U.S. patent application number 17/371261 was filed with the patent office on 2022-02-10 for nucleoside-modified rna for inducing an adaptive immune response.
The applicant listed for this patent is ACUITAS THERAPEUTICS, INC., The Trustees of the University of Pennsylvania. Invention is credited to Michael J. Hope, Norbert Pardi, Ying Tam, Drew Weissman.
Application Number | 20220040285 17/371261 |
Document ID | / |
Family ID | 1000005925591 |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220040285 |
Kind Code |
A1 |
Weissman; Drew ; et
al. |
February 10, 2022 |
Nucleoside-Modified RNA For Inducing an Adaptive Immune
Response
Abstract
The present invention relates to compositions and methods for
inducing an adaptive immune response in a subject. In certain
embodiments, the present invention provides a composition
comprising a nucleoside-modified nucleic acid molecule encoding an
antigen, adjuvant, or a combination thereof. For example, in
certain embodiments, the composition comprises a vaccine comprising
a nucleoside-modified nucleic acid molecule encoding an antigen,
adjuvant, or a combination thereof.
Inventors: |
Weissman; Drew; (Wynnewood,
PA) ; Pardi; Norbert; (Philadelphia, PA) ;
Tam; Ying; (Vancouver, CA) ; Hope; Michael J.;
(Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania
ACUITAS THERAPEUTICS, INC. |
Philadelphia
Vancouver |
PA |
US
CA |
|
|
Family ID: |
1000005925591 |
Appl. No.: |
17/371261 |
Filed: |
July 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15569546 |
Oct 26, 2017 |
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PCT/US16/29572 |
Apr 27, 2016 |
|
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17371261 |
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62153143 |
Apr 27, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/18 20180101;
C07D 295/13 20130101; C07C 219/08 20130101; A61P 35/00 20180101;
A61P 31/16 20180101; A61K 39/0011 20130101; A61K 2039/55555
20130101; A61K 2039/51 20130101; A61K 39/12 20130101; A61K 31/712
20130101; A61K 9/127 20130101; A61K 39/002 20130101; A61K 9/0019
20130101; C12N 15/11 20130101; A61K 39/02 20130101; A61K 31/7115
20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 39/00 20060101 A61K039/00; A61K 39/002 20060101
A61K039/002; A61K 39/02 20060101 A61K039/02; C07C 219/08 20060101
C07C219/08; C07D 295/13 20060101 C07D295/13; A61K 9/127 20060101
A61K009/127; A61P 31/18 20060101 A61P031/18; A61P 31/16 20060101
A61P031/16; A61K 9/00 20060101 A61K009/00; A61K 31/7115 20060101
A61K031/7115; A61K 31/712 20060101 A61K031/712; C12N 15/11 20060101
C12N015/11 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
RO1-AI-090788 awarded by the National Institute of Health. The
government has certain rights in the invention.
Claims
1. A composition for inducing an adaptive immune response in a
subject, the composition comprising at least one
nucleoside-modified RNA encoding at least one antigen.
2. The composition of claim 1, wherein the at least one isolated
nucleoside-modified RNA comprises at least one selected from the
group consisting of pseudouridine and 1-methyl-pseudouridine.
3. (canceled)
4. The composition of claim 1, wherein the at least one antigen
comprises at least one selected from the group consisting of a
viral antigen, a bacterial antigen, a fungal antigen, a parasitic
antigen, a tumor-associated antigen, and a tumor-specific
antigen
5. The composition of claim 1, wherein the at least one antigen
comprises an antigen selected from the group consisting of an HIV
antigen and an influenza antigen.
6. The composition of claim 5, wherein the antigen comprises an
antigen selected from the group consisting of HIV envelope (Env)
and influenza hemagglutinin (HA).
7-8. (canceled)
9. The composition of claim 1, wherein the composition further
comprises an adjuvant.
10. The composition of claim 1, wherein the at least one
nucleoside-modified RNA further encodes at least one adjuvant.
11. The composition of claim 1, further comprising a lipid
nanoparticle (LNP).
12. The composition of claim 11, wherein the at least one
nucleoside-modified RNA is encapsulated within the LNP.
13. The composition of claim 1, wherein the composition is a
vaccine.
14. The composition of claim 11, wherein the LNP comprises a
compound having a structure of Formula (I): ##STR00155## or a
pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer
thereof, wherein: L.sup.1 and L.sup.2 are each independently
--O(C.dbd.O)--, --(C.dbd.O)O-- or a carbon-carbon double bond;
R.sup.1a and R.sup.1b are, at each occurrence, independently either
(a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond; R.sup.2a and R.sup.2b are, at each occurrence,
independently either (a) H or C.sub.1-C.sub.12 alkyl, or (b)
R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b together with
the carbon atom to which it is bound is taken together with an
adjacent R.sup.2b and the carbon atom to which it is bound to form
a carbon-carbon double bond; R.sup.3a and R.sup.3b are, at each
occurrence, independently either (a) H or C.sub.1-C.sub.12 alkyl,
or (b) R.sup.3a is H or C.sub.1-C.sub.12 alkyl, and R.sup.3b
together with the carbon atom to which it is bound is taken
together with an adjacent R.sup.3b and the carbon atom to which it
is bound to form a carbon-carbon double bond; R.sup.4a and R.sup.4b
are, at each occurrence, independently either (a) H or
C.sub.1-C.sub.12 alkyl, or (b) R.sup.4a is H or C.sub.1-C.sub.12
alkyl, and R.sup.4b together with the carbon atom to which it is
bound is taken together with an adjacent R.sup.4b and the carbon
atom to which it is bound to form a carbon-carbon double bond;
R.sup.5 and R.sup.6 are each independently methyl or cycloalkyl;
R.sup.7 is, at each occurrence, independently H or C.sub.1-C.sub.12
alkyl; R.sup.8 and R.sup.9 are each independently unsubstituted
C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the
nitrogen atom to which they are attached, form a 5, 6 or 7-membered
heterocyclic ring comprising one nitrogen atom; a and d are each
independently an integer from 0 to 24; b and c are each
independently an integer from 1 to 24; and e is 1 or 2.
15. The composition of claim 11, wherein the LNP comprises a
compound having a structure of Formula (II): ##STR00156## or a
pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer
thereof, wherein: L.sup.1 and L.sup.2 are each independently
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--,
--S(O).sub.x--, --S--S--, --C(.dbd.O)S--, --SC(.dbd.O)--,
--NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)NR.sup.a, --OC(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)O--, or a direct bond; G.sup.1 is
C.sub.1-C.sub.2 alkylene, --(C.dbd.O)--, --O(C.dbd.O)--,
--SC(.dbd.O)--, --NR.sup.aC(.dbd.O)-- or a direct bond; G.sup.2 is
--C(.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)S--, --C(.dbd.O)NR.sup.a
or a direct bond; G.sup.3 is C.sub.1-C.sub.6 alkylene; R.sup.a is H
or C.sub.1-C.sub.12 alkyl; R.sup.1a and R.sup.1b are, at each
occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl;
or (b) R.sup.1a is H or C.sub.1-C.sub.12 alkyl, and R.sup.1b
together with the carbon atom to which it is bound is taken
together with an adjacent R.sup.1b and the carbon atom to which it
is bound to form a carbon-carbon double bond; R.sup.2a and R.sup.2b
are, at each occurrence, independently either: (a) H or
C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or C.sub.1-C.sub.12
alkyl, and R.sup.2b together with the carbon atom to which it is
bound is taken together with an adjacent R.sup.2b and the carbon
atom to which it is bound to form a carbon-carbon double bond;
R.sup.3a and R.sup.3b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond; R.sup.4a and R.sup.4b are, at each occurrence,
independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b)
R.sup.4a is H or C.sub.1-C.sub.12 alkyl, and R.sup.4b together with
the carbon atom to which it is bound is taken together with an
adjacent R.sup.4b and the carbon atom to which it is bound to form
a carbon-carbon double bond; R.sup.5 and R.sup.6 are each
independently H or methyl; R.sup.7 is C.sub.4-C.sub.20 alkyl;
R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12 alkyl;
or R.sup.8 and R.sup.9, together with the nitrogen atom to which
they are attached, form a 5, 6 or 7-membered heterocyclic ring; a,
b, c and d are each independently an integer from 1 to 24; and x is
0, 1 or 2.
