U.S. patent application number 13/481127 was filed with the patent office on 2013-04-25 for modified nucleosides, nucleotides, and nucleic acids, and uses thereof.
The applicant listed for this patent is Jason P. Schrum. Invention is credited to Jason P. Schrum.
Application Number | 20130102034 13/481127 |
Document ID | / |
Family ID | 45893552 |
Filed Date | 2013-04-25 |
United States Patent
Application |
20130102034 |
Kind Code |
A1 |
Schrum; Jason P. |
April 25, 2013 |
MODIFIED NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS, AND USES
THEREOF
Abstract
The present disclosure provides modified nucleosides,
nucleotides, and nucleic acids, and methods of using thereof.
Inventors: |
Schrum; Jason P.;
(Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Schrum; Jason P. |
Somerville |
MA |
US |
|
|
Family ID: |
45893552 |
Appl. No.: |
13/481127 |
Filed: |
May 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13251840 |
Oct 3, 2011 |
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13481127 |
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61404413 |
Oct 1, 2010 |
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Current U.S.
Class: |
435/91.2 ;
435/194; 435/375; 435/91.3; 435/91.5; 536/25.6; 536/26.22;
536/26.23; 536/26.3; 536/26.8 |
Current CPC
Class: |
A61K 48/0066 20130101;
C12P 21/00 20130101; C12N 5/0602 20130101; C12N 15/102 20130101;
C12N 2310/3341 20130101; C12N 2310/335 20130101; C12N 15/1138
20130101; C07H 19/10 20130101; C07K 16/00 20130101; C07K 16/2887
20130101; G01N 33/559 20130101; C12N 15/67 20130101; C07K 2317/24
20130101; C12N 15/11 20130101; C07H 21/02 20130101; C12N 15/1136
20130101 |
Class at
Publication: |
435/91.2 ;
536/26.3; 536/26.23; 536/26.22; 536/26.8; 536/25.6; 435/194;
435/91.5; 435/91.3; 435/375 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07H 19/10 20060101 C07H019/10 |
Claims
1. A compound comprising a nucleotide that disrupts binding of a
major groove binding partner with a nucleic acid comprising the
nucleotide, wherein the nucleotide has decreased binding affinity
to the major groove binding partner.
2. The compound of claim 1, wherein the nucleotide comprises a
chemical modification located on the major groove face of a
nucleobase portion of the nucleotide.
3. The compound of claim 2, wherein the nucleobase portion
comprises a pyrimidine nucleobase, and wherein the chemical
modification comprises replacing or substituting an atom of the
major groove face of the pyrimidine nucleobase with an amine, an
SH, a methyl, an ethyl, a chloro or a fluoro group.
4. The compound of claim 2, wherein the chemical modification is
located on a sugar portion of the nucleotide.
5. The compound of claim 2, wherein the chemical modification is
located on a phosphate backbone of the nucleotide.
6. The compound of claim 1, having Formula I: ##STR00040## wherein:
Z is O or S; each of Y.sup.1 is independently selected from
--OR.sup.a1, --NR.sup.a1R.sup.b1 and --SR.sup.a1; each of Y.sup.2
is independently selected from O, NR.sup.a, S or a linker
comprising an atom selected from the group consisting of C, O, N,
and S; each of Y.sup.3 is independently selected from O and S;
Y.sup.4 is selected from H, --OR.sup.a, --SR.sup.a, and
--NHR.sup.a; n is 0, 1, 2, or 3; m is 0, 1, 2 or 3; B is a
nucleobase; R.sup.a is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
C.sub.2-20 alkynyl, or C.sub.6-20 aryl; R.sup.a1 and R.sup.b1 are
each independently H or a counterion; and --Y.sup.3--R.sup.c1 is OH
or SH at a pH of about 1 or --Y.sup.3--R.sup.c1 is O.sup.- or
S.sup.- at physiological pH; or --Y.sup.3--R.sup.c1 is C.sub.1-20
alkoxy, C.sub.2-20 --O-alkenyl, or C.sub.1-20 --O-alkynyl; wherein
when B is an unmodified nucleobase selected from cytosine, guanine,
uracil and adenine, then at least one of Z, Y.sup.1 or Y.sup.2 is
not O or OH.
7. The compound of claim 6, wherein B is a nucleobase of Formula
II-a, II-b, or II-c: ##STR00041## wherein: denotes a single or
double bond; X is O or S; V, U and W are each independently C or N;
wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-6
alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.D,
--C(O)OR.sup.S, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c; and wherein
when V is N then R.sup.1 is absent; R.sup.2 is H, --OW, --SR.sup.S,
--NR.sup.aR.sup.b, or halo; or when V is C then R.sup.1 and R.sup.2
together with the carbon atoms to which they are attached can form
a 5- or 6-membered ring optionally substituted with 1-4
substituents selected from halo, --OH, --SH, --NR.sup.aR.sup.b,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, C.sub.1-6
alkoxy, or C.sub.1-6 thioalkyl; R.sup.3 is H or C.sub.1-6 alkyl;
R.sup.4 is H or C.sub.1-6 alkyl; wherein when denotes a double bond
then R.sup.4 is absent, or N--R.sup.4, taken together, forms a
positively charged N substituted with C.sub.1-6 alkyl; R.sup.a and
R.sup.b are each independently H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, or C.sub.6-10 aryl; and R.sup.c is H,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, phenyl, benzyl, a polyethylene
glycol group, or an amino-polyethylene glycol group.
8. The compound of claim 7, wherein B is a nucleobase of Formula
II-al, II-a2, II-a3, II-a4, or II-a5: ##STR00042##
9. The compound of claim 6, wherein B is a nucleobase selected from
the group consisting of cytosine, guanine, adenine, and uracil.
10. The compound of claim 6, having Formula I-a: ##STR00043##
11. The compound of claim 6, having Formula I-b: ##STR00044##
12. The compound of claim 6, having Formula I-c: ##STR00045##
13. The compound of claim 6, selected from the group consisting of:
##STR00046##
14. The compound of claim 6, selected from the group consisting of:
##STR00047##
15. A nucleic acid sequence comprising at least two nucleotides,
the nucleic acid sequence comprising a nucleotide that disrupts
binding of a major groove binding partner with the nucleic acid
sequence, wherein the nucleotide has decreased binding affinity to
the major groove binding partner.
16. The nucleic acid sequence of claim 15, comprising a compound of
Formula I-d: ##STR00048## wherein: Z is O or S; each of Y.sup.1 is
independently selected from --OR.sup.al, --NR.sup.a1 R.sup.b1, and
--SR.sup.a1; each of Y.sup.2 is independently selected from O,
NR.sup.a, S or a linker comprising an atom selected from the group
consisting of C, O, N, and S; B is a nucleobase; R.sup.a is H,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, or
C.sub.6-10 aryl; and R.sup.a1 and R.sup.b1 are each independently H
or a counterion; and --OR.sup.c1 is OH at a pH of about 1 or
--OR.sup.c1 is O.sup.- at physiological pH; wherein when B is an
unmodified nucleobase selected from cytosine, guanine, uracil and
adenine, then at least one of Z, Y.sup.1 or Y.sup.2 is not O or
OH.
17. The nucleic acid sequence of claim 16, wherein B is a
nucleobase of Formula II-a, II-b, or II-c: ##STR00049## wherein:
denotes a single or double bond; X is O or S; V, U and W are each
independently C or N; wherein when V is C then R.sup.1 is H,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, halo, or
--OR.sup.c, wherein C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6
alkynyl are each optionally substituted with --OH,
--NR.sup.aR.sup.b, --SH, --C(O)R.sup.D, --C(O)OR.sup.c,
--NHC(O)R.sup.c, or --NHC(O)OR.sup.c; and wherein when V is N then
R.sup.1 is absent; R.sup.2 is H, --OR.sup.c, --SR.sup.c,
--NR.sup.aR.sup.b, or halo; or when V is C then R.sup.1 and R.sup.2
together with the carbon atoms to which they are attached can form
a 5- or 6-membered ring optionally substituted with 1-4
substituents selected from halo, --OH, --SH, --NR.sup.aR.sup.b,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, C.sub.1-6
alkoxy, or C.sub.1-6 thioalkyl; R.sup.3 is H or C.sub.1-6 alkyl;
R.sup.4 is H or C.sub.1-6 alkyl; wherein when denotes a double bond
then R.sup.4 is absent, or N--R.sup.4, taken together, forms a
positively charged N substituted with C.sub.1-6 alkyl; R.sup.a and
R.sup.b are each independently H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, or C.sub.6-10 aryl; and R.sup.c is H,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, phenyl, benzyl, a polyethylene
glycol group, or an amino-polyethylene glycol group.
18. The nucleic acid sequence of claim 17, wherein B is a
nucleobase of Formula II-al, II-a2, II-a3, II-a4, or II-a5:
##STR00050##
19. The nucleic acid sequence of claim 16, wherein B is a
nucleobase selected from the group consisting of cytosine, guanine,
adenine, and uracil.
20. The nucleic acid sequence of claim 16, wherein the nucleic acid
sequence contains a plurality of structurally unique compounds of
Formula I-d.
21. The nucleic acid sequence of claim 16, wherein at least 25% of
the cytosines are replaced by a compound of Formula I-d and/or
wherein at least 25% of the uracils are replaced by a compound of
Formula I-d.
22. The compound of claim 1, wherein the major groove interacting
partners are selected from the group consisting of: TLRs (Toll-like
Receptors) 3, 7, and 8; RIG-I (retinoic acid-inducible gene I);
MDA5 (melanoma differentiation-associated gene 5); laboratory of
genetics and physiology 2 (LGP2); HIN-200 domain containing
proteins; and Helicase-domain containing proteins.
23. A non-naturally occurring nucleotide comprising one or more
chemical modifications of a naturally occurring nucleotide, wherein
the nucleotide reduces the induction of the cellular innate immune
response of a cell to a modified nucleic acid comprising the
non-naturally occurring nucleotide when the modified nucleic acid
is introduced into the cell, as compared to the induction of the
cellular innate immune in a cell induced by a corresponding
unmodified nucleic acid.
24. The compound of claim 23, wherein the nucleotide reduces the
innate immune response or the secretion of pro-inflammatory
cytokines or both by at least about 10%.
25. The compound of claim 23, wherein the nucleotide reduces the
innate immune response or the secretion of pro-inflammatory
cytokines or both by about 75%.
26. The compound of claim 23, wherein the nucleotide reduces the
innate immune response or the secretion of pro-inflammatory
cytokines or both by at least 90%.
27. The modified nucleic acid comprising the non-naturally
occurring nucleotide of claim 23, further comprising a
translateable region encoding a protein of interest.
28. A composition comprising the modified nucleic acid of claim 27,
in an amount sufficient to increase the production of the protein
of interest when introduced into a target cell, as compared to the
amount of protein produced in a cell containing a corresponding
unmodified nucleic acid encoding the protein of interest.
29. The composition of claim 28, wherein the increase is at least
about 10%.
30. The composition of claim 28, wherein the increase is at least
about 50%.
31. The composition of claim 28, wherein the increase is at least
about 100%.
32. The modified nucleic acid of claim 27, wherein at least 25% of
the cytosines in the nucleic acid are replaced by a compound of
Formula I-d.
33. The modified nucleic acid of claim 27, wherein at least 90% of
the cytosines in the nucleic acid are replaced by a compound of
Formula I-d.
34. The modified nucleic acid of claim 27, wherein about 100% of
the cytosines in the nucleic acid are replaced by a compound of
Formula I-d.
35. The modified nucleic acid of claim 27, wherein at least 25% of
the uracils in the nucleic acid are replaced by a compound of
Formula I-d.
36. The modified nucleic acid of claim 27, wherein at least 90% of
the uracils in the nucleic acid are replaced by a compound of
Formula I-d.
37. The modified nucleic acid of claim 27, wherein about 100% of
the uracils in the nucleic acid are replaced by a compound of
Formula I-d.
38. The modified nucleic acid of claim 27, wherein at least 25% of
the cytosines in the nucleic acid are replaced by a first compound
of Formula I-d and wherein at least 25% of the uracils in the
nucleic acid are replaced by a second compound of Formula I-d.
39. The modified nucleic acid of claim 27, wherein about 100% of
the cytosines in the nucleic acid are replaced by a first compound
of Formula I-d and wherein about 100% of the uracils in the nucleic
acid are replaced by a second compound of Formula I-d.
40. The composition of claim 28, further comprising an RNA
polymerase, a cDNA template, or a combination thereof.
41. The composition of claim 40, further comprising a nucleotide
selected from the group consisting of adenosine, cytosine,
guanosine, and uracil.
42. A method of preparing a nucleic acid sequence comprising a
nucleotide that disrupts binding of a major groove binding partner
with the nucleic acid sequence, wherein the nucleic acid sequence
comprises a compound of Formula I-d: ##STR00051## wherein: the
nucleotide has decreased binding affinity to the major groove
binding partner; Z is O or S; each of Y.sup.1 is independently
selected from --OR.sup.al, --NR.sup.a1R.sup.b1, and --SR.sup.a1;
each of Y.sup.2 is independently selected from O, NR.sup.a, S or a
linker comprising an atom selected from the group consisting of C,
O, N, and S; B is a nucleobase; and R.sup.a1 and R.sup.b1 are each
independently H or a counterion; and --OR.sup.c1 is OH at a pH of
about 1 or --OR.sup.c1 is O.sup.- at physiological pH; wherein when
B is an unmodified nucleobase selected from cytosine, guanine,
uracil and adenine, then at least one of Z, Y.sup.1 or Y.sup.2 is
not O or OH; the method comprising: reacting a compound of Formula
I-c: ##STR00052## with an RNA polymerase, and a cDNA template.
43. The method of claim 42, wherein the reaction is repeated from 1
to about 7,000 times.
