (IVT)
A. Materials and Methods
Unnatural 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 alternative
nucleotides. The open reading frame (ORF) of the gene of interest
may be 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.
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.
For the present invention, NEB DH5-alpha Competent E. coli may be
used. Transformations are performed according to NEB instructions
using 100 ng of plasmid. The protocol is as follows:
Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10
minutes.
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.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at 42.degree. C. for exactly 30 seconds. Do not mix.
Place on ice for 5 minutes. Do not mix.
Pipette 950 .mu.l of room temperature SOC into the mixture.
Place at 37.degree. C. for 60 minutes. Shake vigorously (250 rpm)
or rotate.
Warm selection plates to 37.degree. C.
Mix the cells thoroughly by flicking the tube and inverting.
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.
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.
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.
In order to generate cDNA for In Vitro Transcription (IVT), the
plasmid 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.
IVT Reaction
The in vitro transcription reaction generates mRNA containing
alternative nucleotides or alternative RNA. The input nucleotide
triphosphate (NTP) mix is made in-house using natural and unnatural
NTPs.
A typical in vitro transcription reaction includes the
following:
TABLE-US-00007 Template cDNA 1.0 .mu.g 10x transcription buffer
(400 mM Tris-HCl pH 8.0, 2.0 .mu.l 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 up to 20.0 .mu.l
Incubation at 37.degree. C. for 3 hr-5 hrs.
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.
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 alternative 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.
B. Agarose Gel Electrophoresis of Unnatural mRNA
Individual unnatural 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
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 Unnatural mRNA Quantification and UV Spectral Data
Unnatural mRNAs in TE buffer (1 .mu.l) are used for Nanodrop UV
absorbance readings to quantitate the yield of each alternative
mRNA from an in vitro transcription reaction (UV absorbance traces
are not shown).
Example 3: Enzymatic Capping of mRNA
Capping of the mRNA is performed as follows where the mixture
includes: IVT RNA 60 .mu.g-180 .mu.g and dH.sub.2O 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.
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 (400 U);
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.
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 4: 5'-Guanosine Capping
A. Materials and Methods
The cloning, gene synthesis and vector sequencing may be performed
by DNA2.0 Inc. (Menlo Park, Calif.). The ORF is restriction
digested using XbaI and used for cDNA synthesis using tailed- or
tail-less-PCR. The tailed-PCR cDNA product is used as the template
for the mRNA synthesis reaction using 25 mM each alternative
nucleotide mix (all alternative nucleotides may be custom
synthesized or purchased from TriLink Biotech, San Diego, Calif.
except pyrrolo-C triphosphate which may be purchased from Glen
Research, Sterling Va.; unmodified nucleotides are purchased from
Epicenter Biotechnologies, Madison, Wis.) and CellScript
MEGASCRIPT.TM. (Epicenter Biotechnologies, Madison, Wis.) complete
mRNA synthesis kit.
The in vitro transcription reaction is run for 4 hours at
37.degree. C. Alternative mRNAs incorporating adenosine
alternatives are poly (A) tailed using yeast Poly (A) Polymerase
(Affymetrix, Santa Clara, Calif.). The PCR reaction uses HiFi PCR
2.times. MASTER MIX.TM. (Kapa Biosystems, Woburn, Mass.).
Alternative mRNAs are 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 is run on an agarose gel and visualized. Alternative
mRNA may be purified with Ambion/Applied Biosystems (Austin, Tex.)
MEGAClear RNA.TM. purification kit. The PCR uses PURELINK.TM. PCR
purification kit (Invitrogen, Carlsbad, Calif.). The product is
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 is
resuspended in TE buffer.
B. 5' Capping Alternative Polynucleotide (mRNA) Structure
5'-capping of alternative 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 alternative 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'-O 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'-O
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'-O methyl-transferase.
Enzymes are preferably derived from a recombinant source.
When transfected into mammalian cells, the unnatural 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 5: PolyA Tailing Reaction
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.
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 6: Method of Screening for Protein Expression
A. Electrospray Ionization
A biological sample which may contain proteins encoded by unnatural
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.