16. The composition of claim 11, wherein the LNP comprises a
compound having a structure of Formula (III): ##STR00157## or a
pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer
thereof, wherein: one of L.sup.1 or L.sup.2 is --O(C.dbd.O)--,
--(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--,
--C(.dbd.O)S--, SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, NR.sup.aC(.dbd.O)NR.sup.a--,
--OC(.dbd.O)NR.sup.a-- or --NR.sup.aC(.dbd.O)O--, and the other of
L.sup.1 or L.sup.2 is --O(C.dbd.O)--, --(C.dbd.O)O--,
--C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--, --C(.dbd.O)S--,
SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O-- or a direct bond; G.sup.1 and G.sup.2 are
each independently unsubstituted C.sub.1-C.sub.12 alkylene or
C.sub.1-C.sub.12 alkenylene; G.sup.3 is C.sub.1-C.sub.24 alkylene,
C.sub.1-C.sub.24 alkenylene, C.sub.3-C.sub.8 cycloalkylene,
C.sub.3-C.sub.8 cycloalkenylene; R.sup.a is H or C.sub.1-C.sub.12
alkyl; R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24
alkyl or C.sub.6-C.sub.24 alkenyl; R.sup.3 is H, OR.sup.5, CN,
--C(.dbd.O)OR.sup.4, --OC(.dbd.O)R.sup.4 or
--NR.sup.5C(.dbd.O)R.sup.4; R.sup.4 is C.sub.1-C.sub.12 alkyl;
R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and x is 0, 1 or 2.
17. The composition of claim 11, wherein the LNP comprises a
compound having one of the following structures: ##STR00158##
##STR00159##
18. The composition of claim 11, wherein the LNP comprises a
pegylated lipid having the following structure (IV): ##STR00160##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein: R.sup.10 and R.sup.11 are each independently a
straight or branched, saturated or unsaturated alkyl chain
containing from 10 to 30 carbon atoms, wherein the alkyl chain is
optionally interrupted by one or more ester bonds; and z has a mean
value ranging from 30 to 60.
19. The composition of claim 18, wherein the pegylated lipid has
the following structure (IVa): ##STR00161## wherein n is an integer
selected such that the average molecular weight of the pegylated
lipid is about 2500 g/mol.
20-44. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is continuation of U.S. patent application
Ser. No. 15/569,546, filed Oct. 26, 2017, now allowed, which is a
U.S. National Phase Application filed under 35 U.S.C. .sctn. 371
claiming benefit to International Patent Application No.
PCT/US2016/029572, filed Apr. 27, 2016, which is entitled to
priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent
Application No. 62/153,143, filed Apr. 27, 2015, all of which are
hereby incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Nucleic acid vaccines (NAV) have been under development for
more than two decades. While significant advances have been made in
terms of the use of DNA vaccine strategies, much less progress has
been made with RNA vaccination strategies. Messenger RNA (mRNA)
vaccines have the potential to be developed quickly and may provide
a potent response. mRNA vaccines have the advantage of providing a
response when delivered to the cytoplasm, as compared to DNA
vaccines, which must be delivered to the nucleus.
[0004] However, mRNA vaccine development has been hampered due to
problems with mRNA stability, delivery and immunogenicity directed
against the mRNA itself via the innate immune system. While
optimization of RNA vaccines has proven somewhat effective in
recent years in an ex vivo setting, current methods of producing
mRNA vaccines provide poor antibody and CD8+ T-cell responses when
directly administered in vivo.
[0005] Thus, there is a need in the art for improved compositions
and methods of using RNA encoding an antigen for induction of an
adaptive immune response. The present invention satisfies this
unmet need.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention provides a composition
for inducing an adaptive immune response in a subject, where the
composition comprises at least one nucleoside-modified RNA encoding
at least one antigen. In one embodiment, the at least one isolated
nucleoside-modified RNA comprises pseudouridine. In one embodiment,
the at least one isolated nucleoside-modified RNA comprises
1-methyl-pseudouridine.
[0007] In one embodiment, the at least one antigen encoded by the
nucleoside-modified RNA is a viral antigen, a bacterial antigen, a
fungal antigen, a parasitic antigen, a tumor-associated antigen, or
a tumor-specific antigen. In one embodiment, the at least one
antigen comprises an HIV antigen. In one embodiment, the HIV
antigen comprises Envelope (Env). In one embodiment, the at least
one antigen comprises an influenza antigen. In one embodiment, the
influenza antigen comprises hemagglutinin (HA).
[0008] In one embodiment, the composition further comprises an
adjuvant. In one embodiment, the at least one nucleoside-modified
RNA further encodes at least one adjuvant. In one embodiment, the
composition is a vaccine.
[0009] In one embodiment, the composition further comprises a lipid
nanoparticle (LNP). In one embodiment, the at least one
nucleoside-modified RNA is encapsulated within the LNP. In one
embodiment, the LNP comprises a compound having a structure of
Formula (I):
##STR00001##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0010] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O-- or a carbon-carbon double bond;
[0011] R.sup.1a and R.sup.1b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0012] R.sup.2a and R.sup.2b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0013] R.sup.3a and R.sup.3b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0014] R.sup.4a and R.sup.4b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0015] R.sup.5 and R.sup.6 are each independently methyl or
cycloalkyl;
[0016] R.sup.7 is, at each occurrence, independently H or
C.sub.1-C.sub.12 alkyl;
[0017] R.sup.8 and R.sup.9 are each independently unsubstituted
C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the
nitrogen atom to which they are attached, form a 5, 6 or 7-membered
heterocyclic ring comprising one nitrogen atom;
[0018] a and d are each independently an integer from 0 to 24;
[0019] b and c are each independently an integer from 1 to 24;
and
[0020] e is 1 or 2.
[0021] In one embodiment, the LNP comprises a compound having a
structure of Formula (II):
##STR00002##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0022] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--,
--C(.dbd.O)S--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a,
--OC(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)O--, or a direct
bond;
[0023] G.sup.1 is C.sub.1-C.sub.2 alkylene, --(C.dbd.O)--,
--O(C.dbd.O)--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)-- or a direct
bond;
[0024] G.sup.2 is --C(.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)S--,
--C(.dbd.O)NR.sup.a or a direct bond;
[0025] G.sup.3 is C.sub.1-C.sub.6 alkylene;
[0026] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0027] R.sup.1a and R.sup.1b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0028] R.sup.2a and R.sup.2b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0029] R.sup.3a and R.sup.3b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0030] R.sup.4a and R.sup.4b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0031] R.sup.5 and R.sup.6 are each independently H or methyl;
[0032] R.sup.7 is C.sub.4-C.sub.20 alkyl;
[0033] R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12
alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to
which they are attached, form a 5, 6 or 7-membered heterocyclic
ring;
[0034] a, b, c and d are each independently an integer from 1 to
24; and
[0035] x is 0, 1 or 2.
[0036] In one embodiment, the LNP comprises a compound having a
structure of Formula (III):
##STR00003##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0037] one of L.sup.1 or L.sup.2 is --O(C.dbd.O)--, --(C.dbd.O)O--,
--C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--, --C(.dbd.O)S--,
SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O--, and the other of L.sup.1 or L.sup.2 is
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--,
--S(O).sub.x--, --S--S--, --C(.dbd.O)S--, SC(.dbd.O)--,
--NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O-- or a direct bond;
[0038] G.sup.1 and G.sup.2 are each independently unsubstituted
C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene;
[0039] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24
alkenylene, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8
cycloalkenylene;
[0040] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0041] R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24
alkyl or C.sub.6-C.sub.24 alkenyl;
[0042] R.sup.3 is H, OR.sup.5, CN, --C(.dbd.O)OR.sup.4,
--OC(.dbd.O)R.sup.4 or --NR.sup.5C(.dbd.O)R.sup.4;
[0043] R.sup.4 is C.sub.1-C.sub.12 alkyl;
[0044] R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and
[0045] x is 0, 1 or 2.
[0046] In one embodiment, the LNP comprises a compound having one
of the following structures:
##STR00004## ##STR00005##
[0047] In one embodiment, the LNP comprises a pegylated lipid
having the following structure (IV):
##STR00006##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein:
[0048] R.sup.10 and R.sup.11 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing from 10
to 30 carbon atoms, wherein the alkyl chain is optionally
interrupted by one or more ester bonds; and
[0049] z has a mean value ranging from 30 to 60.
[0050] In one embodiment, the pegylated lipid has the following
structure (IVa):
##STR00007##
[0051] wherein n is an integer selected such that the average
molecular weight of the pegylated lipid is about 2500 g/mol.