44. The method of claim 42, wherein B is a nucleobase of Formula
II-a, II-b, or II-c: ##STR00053## wherein: denotes a single or
double bond; X is O or S; V, U and W are each independently C or N;
wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-6
alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.D,
--C(O)OR.sup.c, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c; and wherein
when V is N then R.sup.1 is absent; R.sup.2 is H, --OR.sup.c,
--SR.sup.c, --NR.sup.aR.sup.b, or halo; or when V is C then R.sup.1
and R.sup.2 together with the carbon atoms to which they are
attached can form a 5- or 6-membered ring optionally substituted
with 1-4 substituents selected from halo, --OH, --SH,
--NR.sup.aR.sup.b, C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6
alkynyl, C.sub.1-6 alkoxy, or C.sub.1-6 thioalkyl; R.sup.3 is H or
C.sub.1-6 alkyl; R.sup.4 is H or C.sub.1-6 alkyl; wherein when
denotes a double bond then R.sup.4 is absent, or N--R.sup.4, taken
together, forms a positively charged N substituted with C.sub.1-6
alkyl; R.sup.a and R.sup.b are each independently H, C.sub.1-6
alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, or C.sub.6-10 aryl;
and R.sup.c is H, C.sub.1-6 alkyl, C.sub.1-6 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
45. The method of claim 44, wherein B is a nucleobase of Formula
II-al, II-a2, II-a3, II-a4, or II-a5: ##STR00054##
46. A method of amplifying a nucleic acid sequence comprising a
nucleotide that disrupts binding of a major groove binding partner
with the nucleic acid sequence, the method comprising: reacting a
compound of Formula I-c: ##STR00055## wherein: the nucleotide has
decreased binding affinity to the major groove binding partner; Z
is O or S; each of Y.sup.1 is independently selected from
--OR.sup.al, --NR.sup.a1R.sup.b1, and --SR.sup.a1; each of Y.sup.2
is independently selected from O, NR.sup.a, S or a linker
comprising an atom selected from the group consisting of C, O, N,
and S; B is a nucleobase; and R.sup.a1 and R.sup.b1 are each
independently H or a counterion; and --OR.sup.c1 is OH at a pH of
about 1 or --OR.sup.c1 is O.sup.- at physiological pH; wherein when
B is an unmodified nucleobase selected from cytosine, guanine,
uracil and adenine, then at least one of Z, Y.sup.1 or Y.sup.2 is
not O or OH; with a primer, a cDNA template, and an RNA
polymerase.
47. The method of claim 46, wherein B is a nucleobase of Formula
II-a, II-b, or II-c: ##STR00056## wherein: denotes a single or
double bond; X is O or S; V, U and W are each independently C or N;
wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-6
alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.D,
--C(O)OR.sup.c, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c; and wherein
when V is N then R.sup.1 is absent; R.sup.2 is H, --OR.sup.c,
--SR.sup.c, --NR.sup.aR.sup.b, or halo; or when V is C then R.sup.1
and R.sup.2 together with the carbon atoms to which they are
attached can form a 5- or 6-membered ring optionally substituted
with 1-4 substituents selected from halo, --OH, --SH,
--NR.sup.aR.sup.b, C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6
alkynyl, C.sub.1-6 alkoxy, or C.sub.1-6 thioalkyl; R.sup.3 is H or
C.sub.1-6 alkyl; R.sup.4 is H or C.sub.1-6 alkyl; wherein when
denotes a double bond then R.sup.4 is absent, or N--R.sup.4, taken
together, forms a positively charged N substituted with C.sub.1-6
alkyl; R.sup.a and R.sup.b are each independently H, C.sub.1-6
alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, or C.sub.6-10 aryl;
and R.sup.c is H, C.sub.1-6 alkyl, C.sub.1-6 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
48. The method of claim 46, wherein B is a nucleobase of Formula
II-al, II-a2, II-a3, II-a4, or II-a5: ##STR00057##
48. A method of synthesizing a pharmaceutical nucleic acid,
comprising the steps of: a) providing a complementary
deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein
of interest; b) selecting a nucleotide that is known to disrupt a
binding of a major groove binding partner with a nucleic acid,
wherein the nucleotide has decreased binding affinity to the major
groove binding partner; and c) contacting the provided cDNA and the
selected nucleotide with an RNA polymerase, under conditions such
that the pharmaceutical nucleic is synthesized.
49. The method of claim 48, wherein the pharmaceutical nucleic acid
is a ribonucleic acid (RNA).
50. A method of inducing a physiological change in a target cell
population, comprising the steps of: a) providing a first nucleic
acid comprising i) a translatable region encoding a protein of
interest and ii) a nucleic acid modification, wherein the first
nucleic acid is substantially resistant to cellular degradation;
and b) contacting an effective amount of the first nucleic acid to
a producer cell under conditions such that the protein of interest
is produced in the producer cell and secreted therefrom, wherein
the secreted protein of interest contacts the target cell
population and induces a physiological change therein.
51. The method of claim 50, wherein the protein of interest is
capable of binding to a receptor on the surface of at least one
cell present in the target cell population.
52. The method of claim 50, wherein the secreted protein is capable
of interacting with a receptor on the surface of at least one cell
present in the target cell population.
53. The method of claim 50, wherein the secreted protein is
Granulocyte-Colony Stimulating Factor (G-CSF).
53. The method of claim 51, wherein the target cell population
comprises one or more cells that express the G-CSF receptor.
54. A compound comprising a nucleic acid comprising one or more
nucleotides having Formula I: ##STR00058## wherein: Z is O or S;
each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.a1; each of Y.sup.2 is
independently selected from O, NR.sup.a, S or a linker comprising
an atom selected from the group consisting of C, O, N, and S; n is
0, 1, 2, or 3; m is 0, 1, 2 or 3; B is a nucleobase; R.sup.a is H,
C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, or
C.sub.6-10 aryl; R.sup.a1 and R.sup.b1 are each independently H or
a counterion; and --OR.sup.a1 is OH at a pH of about 1 or
--OR.sup.c1 is O.sup.- at physiological pH; wherein when B is an
unmodified nucleobase selected from cytosine, guanine, uracil and
adenine, then at least one of Z, Y.sup.1 or Y.sup.2 is not O or OH,
and wherein a cell comprising the nucleic acid is characterized by:
i) Decreased cellular secretion of s pro-inflammatory cytokine; ii)
decreased activation of a cellular innate immune responder; iii)
decreased suspectibility to a cellular nuclease; iv) decreased
binding to a negative regulator of gene expression; v) decreased
binding to a nucleic acid; vi) increased protein translation
efficiency; vii) increased half-life; viii) or a combination
thereof.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/404,413, filed on Oct. 1, 2010, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] Naturally occurring RNAs are synthesized from four basic
ribonucleotides: ATP, CTP, UTP and GTP, but may contain
post-transcriptionally modified nucleotides. Further, approximately
one hundred different nucleoside modifications have been identified
in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA
Modification Database: 1999 update. Nucl Acids Res 27: 196-197).
The role of nucleoside modifications on the immuno-stimulatory
potential, stability, and on the translation efficiency of RNA, and
the consequent benefits to this for enhancing protein expression
and producing therapeutics however, is unclear.
[0003] There are multiple problems with prior methodologies of
effecting protein expression. For example, heterologous
deoxyribonucleic acid (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.
[0004] There is a need in the art for biological modalities to
address the modulation of intracellular translation of nucleic
acids.
SUMMARY
[0005] The present disclosure provides, inter alia, modified
nucleosides, modified nucleotides, and modified nucleic acids which
can exhibit a reduced innate immune response when introduced into a
population of cells, both in vivo and ex vivo. Further, these
modified nucleosides, modified nucleotides, and modified nucleic
acids described herein can disrupt binding of a major groove
interacting partner with the nucleic acid. Because of the reduced
immunogenicity and the decrease in major groove interactions, these
modified nucleosides, modified nucleotides, and modified nucleic
acids can be more efficient during protein production than, e.g.,
unmodified nucleic acids.
[0006] Thus, the present disclosure provides compounds comprising
nucleotides that can disrupt binding of a major groove binding
partner with a nucleic acid, wherein the nucleotide has decreased
binding affinity to the major groove binding partner.
[0007] The present disclosure further provides compounds having
Formula I:
##STR00001##
wherein constituent variables are provided herein.
[0008] The present disclosure further provides nucleic acid
sequences of at least two nucleotides comprising a compound of
Formula I-d:
##STR00002##
wherein constituent variables are provided herein.
[0009] The present disclosure further provides compositions
comprising at least one compound of Formula I.
[0010] The present disclosure further provides pharmaceutical
compositions comprising a compound of Formula I.
[0011] The present disclosure further provides methods of preparing
nucleic acid sequences of at least two nucleotides of a compound of
Formula I-d.
[0012] The present disclosure further provides methods of
amplifying nucleic acid sequences of at least two nucleotides of a
compound of Formula I-d.
[0013] The present disclosure further provides kits comprising a
compound of Formula I.
[0014] 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
invention; 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.
[0015] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIGS. 1A and 1B depict images of non-denaturing agarose gels
of each in vitro-transcribed modified RNA.
[0017] FIGS. 2A and 2B depict images of an Enzyme-linked
immunosorbent assay (ELISA) for Human Granulocyte-Colony
Stimulating Factor (G-CSF) of in vitro transfected Human
Keratinocyte cells with each indicated modRNA encoding human G-CSF
and the line indicates a saturating level of maximum detectable
limit of secreted G-CSF in the assay.
[0018] FIGS. 3A-N depict line graphs of a series of Enzyme-linked
immunosorbent assays (ELISA) for Human Granulocyte-Colony
Stimulating Factor (G-CSF) secreted from in vitro-transfected Human
Keratinocyte cells at different time points with each indicated
human G-CSF-encoding modRNA at the indicated doses. The line
indicates a saturating level of maximum detectable limit of
secreted G-CSF in the assay.
[0019] FIGS. 4A and 4B depict bar graphs of a series of
Enzyme-linked immunosorbent assays (ELISA) for endogenous cellular
human Tumor Necrosis Factor-.alpha. (TNF-.alpha.) secreted from in
vitro-transfected Human Keratinocyte cells at 24 hours with each
indicated hu-G-CSF-encoding modRNA at increasing doses.
[0020] FIGS. 4C and 4D depict bar graphs of a series of
Enzyme-linked immunosorbent assays (ELISA) for endogenous cellular
human Interferon-.beta. (IFN-.beta.) secreted from in
vitro-transfected Human Keratinocyte cells at 24 hours with each
indicated hu-G-CSF-encoding modRNA at increasing doses.
[0021] FIGS. 4E and 4F depict bar graphs of a series of
Enzyme-linked immunosorbent assays (ELISA) for human-G-CSF secreted
from in vitro-transfected Human Keratinocyte cells at 24 hours with
each indicated hu-G-CSF-encoding modRNA at increasing doses.
[0022] FIG. 5A is a table showing results from an Enzyme-linked
immunosorbent assay (ELISA) for human-G-CSF secreted from in
vitro-transfected Human Keratinocyte cells sampled from individual
wells in a co-culture 24-well tissue culture plate 42 hours
post-transfection with 750 ng of each indicated hu-G-CSF-encoding
modRNA.
[0023] FIG. 5B depicts an image of an agarose gel of RT-PCR
hu-G-CSF modRNA products from co-culture cell extracts 42 hours
post-transfection of the human keratinocyte feeder layer with
hu-G-CSG modRNA and the un-transfected Kasumi-1 and KG-1 insert
culture cells.
[0024] FIGS. 5C and 5D depict graphs of results from a
hu-G-CSF-modRNA-induced cell proliferation assay of Kasumi-1 (FIG.
5C) and KG-1 (FIG. 5D) cells normalized to untransfected cells.
Hu-G-CSF modRNA identity transfected into human keratinocyte feeder
cells is indicated.
[0025] FIGS. 6A-L depict graphs of the UV absorbance spectra for
exemplary modRNA molecules that incorporate the indicated modified
nucleotide.
DETAILED DESCRIPTION
[0026] The present disclosure provides, inter alia, modified
nucleosides, modified nucleotides, and modified nucleic acids that
exhibit a reduced innate immune response when introduced into a
population of cells. The modified nucleosides, modified
nucleotides, and modified nucleic acids can be chemically modified
on the major groove face, thereby disrupting major groove binding
partner interactions, which cause innate immune responses.
[0027] In general, exogenous unmodified nucleic acids, particularly
viral nucleic acids, introduced into cells induce an innate immune
response, resulting in cytokine and interferon (IFN) production and
cell death. However, it is of great interest for therapeutics,
diagnostics, reagents and for biological assays to deliver a
nucleic acid, e.g., a ribonucleic acid (RNA) inside a cell, either
in vivo or ex vivo, such as to cause intracellular translation of
the nucleic acid and production of the encoded protein. Of
particular importance is the delivery and function of a
non-integrative nucleic acid, as nucleic acids characterized by
integration into a target cell are generally imprecise in their
expression levels, deleteriously transferable to progeny and
neighbor cells, and suffer from the substantial risk of causing
mutation. Provided herein in part are nucleic acids encoding useful
polypeptides capable of modulating a cell's function and/or
activity, and methods of making and using these nucleic acids and
polypeptides. As described herein, these nucleic acids are capable
of reducing the innate immune activity of a population of cells
into which they are introduced, thus increasing the efficiency of
protein production in that cell population. Further, one or more
additional advantageous activities and/or properties of the nucleic
acids and proteins of the present disclosure are described.
[0028] Further, the modified nucleosides, modified nucleotides, and
modified nucleic acids described herein can be modified on the
major groove face. These major groove modifications can allow for
alterations, e.g. a decrease, in the interaction of the modified
nucleosides, modified nucleotides, and modified nucleic acids with
a binding groove partner.
[0029] Accordingly, in a first aspect, the present disclosure
provides compounds comprising a nucleotide that can disrupts
binding of a major groove interacting, e.g. binding, partner with a
nucleic acid, wherein the nucleotide has decreased binding affinity
to major groove interacting, e.g. binding, partners.
[0030] In another aspect, the present disclosure provides compounds
comprising a nucleotide that contains chemical modifications,
wherein the nucleotide can have altered binding to major groove
interacting, e.g. binding, partners.
[0031] In some embodiments, the chemical modifications are located
on the major groove face of the nucleobase, and wherein the
chemical modifications can include replacing or substituting an
atom of a pyrimidine nucleobase with an amine, an SH, an alkyl
(e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro).
[0032] In some embodiments, the chemical modifications can be
located on the major groove face of the nucleobase, and wherein the
chemical modification can include replacing or substituting an atom
of a pyrimidine nucleobase with an amine, an SH, a methyl or ethyl,
or a chloro or fluoro.
[0033] In some embodiments, the chemical modifications can be
located on the sugar moiety of the nucleotide.
[0034] In some embodiments, the chemical modifications can be
located on the phosphate backbone of the nucleotide.
[0035] In some embodiments, the chemical modifications can alter
the electrochemistry on the major groove face of the
nucleotide.
[0036] In another aspect, the present disclosure provides
nucleotides that contain chemical modifications, wherein the
nucleotide reduces the cellular innate immune response, as compared
to the cellular innate immune induced by a corresponding unmodified
nucleic acid.