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
A biological sample which may contain proteins encoded by unnatural
RNA administered to the subject is prepared and analyzed according
to the manufacturer protocol for matrix-assisted laser
desorption/ionization (MALDI).
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
A biological sample, which may contain proteins encoded by
unnatural 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.
Patterns of protein fragments, or whole proteins, are compared to
known controls for a given protein and identity is determined by
comparison.
Example 7: Transfection
A. Reverse Transfection
For experiments performed in a 24-well collagen-coated tissue
culture plate, Keratinocytes or other cells 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 alternative mRNA to be
transfected, alternative 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
In a 24-well collagen-coated tissue culture plate, cells 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,
if used, are seeded at a cell density of 0.3.times.10.sup.5. Cells
are then grown to a confluency of >70% for over 24 hours. For
each alternative mrna to be transfected, alternative 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. Translation Screen: ELISA
Cells are grown in EpiLife medium with Supplement S7 from
Invitrogen at a confluence of >70%. Cells are reverse
transfected with 300 ng of the indicated alternative mRNA complexed
with RNAIMAX.TM. from Invitrogen. Alternatively, cells are forward
transfected with 300 ng alternative 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.
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 polypeptide concentration in the culture medium is
measured at 18 hours post-transfection for each of the unnatural
mRNAs in triplicate. Secretion of the polypeptide of interest from
transfected human cells is quantified using an ELISA kit from
Invitrogen or R&D Systems (Minneapolis, Minn.) following the
manufacturers recommended instructions.
D. Dose and Duration: ELISA
Cells are grown in EPILIFE.RTM. medium with Supplement S7 from
Invitrogen at a confluence of >70%. Cells are reverse
transfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750
ng, or 1500 ng alternative mRNA complexed with RNAIMAX.TM. from
Invitrogen. The alternative mRNA: RNAIMAX.TM. complex is formed as
described. Secreted polypeptide concentration in the culture medium
is measured at 0, 6, 12, 24, and 48 hours post-transfection for
each concentration of each alternative mRNA in triplicate.
Secretion of the polypeptide of interest from transfected human
cells is quantified using an ELISA kit from Invitrogen or R&D
Systems following the manufacturers recommended instructions.
Example 8: Cellular Innate Immune Response: IFN-Beta ELISA and
TNF-Alpha ELISA
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.
Cells are grown in EPILIFE.RTM. medium with Human Growth Supplement
in the absence of hydrocortisone from Invitrogen at a confluence of
>70%. Cells are reverse transfected with 0 ng, 93.75 ng, 187.5
ng, 375 ng, 750 ng, 1500 ng or 3000 ng of the indicated chemically
alternative 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 unnatural
mRNAs using an ELISA kit from Invitrogen according to the
manufacturer protocols.
Secreted IFN-.beta. is measured 24 hours post-transfection for each
of the unnatural 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 alternative
mRNAs. Secretion of the polypeptide of interest from transfected
human cells is quantified using an ELISA kit from Invitrogen or
R&D Systems (Minneapolis, Minn.) following the manufacturers
recommended instructions. These data indicate which unnatural mRNA
are capable eliciting a reduced cellular innate immune response in
comparison to natural and other alternative polynucleotides or
reference compounds by measuring exemplary type 1 cytokines such as
TNF-alpha and IFN-beta.
Example 9: Cytotoxicity and Apoptosis
This experiment demonstrates cellular viability, cytotoxity and
apoptosis for distinct alternative 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 0 ng, 46.875 ng, 93.75
ng, 187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of
unnatural mRNA complexed with RNAIMAX.TM. from Invitrogen. The
unnatural 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
unnatural mRNA in triplicate. Secretion of the polypeptide of
interest 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.
OTHER EMBODIMENTS
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 LISTINGS
1
4110DNAHomo sapiens 1tttttctttt 10211DNAHomo sapiens 2ttttgctttt t
11310DNAHomo sapiens 3ttttgctttt 10411DNAHomo sapiens 4aaaaagcaaa a
11
* * * * *
References