[0052] In one aspect, the present invention provides a method of
inducing an adaptive immune response in a subject. The method
comprises administering to the subject an effective amount of a
composition comprising at least one nucleoside-modified RNA
encoding at least one antigen. In one embodiment, the at least one
isolated nucleoside-modified RNA comprises pseudouridine. In one
embodiment, the at least one isolated nucleoside-modified RNA
comprises 1-methyl-pseudouridine.
[0053] In one embodiment, the at least one antigen encoded by the
nucleoside-modified RNA is a viral antigen, a bacterial antigen, a
fungal antigen, a parasitic antigen, a tumor-associated antigen, or
a tumor-specific antigen. In one embodiment, the at least one
antigen comprises an HIV antigen. In one embodiment, the HIV
antigen comprises Envelope (Env). In one embodiment, the at least
one antigen comprises an influenza antigen. In one embodiment, the
influenza antigen comprises hemagglutinin (HA).
[0054] In one embodiment, the composition further comprises an
adjuvant. In one embodiment, the at least one nucleoside-modified
RNA further encodes at least one adjuvant. In one embodiment, the
composition is a vaccine.
[0055] In one embodiment, the composition further comprises a lipid
nanoparticle (LNP). In one embodiment, the at least one
nucleoside-modified RNA is encapsulated within the LNP. In one
embodiment, the LNP comprises a compound having a structure of
Formula (I):
##STR00008##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0056] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O-- or a carbon-carbon double bond;
[0057] R.sup.1a and R.sup.1b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0058] R.sup.2a and R.sup.2b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0059] R.sup.3a and R.sup.3b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0060] R.sup.4a and R.sup.4b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0061] R.sup.5 and R.sup.6 are each independently methyl or
cycloalkyl;
[0062] R.sup.7 is, at each occurrence, independently H or
C.sub.1-C.sub.12 alkyl;
[0063] R.sup.8 and R.sup.9 are each independently unsubstituted
C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the
nitrogen atom to which they are attached, form a 5, 6 or 7-membered
heterocyclic ring comprising one nitrogen atom;
[0064] a and d are each independently an integer from 0 to 24;
[0065] b and c are each independently an integer from 1 to 24;
and
[0066] e is 1 or 2.
[0067] In one embodiment, the LNP comprises a compound having a
structure of Formula (II):
##STR00009##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0068] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--,
--C(.dbd.O)S--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a,
--OC(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)O--, or a direct
bond;
[0069] G.sup.1 is C.sub.1-C.sub.2 alkylene, --(C.dbd.O)--,
--O(C.dbd.O)--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)-- or a direct
bond;
[0070] G.sup.2 is --C(.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)S--,
--C(.dbd.O)NR.sup.a or a direct bond;
[0071] G.sup.3 is C.sub.1-C.sub.6 alkylene;
[0072] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0073] R.sup.1a and R.sup.1b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0074] R.sup.2a and R.sup.2b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0075] R.sup.3a and R.sup.3b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0076] R.sup.4a and R.sup.4b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0077] R.sup.5 and R.sup.6 are each independently H or methyl;
[0078] R.sup.7 is C.sub.4-C.sub.20 alkyl;
[0079] R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12
alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to
which they are attached, form a 5, 6 or 7-membered heterocyclic
ring;
[0080] a, b, c and d are each independently an integer from 1 to
24; and
[0081] x is 0, 1 or 2.
[0082] In one embodiment, the LNP comprises a compound having a
structure of Formula (III):
##STR00010##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0083] one of L.sup.1 or L.sup.2 is --O(C.dbd.O)--, --(C.dbd.O)O--,
--C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--, --C(.dbd.O)S--,
SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O--, and the other of L.sup.1 or L.sup.2 is
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--,
--S(O).sub.x--, --S--S--, --C(.dbd.O)S--, SC(.dbd.O)--,
--NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O-- or a direct bond;
[0084] G.sup.1 and G.sup.2 are each independently unsubstituted
C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene;
[0085] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24
alkenylene, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8
cycloalkenylene;
[0086] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0087] R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24
alkyl or C.sub.6-C.sub.24 alkenyl;
[0088] R.sup.3 is H, OR.sup.5, CN, --C(.dbd.O)OR.sup.4,
--OC(.dbd.O)R.sup.4 or --NR.sup.5C(.dbd.O)R.sup.4;
[0089] R.sup.4 is C.sub.1-C.sub.12 alkyl;
[0090] R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and
[0091] x is 0, 1 or 2.
[0092] In one embodiment, the LNP comprises a compound having one
of the following structures:
##STR00011## ##STR00012##
[0093] In one embodiment, the LNP comprises a pegylated lipid
having the following structure (IV):
##STR00013##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein:
[0094] R.sup.10 and R.sup.11 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing from 10
to 30 carbon atoms, wherein the alkyl chain is optionally
interrupted by one or more ester bonds; and
[0095] z has a mean value ranging from 30 to 60.
[0096] In one embodiment, the pegylated lipid has the following
structure (IVa):
##STR00014##
[0097] wherein n is an integer selected such that the average
molecular weight of the pegylated lipid is about 2500 g/mol.
[0098] In one embodiment, the composition is administered by
intradermal, subcutaneous, or intramuscular delivery. In one
embodiment, the method comprises a single administration of the
composition. In one embodiment, the method comprises a multiple
administrations of the composition.
[0099] In one embodiment, the method treats or prevents at least
one selected from the group consisting of a viral infection, a
bacterial infections, a fungal infection, a parasitic infection,
and cancer. In one embodiment, the method treats or prevents HIV
infection. In one embodiment, the method treats or prevents
influenza infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0100] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0101] FIG. 1 is a schematic illustrating the experimental setup
for ENV-LNP immunization that applies to FIG. 2-FIG. 11. Animals
received two intradermal injections of either 3 .mu.g, 10 .mu.g or
30 .mu.g of HIV-1 CD4-independent R3A envelope encoding mRNA
encapsulated into lipid nanoparticles (LNP). Control mice were
injected with 30 .mu.g firefly luciferase (LUC) encoding mRNA
complexed into LNP. There was a 4-week interval between mRNA-LNP
injections and animals were sacrificed 14 days after the second
injection.
[0102] FIG. 2 is a set of graphs illustrating that two
immunizations with ENV-LNPs elicit robust CD4+ T cell responses.
The graphs depict IFN-.gamma. (left) and TNF-.alpha. (right),
production by antigen specific CD4+ T cells. Cytokine production of
individual animals is displayed as the percent of total CD4+ T
cells in the spleen. IFN=interferon, TNF=tumor necrosis factor.
Luc=control mice injected with 30 .mu.g of control luciferase
encoding mRNA-LNPs injected intradermally (ID). All intracellular
cytokine measurements were performed using multicolor flow
cytometry after stimulation with peptide pools of 15-mers
overlapping by 11 amino acids of the complete envelope sequence.
Standard error of the mean is indicated.
[0103] FIG. 3 is a graph illustrating that two immunizations with
ENV-LNPs elicit robust CD4+ T cell responses. The graphs depict
IL-2 production by antigen specific CD4+ T cells. Cytokine
production of individual animals is displayed as the percent of
total CD4+ T cells in the spleen. IL-2=interleukin 2. Luc=control
mice injected with 30 .mu.g of control luciferase encoding
mRNA-LNPs injected intradermally (ID). All intracellular cytokine
measurements were performed using multicolor flow cytometry after
stimulation with peptide pools of 15-mers overlapping by 11 amino
acids of the complete envelope sequence. Standard error of the mean
is indicated.
[0104] FIG. 4 is a graph illustrating that two immunizations with
ENV-LNPs elicit robust multifunctional CD4+ T cell responses. The
graphs depict the distribution of mono, - bi,- and trifunctional
antigen specific CD4+ T cells in vaccinated animals 14 days after
the second intradermal immunization. The bar graph shows the
percentage of antigen specific CD4+ T cells producing one, two or
three cytokines, as indicated. All intracellular cytokine
measurements were performed using multicolor flow cytometry after
stimulation with peptide pools of 15-mers overlapping by 11 amino
acids of the complete envelope sequence. Standard error of the mean
is indicated on bars.
[0105] FIG. 5 is a graph illustrating that two immunizations with
ENV-LNP results in a significant increase in total T follicular
helper (Tfh) cell numbers. The graph depicts the frequency of
splenic Tfh cells in vaccinated animals. CD4, CXCR5 and PD-1
markers were used to determine Tfh cells. E10=10 .mu.g of iR3A
envelope encoding mRNA injected ID. E30=30 .mu.g of iR3A envelope
encoding mRNA injected ID. Luc=30 .mu.g of control luciferase
encoding mRNA injected ID. Naive: uninjected animal.