[0037] In another aspect, the present disclosure provides nucleic
acid sequences comprising at least two nucleotides, the nucleic
acid sequence comprising a nucleotide that disrupts binding of a
major groove interacting partner with the nucleic acid sequence,
wherein the nucleotide has decreased binding affinity to the major
groove binding partner.
[0038] In another aspect, the present disclosure provides
compositions comprising a compound as described herein.
[0039] In some embodiments, the composition is a reaction
mixture.
[0040] In some embodiments, the composition is a pharmaceutical
composition.
[0041] In some embodiments, the composition is a cell culture.
[0042] In some embodiments, the compositions further comprise an
RNA polymerase and a cDNA template.
[0043] In some embodiments, the compositions further comprise a
nucleotide selected from the group consisting of adenosine,
cytosine, guanosine, and uracil.
[0044] In a further aspect, the present disclosure provides for
methods of synthesizing a pharmaceutical nucleic acid, comprising
providing a complementary deoxyribonucleic acid (cDNA) that encodes
a pharmaceutical protein of interest; selecting a nucleotide that
is known to disrupt a binding of a major groove binding partner
with a nucleic acid, wherein the nucleotide has decreased binding
affinity to the major groove binding partner; and contacting the
provided cDNA and the selected nucleotide with an RNA polymerase,
under conditions such that the pharmaceutical nucleic acid is
synthesized.
[0045] In some embodiments, the pharmaceutical nucleic acid is a
ribonucleic acid (RNA).
[0046] In a further aspect, the present disclosure provides for
methods of making a pharmaceutical formulation comprising a
physiologically active secreted protein, comprising transfecting a
first population of human cells with a pharmaceutical nucleic acid
made by the methods described herein, wherein the secreted protein
is active upon a second population of human cells.
[0047] In some embodiments, the secreted protein is capable of
interacting, e.g. binding, with a receptor on the surface of at
least one cell present in the second population.
[0048] In some embodiments, the secreted protein is
Granulocyte-Colony Stimulating Factor (G-CSF).
[0049] In some embodiments, the second population contains
myeloblast cells that express the G-CSF receptor.
[0050] In a further aspect, the present disclosure provides for
methods of making a pharmaceutical formulation comprising human
cells comprising a physiologically active secreted protein,
comprising transfecting a first population of human cells with a
pharmaceutical nucleic acid made by the methods described herein,
wherein the secreted protein is active upon a second population of
human cells.
DEFINITIONS
[0051] 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.
[0052] It is further intended that the compounds of the present
disclosure are 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.
[0053] 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.
[0054] As used herein, the term "alkyl" is meant to refer to a
saturated hydrocarbon group which is straight-chained or branched.
Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g.,
n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl),
pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An
alkyl group can contain from 1 to about 20, from 2 to about 20,
from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to
about 4, or from 1 to about 3 carbon atoms.
[0055] As used herein, "alkenyl" refers to an alkyl group having
one or more double carbon-carbon bonds. Example alkenyl groups
include ethenyl, propenyl, and the like.
[0056] As used herein, "alkoxy" refers to an --O-alkyl group.
Example alkoxy groups include methoxy, ethoxy, propoxy (e.g.,
n-propoxy and isopropoxy), t-butoxy, and the like.
[0057] As used herein, "alkynyl" refers to an alkyl group having
one or more triple carbon-carbon bonds. Example alkynyl groups
include ethynyl, propynyl, and the like.
[0058] As used herein, "aryl" refers to monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as,
for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl,
indenyl, and the like. In some embodiments, aryl groups have from 6
to about 20 carbon atoms.
[0059] As used herein, "halo" or "halogen" includes fluoro, chloro,
bromo, and iodo.
[0060] As used herein, "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.
[0061] As used herein, "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.
[0062] As used herein, "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).
[0063] As used herein, "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.
[0064] As used herein, "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, where a nucleic acid is biologically active, a portion
of that nucleic acid that shares at least one biological activity
of the whole nucleic acid is typically referred to as a
"biologically active" portion.
[0065] As used herein, "conserved" refers to nucleotides or amino
acid residues of a polynucleotide sequence or amino acid sequence,
respectively, that are those that occur unaltered in the same
position of two or more related 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.
[0066] 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.
[0067] 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.
[0068] As used herein, "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).
[0069] As used herein, "in vivo" refers to events that occur within
an organism (e.g., animal, plant, or microbe).
[0070] As used herein, "isolated" refers to a substance or entity
that has been (1) separated from at least some of the components
with which it was associated when initially produced (whether in
nature or in an experimental setting), and/or (2) produced,
prepared, and/or manufactured by the hand of man. 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.
[0071] As used herein, "subject" or "patient" refers to any
organism to which a composition in accordance with the present
disclosure 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.
[0072] As used herein, "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.
[0073] 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.
[0074] 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. In some
embodiments, an individual who is susceptible to a disease,
disorder, and/or condition (for example, cancer) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development of the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0075] As used herein, "therapeutically effective amount" means an
amount of an agent to be delivered (e.g., nucleic acid, drug,
therapeutic agent, diagnostic agent, prophylactic agent, etc.) that
is sufficient, when administered to a subject suffering from or
susceptible to a disease, disorder, and/or condition, to treat,
improve symptoms of, diagnose, prevent, and/or delay the onset of
the disease, disorder, and/or condition.
[0076] As used herein, "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.
[0077] As used herein, "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
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.
In some embodiments, treatment comprises delivery of a protein
associated with a therapeutically active nucleic acid to a subject
in need thereof.
[0078] As used herein, "unmodified" refers to a nucleic acid prior
to being modified, e.g. adenosine, guanosine, cytosine, thymidine,
and uracil, or a naturally occurring amino acid. 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.
[0079] Compounds of the present disclosure also include tautomeric
forms. Tautomeric forms result from the swapping of a single bond
with an adjacent double bond together with the concomitant
migration of a proton. Tautomeric forms include prototropic
tautomers which are isomeric protonation states having the same
empirical formula and total charge. Example 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, for example, 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.
[0080] Compounds of the present disclosure can also include all
isotopes of atoms occurring in the intermediates or final
compounds. Isotopes include those atoms having the same atomic
number but different mass numbers. For example, isotopes of
hydrogen include tritium and deuterium.
[0081] The term "compound," as used herein, is meant to include all
stereoisomers, geometric isomers, tautomers, and isotopes of the
structures depicted.
[0082] In some embodiments, the compounds of the present disclosure
are substantially isolated. By "substantially isolated" is meant
that the compound is at least partially or 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.
[0083] The compounds of the present disclosure, and salts thereof,
can also be prepared in combination with solvent or water molecules
to form solvates and hydrates by routine methods.
[0084] 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.
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. 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 and Journal of Pharmaceutical
Science, 66, 2 (1977), each of which is incorporated herein by
reference in its entirety.
[0085] 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.
[0086] The present disclosure also includes prodrugs of the
compounds described herein. As used herein, "prodrugs" refer to any
carriers, typically covalently bonded, which release the active
parent drug when 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. Examples of prodrugs include, but are
not limited to, acetate, formate and benzoate derivatives of
alcohol and amine functional groups in the compounds of the present
disclosure. 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.
Modified Nucleosides and Nucleotides
[0087] The present disclosure provides for modified nucleosides and
nucleotides. As described herein "nucleoside" is defined as a
compound containing a five-carbon sugar molecule (a pentose or
ribose) or derivative thereof, and an organic base, purine or
pyrimidine, or a derivative thereof. As described herein,
"nucleotide" is defined as a nucleoside consisting of a phosphate
group. The nucleosides and nucleotides described herein are
generally chemically modified on the major groove face. In some
embodiments, the major groove chemical modifications can include an
amino group, a thiol group, an alkyl group, or a halo group.
[0088] Table 1 below identifies the chemical faces of each
canonical nucleotide. Circles identify the atoms comprising the
respective chemical regions.
TABLE-US-00001 Major Groove Face Pyrimi- dines Cyti- dine:
##STR00003## Uri- dine: ##STR00004## Purines Adeno- sine:
##STR00005## Guano- sine: ##STR00006## Minor Groove Face Pyrimi-
dines Cyti- dine: ##STR00007## Uri- dine: ##STR00008## Purines
Adeno- sine: ##STR00009## Guano- sine: ##STR00010## Watson-Crick
Base-pairing Face Pyrimi- dines Cyti- dine: ##STR00011## Uri- dine:
##STR00012## Purines Adeno- sine: ##STR00013## Guano- sine:
##STR00014##
[0089] In some embodiments, modified nucleosides include
pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,
2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,
5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudouridine, 5-propynyl-uridine,
1-propynyl-pseudouridine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine.
[0090] In some embodiments, modified nucleosides include
5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine,
N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine,
5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,
pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine.
[0091] In other embodiments, modified nucleosides include
2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyl adenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine.
[0092] In some embodiments, modified nucleosides include inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
[0093] In some embodiments, the nucleotide can be modified on the
major groove face and can include replacement of the hydrogen on
C-5 of uracil with a methyl group or a halo group.
[0094] In some embodiments, the nucleoside and nucleotide can be a
compound of Formula I:
##STR00015##
wherein:
[0095] Z is O or S;
[0096] each of Y.sup.1 is independently selected from --OR.sup.a1,
it and --SR.sup.a1;
[0097] each of Y.sup.2 is independently selected from O, NR.sup.a,
S or a linker comprising an atom selected from the group consisting
of C, O, N, and S;
[0098] each of Y.sup.3 is independently selected from O and S;
[0099] Y.sup.4 is selected from H, --SR.sup.a, and --NHR.sup.a;
[0100] n is 0, 1, 2, or 3;
[0101] m is 0, 1, 2 or 3;
[0102] B is a nucleobase;
[0103] R.sup.a is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
[0104] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0105] --Y.sup.3--R.sup.c1 is OH or SH at a pH of about 1 or
--Y.sup.3--R.sup.c1 is O.sup.- or S.sup.- at physiological pH;
[0106] or --Y.sup.3--R.sup.c1 is C.sub.1-20 alkoxy, C.sub.2-20
--O-alkenyl, or C.sub.1-20 --O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine,
guanine, uracil and adenine, then at least one of Z, Y.sup.1 or
Y.sup.2 is not O or OH.
[0107] In some embodiments, B is a nucleobase of Formula II-a,
II-b, or II-c:
##STR00016##
wherein:
[0108] denotes a single or double bond;
[0109] X is O or S;
[0110] U and W are each independently C or N;
[0111] V is O, S, C or N;
wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.c,
--C(O)OR.sup.c, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c;
[0112] and wherein when V is O, S, or N then R.sup.1 is absent;
[0113] R.sup.2 is H, --NR.sup.aR.sup.b, or halo;
or when V is C then R.sup.1 and R.sup.2 together with the carbon
atoms to which they are attached can form a 5- or 6-membered ring
optionally substituted with 1-4 substituents selected from halo,
--OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0114] R.sup.3 is H or C.sub.1-20 alkyl;
[0115] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0116] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0117] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0118] In some embodiments, B is a nucleobase of Formula II-al,
II-a2, II-a3, II-a4, or II-a5:
##STR00017##
[0119] In some embodiments, B is a nucleobase selected from the
group consisting of cytosine, guanine, adenine, and uracil.
[0120] In some embodiments, B is a pyrimidine or derivative
thereof.
[0121] In some embodiments the nucleotide is a compound of Formula
I-a:
##STR00018##
[0122] In some embodiments the nucleotide is a compound of Formula
I-b:
##STR00019##
[0123] In some embodiments the nucleotide is a compound of Formula
I-c:
##STR00020##
[0124] In some embodiments, the nucleotide is selected from the
group consisting of:
##STR00021## ##STR00022##
[0125] In some embodiments, the nucleotide is selected from the
group consisting of:
##STR00023##
[0126] For example, the modified nucleotide can be:
##STR00024##
[0127] In some embodiments, the major groove chemical modification
can include replacement of the C--H group at C-5 with an --NH--
group or a --NH(CH.sub.3)-- group.
[0128] For example, the modified nucleotide can be:
##STR00025##
[0129] In another embodiment, the major groove chemical
modification can include replacement of the hydrogen at C-5 of
cytosine with a halo group or a methyl group.
[0130] For example, the modified nucleotide can be:
##STR00026##
[0131] In yet a further embodiment, the major groove chemical
modification can include a fused ring that is formed by the
NH.sub.2 at the C-4 position and the carbon atom at the C-5
position.
[0132] For example, the modified nucleotide can be:
##STR00027##
[0133] In some embodiments, a modified nucleotide is
5'-O-(1-Thiophosphate)-Adenosine, 5'-O-(1-Thiophosphate)-Cytidine,
5'-O-(1-Thiophosphate)-Guanosine, 5'-O-(1-Thiophosphate)-Uridine or
5'-O-(1-Thiophosphate)-Pseudouridine.
5'-O-(1-Thiophosphate)-Adenosine
5'-O-(1-Thiophosphate)-Cytidine
5'-O-(1-Thiophosphate)-Guanosine
5'-O-(1-Thiophosphate)-Uridine
5'-O-(1-Thiophosphate)-Pseudouridine
[0134] The .alpha.-thio substituted phosphate moiety is provided to
confer stability to RNA and DNA polymers through the unnatural
phosphorothioate backbone linkages. Phosphorothioate DNA and RNA
have increased nuclease resistance and subsequently a longer
half-life in a cellular environment. Phosphorothioate linked
nucleic acids are expected to also reduce the innate immune
response through weaker binding/activation of cellular innate
immune molecules.
[0135] Further examples of modified nucleotides and modified
nucleotide combinations are provided below in Table 2.