[0106] FIG. 6 is a set of graphs illustrating that two intradermal
immunizations with ENV-LNPs elicits robust CD8+ T cell responses.
The graphs depict IFN-7 (left) and TNF-.alpha. (right) production
by antigen specific CD8+ T cells. Cytokine production of individual
animals is displayed. E10=10 .mu.g of iR3A envelope encoding mRNA
injected ID. E30=30 .mu.g of iR3A envelope encoding mRNA injected
ID. Luc=30 .mu.g of control luciferase encoding mRNA injected ID.
All intracellular cytokine measurements were performed using
multicolor flow cytometry after stimulation with peptide pools of
15-mers overlapping by 11 amino acids of the complete envelope
sequence. Standard error of the mean is indicated.
[0107] FIG. 7 is a set of graphs illustrating that two intradermal
immunizations with ENV-LNPs elicit robust CD8+ T cell responses.
The graphs depict IL-2 (left) and CD107a (right) production of
antigen specific CD8+ T cells. TL-2 and CD107a production of
individual animals is displayed. E10=10 .mu.g of iR3A envelope
encoding mRNA injected ID. E30=30 .mu.g of iR3A envelope encoding
mRNA injected ID. Luc=30 .mu.g of control luciferase encoding mRNA
injected ID. All intracellular cytokine measurements were performed
using multicolor flow cytometry after stimulation with peptide
pools of 15-mers overlapping by 11 amino acids of the complete
envelope sequence. Standard error of the mean is indicated.
[0108] FIG. 8 is a set of graphs illustrating that two intradermal
immunizations with ENV-LNPs elicit robust multifunctional CD8+ T
cell responses. The graphs depict the distribution of mono-, bi-,
and trifunctional antigen specific CD8+ T cells in vaccinated
animals. Pie charts show the distribution of antigen specific CD8+
T cells producing one, two or three cytokines. The bar graph shows
the frequency of antigen specific CD8+ T cells producing one, two
or three cytokines. ENV10=10 .mu.g of iR3A envelope encoding mRNA
injected ID. ENV30=30 .mu.g of iR3A envelope encoding mRNA injected
ID. Luc=30 .mu.g of control luciferase encoding mRNA injected ID.
All intracellular cytokine measurements were performed using
multicolor flow cytometry after stimulation with peptide pools of
15-mers overlapping by 11 amino acids of the complete envelope
sequence. Standard error of the mean is indicated on bars. G=INF-7,
T=TNF-.alpha., 107=CD107a.
[0109] FIG. 9A and FIG. 9B depict a set of graphs illustrating that
immunization with ENV-LNPs elicit robust B cell responses. FIG. 9A
depicts antigen-specific antibody responses as measured by ELISA
assays. Experiments were conducted to measure HIV-1 gp120 specific
IgG titers after two intradermal injections of mRNA-LNPs. Titers
were measured by a gp120 specific ELISA assay where gp120 coated
the plate and gp120-specific IgG was measured with a peroxidase
labeled goat andi-mouse IgG. Standard error of the mean is
indicated on bars. FIG. 9B depicts a set of graphs demonstrating
that similar amounts of Env-specific IgG1 and IgG2 are produced two
weeks after two immunizations with mRNA-LNP.
[0110] FIG. 10 is a graph depicting the results of example
experiments. Mice were immunized 2 times with 30 .mu.g of LNP
complexed 1-methyl-pseudouridine-mRNA encoding luciferase (luc), or
10 or 30 .mu.g of 1-methyl-pseudouridine modified mRNA encoding HIV
envelope iR3A complexed by the intradermal route at 1 month
intervals. Serum was analyzed for the ability to neutralize HIV
infection by the tier 1 MN.3 strain and the control MLV. Serum was
sequentially diluted and the dilution for 50% inhibition is shown.
Each symbol represents an individual mouse.
[0111] FIG. 11 is a graph depicting the results of example
experiments. Mice were immunized 2 times with 10 or 30 .mu.g of
1-methyl-pseudouridine modified mRNA encoding HIV envelope iR3A
complexed to LNPs by the intradermal route at 1 month intervals.
Serum was analyzed for the ability to neutralize HIV infection by
the tier 2 X2278_C2_B6 strain and the control MLV. Serum was
sequentially diluted and the dilution for 50% inhibition is
shown.
[0112] FIG. 12 is a schematic illustrating the experimental setup
for ENV mRNA-LNP immunization. Animals received a single
intradermal injection of 30 .mu.g HIV-1 CD4-independent R3A
envelope encoding mRNA encapsulated into lipid nanoparticles (ENV).
Control mice were injected with 30 .mu.g firefly luciferase
encoding mRNA complexed into LNP. Animals were sacrificed 14 days
after mRNA administration.
[0113] FIG. 13 is a set of graphs illustrating that a single
injection with 30 .mu.g ENV mRNA-LNPs elicits robust CD4+ T cell
responses. The graphs depict IFN-7 (left) and TNF-.alpha. (right)
production of antigen specific CD4+ T cells. Cytokine production of
individual animals is displayed. ENV=30 .mu.g of iR3A envelope
encoding mRNA injected ID. Luc=30 ug of control luciferase encoding
mRNA injected ID. All intracellular cytokine measurements were
performed using multicolor flow cytometry after stimulation with
peptide pools of 15-mers overlapping by 11 amino acids of the
complete envelope sequence. The percent of total spleen cells
expressing cytokine after peptide stimulation is expressed.
Standard error of the mean is indicated.
[0114] FIG. 14 is a set of graphs illustrating that a single
injection with 30 .mu.g ENV mRNA-LNPs elicits robust CD4+ T cell
responses. The graphs depict IL-2 (left) and CD107a production
(right) of antigen specific CD4+ T cells. IL-2 and CD107a
production of individual animals is displayed. ENV=30 .mu.g of iR3A
envelope encoding mRNA injected ID. Luc=30 .mu.g of control
luciferase encoding mRNA injected ID. All intracellular cytokine
measurements were performed using multicolor flow cytometry after
stimulation with peptide pools of 15-mers overlapping by 11 amino
acids of the complete envelope sequence. Standard error of the mean
is indicated.
[0115] FIG. 15 is a set of graphs illustrating that a single
injection with 30 .mu.g ENV mRNA-LNPs elicit robust polyfunctional
CD4+ T cell responses. The graphs depict the distribution of mono-,
bi- and trifunctional antigen specific CD4+ T cells in vaccinated
animals. Pie charts show the distribution of antigen specific CD4+
T cells producing one, two or three cytokines. The bar graph shows
the frequency of antigen specific CD4+ T cells producing one, two
or three cytokines. ENV=30 .mu.g of iR3A envelope encoding mRNA
injected ID. Luc=30 .mu.g of control luciferase encoding mRNA
injected ID. All intracellular cytokine measurements were performed
using multicolor flow cytometry after stimulation with peptide
pools of 15-mers overlapping by 11 amino acids of the complete
envelope sequence. Standard error of the mean is indicated on bars.
G=INF-7, T=TNF-.alpha., 2=IL-2
[0116] FIG. 16 is a graph illustrating that a single injection with
30 .mu.g ENV mRNA-LNPs results in a significant increase in total
Tfh cell numbers in the spleen. The graph depicts the frequency of
Tfh cells in vaccinated animals. CD4, CXCR5 and PD-1 markers were
used to determine Tfh cells. ENV=30 .mu.g of iR3A envelope encoding
mRNA injected ID. Luc=30 .mu.g of control luciferase encoding mRNA
injected ID. Standard error of the mean is indicated on the bars.
Naive: uninjected animals
[0117] FIG. 17 is a set of graphs illustrating that a single
injection with 30 .mu.g ENV-LNPs elicit robust antigen specific Tfh
cell immune responses. The graphs depict IFN-.gamma. (top left),
TNF-.alpha. (top right), and IL-2 (bottom) production of antigen
specific Tfh CD4+ T cells. Tfh cells were identified by expression
of nuclear Bcl6. Cytokine production of individual animals is
displayed. ENV=30 .mu.g of iR3A envelope encoding mRNA injected ID.
Luc=30 .mu.g of control luciferase encoding mRNA injected ID. All
intracellular cytokine measurements were performed using multicolor
flow cytometry after stimulation with peptide pools of 15-mers
overlapping by 11 amino acids of the complete envelope sequence.
Standard error of the mean is indicated.
[0118] FIG. 18 is a set of graphs illustrating that a single
injection with 30 .mu.g ENV-LNPs elicit robust polyfunctional Tfh
cell immune responses. The graphs depict the distribution of mono-,
bi,- and trifunctional antigen specific Tfh cells in vaccinated
animals. Pie charts show the distribution of antigen specific Tfh
cells producing one, two or three cytokines. Tfh cells were
identified by expression of nuclear Bcl6. The bar graph shows the
frequency of antigen specific Tfh cells producing one, two or three
cytokines. ENV=30 .mu.g of iR3A envelope encoding mRNA injected ID.
Luc=30 .mu.g of control luciferase encoding mRNA injected ID. All
intracellular cytokine measurements were performed using multicolor
flow cytometry after stimulation with peptide pools of 15-mers
overlapping by 11 amino acids of the complete envelope sequence.
Standard error of the mean is indicated on bars. G=INF-.gamma.,
T=TNF-.alpha., 2=IL-2.
[0119] FIG. 19 is a graph illustrating that a single injection with
30 .mu.g ENV mRNA-LNPs elicits IgG producing B cell responses. The
graph depicts antigen specific antibody responses measured by ELISA
assays. Experiments were conducted to measure HIV-1 gp120 specific
IgG titers after a single injection with mRNA-LNPs. ENV=30 .mu.g of
iR3A envelope encoding mRNA injected ID. Luc=30 .mu.g of control
luciferase encoding mRNA injected ID. Naive=uninjected animals.
Standard error of the mean is indicated on bars.
[0120] FIG. 20 is a graph depicting the results of example
experiments demonstrating the benefits of nucleoside modification
and LNP complexing. Mice were immunized 2 times with 10 .mu.g of
unmodified, 1-methyl-pseudouridine, or 1-methyl-pseudouridine-LNP
complexed mRNA encoding iR3A HIV envelope by the intradermal route
at 1 month intervals. Spleen cells obtained 14 days after the
second immunization were analyzed by a 6 hour stimulation with
envelope overlapping peptides and analyzed for expression of CD107A
or intracellular IFN-7, TNF-.alpha., and IL-2 by CD3+, CD8+ T
cells. Control (medium)stimulated responses were subtracted for
each mouse. Groups of 6 mice were averaged. Modified mRNA-LNP
responses were significantly greater (p<0.01) than uncomplexed
modified or unmodified mRNA or control (luciferase modified mRNA)
treated mice.
[0121] FIG. 21 is a graph depicting the results of example
experiments that demonstrate the benefits of nucleoside
modification and LNP complexing. Mice were immunized 2 times with
10 .mu.g of unmodified mRNA, 1-methyl-pseudouridine mRNA, or
1-methyl-pseudouridine-mRNA-LNP complexed all encoding iR3A HIV
envelope by the intradermal route at 1 month intervals. Spleen
cells were analyzed by a 6 hour stimulation with envelope
overlapping peptides and analyzed for expression of intracellular
IFN-7, TNF-.alpha., and IL-2 by CD3+, CD4+ T cells. Control
(medium) stimulated responses were subtracted for each mouse.
Groups of 6 mice were averaged. Modified mRNA-LNP responses were
significantly greater (p<0.01) than uncomplexed modified or
unmodified mRNA or control (luciferase modified mRNA) treated
mice.
[0122] FIG. 22 is a graph depicting the results of example
experiments that demonstrate the benefits of nucleoside
modification and LNP complexing. Mice were immunized 2 times with
10 .mu.g of uncomplexed 1-methyl-pseudouridine modified mRNA
encoding HIV envelope iR3A (naked iR3A), 1-methyl-pseudouridine
mRNA-LNPs encoding luciferase (luc-LNP), or iR3A mRNA complexed
LNPs by the intradermal route at 1 month intervals. Serum was
analyzed for envelope (gp120) specific responses by ELISA. Serum
was diluted 1:1000 and analyzed. A monoclonal antibody specific for
gp120 was used to determine concentration in serum.
[0123] FIG. 23 is a graph depicting the results of example
experiments measuring CD4+ T cell responses, as measured by
IFN-.gamma. (left), TNF-.alpha. (center), and IL-2 (right) positive
CD4+ T cells detected 10 days after a single administration of 30
.mu.g of PR8 HA encoding mRNA-LNP. All intracellular cytokine
measurements were performed using multicolor flow cytometry after
stimulation with peptide pools of 15-mers overlapping by 11 amino
acids of the complete hemagglutinin sequence. Standard error of the
mean is indicated on bars.
[0124] FIG. 24 is a set of graphs depicting the results of example
experiments examining polyfunctional CD4+ T cell responses after
single immunization of PR8 HA encoding mRNA-LNP. Pie charts show
the distribution of antigen specific CD4+ T cells producing one,
two or three cytokines. The bar graph shows the ratio of antigen
specific CD4+ T cells producing one, two or three cytokines.
G=INF-.gamma., T=TNF-.alpha., 2=IL-2. All intracellular cytokine
measurements were performed using multicolor flow cytometry after
stimulation with peptide pools of 15-mers overlapping by 11 amino
acids of the complete hemagglutinin sequence. Standard error of the
mean is indicated on bars.
[0125] FIG. 25 is a graph depicting the results of example
experiments measuring CD8+ T cell responses, as measured by
IFN-.gamma. (left) and TNF-.alpha. (right) positive CD8+ T cells
detected 14 days after a single administration of 30 .mu.g of PR8
HA encoding mRNA-LNP. All intracellular cytokine measurements were
performed using multicolor flow cytometry after stimulation with
peptide pools of 15-mers overlapping by 11 amino acids of the
complete hemagglutinin sequence. Standard error of the mean is
indicated on bars.
[0126] FIG. 26 is a set of graphs depicting the results of example
experiments depicting HI titer 14 days and 28 days after
administration of either 10 .mu.g or 30 .mu.g of PR8 HA mRNA-LNP.
Titers were measured by the standard hemaglutinin inhibition assay,
where turkey red blood cells were coated with PR8 hemagglutinin.
Serum at 2-fold increasing dilutions was added to the RBCs and the
titer where hemagglutination was lost was measured.
[0127] FIG. 27 is a set of graphs depicting the results of example
experiments demonstrating that a single administration of PR8 HA
encoding mRNA-LNP results in increased germinal center (GC) B
cells. GC B cells were defined as IgD.sup.-, B220.sup.+,
CD138.sup.-, CD19.sup.+, IgM.sup.-], CD3- and CD14-. The total
number of cells in the spleen was calculated by counting the number
of spleen cells and multiplying that by the % GC B cells.
[0128] FIG. 28 is a set of graphs depicting the results of example
experiments demonstrating that a single administration of PR8 HA
encoding mRNA-LNP results in increased total memory B cells in the
spleen. Memory B cells were defined as CD3-, CD14-, CD11c+, T-bet+.
The total number of cells in the spleen was calculated by counting
the number of spleen cells and multiplying that by the % memory B
cells.
[0129] FIG. 29 is a set of graphs depicting the results of example
experiments demonstrating that a single administration of PR8 HA
encoding mRNA-LNP results in increased total Tfh cells in the
spleen. Tfh cells were defined as CD4+, memory+, CXCR5+, PD-1+
cells. The total number of cells in the spleen was calculated by
counting the number of spleen cells and multiplying that by the %
Tfh cells.
[0130] FIG. 30 is a graph depicting the results of example
experiments measuring CD8+ Tfh cell responses, as measured by
IFN-.gamma. (left) and IL-2 (right) positive CD8+ T cells detected
10 days after a single administration of 30 .mu.g of PR8 HA
encoding mRNA-LNPs. Tfh cells were identified by expression of
Bcl6.
[0131] FIG. 31 is a graph depicting the results of example
experiments demonstrating the relative amount of cytokine
expression in Tfh cells compared to total T cells purified from the
spleens of mice immunized with PR8 HA encoding mRNA-LNP. All T
cells were selected by negative selection. Tfh cells were further
purified by flow cytometric sorting selecting memory+, CXCR5+,
PD-1+ cells. mRNA was isolated from the T cell populations and
analyzed by real time PCR using specific primers. Values are
expressed as compared to universal mRNA.
[0132] FIG. 32 is a graph depicting the results of example
experiments demonstrating that T follicular regulatory cells are
not increased by administration of modified mRNA-LNP. Tfh cells
were identified as memory+, CXCR5+, PD-1+, Bcl6+ and T follicular
regulatory cells were identified as memory+, CXCR5+, PD-1+, Bcl6+,
FoxP3+. Data is expressed as the percentage of Tfh cells that were
T follicular regulatory cells.