TABLE-US-00002 TABLE 2 Modified Nucleotide Modified Nucleotide
Combination 6-aza-cytidine .alpha.-thio-cytidine/5-iodo-uridine
2-thio-cytidine .alpha.-thio-cytidine/N1-methyl-pseudo-uridine
.alpha.-thio-cytidine .alpha.-thio-cytidine/.alpha.-thio-uridine
Pseudo-iso-cytidine .alpha.-thio-cytidine/5-methyl-uridine
5-aminoallyl-uridine .alpha.-thio-cytidine/pseudo-uridine
5-iodo-uridine Pseudo-iso-cytidine/5-iodo-uridine N1-methyl-
Pseudo-iso-cytidine/N1-methyl-pseudo-uridine pseudouridine
5,6-dihydrouridine Pseudo-iso-cytidine/.alpha.-thio-uridine
.alpha.-thio-uridine Pseudo-iso-cytidine/5-methyl-uridine
4-thio-uridine Pseudo-iso-cytidine/Pseudo-uridine 6-aza-uridine
Pyrrolo-cytidine 5-hydroxy-uridine Pyrrolo-cytidine/5-iodo-uridine
Deoxy-thymidine Pyrrolo-cytidine/N1-methyl-pseudo-uridine
Pseudo-uridine Pyrrolo-cytidine/.alpha.-thio-uridine Inosine
Pyrrolo-cytidine/5-methyl-uridine .alpha.-thio-guanosine
Pyrrolo-cytidine/Pseudo-uridine 8-oxo-guanosine
5-methyl-cytidine/5-iodo-uridine O6-methyl-guanosine
5-methyl-cytidine/N1-methyl-pseudo-uridine 7-deaza-guanosine
5-methyl-cytidine/.alpha.-thio-uridine No modification
5-methyl-cytidine/5-methyl-uridine N1-methyl-adenosine
5-methyl-cytidine/Pseudo-uridine 2-amino-6-Chloro-
5-methyl-cytidine purine N6-methyl-2-amino- 25% Pseudo-iso-cytidine
purine 6-Chloro-purine 25% N1-methyl-pseudo-uridine
N6-methyl-adenosine 25% N1-Methyl-pseudo-uridine/75%-pseudo-uridine
.alpha.-thio-adenosine 5-methyl-uridine 8-azido-adenosine
5-iodo-cytidine 7-deaza-adenosine
Synthesis of Modified Nucleotides
[0136] The modified nucleosides and nucleotides disclosed herein
can be prepared from readily available starting materials using the
following general methods and procedures. It is understood that
where typical or preferred process conditions (i.e., reaction
temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given; other process conditions can also be used unless
otherwise stated. Optimum reaction conditions may vary with the
particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization
procedures.
[0137] 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.
[0138] Preparation of modified nucleosides and nucleotides can
involve the protection and deprotection of various chemical groups.
The need for protection and deprotection, and the selection of
appropriate protecting groups can be readily determined by one
skilled in the art. The chemistry of protecting groups can be
found, for example, in Greene, et al., Protective Groups in Organic
Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated
herein by reference in its entirety.
[0139] 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
modified nucleosides and nucleotides can be carried out by any of
numerous methods known in the art. An example method includes
fractional recrystallization using a "chiral resolving acid" which
is an optically active, salt-forming organic acid. Suitable
resolving agents for fractional recrystallization methods are, for
example, optically active acids, such as the D and L forms of
tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids. Resolution of racemic mixtures can
also be carried out by elution on a column packed with an optically
active resolving agent (e.g., dinitrobenzoylphenylglycine).
Suitable elution solvent composition can be determined by one
skilled in the art.
[0140] Exemplary syntheses of modified nucleotides are provided
below in Schemes 1 and 2.
##STR00028##
##STR00029##
[0141] Modified nucleosides and nucleotides can also be prepared
according to the synthetic methods described in Ogata et al.
Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al.
Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al.
Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9):
1729-1740, 1992, each of which are incorporated by reference in
their entirety.
Modified Nucleic Acids
[0142] The present disclosure provides nucleic acids, including
RNAs such as mRNAs that contain one or more modified nucleosides
(termed "modified nucleic acids") or nucleotides as described
herein, which have useful properties including the significant
decreast or lack of a substantial induction of the innate immune
response of a cell into which the mRNA is introduced, or the
suppression thereof. Because these modified nucleic acids enhance
the efficiency of protein production, intracellular retention of
nucleic acids, and viability of contacted cells, as well as possess
reduced immunogenicity, of these nucleic acids compared to
unmodified nucleic acids, having these properties are termed
"enhanced nucleic acids" herein.
[0143] In addition, the present disclosure provides nucleic acids,
which have decreased binding affinity to a major groove
interacting, e.g. binding, partner. For example, the nucleic acids
are comprised of at least one nucleotide that has been chemically
modified on the major groove face as described herein.
[0144] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that is or can be incorporated into an
oligonucleotide chain. Exemplary nucleic acids for use in
accordance with the present disclosure include, but are not limited
to, one or more of DNA, RNA including messenger mRNA (mRNA),
hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs,
miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce
triple helix formation, aptamers, vectors, etc., described in
detail herein.
[0145] Provided are modified nucleic acids containing a
translatable region and one, two, or more than two different
nucleoside modifications. In some embodiments, the modified nucleic
acid exhibits reduced degradation in a cell into which the nucleic
acid is introduced, relative to a corresponding unmodified nucleic
acid. Exemplary nucleic acids include ribonucleic acids (RNAs),
deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol
nucleic acids (GNAs), locked nucleic acids (LNAs) or a hybrid
thereof. In preferred embodiments, the modified nucleic acid
includes messenger RNAs (mRNAs). As described herein, the nucleic
acids of the present disclosure do not substantially induce an
innate immune response of a cell into which the mRNA is
introduced.
[0146] In certain embodiments, it is desirable to intracellularly
degrade a modified nucleic acid introduced into the cell, for
example if precise timing of protein production is desired. Thus,
the present disclosure provides a modified nucleic acid containing
a degradation domain, which is capable of being acted on in a
directed manner within a cell.
[0147] Other components of nucleic acid are optional, and are
beneficial in some embodiments. For example, a 5' untranslated
region (UTR) and/or a 3'UTR are provided, wherein either or both
may independently contain one or more different nucleoside
modifications. In such embodiments, nucleoside modifications may
also be present in the translatable region. Also provided are
nucleic acids containing a Kozak sequence.
[0148] Additionally, provided are nucleic acids containing one or
more intronic nucleotide sequences capable of being excised from
the nucleic acid.
[0149] Further, provided are nucleic acids containing an internal
ribosome entry site (IRES). An IRES may act as the sole ribosome
binding site, or may serve as one of multiple ribosome binding
sites of an mRNA. An mRNA containing more than one functional
ribosome binding site may encode several peptides or polypeptides
that are translated independently by the ribosomes ("multicistronic
mRNA"). When nucleic acids are provided with an IRES, further
optionally provided is a second translatable region. Examples of
IRES sequences that can be used according to the present disclosure
include without limitation, those from picornaviruses (e.g. FMDV),
pest viruses (CFFV), polio viruses (PV), encephalomyocarditis
viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C
viruses (HCV), classical swine fever viruses (CSFV), murine
leukemia virus (MLV), simian immune deficiency viruses (SIV) or
cricket paralysis viruses (CrPV).
[0150] In some embodiments, the nucleic acid sequences comprise a
compound of Formula I-d:
##STR00030##
wherein:
[0151] Z is O or S;
[0152] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.al;
[0153] each of Y.sup.2 is independently selected from O, NR.sup.a,
S or a linker comprising an atom selected from the group consisting
of C, O, N, and S;
[0154] each of Y.sup.3 is independently selected from O and S;
[0155] Y.sup.4 is selected from H, --OR.sup.a, --SR.sup.a, and
--NHR.sup.a;
[0156] B is a nucleobase;
[0157] R.sup.a is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
[0158] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0159] --Y.sup.3--R.sup.c1 is OH or SH at a pH of about 1 or
--Y.sup.3--R.sup.c1 is O.sup.- or S.sup.- at physiological pH;
[0160] or --Y.sup.3--R.sup.c1 is C.sub.1-20 alkoxy, C.sub.2-20
--O-alkenyl, or C.sub.1-20 --O-alkynyl;
[0161] wherein when B is an unmodified nucleobase selected from
cytosine, guanine, thymidine, uracil and adenine, then at least one
of Z, Y' or Y.sup.2 is not O or OH.
[0162] In some embodiments, B is a nucleobase of Formula II-a,
II-b, or II-c:
##STR00031##
wherein:
[0163] denotes a single or double bond;
[0164] X is O or S;
[0165] U and W are each independently C or N;
[0166] V is O, S, C or N;
wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.c,
--C(O)OR.sup.c, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c;
[0167] and wherein when V is O, S, or N then R.sup.1 is absent;
[0168] R.sup.2 is H, --NR.sup.aR.sup.b, or halo;
or when V is C then R.sup.1 and R.sup.2 together with the carbon
atoms to which they are attached can form a 5- or 6-membered ring
optionally substituted with 1-4 substituents selected from halo,
--OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0169] R.sup.3 is H or C.sub.1-20 alkyl;
[0170] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0171] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0172] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0173] In some embodiments, B is a nucleobase of Formula II-al,
II-a2, II-a3, II-a4, or II-a5:
##STR00032##
[0174] In some embodiments, at least 25% of the cytosines are
replaced by a compound of Formula I-a (e.g., at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or about
100%).
[0175] In some embodiments, at least 25% of the uracils are
replaced by a compound of Formula I-a (e.g., at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or about
100%).
[0176] In some embodiments, at least 25% of the cytosines and 25%
of the uracils are replaced by a compound of Formula I-a (e.g., at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or about 100%).
[0177] In some embodiments, the nucleic acid is translatable.
Major Groove Interacting Partners
[0178] As described herein, the phrase "major groove interacting
partner" refers RNA recognition receptors that detect and respond
to RNA ligands through interactions, e.g. binding, with the major
groove face of a nucleotide or nucleic acid. As such, RNA ligands
comprising modified nucleotides or nucleic acids as described
herein decrease interactions with major groove binding partners,
and therefore decrease an innate immune response, or expression and
secretion of pro-inflammatory cytokines, or both.
[0179] 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
Activation Using Modified Nucleic Acids
[0180] The term "innate immune response" includes a cellular
response to exogenous nucleic acids, including single stranded
nucleic acids, generally of viral or bacterial origin, which
involves the induction of cytokine expression and release,
particularly the interferons, and cell death. Protein synthesis is
also reduced during the innate cellular immune response. While it
is advantageous to eliminate the innate immune response in a cell,
the present disclosure provides modified mRNAs that substantially
reduce the immune response, including interferon signaling, without
entirely eliminating such a response. In some embodiments, the
immune response is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as compared to the
immune response induced by a corresponding unmodified nucleic acid.
Such a reduction can be measured by expression or activity level of
Type 1 interferons or the expression of interferon-regulated genes
such as the toll-like receptors (e.g., TLR7 and TLR8). Reduction of
innate immune response can also be measured by decreased cell death
following one or more administrations of modified RNAs to a cell
population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%,
or over 95% less than the cell death frequency observed with a
corresponding unmodified nucleic acid. Moreover, cell death may
affect fewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or
fewer than 0.01% of cells contacted with the modified nucleic
acids.
[0181] The present disclosure provides for the repeated
introduction (e.g., transfection) of modified nucleic acids into a
target cell population, e.g., in vitro, ex vivo, or in vivo. The
step of contacting the cell population may be repeated one or more
times (such as two, three, four, five or more than five times). In
some embodiments, the step of contacting the cell population with
the modified nucleic acids is repeated a number of times sufficient
such that a predetermined efficiency of protein translation in the
cell population is achieved. Given the reduced cytotoxicity of the
target cell population provided by the nucleic acid modifications,
such repeated transfections are achievable in a diverse array of
cell types.
Polypeptide Variants
[0182] Provided are nucleic acids that encode variant polypeptides,
which have a certain identity with a reference polypeptide
sequence. The term "identity" as known in the art, refers to a
relationship between the sequences of two or more peptides, as
determined by comparing the sequences. In the art, "identity" also
means the degree of sequence relatedness between peptides, as
determined by the number of matches between strings of two or more
amino acid residues. "Identity" measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model or
computer program (i.e., "algorithms"). Identity of related peptides
can be readily calculated by known methods. Such methods include,
but are not limited to, those described in Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
[0183] In some embodiments, the polypeptide variant has the same or
a similar activity as the reference polypeptide. Alternatively, the
variant has an altered activity (e.g., increased or decreased)
relative to a reference polypeptide. Generally, variants of a
particular polynucleotide or polypeptide of the present disclosure
will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to that particular reference polynucleotide or
polypeptide as determined by sequence alignment programs and
parameters described herein and known to those skilled in the
art.
[0184] 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
[0185] Also provided are polynucleotide libraries containing
nucleoside modifications, wherein the polynucleotides individually
contain a first nucleic acid sequence encoding a polypeptide, such
as an antibody, protein binding partner, scaffold protein, and
other polypeptides known in the art. Preferably, the
polynucleotides are mRNA in a form suitable for direct introduction
into a target cell host, which in turn synthesizes the encoded
polypeptide.
[0186] In certain embodiments, multiple variants of a protein, each
with different amino acid modification(s), are produced and tested
to determine the best variant in terms of pharmacokinetics,
stability, biocompatibility, and/or biological activity, or a
biophysical property such as expression level. Such a library may
contain 10, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, or over 10.sup.9 possible variants
(including substitutions, deletions of one or more residues, and
insertion of one or more residues).
Polypeptide-Nucleic Acid Complexes
[0187] Proper protein translation involves the physical aggregation
of a number of polypeptides and nucleic acids associated with the
mRNA. Provided by the present disclosure are protein-nucleic acid
complexes, containing a translatable mRNA having one or more
nucleoside modifications (e.g., at least two different nucleoside
modifications) and one or more polypeptides bound to the mRNA.
Generally, the proteins are provided in an amount effective to
prevent or reduce an innate immune response of a cell into which
the complex is introduced.
Untranslatable Modified Nucleic Acids
[0188] As described herein, provided are mRNAs having sequences
that are substantially not translatable. Such mRNA is effective as
a vaccine when administered to a mammalian subject.
[0189] Also provided are modified nucleic acids that contain one or
more noncoding regions. Such modified nucleic acids are generally
not translated, but are capable of binding to and sequestering one
or more translational machinery component such as a ribosomal
protein or a transfer RNA (tRNA), thereby effectively reducing
protein expression in the cell. The modified nucleic acid may
contain a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small
interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
Synthesis of Modified Nucleic Acids
[0190] Nucleic acids for use in accordance with the present
disclosure may be prepared according to any available technique
including, but not limited to chemical synthesis, enzymatic
synthesis, which is generally termed in vitro transcription,
enzymatic or chemical cleavage of a longer precursor, etc. Methods
of synthesizing RNAs are known in the art (see, e.g., Gait, M. J.
(ed.) Oligonucleotide synthesis: a practical approach, Oxford
[Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P.
(ed.) Oligonucleotide synthesis: methods and applications, Methods
in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana
Press, 2005; both of which are incorporated herein by
reference).
[0191] The modified nucleosides and nucleotides disclosed herein
can be prepared from readily available starting materials using the
following general methods and procedures. It is understood that
where typical or preferred process conditions (i.e., reaction
temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given; other process conditions can also be used unless
otherwise stated. Optimum reaction conditions may vary with the
particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization
procedures.