[0133] FIG. 33 is a graph depicting the results of example
experiments demonstrating the weight loss as a measure of illness
after influenza challenge in HA mRNA-LNP or control single
immunized mice.
[0134] FIG. 34 is a set of graphs depicting the results of example
experiments demonstrating HA binding at 2 weeks (center) and 4
weeks (right) to hemagglutinin where both the head and stalk are
derived from H1.
[0135] FIG. 35 is a set of graphs depicting the results of example
experiments demonstrating specific binding to the stalk region of
hemagglutinin. HA binding at 2 weeks (center) and 4 weeks (right)
to hybrid hemagglutinin containing H5-head/H1-stalk HA (top) and
H5-head/H3-stalk (bottom).
[0136] FIG. 36 is a set of graphs depicting the results of example
experiments examining binding to whole HA (left) and HA stalk
(right) binding over time. It is demonstrated that stalk binding
increases over time post immunization.
[0137] FIG. 37 is a graph depicting the results of experiments
demonstrating that the neutralization titer as measured by
hemagglutinin inhibition after a single administration of PR8 HA
encoding modified mRNA-LNP remains unchanged 6 months after
administration.
[0138] FIG. 38 is a graph depicting the results of experiments
depicting HA inhibition titer measured 2 weeks after single
administration of Cal/7/2009 HA encoding mRNA-LNPs.
[0139] FIG. 39 is a set of graphs depicting the results of example
experiments measuring CD4+ T cell responses, as measured by
IFN-.gamma. (top left), TNF-.alpha. (top right), and IL-2 (bottom)
positive CD4+ T cells detected 2 weeks after a single
administration of CA09 HA encoding mRNA-LNP. Cytokine production of
individual animals is displayed as the percent of total CD4+ T
cells in the spleen. Poly(C)=control mice injected with 30 .mu.g of
control poly(C) mRNA-LNPs injected intradermally (ID). All
intracellular cytokine measurements were performed using multicolor
flow cytometry after stimulation with peptide pools of 15-mers
overlapping by 11 amino acids of the complete HA sequence. Standard
error of the mean is indicated.
[0140] FIG. 40 is a graph depicting the results of example
experiments measuring Tfh cell response 2 weeks after single
administration of CA09 HA encoding mRNA-LNP. Tfh cells were defined
as CD4+, memory+, CXCR5+, PD-1+ cells. Error bars are standard
error of the mean.
[0141] FIG. 41 is a set of graphs depicting the results of example
experiments measuring HA inhibition titers after single
administration of CA09 HA encoding mRNA administered by
intramuscular injection (left) and intradermal injection
(right).
[0142] FIG. 42 is a graph depicting the results of example
experiments demonstrating INF-.alpha. production induced by codon
optimized unmodified HA mRNA with none by m1.psi. modified mRNA,
demonstrating that unmodified codon optimized HA encoding mRNA
induces an innate immune response.
[0143] FIG. 43 is a set of graphs depicting the results of example
experiments demonstrating that intravenous injection of HPLC
purified nucleoside modified mRNA-LNP does not induce
proinflammatory cytokines or type I interferons.
[0144] FIG. 44 is a set of graphs depicting the results of example
experiments demonstrating that administration of nucleoside
modified HA encoding mRNA induces significantly better CD4+ T cell
response, as measured by IFN-.gamma. (left), TNF-.alpha. (center),
and IL-2 (right) production, as compared to unmodified HA encoding
mRNA.
[0145] FIG. 45 is a graph depicting the results of example
experiments demonstrating that administration of nucleoside
modified HA encoding mRNA results in increased numbers of Tfh cells
in the spleen, as compared to unmodified HA-encoding mRNA.
[0146] FIG. 46 is a graph depicting the results of example
experiments demonstrating that administration of nucleoside
modified HA encoding mRNA results in increased frequencies of
antigen-specific Tfh cells response, as compared to unmodified
HA-encoding mRNA.
[0147] FIG. 47 is a graph depicting the results of example
experiments demonstrating that administration of nucleoside
modified HA encoding mRNA results in increased HA inhibition titers
measured 10 days after a single administration of unmodified codon
optimized or m1.psi., as compared to unmodified HA-encoding
mRNA.
[0148] FIG. 48 is a set of graphs depicting the results of example
experiments examining mRNA translation of luciferase encoding
m1.psi. modified mRNA administered as complexed in LNP (left) and
naked (right). Translation was measured by injecting luciferase
encoding m1.psi.-mRNA and then 4 hours later, administering
D-luciferin, and imaging on an IVIS spectrum. Activity was
quantitated by selecting regions of increased signal and using IVIS
software.
[0149] FIG. 49 depicts the results of example experiments which
compare different LNP formulations intradermally injected into
mice.
DETAILED DESCRIPTION
[0150] The present invention relates to compositions and methods
for inducing an adaptive immune response in a subject. In certain
embodiments, the invention provides a composition comprising at
least one nucleoside-modified RNA encoding at least one antigen,
adjuvant, or a combination thereof. For example, in one embodiment,
the composition is a vaccine comprising at least one
nucleoside-modified RNA encoding at least one antigen, adjuvant, or
a combination thereof, where the vaccine induces immunity in the
subject to the at least one antigen, and therefore induces immunity
in the subject to a pathogen or pathology associated with the at
least one antigen. In certain embodiments, the at least one
nucleoside-modified RNA is encapsulated in a lipid nanoparticle
(LNP).
Definitions
[0151] Unless defined otherwise, 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. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0152] As used herein, each of the following terms has the meaning
associated with it in this section.
[0153] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0154] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of 20% or +10%, more preferably +5%, even more
preferably +1%, and still more preferably +0.1% from the specified
value, as such variations are appropriate to perform the disclosed
methods.
[0155] The term "antibody," as used herein, refers to an
immunoglobulin molecule, which specifically binds with an antigen.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive
portions of intact immunoglobulins. Antibodies are typically
tetramers of immunoglobulin molecules. The antibodies in the
present invention may exist in a variety of forms including, for
example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and
F(ab).sub.2, as well as single chain antibodies and humanized
antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al.,
1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,
N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0156] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, scFv antibodies, and multispecific
antibodies formed from antibody fragments.
[0157] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
[0158] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations. K
and k light chains refer to the two major antibody light chain
isotypes.
[0159] By the term "synthetic antibody" as used herein, is meant an
antibody, which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage. The term
should also be construed to mean an antibody which has been
generated by the synthesis of a DNA molecule encoding the antibody
and which DNA molecule expresses an antibody protein, or an amino
acid sequence specifying the antibody, wherein the DNA or amino
acid sequence has been obtained using synthetic DNA or amino acid
sequence technology which is available and well known in the art.
The term should also be construed to mean an antibody, which has
been generated by the synthesis of an RNA molecule encoding the
antibody. The RNA molecule expresses an antibody protein, or an
amino acid sequence specifying the antibody, wherein the RNA has
been obtained by transcribing DNA (synthetic or cloned) or other
technology, which is available and well known in the art.
[0160] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an adaptive immune response. This immune
response may involve either antibody production, or the activation
of specific immunogenically-competent cells, or both. The skilled
artisan will understand that any macromolecule, including virtually
all proteins or peptides, can serve as an antigen. Furthermore,
antigens can be derived from recombinant or genomic DNA or RNA. A
skilled artisan will understand that any DNA or RNA, which
comprises a nucleotide sequences or a partial nucleotide sequence
encoding a protein that elicits an adaptive immune response
therefore encodes an "antigen" as that term is used herein.
Furthermore, one skilled in the art will understand that an antigen
need not be encoded solely by a full length nucleotide sequence of
a gene. It is readily apparent that the present invention includes,
but is not limited to, the use of partial nucleotide sequences of
more than one gene and that these nucleotide sequences are arranged
in various combinations to elicit the desired immune response.
Moreover, a skilled artisan will understand that an antigen need
not be encoded by a "gene" at all. It is readily apparent that an
antigen can be generated synthesized or can be derived from a
biological sample. Such a biological sample can include, but is not
limited to a tissue sample, a tumor sample, a cell or a biological
fluid.
[0161] The term "adjuvant" as used herein is defined as any
molecule to enhance an antigen-specific adaptive immune
response.
[0162] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0163] An "effective amount" as used herein, means an amount which
provides a therapeutic or prophylactic benefit.
[0164] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0165] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) RNA, and viruses (e.g.,
lentiviruses, retroviruses, adenoviruses, and adeno-associated
viruses) that incorporate the recombinant polynucleotide.