[0192] 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.
[0193] Preparation of modified nucleosides and nucleotides can
involve the protection and deprotection of various chemical groups.
The need for protection and deprotection, and the selection of
appropriate protecting groups can be readily determined by one
skilled in the art. The chemistry of protecting groups can be
found, for example, in Greene, et al., Protective Groups in Organic
Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated
herein by reference in its entirety.
[0194] 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.
[0195] Resolution of racemic mixtures of modified nucleosides and
nucleotides can be carried out by any of numerous methods known in
the art. An example method includes fractional recrystallization
using a "chiral resolving acid" which is an optically active,
salt-forming organic acid. Suitable resolving agents for fractional
recrystallization methods are, for example, optically active acids,
such as the D and L forms of tartaric acid, diacetyltartaric acid,
dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or
the various optically active camphorsulfonic acids. Resolution of
racemic mixtures can also be carried out by elution on a column
packed with an optically active resolving agent (e.g.,
dinitrobenzoylphenylglycine). Suitable elution solvent composition
can be determined by one skilled in the art. Modified nucleic acids
need not be uniformly modified along the entire length of the
molecule. Different nucleotide modifications and/or backbone
structures may exist at various positions in the nucleic acid. One
of ordinary skill in the art will appreciate that the nucleotide
analogs or other modification(s) may be located at any position(s)
of a nucleic acid such that the function of the nucleic acid is not
substantially decreased. A modification may also be a 5' or 3'
terminal modification. The nucleic acids may contain at a minimum
one and at maximum 100% modified nucleotides, or any intervening
percentage, such as at least 5% modified nucleotides, at least 10%
modified nucleotides, at least 25% modified nucleotides, at least
50% modified nucleotides, at least 80% modified nucleotides, or at
least 90% modified nucleotides. For example, the nucleic acids may
contain a modified pyrimidine such as uracil or cytosine. In some
embodiments, at least 5%, at least 10%, at least 25%, at least 50%,
at least 80%, at least 90% or 100% of the uracil in the nucleic
acid is replaced with a modified uracil. The modified uracil can be
replaced by a compound having a single unique structure, or can be
replaced by a plurality of compounds having different structures
(e.g., 2, 3, 4 or more unique structures). In some embodiments, at
least 5%, at least 10%, at least 25%, at least 50%, at least 80%,
at least 90% or 100% of the cytosine in the nucleic acid is
replaced with a modified cytosine. The modified cytosine can be
replaced by a compound having a single unique structure, or can be
replaced by a plurality of compounds having different structures
(e.g., 2, 3, 4 or more unique structures).
[0196] Generally, the shortest length of a modified mRNA of the
present disclosure can be the length of an mRNA sequence that is
sufficient to encode for a dipeptide. In another embodiment, the
length of the mRNA sequence is sufficient to encode for a
tripeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a tetrapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
pentapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a hexapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
heptapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for an octapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
nonapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a decapeptide.
[0197] Examples of dipeptides that the modified nucleic acid
sequences can encode for include, but are not limited to, carnosine
and anserine.
[0198] 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.
[0199] The present disclosure provides methods of preparing a
nucleic acid sequence comprising a nucleotide that disrupts binding
of a major groove interacting partner with the nucleic acid
sequence, wherein the nucleic acid sequence comprises a compound of
Formula I-d:
##STR00033##
wherein:
[0200] Z is O or S;
[0201] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.a1;
[0202] each of Y.sup.2 is independently selected from O, NR.sup.a,
S or a linker comprising an atom selected from the group consisting
of C, O, N, and S;
[0203] each of Y.sup.3 is independently selected from O and S;
[0204] Y.sup.4 is selected from H, --OR.sup.a, --SR.sup.a, and
--NHR.sup.a;
[0205] B is a nucleobase;
[0206] R.sup.a is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
[0207] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0208] --Y.sup.3--R.sup.c1 is OH or SH at a pH of about 1 or
--Y.sup.3--R.sup.c1 is O.sup.- or S.sup.- at physiological pH;
[0209] or --Y.sup.3--R.sup.c1 is C.sub.1-20 alkoxy, C.sub.2-20
--O-alkenyl, or C.sub.1-20 --O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine,
guanine, uracil and adenine, then at least one of Z, Y.sup.1 or
Y.sup.2 is not O or OH; the method comprising: reacting a compound
of Formula I-c:
##STR00034##
with an RNA polymerase, and a cDNA template.
[0210] In some embodiments, the reaction is repeated from 1 to
about 7,000 times.
[0211] In some embodiments, B is a nucleobase of Formula II-a, Mb,
or II-c:
##STR00035##
wherein:
[0212] denotes a single or double bond;
[0213] X is O or S;
[0214] U and W are each independently C or N;
[0215] V is O, S, C or N;
wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each optionally
substituted with --OH, --NR.sup.aR.sup.b, --SH, --C(O)R.sup.c,
--C(O)OR.sup.c, --NHC(O)R.sup.c, or --NHC(O)OR.sup.c;
[0216] and wherein when V is O, S, or N then R.sup.1 is absent;
[0217] R.sup.2 is H, --OR.sup.c, --SR.sup.c, --NR.sup.aR.sup.b, or
halo;
or when V is C then R.sup.1 and R.sup.2 together with the carbon
atoms to which they are attached can form a 5- or 6-membered ring
optionally substituted with 1-4 substituents selected from halo,
--OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0218] R.sup.3 is H or C.sub.1-20 alkyl;
[0219] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0220] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0221] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0222] In some embodiments, B is a nucleobase of Formula II-al,
II-a2, II-a3, II-a4, or II-a5:
##STR00036##
[0223] In some embodiments, the methods further comprise a
nucleotide selected from the group consisting of adenosine,
cytosine, guanosine, and uracil.
[0224] In some embodiments, the nucleobase is a pyrimidine or
derivative thereof.
[0225] In a further aspect, the present disclosure provides methods
of amplifying a nucleic acid sequence comprising a nucleotide that
disrupts binding of a major groove binding partner with the nucleic
acid sequence, the method comprising:
reacting a compound of Formula I-c:
##STR00037##
[0226] Z is O or S;
[0227] each of Y.sup.1 is independently selected from --OR.sup.a1,
--NR.sup.a1R.sup.b1, and --SR.sup.a1;
[0228] each of Y.sup.2 is independently selected from O, NR.sup.a,
S or a linker comprising an atom selected from the group consisting
of C, O, N, and S;
[0229] each of Y.sup.3 is independently selected from O and S;
[0230] Y.sup.4 is selected from H, --OR.sup.a, --SR.sup.a, and
--NHR.sup.a;
[0231] B is a nucleobase;
[0232] R.sup.a is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
[0233] R.sup.a1 and R.sup.b1 are each independently H or a
counterion; and
[0234] --Y.sup.3--R.sup.c1 is OH or SH at a pH of about 1 or
--Y.sup.3--R.sup.c1 is O.sup.- or S.sup.- at physiological pH;
[0235] or --Y.sup.3--R.sup.c1 is C.sub.1-20 alkoxy, C.sub.2-20
--O-alkenyl, or C.sub.1-20 --O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine,
guanine, uracil and adenine, then at least one of Z, Y.sup.1 or
Y.sup.2 is not O or OH; with a primer, a cDNA template, and an RNA
polymerase.
[0236] In some embodiments, B is a nucleobase of Formula II-a,
II-b, or II-c:
##STR00038##
wherein:
[0237] denotes a single or double bond;
[0238] X is O or S;
[0239] U and W are each independently C or N;
[0240] V is O, S, C or N;
[0241] wherein when V is C then R.sup.1 is H, C.sub.1-6 alkyl,
C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, halo, or --OR.sup.c, wherein
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl are each
optionally substituted with --OH, --NR.sup.aR.sup.b, --SH,
--C(O)R', --C(O)OR', --NHC(O)R.sup.c, or --NHC(O)OR.sup.c;
[0242] and wherein when V is O, S, or N then R.sup.1 is absent;
[0243] R.sup.2 is H, --OR.sup.c, --SR', --NR.sup.aR.sup.b, or
halo;
or when V is C then R.sup.1 and R.sup.2 together with the carbon
atoms to which they are attached can form a 5- or 6-membered ring
optionally substituted with 1-4 substituents selected from halo,
--OH, --SH, --NR.sup.aR.sup.b, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, C.sub.2-20 alkynyl, C.sub.1-20 alkoxy, or C.sub.1-20
thioalkyl;
[0244] R.sup.3 is H or C.sub.1-20 alkyl;
[0245] R.sup.4 is H or C.sub.1-20 alkyl; wherein when denotes a
double bond then R.sup.4 is absent, or N--R.sup.4, taken together,
forms a positively charged N substituted with C.sub.1-20 alkyl;
[0246] R.sup.a and R.sup.b are each independently H, C.sub.1-20
alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, or C.sub.6-20 aryl;
and
[0247] R.sup.c is H, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, phenyl,
benzyl, a polyethylene glycol group, or an amino-polyethylene
glycol group.
[0248] In some embodiments, B is a nucleobase of Formula II-al,
II-a2, II-a3, II-a4, or II-a5:
##STR00039##
[0249] In some embodiments, the methods further comprise a
nucleotide selected from the group consisting of adenosine,
cytosine, guanosine, and uracil.
[0250] In some embodiments, the nucleobase is a pyrimidine or
derivative thereof.
Uses of Modified Nucleic Acids
Therapeutic Agents
[0251] The modified nucleic acids and the proteins translated from
the modified nucleic acids described herein can be used as
therapeutic agents. For example, a modified nucleic acid described
herein can be administered to a subject, wherein the modified
nucleic acid is translated in vivo to produce a therapeutic peptide
in the subject. Accordingly, provided herein are compositions,
methods, kits, and reagents for treatment or prevention of disease
or conditions in humans and other mammals. The active therapeutic
agents of the present disclosure include modified nucleic acids,
cells containing modified nucleic acids or polypeptides translated
from the modified nucleic acids, polypeptides translated from
modified nucleic acids, and cells contacted with cells containing
modified nucleic acids or polypeptides translated from the modified
nucleic acids.
[0252] In certain embodiments, provided are combination
therapeutics containing one or more modified nucleic acids
containing translatable regions that encode for a protein or
proteins that boost a mammalian subject's immunity along with a
protein that induces antibody-dependent cellular toxitity. For
example, provided are therapeutics containing one or more nucleic
acids that encode trastuzumab and granulocyte-colony stimulating
factor (G-CSF). In particular, such combination therapeutics are
useful in Her2+ breast cancer patients who develop induced
resistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy.
2(6):795-8 (2010)).
[0253] Provided are methods of inducing translation of a
recombinant polypeptide in a cell population using the modified
nucleic acids described herein. Such translation can be in vivo, ex
vivo, in culture, or in vitro. The cell population is contacted
with an effective amount of a composition containing a nucleic acid
that has at least one nucleoside modification, and a translatable
region encoding the recombinant polypeptide. The population is
contacted under conditions such that the nucleic acid is localized
into one or more cells of the cell population and the recombinant
polypeptide is translated in the cell from the nucleic acid.
[0254] An effective amount of the composition is provided based, at
least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the nucleic acid (e.g.,
size, and extent of modified nucleosides), and other determinants.
In general, an effective amount of the composition provides
efficient protein production in the cell, preferably more efficient
than a composition containing a corresponding unmodified nucleic
acid. Increased efficiency may be demonstrated by increased cell
transfection (i.e., the percentage of cells transfected with the
nucleic acid), increased protein translation from the nucleic acid,
decreased nucleic acid degradation (as demonstrated, e.g., by
increased duration of protein translation from a modified nucleic
acid), or reduced innate immune response of the host cell.
[0255] Aspects of the present disclosure are directed to methods of
inducing in vivo translation of a recombinant polypeptide in a
mammalian subject in need thereof. Therein, an effective amount of
a composition containing a nucleic acid that has at least one
nucleoside modification and a translatable region encoding the
recombinant polypeptide is administered to the subject using the
delivery methods described herein. The nucleic acid is provided in
an amount and under other conditions such that the nucleic acid is
localized into a cell of the subject and the recombinant
polypeptide is translated in the cell from the nucleic acid. The
cell in which the nucleic acid is localized, or the tissue in which
the cell is present, may be targeted with one or more than one
rounds of nucleic acid administration.
[0256] Other aspects of the present disclosure relate to
transplantation of cells containing modified nucleic acids to a
mammalian subject. Administration of cells to mammalian subjects is
known to those of ordinary skill in the art, such as local
implantation (e.g., topical or subcutaneous administration), organ
delivery or systemic injection (e.g., intravenous injection or
inhalation), as is the formulation of cells in pharmaceutically
acceptable carrier. Compositions containing modified nucleic acids
are formulated for administration intramuscularly, transarterially,
intraperitoneally, intravenously, intranasally, subcutaneously,
endoscopically, transdermally, or intrathecally. In some
embodiments, the composition is formulated for extended
release.
[0257] 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.
[0258] In certain embodiments, the administered modified nucleic
acid directs production of one or more recombinant polypeptides
that provide a functional activity which is substantially absent in
the cell in which the recombinant polypeptide is translated. For
example, the missing functional activity may be enzymatic,
structural, or gene regulatory in nature.
[0259] In other embodiments, the administered modified nucleic acid
directs production of one or more recombinant polypeptides that
replace a polypeptide (or multiple polypeptides) that is
substantially absent in the cell in which the recombinant
polypeptide is translated. Such absence may be due to genetic
mutation of the encoding gene or regulatory pathway thereof.
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, do
to mutation of the endogenous protein resulting in altered activity
or localization. Additionally, the recombinant polypeptide
antagonizes, directly or indirectly, the activity of a biological
moiety present in, on the surface of, or secreted from the cell.
Examples of antagonized biological moieties include lipids (e.g.,
cholesterol), a lipoprotein (e.g., low density lipoprotein), a
nucleic acid, a carbohydrate, or a small molecule toxin.
[0260] 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.
[0261] As described herein, a useful feature of the modified
nucleic acids of the present disclosure is the capacity to reduce
the innate immune response of a cell to an exogenous nucleic acid.
Provided are methods for performing the titration, reduction or
elimination of the immune response in a cell or a population of
cells. In some embodiments, the cell is contacted with a first
composition that contains a first dose of a first exogenous nucleic
acid including a translatable region and at least one nucleoside
modification, and the level of the innate immune response of the
cell to the first exogenous nucleic acid is determined.