[0166] "Homologous" refers to the sequence similarity or sequence
identity between two polypeptides or between two nucleic acid
molecules. When a position in both of the two compared sequences is
occupied by the same base or amino acid monomer subunit, e.g., if a
position in each of two DNA molecules is occupied by adenine, then
the molecules are homologous at that position. The percent of
homology between two sequences is a function of the number of
matching or homologous positions shared by the two sequences
divided by the number of positions compared.times.100. For example,
if 6 of 10 of the positions in two sequences are matched or
homologous then the two sequences are 60% homologous. By way of
example, the DNA sequences ATTGCC and TATGGC share 50% homology.
Generally, a comparison is made when two sequences are aligned to
give maximum homology.
[0167] "Immunogen" refers to any substance introduced into the body
in order to generate an immune response. That substance can a
physical molecule, such as a protein, or can be encoded by a
vector, such as DNA, mRNA, or a virus.
[0168] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
peptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0169] In the context of the present invention, the following
abbreviations for the commonly occurring nucleosides (nucleobase
bound to ribose or deoxyribose sugar via N-glycosidic linkage) are
used. "A" refers to adenosine, "C" refers to cytidine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0170] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0171] By the term "modulating," as used herein, is meant mediating
a detectable increase or decrease in the level of a response in a
subject compared with the level of a response in the subject in the
absence of a treatment or compound, and/or compared with the level
of a response in an otherwise identical but untreated subject. The
term encompasses perturbing and/or affecting a native signal or
response thereby mediating a beneficial therapeutic response in a
subject, preferably, a human.
[0172] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. Nucleotide sequences that encode proteins and RNA
may include introns. In addition, the nucleotide sequence may
contain modified nucleosides that are capable of being translation
by translational machinery in a cell. For example, an mRNA where
all of the uridines have been replaced with pseudouridine, 1-methyl
psuedouridien, or another modified nucleoside.
[0173] The term "operably linked" refers to functional linkage
between a regulatory sequence and a heterologous nucleic acid
sequence resulting in expression of the latter. For example, a
first nucleic acid sequence is operably linked with a second
nucleic acid sequence when the first nucleic acid sequence is
placed in a functional relationship with the second nucleic acid
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence. Generally, operably linked DNA or RNA
sequences are contiguous and, where necessary to join two protein
coding regions, in the same reading frame.
[0174] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0175] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR.TM., and the
like, and by synthetic means.
[0176] In certain instances, the polynucleotide or nucleic acid of
the invention is a "nucleoside-modified nucleic acid," which refers
to a nucleic acid comprising at least one modified nucleoside. A
"modified nucleoside" refers to a nucleoside with a modification.
For example, over one hundred different nucleoside modifications
have been identified in RNA (Rozenski, et al., 1999, The RNA
Modification Database: 1999 update. Nucl Acids Res 27:
196-197).
[0177] In certain embodiments, "pseudouridine" refers, in another
embodiment, to m.sup.1acp.sup.3.PSI.
(1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another
embodiment, the term refers to m.sup.1.PSI.
(1-methylpseudouridine). In another embodiment, the term refers to
.PSI.m (2'-O-methylpseudouridine. In another embodiment, the term
refers to m.sup.5D (5-methyldihydrouridine). In another embodiment,
the term refers to m.sup.3.PSI. (3-methylpseudouridine). In another
embodiment, the term refers to a pseudouridine moiety that is not
further modified. In another embodiment, the term refers to a
monophosphate, diphosphate, or triphosphate of any of the above
pseudouridines. In another embodiment, the term refers to any other
pseudouridine known in the art. Each possibility represents a
separate embodiment of the present invention.
[0178] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0179] The term "promoter" as used herein is defined as a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a polynucleotide sequence. For example, the
promoter that is recognized by bacteriophage RNA polymerase and is
used to generate the mRNA by in vitro transcription.
[0180] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more other species. But, such
cross-species reactivity does not itself alter the classification
of an antibody as specific. In another example, an antibody that
specifically binds to an antigen may also bind to different allelic
forms of the antigen. However, such cross reactivity does not
itself alter the classification of an antibody as specific. In some
instances, the terms "specific binding" or "specifically binding,"
can be used in reference to the interaction of an antibody, a
protein, or a peptide with a second chemical species, to mean that
the interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0181] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, diminution, remission, or eradication of at least one
sign or symptom of a disease or disorder state.
[0182] The term "therapeutically effective amount" refers to the
amount of the subject compound that will elicit the biological or
medical response of a tissue, system, or subject that is being
sought by the researcher, veterinarian, medical doctor or other
clinician. The term "therapeutically effective amount" includes
that amount of a compound that, when administered, is sufficient to
prevent development of, or alleviate to some extent, one or more of
the signs or symptoms of the disorder or disease being treated. The
therapeutically effective amount will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the subject to be treated.
[0183] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject.
[0184] The term "transfected" or "transformed" or "transduced" as
used herein refers to a process by which exogenous nucleic acid is
transferred or introduced into the host cell. A "transfected" or
"transformed" or "transduced" cell is one which has been
transfected, transformed or transduced with exogenous nucleic acid.
The cell includes the primary subject cell and its progeny.
[0185] The phrase "under transcriptional control" or "operatively
linked" as used herein means that the promoter is in the correct
location and orientation in relation to a polynucleotide to control
the initiation of transcription by RNA polymerase and expression of
the polynucleotide.
[0186] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the
like.
[0187] "Alkyl" refers to a straight or branched hydrocarbon chain
radical consisting solely of carbon and hydrogen atoms, which is
saturated or unsaturated (i.e., contains one or more double and/or
triple bonds), having from one to twenty-four carbon atoms
(C.sub.1-C.sub.24 alkyl), one to twelve carbon atoms
(C.sub.1-C.sub.12 alkyl), one to eight carbon atoms
(C.sub.1-C.sub.8 alkyl) or one to six carbon atoms (C.sub.1-C.sub.6
alkyl) and which is attached to the rest of the molecule by a
single bond, e.g., methyl, ethyl, n propyl, 1-methylethyl (iso
propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3
methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-1-enyl,
pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl,
pentynyl, hexynyl, and the like. Unless specifically stated
otherwise, an alkyl group is optionally substituted.
[0188] "Alkylene" or "alkylene chain" refers to a straight or
branched divalent hydrocarbon chain linking the rest of the
molecule to a radical group, consisting solely of carbon and
hydrogen, which is saturated or unsaturated (i.e., contains one or
more double (alkenylene) and/or triple bonds (alkynylene)), and
having, for example, from one to twenty-four carbon atoms
(C.sub.1-C.sub.24 alkylene), one to fifteen carbon atoms
(C.sub.1-C.sub.15 alkylene), one to twelve carbon atoms
(C.sub.1-C.sub.12 alkylene), one to eight carbon atoms
(C.sub.1-C.sub.8 alkylene), one to six carbon atoms
(C.sub.1-C.sub.6 alkylene), two to four carbon atoms
(C.sub.2-C.sub.4 alkylene), one to two carbon atoms
(C.sub.1-C.sub.2 alkylene), e.g., methylene, ethylene, propylene,
n-butylene, ethenylene, propenylene, n-butenylene, propynylene,
n-butynylene, and the like. The alkylene chain is attached to the
rest of the molecule through a single or double bond and to the
radical group through a single or double bond. The points of
attachment of the alkylene chain to the rest of the molecule and to
the radical group can be through one carbon or any two carbons
within the chain. Unless stated otherwise specifically in the
specification, an alkylene chain may be optionally substituted.
[0189] "Cycloalkyl" or "carbocyclic ring" refers to a stable non
aromatic monocyclic or polycyclic hydrocarbon radical consisting
solely of carbon and hydrogen atoms, which may include fused or
bridged ring systems, having from three to fifteen carbon atoms,
preferably having from three to ten carbon atoms, and which is
saturated or unsaturated and attached to the rest of the molecule
by a single bond. Monocyclic radicals include, for example,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl. Polycyclic radicals include, for example, adamantyl,
norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the
like. Unless specifically stated otherwise, a cycloalkyl group is
optionally substituted.
[0190] "Cycloalkylene" is a divalent cycloalkyl group. Unless
otherwise stated specifically in the specification, a cycloalkylene
group may be optionally substituted.