Subsequently, the cell is contacted with a second composition,
which includes a second dose of the first exogenous nucleic acid,
the second dose containing a lesser amount of the first exogenous
nucleic acid as compared to the first dose. Alternatively, the cell
is contacted with a first dose of a second exogenous nucleic acid.
The second exogenous nucleic acid may contain one or more modified
nucleosides, which may be the same or different from the first
exogenous nucleic acid or, alternatively, the second exogenous
nucleic acid may not contain modified nucleosides. The steps of
contacting the cell with the first composition and/or the second
composition may be repeated one or more times. Additionally,
efficiency of protein production (e.g., protein translation) in the
cell is optionally determined, and the cell may be re-transfected
with the first and/or second composition repeatedly until a target
protein production efficiency is achieved.
Therapeutics for Diseases and Conditions
[0262] Provided are methods for treating or preventing a symptom of
diseases characterized by missing or aberrant protein activity, by
replacing the missing protein activity or overcoming the aberrant
protein activity. Because of the rapid initiation of protein
production following introduction of modified mRNAs, as compared to
viral DNA vectors, the compounds of the present disclosure are
particularly advantageous in treating acute diseases such as
sepsis, stroke, and myocardial infarction. Moreover, the lack of
transcriptional regulation of the modified mRNAs of the present
disclosure is advantageous in that accurate titration of protein
production is achievable.
[0263] Diseases characterized by dysfunctional or aberrant protein
activity include, but not limited to, cancer and proliferative
diseases, genetic diseases (e.g., cystic fibrosis), autoimmune
diseases, diabetes, neurodegenerative diseases, cardiovascular
diseases, and metabolic diseases. The present disclosure provides a
method for treating such conditions or diseases in a subject by
introducing nucleic acid or cell-based therapeutics containing the
modified nucleic acids provided herein, wherein the modified
nucleic acids encode for a protein that antagonizes or otherwise
overcomes the aberrant protein activity present in the cell of the
subject. Specific examples of a dysfunctional protein are the
missense mutation variants of the cystic fibrosis transmembrane
conductance regulator (CFTR) gene, which produce a dysfunctional
protein variant of CFTR protein, which causes cystic fibrosis.
[0264] 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, or are
essentially non-functional. The present disclosure provides a
method for treating such conditions or diseases in a subject by
introducing nucleic acid or cell-based therapeutics containing the
modified nucleic acids provided herein, wherein the modified
nucleic acids encode for a protein that replaces the protein
activity missing from the target cells of the subject. Specific
examples of a dysfunctional protein are the nonsense mutation
variants of the cystic fibrosis transmembrane conductance regulator
(CFTR) gene, which produce a nonfunctional protein variant of CFTR
protein, which causes cystic fibrosis.
[0265] Thus, provided are methods of treating cystic fibrosis in a
mammalian subject by contacting a cell of the subject with a
modified nucleic acid having a translatable region that encodes a
functional CFTR polypeptide, under conditions such that an
effective amount of the CTFR polypeptide is present in the cell.
Preferred target cells are epithelial cells, such as the lung, and
methods of administration are determined in view of the target
tissue; i.e., for lung delivery, the RNA molecules are formulated
for administration by inhalation.
[0266] In another embodiment, the present disclosure provides a
method for treating hyperlipidemia in a subject, by introducing
into a cell population of the subject with a modified mRNA molecule
encoding Sortilin, a protein recently characterized by genomic
studies, thereby ameliorating the hyperlipidemia in a subject. The
SORT1 gene encodes a trans-Golgi network (TGN) transmembrane
protein called Sortilin. Genetic studies have shown that one of
five individuals has a single nucleotide polymorphism, rs12740374,
in the 1p13 locus of the SORT I gene that predisposes them to
having low levels of low-density lipoprotein (LDL) and
very-low-density lipoprotein (VLDL). Each copy of the minor allele,
present in about 30% of people, alters LDL cholesterol by 8 mg/dL,
while two copies of the minor allele, present in about 5% of the
population, lowers LDL cholesterol 16 mg/dL. Carriers of the minor
allele have also been shown to have a 40% decreased risk of
myocardial infarction. Functional in vivo studies in mice describes
that overexpression of SORT1 in mouse liver tissue led to
significantly lower LDL-cholesterol levels, as much as 80% lower,
and that silencing SORT1 increased LDL cholesterol approximately
200% (Musunuru K et al. From noncoding variant to phenotype via
SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:
714-721).
Methods of Cellular Nucleic Acid Delivery
[0267] Methods of the present disclosure enhance nucleic acid
delivery into a cell population, in vivo, ex vivo, or in culture.
For example, a cell culture containing a plurality of host cells
(e.g., eukaryotic cells such as yeast or mammalian cells) is
contacted with a composition that contains an enhanced nucleic acid
having at least one nucleoside modification and, optionally, a
translatable region. The composition also generally contains a
transfection reagent or other compound that increases the
efficiency of enhanced nucleic acid uptake into the host cells. The
enhanced nucleic acid exhibits enhanced retention in the cell
population, relative to a corresponding unmodified nucleic acid.
The retention of the enhanced nucleic acid is greater than the
retention of the unmodified nucleic acid. In some embodiments, it
is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than
200% greater than the retention of the unmodified nucleic acid.
Such retention advantage may be achieved by one round of
transfection with the enhanced nucleic acid, or may be obtained
following repeated rounds of transfection.
[0268] In some embodiments, the enhanced nucleic acid is delivered
to a target cell population with one or more additional nucleic
acids. Such delivery may be at the same time, or the enhanced
nucleic acid is delivered prior to delivery of the one or more
additional nucleic acids. The additional one or more nucleic acids
may be modified nucleic acids or unmodified nucleic acids. It is
understood that the initial presence of the enhanced nucleic acids
does not substantially induce an innate immune response of the cell
population and, moreover, that the innate immune response will not
be activated by the later presence of the unmodified nucleic acids.
In this regard, the enhanced nucleic acid may not itself contain a
translatable region, if the protein desired to be present in the
target cell population is translated from the unmodified nucleic
acids.
Targeting Moieties
[0269] In some embodiments, modified nucleic acids are provided to
express a protein-binding partner or a receptor on the surface of
the cell, which functions to target the cell to a specific tissue
space or to interact with a specific moiety, either in vivo or in
vitro. Suitable protein-binding partners include antibodies and
functional fragments thereof, scaffold proteins, or peptides.
Additionally, modified nucleic acids can be employed to direct the
synthesis and extracellular localization of lipids, carbohydrates,
or other biological moieties.
Permanent Gene Expression Silencing
[0270] A method for epigenetically silencing gene expression in a
mammalian subject, comprising a nucleic acid where the translatable
region encodes a polypeptide or polypeptides capable of directing
sequence-specific histone H3 methylation to initiate
heterochromatin formation and reduce gene transcription around
specific genes for the purpose of silencing the gene. For example,
a gain-of-function mutation in the Janus Kinase 2 gene is
responsible for the family of Myeloproliferative Diseases.
Pharmaceutical Compositions
[0271] The present disclosure provides proteins generated from
modified mRNAs. Pharmaceutical compositions may optionally comprise
one or more additional therapeutically active substances. In
accordance with some embodiments, a method of administering
pharmaceutical compositions comprising 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 or protein-containing complex as
described herein.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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..
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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).
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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).
[0310] 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.
[0311] 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.
[0312] 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
[0313] 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.
[0314] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a nucleic acid modification, wherein the
nucleic acid is capable of evading an innate immune response of a
cell into which the first isolated nucleic acid is introduced, and
packaging and instructions.
[0315] In one aspect, the disclosure provides kits for protein
production, comprising: a first isolated 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.
[0316] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a nucleoside modification, wherein the
nucleic acid exhibits reduced degradation by a cellular nuclease,
and packaging and instructions.
[0317] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and at least two different nucleoside
modifications, wherein the nucleic acid exhibits reduced
degradation by a cellular nuclease, and packaging and
instructions.
[0318] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and at least one nucleoside modification,
wherein the nucleic acid exhibits reduced degradation by a cellular
nuclease; a second nucleic acid comprising an inhibitory nucleic
acid; and packaging and instructions.
[0319] In some embodiments, the first isolated nucleic acid
comprises messenger RNA (mRNA). In some embodiments the mRNA
comprises at least one nucleoside selected from the group
consisting of pyridin-4-one ribonucleoside, 5-aza-uridine,
2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,
5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyluridine, 1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine,
5-methyl-uridine, 1-methyl-pseudouridine,
4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine.
[0320] In some embodiments, the mRNA comprises at least one
nucleoside selected from the group consisting of 5-aza-cytidine,
pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,
5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine.
[0321] In some embodiments, the mRNA comprises at least one
nucleoside selected from the group consisting of 2-aminopurine,
2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,
7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,
7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,
1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine.
[0322] In some embodiments, the mRNA comprises at least one
nucleoside selected from the group consisting of inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
[0323] In another aspect, the disclosure provides compositions for
protein production, comprising a first isolated nucleic acid
comprising a translatable region and a nucleoside modification,
wherein the nucleic acid exhibits reduced degradation by a cellular
nuclease, and a mammalian cell suitable for translation of the
translatable region of the first nucleic acid.
EXAMPLES
[0324] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Modified mRNA In Vitro Transcription
Materials and Methods
[0325] Modified mRNAs (modRNAs) were made using standard laboratory
methods and materials for in vitro transcription with the exception
that the nucleotide mix contained modified nucleotides. The open
reading frame (ORF) of the gene of interest is flanked by a 5'
untranslated region (UTR) containing a strong Kozak translational
initiation signal and an alpha-globin 3' UTR terminating with an
oligo(dT) sequence for templated addition of a polyA tail for
modRNAs not incorporating Adenosine analogs. Adenosine-containing
modRNAs were synthesized without an oligo (dT) sequence to allow
for post-transcription poly (A) polymerase poly-(A) tailing. The
modRNAs were modified by incorporating chemically modified
nucleotides indicated in Table 3 (below) during the in vitro
transcription with 100% replacement of the corresponding natural
nucleotide or partial replacement of the corresponding natural
nucleotide at the indicated percentage.
[0326] Table 3 indicates the chemical identity of each
chemically-distinct modified nucleotide incorporated into a
modified mRNA with the given chemistry designation number.
TABLE-US-00003 TABLE 3 Modified Nucleotide Chemistry # Modified
Nucleotide Combination Chemistry # 6-aza-cytidine Chem 1
.alpha.-thio-cytidine/5-iodo-uridine Chem 29 2-thio-cytidine Chem 2
.alpha.-thio-cytidine/N1-methyl-pseudo-uridine Chem 30
.alpha.-thio-cytidine Chem 3
.alpha.-thio-cytidine/.alpha.-thio-uridine Chem 31
Pseudo-iso-cytidine Chem 4 .alpha.-thio-cytidine/5-methyl-uridine
Chem 32 5-aminoallyl-uridine Chem 5
.alpha.-thio-cytidine/pseudo-uridine Chem 33 5-iodo-uridine Chem 6
Pseudo-iso-cytidine/5-iodo-uridine Chem 34 N1-methyl-pseudouridine
Chem 7 Pseudo-iso-cytidine/N1-methyl-pseudo-uridine Chem 35
5,6-dihydrouridine Chem 8 Pseudo-iso-cytidine/.alpha.-thio-uridine
Chem 36 .alpha.-thio-uridine Chem 9
Pseudo-iso-cytidine/5-methyl-uridine Chem 37 4-thio-uridine Chem 10
Pseudo-iso-cytidine/Pseudo-uridine Chem 38 6-aza-uridine Chem 11
Pyrrolo-cytidine Chem 39 5-hydroxy-uridine Chem 12
Pyrrolo-cytidine/5-iodo-uridine Chem 40 Deoxy-thymidine Chem 13
Pyrrolo-cytidine/N1-methyl-pseudo-uridine Chem 41 Pseudo-uridine
Chem 14 Pyrrolo-cytidine/.alpha.-thio-uridine Chem 42 Inosine Chem
15 Pyrrolo-cytidine/5-methyl-uridine Chem 43 .alpha.-thio-guanosine
Chem 16 Pyrrolo-cytidine/Pseudo-uridine Chem 44 8-oxo-guanosine
Chem 17 5-methyl-cytidine/5-iodo-uridine Chem 45
O6-methyl-guanosine Chem 18
5-methyl-cytidine/N1-methyl-pseudo-uridine Chem 46
7-deaza-guanosine Chem 19 5-methyl-cytidine/.alpha.-thio-uridine
Chem 47 No modification Chem 20 5-methyl-cytidine/5-methyl-uridine
Chem 48 N1-methyl-adenosine Chem 21
5-methyl-cytidine/Pseudo-uridine Chem 49 2-amino-6-Chloro-purine
Chem 22 5-methyl-cytidine Chem 50 N6-methyl-2-amino- Chem 23 25%
Pseudo-iso-cytidine Chem 51 purine 25% N1-methyl-pseudo-uridine
Chem 52 6-Chloro-purine Chem 24 25%
N1-Methyl-pseudo-uridine/75%-pseudo- Chem 53 N6-methyl-adenosine
Chem 25 uridine .alpha.-thio-adenosine Chem 26 5-methyl-uridine
Chem 54 8-azido-adenosine Chem 27 5-iodo-cytidine Chem 55
7-deaza-adenosine Chem 28
[0327] Agarose Gel Electrophoresis of modRNA:
[0328] Individual modRNAs (200-400 ng in a 20 .mu.l volume) were
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 (FIG. 1A). Tables 4 and 5 below
indicate the modified nucleotide (Table 4) or nucleic acid (Table
5) loaded in each lane. These data indicate which chemically
modified nucleotides were transcribed into chemically-modified
mRNAs and the quality of each individual modRNA. These data
demonstrate that nucleotides with chemical modifications on the
major groove and minor groove face of the nucleotide were capable
of being transcribed into a modRNA.
TABLE-US-00004 TABLE 4 Lane Modified NTP 1 .alpha.-thio-cytidine 2
Pseudo-iso-cytidine 3 5-aminoallyl-uridine 4 5-iodo-uridine 5
N1-methyl-pseudo-uridine 6 .alpha.-thio-uridine 7 4-thio-uridine 8
5-hydroxy-uridine 9 Deoxy-thymidine 10 Pseudo-uridine 11 Inosine 12
.alpha.-thio-guanosine 13 8-oxo-guanosine 14 N1-methyl-guanosine 15
O6-methyl-guanosine 16 No modification 17 N1-methyl-adenosine 18
2-amino-6-Chloro-purine 19 N6-methyl-2-amino-purine 20
6-Chloro-purine 21 .alpha.-thio-adenosine 22 8-azido-adenosine 23
7-deaza-adenosine 24 6-aza-cytidine 25 2-thio-cytidine 26
5,6-dihydro-uridine 27 6-aza-uridine 28 7-deaza-guanosine 29
N6-methyl-adenosine
TABLE-US-00005 TABLE 5 Lane Modified NTP combination 1
.alpha.-thio-cytidine/5-iodo-uridine 2
.alpha.-thio-cytidine/N1-methyl-pseudouridine 3
.alpha.-thio-cytidine/.alpha.-thio-uridine 4
.alpha.-thio-cytidine/5-methyl-uridine 5
.alpha.-thio-cytidine/pseudouridine 6
5-iodo-cytidine/5-iodo-uridine 7
5-iodo-cytidine/N1-methyl-pseudouridine 8
5-iodo-cytidine/.alpha.-thio-uridine 9
5-iodo-cytidine/5-methyl-uridine 10 5-iodo-cytidine/pseudouridine
11 Pseudo-iso-cytidine/5-iodo-uridine 12 Pyrrolo-cytidine 13
Pyrrolo-cytidine/5-iodo-uridine 14
Pyrrolo-cytidine/N1-methyl-pseudouridine 15
Pyrrolo-cytidine/.alpha.-thio-uridine 16
Pyrrolo-cytidine/5-methyl-uridine 17 Pyrrolo-cytidine/pseudouridine
18 5-methyl-cytidine/5-iodo-uridine 19
5-methyl-cytidine/N1-methyl-uridine 20
5-methyl-cytidine/.alpha.-thio-uridine 21
5-methyl-cytidine/5-methyl-uridine 22
5-methyl-cytidine/pseudouridine 23
Pseudo-iso-cytidine/N1-methyl-pseudouridine 24
Pseudo-iso-cytidine/.alpha.-thio-uridine 25
Pseudo-iso-cytidine/5-methyl-uridine 26
Pseudo-iso-cytidine/pseudouridine 27 5-methyl-cytidine 28 25%
pseudo-iso-cytidine 29 25% N1-methyl-pseudouridine 30 25%
N1-methyl-pseudouridine/75% pseudouridine
[0329] Agarose Gel Electrophoresis of RT-PCR Products:
[0330] Individual reverse transcribed-PCR products (200-400 ng)
were 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 (FIG. 1B). Table 5 below indicates the
modified nucleotide loaded in each lane.
[0331] Nanodrop modRNA Quantification and UV Spectral Data:
[0332] modRNAs in TE buffer (1 .mu.l) were used for Nanodrop UV
absorbance readings to quantitate the yield of each modRNA from an
in vitro transcription reaction (UV absorbance traces are shown in
FIGS. 6A-6L). These data indicate which chemically modified
nucleotides were transcribed into chemically-modified mRNAs. These
data also demonstrate that nucleotides with chemical modifications
on the major groove and minor groove face of the nucleotide were
capable of being transcribed into a modRNA. These data further
demonstrate that the nucleotides of the present invention are
transcription-competent and compatible with incorporation into a
modRNA, which may have altered UV spectra due to the presence of a
given modified nucleotide. For example, Pyrrolo-C containing
modRNAs have an increase in UV absorbance at a lower wavelength due
to the presence of the pyrrolo ring of the modified C nucleotide.
In another example, 2-amino-adenine nucleotide-containing modRNAs
have an increase in UV absorbance at a higher wavelength due to the
presence of an exocyclic amine off the purine ring. Nucleotides
that are not transcription-competent and cannot be incorporated
into a modRNA have a scrambled UV spectrum indicating no product
from the transcription reaction.
Example 2
Modified RNA Transfection
[0333] Reverse Transfection:
[0334] For experiments performed in a 24-well collagen-coated
tissue culture plate, Keratinocytes were seeded at a cell density
of 1.times.10.sup.5. For experiments performed in a 96-well
collagen-coated tissue culture plate, Keratinocytes were seeded at
a cell density of 0.5.times.10.sup.5. For each modRNA to be
transfected, modRNA: RNAiMAX was prepared as described and mixed
with the cells in the multi-well plate within a period of time,
e.g., 6 hours, of cell seeding before cells had adhered to the
tissue culture plate.
[0335] Forward Transfection:
[0336] In a 24-well collagen-coated tissue culture plate,
Keratinocytes were 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 were seeded at a cell density of
0.3.times.10.sup.5. Keratinocytes were then grown to a confluency
of >70% for over 24 hours. For each modRNA to be transfected,
modRNA: RNAiMAX was 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.
[0337] modRNA Translation Screen: G-CSF ELISA
[0338] FIGS. 2A and 2B show an Enzyme-linked immunosorbent assay
(ELISA) for Human Granulocyte-Colony Stimulating Factor (hu-G-CSF)
of in vitro transfected Human Keratinocyte cells. Keratinocytes
were grown in EpiLife medium with Supplement S7 from Invitrogen at
a confluence of >70%. FIG. 2A keratinocytes were reverse
transfected with 300 ng of the indicated chemically modified mRNA
complexed with RNAiMAX from Invitrogen. FIG. 2B keratinocytes were
forward transfected with 300 ng modRNA complexed with RNAiMAX from
Invitrogen. The RNA:RNAiMAX complex was formed by first incubating
the RNA with Supplement-free EpiLife media in a 5.times. volumetric
dilution for 10 minutes at room temperature. In a second vial,
RNAiMAX reagent was incubated with Supplement-free EpiLife Media in
a 10.times. volumetric dilution for 10 minutes at room temperature.
The RNA vial was then mixed with the RNAiMAX vial and incubated for
20-30 at room temperature before being added to the cells in a
drop-wise fashion. Secreted huG-CSF concentration in the culture
medium was measured at 18 hours post-transfection for each of the
chemically modified mRNAs in triplicate. Secretion of Human
Granulocyte-Colony Stimulating Factor (G-CSF) from transfected
human keratinocytes was quantified using an ELISA kit from
Invitrogen or R&D Systems (Minneapolis, Minn.) following the
manufacturers recommended instructions. These data show that
huG-CSF modRNAs comprised of chemically distinct nucleotide analogs
(SEQ ID NO: 2) is capable of being translated in Human Keratinocyte
cells and that huG-CSF is transported out of the cells and released
into the extracellular environment. These data indicate which
modified nucleotides were translated into protein when incorporated
into a chemically modified mRNA. These data show that modified RNA
containing nucleotides with chemical modifications on the major
groove face of pyrimidine analogs have the highest levels of
secreted hu-G-CSF into the cell culture medium.
[0339] modRNA Dose and Duration: G-CSF ELISA
[0340] FIGS. 3A-N show Enzyme-linked immunosorbent assays (ELISA)
for Human Granulocyte-Colony Stimulating Factor (G-CSF) of in vitro
transfected Human Keratinocyte cells. Keratinocytes were grown in
EpiLife medium with Supplement S7 from Invitrogen at a confluence
of >70%. Keratinocytes were reverse transfected with 0 ng,
46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng modRNA
complexed with RNAiMAX from Invitrogen. The modRNA:RNAiMAX complex
was formed as described. Secreted huG-CSF concentration in the
culture medium was measured at 0, 6, 12, 24, and 48 hours
post-transfection for each concentration of each modRNA in
triplicate. Secretion of Human Granulocyte-Colony Stimulating
Factor (G-CSF) from transfected human keratinocytes was quantified
using an ELISA kit from Invitrogen or R&D Systems following the
manufacturers recommended instructions. These data show that
huG-CSF modRNAs comprised of chemically distinct nucleotide analogs
(SEQ ID NO: X and Table 6) secreted hu-G-CSF protein in a modRNA
dose-dependent manner from Human Keratinocyte cells and that
huG-CSF is transported out of the cells and released into the
extracellular environment. These data indicate which modified RNAs
containing modified nucleotide analogs sustain hu-G-CSF expression
for the longest and at the highest levels. These data show that
modified RNA containing modified nucleotides with chemical
modifications on the major groove face of pyrimidine analogs have
the highest levels of secreted hu-G-CSF into the cell culture
medium and that 750 ng of modRNA elicits the highest level of
secreted hu-G-CSF.
Example 3
Cellular Innate Immune Response to modRNA
[0341] IFN-.beta. ELISA and TNF-.alpha. ELISA:
[0342] FIGS. 4A-F show an Enzyme-linked immunosorbent assay (ELISA)
for Human Tumor Necrosis Factor-.alpha. (TNF-.alpha.) (FIGS. 4A and
4B); Human Interferon-.beta. (IFN-.beta.) (FIGS. 4C and 4D); and
Human Granulocyte-Colony Stimulating Factor (G-CSF) (FIGS. 4E and
4F) secreted from in vitro-transfected Human Keratinocyte cells.
Keratinocytes were grown in EpiLife medium with Human Keratinocyte
Growth Supplement in the absence of hydrocortisone from Invitrogen
at a confluence of >70%. In FIGS. 4A and 4B, keratinocytes were
reverse transfected with Ong, 93.75 ng, 187.5 ng, 375 ng, 750 ng,
1500 ng or 3000 ng of the indicated chemically modified mRNA
complexed with RNAiMAX from Invitrogen as described in triplicate.
Secreted TNF-.alpha. in the culture medium was measured 24 hours
post-transfection for each of the chemically modified mRNAs using
an ELISA kit from Invitrogen according to the manufacturer
protocols.
[0343] In FIGS. 4C and 4D, secreted IFN-.beta. in the same culture
medium was measured 24 hours post-transfection for each of the
chemically modified mRNAs using an ELISA kit from Invitrogen
according to the manufacturer protocols. In FIGS. 4E and 4F,
secreted hu-G-CSF concentration in the same culture medium was
measured at 24 hours post-transfection for each of the chemically
modified mRNAs. Secretion of Human Granulocyte-Colony Stimulating
Factor (G-CSF) from transfected human keratinocytes was quantified
using an ELISA kit from Invitrogen or R&D Systems (Minneapolis,
Minn.) following the manufacturers recommended instructions. These
data indicate which modified RNAs containing modified nucleotides
were capable of eliciting a reduced cellular innate immune response
in comparison to natural and other chemically modified nucleotides
by measuring exemplary type I cytokines TNF-.alpha. and IFN-.beta..
These data show that modified RNAs containing modified nucleotides
with chemical modifications on the major groove face of pyrimidine
analogs have the lowest levels of secreted TNF-.alpha. and
IFN-.beta. into the cell culture medium while maintaining high
levels of modRNA-encoding hu-G-CSF secretion into the cell culture
medium.
Example 4
Human Granulocyte-Colony Stimulating Factor-Modified RNA-Induced
Cell Proliferation Assay
[0344] FIGS. 5A-D show modRNA-encoding hu-G-CSF produced by a human
keratinocyte feeder cell layer induced the proliferation of both
human myeloblast cells KG-1 and Kasumi-1 that express the
G-CSF-receptor where the cell populations are separated by a
semi-permeable membrane.
[0345] Human keratinocytes were grown in EpiLife medium with
Supplement S7 from Invitrogen at a confluence of >70% in a
24-well collagen-coated Transwell.RTM. (Corning, Lowell, Mass.)
co-culture tissue culture plate. Keratinocytes were reverse
transfected with 750 ng of the indicated chemically modified mRNA
complexed with RNAiMAX from Invitrogen as described in triplicate.
The modRNA:RNAiMAX complex was formed as described. Keratinocyte
media was exchanged 6-8 hours post-transfection. 42-hours
post-transfection, the 24-well Transwell.RTM. plate insert with a
0.4 .mu.m-pore semi-permeable polyester membrane was placed into
the hu-G-CSF modRNA-transfected keratinocyte containing culture
plate. FIG. 5A is a table showing the results from an Enzyme-linked
immunosorbent assay (ELISA) for human-G-CSF secreted from in
vitro-transfected Human Keratinocyte cells sampled from individual
wells in a co-culture 24-well tissue culture plate 42 hours
post-transfection with 750 ng of each indicated hu-G-CSF-encoding
modRNA.
[0346] Human myeloblast cells, Kasumi-1 cells (FIG. 5C) or KG-1
(FIG. 5D) (0.2.times.10.sup.5 cells), were seeded into the insert
well and cell proliferation was quantified 42 hours post-co-culture
initiation using the CyQuant Direct Cell Proliferation Assay
(Invitrogen) in a 100-120 .mu.l volume in a 96-well plate.
modRNA-encoding hu-G-CSF-induced myeloblast cell proliferation was
expressed as a percent cell proliferation normalized to
untransfected keratinocyte/myeloblast co-culture control wells.
Secreted hu-G-CSF concentration in both the keratinocyte and
myeloblast insert co-culture wells was measured at 42 hours
post-co-culture initiation for each modRNA in duplicate. Secretion
of Human Granulocyte-Colony Stimulating Factor (G-CSF) was
quantified using an ELISA kit from Invitrogen following the
manufacturers recommended instructions.
[0347] Transfected hu-G-CSF modRNA in human keratinocyte feeder
cells and untransfected human myeloblast cells were detected by
RT-PCR. Total RNA from sample cells was extracted and lysed using
RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer
instructions. Extracted total RNA was submitted to RT-PCR for
specific amplification of modRNA-G-CSF using ProtoScript.RTM.
M-MuLV Taq RT-PCR kit (New England BioLabs, Ipswich, Mass.)
according to the manufacturer instructions with hu-G-CSF-specific
primers (see below). RT-PCR products were visualized by 1.2%
agarose gel electrophoresis (FIG. 5B). Table 6 below shows which
modRNAs were run on the agarose gel.
TABLE-US-00006 TABLE 6 RT-PCR hu-G-CSF Lane Cell type modRNA Target
1 Keratinocyte KG-1 Feeder Vehicle 2 Keratinocyte KG-1 Feeder
Scramble RNA 3 Keratinocyte KG-1 Feeder No Modification 4
Keratinocyte KG-1 Feeder Chem 7 5 Keratinocyte KG-1 Feeder Chem 6 6
Keratinocyte KG-1 Feeder Chem 37 7 Keratinocyte Kasumi-1 Feeder
Vehicle 8 Keratinocyte Kasumi-1 Feeder Scramble RNA 9 Keratinocyte
Kasumi-1 Feeder No Modification 10 Keratinocyte Kasumi-1 Feeder
Chem 7 11 Keratinocyte Kasumi-1 Feeder Chem 6 12 Keratinocyte
Kasumi-1 Feeder Chem 37 13 Keratinocyte KG-1 Feeder Chem 46 14
Keratinocyte KG-1 Feeder Chem 48 15 Keratinocyte KG-1 Feeder Chem
49 16 Keratinocyte KG-1 Feeder Chem 53 17 Keratinocyte Kasumi-1
Feeder Chem 46 18 Keratinocyte Kasumi-1 Feeder Chem 48 19
Keratinocyte Kasumi-1 Feeder Chem 49 20 Keratinocyte Kasumi-1
Feeder Chem 53 21 Kasumi-1 Vehicle 22 KG-1 Vehicle 23 Kasumi-1
Vehicle 24 Kasumi-1 Scramble RNA 25 Kasumi-1 No Modification 26
Kasumi-1 Chem 7 27 Kasumi-1 Chem 6 28 Kasumi-1 Chem 37 29 Kasumi-1
Chem 46 30 Kasumi-1 Chem 48 31 Kasumi-1 Chem 49 32 Kasumi-1 Chem 53
33 KG-1 Vehicle 34 KG-1 Scramble RNA 35 KG-1 No Modification 36
KG-1 Chem 7 37 Empty Empty 38 Empty Empty 39 Empty Empty 40 Empty
Empty 41 Empty Empty 42 Empty Empty 43 Empty Empty 44 Empty
Empty
[0348] These data show that human keratinocyte cells containing
hu-G-CSF modRNAs comprised of chemically distinct nucleotide
analogs secreted hu-G-CSF protein and that the secreted hu-G-CSF
was physiologically-active in inducing the proliferation of human
myeloblast cells expressing the G-CSF receptor. These data also
show the secreted hu-G-CSF protein was permeable across a
semi-permeable membrane and acted on a different
non-G-CSF-producing cell population. Additionally, these data show
that hu-G-CSF modRNA-transfected into human keratinocyte cells in a
co-culture environment was present in only the transfected
keratinocyte cells and not the un-transfected myeloblast cells.
Further, these data show that the modified nucleotide chemical
composition of hu-G-CSF modRNA did not affect resultant protein
activity.
Example 5
The Effect of modRNA on Cellular Viability
[0349] Cytotoxicity and Apoptosis:
[0350] This experiment demonstrates cellular viability, cytotoxity
and apoptosis for distinct modRNA-in vitro transfected Human
Keratinocyte cells. Keratinocytes are grown in EpiLife medium with
Human Keratinocyte Growth Supplement in the absence of
hydrocortisone from Invitrogen at a confluence of >70%.
Keratinocytes are reverse transfected with Ong, 46.875 ng, 93.75
ng, 187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of
modRNA complexed with RNAiMAX from Invitrogen. The modRNA:RNAiMAX
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 modRNA in triplicate. Secretion of
Human Granulocyte-Colony Stimulating Factor (G-CSF) from
transfected human keratinocytes is quantified using an ELISA kit
from Invitrogen or R&D Systems following the manufacturers
recommended instructions. Cellular viability, cytotoxicity and
apoptosis is measured at 0, 12, 48, 96, and 192 hours
post-transfection using the ApoToxGlo kit from Promega (Madison,
Wis.) according to manufacturer instructions.
Example 6
Co-Culture
[0351] The modified mRNA comprised of chemically-distinct modified
nucleotides encoding human Granulocyte-Colony Stimulating Factor
(G-CSF) may stimulate the cellular proliferation of a transfection
incompetent cell in co-culture environment. The co-culture includes
a highly transfectable cell type such as a human keratinocyte and a
transfection incompetent cell type such as a white blood cell
(WBC). The modified mRNA encoding G-CSF may be transfected into the
highly transfectable cell allowing for the production and secretion
of G-CSF protein into the extracellular environment where G-CSF
acts in a paracrine-like manner to stimulate the white blood cell
expressing the G-CSF receptor to proliferate. The expanded WBC
population may be used to treat immune-compromised patients or
partially reconstitute the WBC population of an immunosuppressed
patient and thus reduce the risk of opportunistic infections.
[0352] Another example, a highly transfectable cell such as a
fibroblast may be transfected with certain growth factors to
support and simulate the growth, maintenance, or differentiation of
poorly transfectable embryonic stem cells or induced pluripotent
stem cells.
Example 7
5'-Guanosine Capping on Modified Nucleic Acids (modRNAs)
[0353] The cloning, gene synthesis and vector sequencing was
performed by DNA2.0 Inc. (Menlo Park, Calif.). Sequence and insert
sequence are set forth herein. The ORF was restriction digested
using XbaI and used for cDNA synthesis using tailed- or
tail-less-PCR. The tailed-PCR cDNA product was used as the template
for the modified mRNA synthesis reaction using 25 mM mixture each
modified nucleotide (all modified nucleotides were custom
synthesized or purchased from TriLink Biotech, San Diego, Calif.
except pyrrolo-C triphosphate purchased from Glen Research,
Sterling Va.; unmodified nucleotides were purchased from Epicenter
Biotechnologies, Madison, Wis.) and CellScript MegaScript.TM.
(Epicenter Biotechnologies, Madison, Wis.) complete mRNA synthesis
kit. The in vitro transcription reaction was run for 4 hours at
37.degree. C. modRNAs incorporating adenosine analogs were poly (A)
tailed using yeast Poly (A) Polymerase (Affymetrix, Santa Clara,
Calif.). PCR reaction used HiFi PCR 2.times. Master Mix.TM. (Kapa
Biosystems, Woburn, Mass.). modRNAs were post-transcriptionally
capped using recombinant Vaccinia Virus Capping Enzyme (New England
BioLabs, Ipswich, Mass.) and a recombinant 2'-O-methyltransferase
(Epicenter Biotechnologies, Madison, Wis.) to generate the
5'-guanosine Cap 1 structure. Cap 2 structure and Cap 3 structures
may be generated using additional 2'-O-methyltransferases. The in
vitro transcribed mRNA product was run on an agarose gel and
visualized. modRNA was purified with Ambion/Applied Biosystems
(Austin, Tex.) MEGAClear RNA.TM. purification kit. PCR used
PureLink.TM. PCR purification kit (Invitrogen, Carlsbad, Calif.).
The product was quantified on Nanodrop.TM. UV Absorbance
(ThermoFisher, Waltham, Mass.). Quality, UV absorbance quality and
visualization of the product was performed on an 1.2% agarose gel.
The product was resuspended in TE buffer.
5' Capping Modified Nucleic Acid (mRNA) Structure:
[0354] 5'-modRNA capping 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'-O-Me-m.sup.7G(5')ppp(5')G;
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'-modRNA capping 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.
TABLE-US-00007 Sequences: G-CSF cDNA: (SEQ ID NO: 1)
agcttttggaccctcgtacagaagctaatacgactcactatagggaaata
agagagaaaagaagagtaagaagaaatataagagccaccatggccggtcc
cgcgacccaaagccccatgaaacttatggccctgcagttgctgctttggc
actcggccctctggacagtccaagaagcgactcctcteggacctgcctca
tcgttgccgcagtcattccttttgaagtgtctggagcaggtgcgaaagat
tcagggcgatggagccgcactccaagagaagctctgcgcgacatacaaac
tttgccatcccgaggagctcgtactgctcgggcacagcttggggattccc
tgggctcctctctcgtcctgtccgtcgcaggctttgcagttggcagggtg
cctttcccagctccactccggtttgttcttgtatcagggactgctgcaag
cccttgagggaatctcgccagaattgggcccgacgctggacacgttgcag
ctcgacgtggcggatttcgcaacaaccatctggcagcagatggaggaact
ggggatggcacccgcgctgcagcccacgcagggggcaatgccggcctttg
cgtccgcgatcagcgcagggegggtggagtcctcgtagcgagccaccttc
aatcatttttggaagtctcgtaccgggtgctgagacatcttgcgcagccg
tgaagcgctgccttctgcggggcttgccttctggccatgcccttcttctc
tcccttgcacctgtacctcttggtctttgaataaagcctgagtaggaagg
cggccgctcgagcatgcatctagagggcccaattcgccctattcgaagtc g G-CSF mRNA:
(SEQ ID NO: 2) agcuuuuggacccucguacagaagcuaauacgacucacuauagggaaaua
agagagaaaagaagaguaagaagaaauauaagagccaccauggccggucc
cgcgacccaaagccccaugaaacuuauggcccugcaguugcugcuuuggc
acucggcccucuggacaguccaagaagcgacuccucucggaccugccuca
ucguugccgcagucauuccuuuugaagugucuggagcaggugcgaaagau
ucagggcgauggagccgcacuccaagagaagcucugcgcgacauacaaac
uuugccaucccgaggagcucguacugcucgggcacagcuuggggauuccc
ugggcuccucucucguccuguccgucgcaggcuuugcaguuggcagggug
ccuuucccagcuccacuccgguuuguucuuguaucagggacugcugcaag
cccuugagggaaucucgccagaauugggcccgacgcuggacacguugcag
cucgacguggcggauuucgcaacaaccaucuggcagcagauggaggaacu
ggggauggcacccgcgcugcagcccacgcagggggcaaugccggccuuug
cguccgcguuucagcgcagggcggguggaguccucguagcgagccaccuu
caaucauuuuuggaagucucguaccgggugcugagacaucuugcgcagcc
gugaagcgcugccuucugcggggcuugccuucuggccaugcccuucuucu
cucccuugcaccuguaccucuuggucuuugaauaaagccugaguaggaag
gcggccgcucgagcaugcaucuagagggcccaauucgcccuauucgaagu cg G-CSF
protein: (SEQ ID NO: 3)
MAGPATQSPMKLMALQLLLWHSALWTVQEATPLGPASSLPQSFLLKCLEQ
VRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQ
ALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTI
WQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRV LRHLAQP cDNA
synthesis primers: Forward Primer: (SEQ ID NO: 4) 5'-TTG GAC CCT
CGT ACA GAA GCT AAT ACG Reverse Primer for template Poly (A)
tailing: (SEQ ID NO: 5) 5'-T(.sub.120)CT TCC TAC TCA GGC TTT ATT
CAA AGA CCA Reverse Primer for post-transcriptional Poly (A)
Polymerase tailing: (SEQ ID NO: 6) 5'-CTT CCT ACT CAG GCT TTA TTC
AAA GAC CA G-CSF modRNA RT-PCR primers: Forward Primer: (SEQ ID NO:
7) 5'-TGG CCG GTC CCG CGA CCC AA Reverse Primer: (SEQ ID NO: 8)
5'-GCT TCA CGG CTG CGC AAG AT
Other Embodiments
[0355] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
81852DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1agcttttgga ccctcgtaca gaagctaata
cgactcacta tagggaaata agagagaaaa 60gaagagtaag aagaaatata agagccacca
tggccggtcc cgcgacccaa agccccatga 120aacttatggc cctgcagttg
ctgctttggc actcggccct ctggacagtc caagaagcga 180ctcctctcgg
acctgcctca tcgttgccgc agtcattcct tttgaagtgt ctggagcagg
240tgcgaaagat tcagggcgat ggagccgcac tccaagagaa gctctgcgcg
acatacaaac 300tttgccatcc cgaggagctc gtactgctcg ggcacagctt
ggggattccc tgggctcctc 360tctcgtcctg tccgtcgcag gctttgcagt
tggcagggtg cctttcccag ctccactccg 420gtttgttctt gtatcaggga
ctgctgcaag cccttgaggg aatctcgcca gaattgggcc 480cgacgctgga
cacgttgcag ctcgacgtgg cggatttcgc aacaaccatc tggcagcaga
540tggaggaact ggggatggca cccgcgctgc agcccacgca gggggcaatg
ccggcctttg 600cgtccgcgtt tcagcgcagg gcgggtggag tcctcgtagc
gagccacctt caatcatttt 660tggaagtctc gtaccgggtg ctgagacatc
ttgcgcagcc gtgaagcgct gccttctgcg 720gggcttgcct tctggccatg
cccttcttct ctcccttgca cctgtacctc ttggtctttg 780aataaagcct
gagtaggaag gcggccgctc gagcatgcat ctagagggcc caattcgccc
840tattcgaagt cg 8522852RNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 2agcuuuugga cccucguaca
gaagcuaaua cgacucacua uagggaaaua agagagaaaa 60gaagaguaag aagaaauaua
agagccacca uggccggucc cgcgacccaa agccccauga 120aacuuauggc
ccugcaguug cugcuuuggc acucggcccu cuggacaguc caagaagcga
180cuccucucgg accugccuca ucguugccgc agucauuccu uuugaagugu
cuggagcagg 240ugcgaaagau ucagggcgau ggagccgcac uccaagagaa
gcucugcgcg acauacaaac 300uuugccaucc cgaggagcuc guacugcucg
ggcacagcuu ggggauuccc ugggcuccuc 360ucucguccug uccgucgcag
gcuuugcagu uggcagggug ccuuucccag cuccacuccg 420guuuguucuu
guaucaggga cugcugcaag cccuugaggg aaucucgcca gaauugggcc
480cgacgcugga cacguugcag cucgacgugg cggauuucgc aacaaccauc
uggcagcaga 540uggaggaacu ggggauggca cccgcgcugc agcccacgca
gggggcaaug ccggccuuug 600cguccgcguu ucagcgcagg gcggguggag
uccucguagc gagccaccuu caaucauuuu 660uggaagucuc guaccgggug
cugagacauc uugcgcagcc gugaagcgcu gccuucugcg 720gggcuugccu
ucuggccaug cccuucuucu cucccuugca ccuguaccuc uuggucuuug
780aauaaagccu gaguaggaag gcggccgcuc gagcaugcau cuagagggcc
caauucgccc 840uauucgaagu cg 8523207PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 1
5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr
Pro 20 25 30 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
Lys Cys Leu 35 40 45 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys 50 55 60 Leu Val Ser Glu Cys Ala Thr Tyr Lys
Leu Cys His Pro Glu Glu Leu 65 70 75 80 Val Leu Leu Gly His Ser Leu
Gly Ile Pro Trp Ala Pro Leu Ser Ser 85 90 95 Cys Pro Ser Gln Ala
Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 100 105 110 Ser Gly Leu
Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 115 120 125 Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 130 135
140 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
145 150 155 160 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala 165 170 175 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala
Ser His Leu Gln Ser 180 185 190 Phe Leu Glu Val Ser Tyr Arg Val Leu
Arg His Leu Ala Gln Pro 195 200 205 427DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4ttggaccctc gtacagaagc taatacg 275149DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
60tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
120cttcctactc aggctttatt caaagacca 149629DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6cttcctactc aggctttatt caaagacca 29720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7tggccggtcc cgcgacccaa 20820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 8gcttcacggc tgcgcaagat 20
* * * * *