[0191] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3-
to 18-membered non-aromatic ring radical which consists of two to
twelve carbon atoms and from one to six heteroatoms selected from
the group consisting of nitrogen, oxygen and sulfur. Unless stated
otherwise specifically in the specification, the heterocyclyl
radical may be a monocyclic, bicyclic, tricyclic or tetracyclic
ring system, which may include fused or bridged ring systems; and
the nitrogen, carbon or sulfur atoms in the heterocyclyl radical
may be optionally oxidized; the nitrogen atom may be optionally
quaternized; and the heterocyclyl radical may be partially or fully
saturated. Examples of such heterocyclyl radicals include, but are
not limited to, dioxolanyl, thienyl[1,3]dithianyl,
decahydroisoquinolyl, imidazolinyl, imidazolidinyl,
isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl,
octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl,
2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl,
4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,
thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,
thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and
1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise, a
heterocyclyl group may be optionally substituted.
[0192] The term "substituted" used herein means any of the above
groups (e.g., alkyl, cycloalkyl or heterocyclyl) wherein at least
one hydrogen atom is replaced by a bond to a non-hydrogen atoms
such as, but not limited to: a halogen atom such as F, Cl, Br, and
I; oxo groups (.dbd.O); hydroxyl groups (--OH); alkoxy groups
(--OR.sup.a, where R.sup.a is C.sub.1-C.sub.12 alkyl or
cycloalkyl); carboxyl groups (--OC(.dbd.O)R.sup.a or
--C(.dbd.O)OR.sup.a, where R.sup.a is H, C.sub.1-C.sub.12 alkyl or
cycloalkyl); amine groups (--NR.sup.aR.sup.b, where R.sup.a and
R.sup.b are each independently H, C.sub.1-C.sub.12 alkyl or
cycloalkyl); C.sub.1-C.sub.12 alkyl groups; and cycloalkyl groups.
In some embodiments the substituent is a C.sub.1-C.sub.12 alkyl
group. In other embodiments, the substituent is a cycloalkyl group.
In other embodiments, the substituent is a halo group, such as
fluoro. In other embodiments, the substituent is a oxo group. In
other embodiments, the substituent is a hydroxyl group. In other
embodiments, the substituent is an alkoxy group. In other
embodiments, the substituent is a carboxyl group. In other
embodiments, the substituent is an amine group.
[0193] "Optional" or "optionally" (e.g., optionally substituted)
means that the subsequently described event of circumstances may or
may not occur, and that the description includes instances where
said event or circumstance occurs and instances in which it does
not. For example, "optionally substituted alkyl" means that the
alkyl radical may or may not be substituted and that the
description includes both substituted alkyl radicals and alkyl
radicals having no substitution.
[0194] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Description
[0195] The present invention relates to compositions and methods
for inducing an adaptive immune response in a subject. In certain
embodiments, the present invention provides a composition
comprising a nucleic acid molecule encoding an antigen, where the
antigen induces an adaptive immune response in the subject. For
example, in certain embodiments, the composition comprises a
vaccine comprising a nucleic acid molecule encoding an antigen.
[0196] In one embodiment, the composition of the invention
comprises in vitro transcribed (IVT) RNA. For example, in certain
embodiments, the composition of the invention comprises IVT RNA
which encodes an antigen, where the antigen induces an adaptive
immune response. In certain embodiments, the antigen is at least
one of a viral antigen, bacterial antigen, fungal antigen,
parasitic antigen, tumor-specific antigen, or tumor-associated
antigen. However, the present invention is not limited to any
particular antigen or combination of antigens.
[0197] In certain embodiments, the antigen-encoding nucleic acid of
the present composition is a nucleoside-modified RNA. The present
invention is based in part on the finding that nucleoside-modified
RNA encoding an antigen induces robust CD4+ T-cell, CD8+ T-cell, or
Tfh cell antigen-specific immune responses. Further, the
antigen-encoding nucleoside-modified RNA was observed to induce
antigen-specific antibody production. The nucleoside-modified RNA
is demonstrated to induce adaptive immune responses that are
comparable or superior to current prime-boost vaccine regimens and
viral vector based regimens.
[0198] In certain embodiments, the composition comprises a lipid
nanoparticle (LNP). For example, in one embodiment, the composition
comprises an antigen-encoding nucleic acid molecule encapsulated
within a LNP. In certain instances the LNP enhances cellular uptake
of the nucleic acid molecule.
[0199] In certain embodiments, the composition comprises an
adjuvant. In certain embodiments, the composition comprises a
nucleic acid molecule encoding an adjuvant. For example, in one
embodiment, the composition comprises a nucleoside-modified RNA
encoding an adjuvant. In one embodiment, the composition comprises
a nucleoside-modified RNA encoding an antigen and an adjuvant. In
one embodiment, the composition comprises a first
nucleoside-modified RNA, which encodes an antigen, and a second
nucleoside-modified RNA, which encodes an adjuvant.
[0200] In one embodiment, the present invention provides a method
for inducing an adaptive immune response in a subject. For example,
the method can be used to provide immunity in the subject against a
virus, bacteria, fungus, parasite, cancer, or the like. In some
embodiments, the method comprises administering to the subject a
composition comprising one or more nucleoside-modified RNA encoding
an antigen, adjuvant, or a combination thereof.
[0201] In one embodiment, the method comprises the systemic
administration of the composition into the subject, including for
example intradermal administration. In certain embodiments, the
method comprises administering a plurality of doses to the subject.
In another embodiment, the method comprises administering a single
dose of the composition, where the single dose is effective in
inducing an adaptive immune response.
Vaccine
[0202] In one embodiment, the present invention provides an
immunogenic composition for inducing an adaptive immune response in
a subject. For example, in one embodiment, the immunogenic
composition is a vaccine. For a composition to be useful as a
vaccine, the composition must induce an adaptive immune response to
the antigen in a cell, tissue or mammal (e.g., a human). In certain
instances, the vaccine induces a protective immune response in the
mammal. As used herein, an "immunogenic composition" may comprise
an antigen (e.g., a peptide or polypeptide), a nucleic acid
encoding an antigen, a cell expressing or presenting an antigen or
cellular component, or a combination thereof. In particular
embodiments the composition comprises or encodes all or part of any
peptide antigen described herein, or an immunogenically functional
equivalent thereof. In other embodiments, the composition is in a
mixture that comprises an additional immunostimulatory agent or
nucleic acids encoding such an agent. Immunostimulatory agents
include but are not limited to an additional antigen, an
immunomodulator, an antigen presenting cell or an adjuvant. In
other embodiments, one or more of the additional agent(s) is
covalently bonded to the antigen or an immunostimulatory agent, in
any combination. In certain embodiments, the antigenic composition
is conjugated to or comprises an HLA anchor motif amino acids.
[0203] In the context of the present invention, the term "vaccine"
refers to a substance that induces immunity upon inoculation into
animals.
[0204] A vaccine of the present invention may vary in its
composition of nucleic acid and/or cellular components. In a
non-limiting example, a nucleic acid encoding an antigen might also
be formulated with an adjuvant. Of course, it will be understood
that various compositions described herein may further comprise
additional components. For example, one or more vaccine components
may be comprised in a lipid, liposome, or lipid nanoparticle. In
another non-limiting example, a vaccine may comprise one or more
adjuvants. A vaccine of the present invention, and its various
components, may be prepared and/or administered by any method
disclosed herein or as would be known to one of ordinary skill in
the art, in light of the present disclosure.
[0205] The induction of the immunity by the expression of the
antigen can be detected by observing in vivo or in vitro the
response of all or any part of the immune system in the host
against the antigen.
[0206] For example, a method for detecting the induction of
cytotoxic T lymphocytes is well known. A foreign substance that
enters the living body is presented to T cells and B cells by the
action of APCs. T cells that respond to the antigen presented by
APC in an antigen specific manner differentiate into cytotoxic T
cells (also referred to as cytotoxic T lymphocytes or CTLs) due to
stimulation by the antigen. These antigen stimulated cells then
proliferate. This process is referred to herein as "activation" of
T cells. Therefore, CTL induction by an epitope of a polypeptide or
peptide or combinations thereof can be evaluated by presenting an
epitope of a polypeptide or peptide or combinations thereof to a T
cell by APC, and detecting the induction of CTL. Furthermore, APCs
have the effect of activating B cells, CD4+ T cells, CD8+ T cells,
macrophages, eosinophils and NK cells.
[0207] A method for evaluating the inducing action of CTL using
dendritic cells (DCs) as APC is well known in the art. DC is a
representative APC having a robust CTL inducing action among APCs.
In the methods of the invention, the epitope of a polypeptide or
peptide or combinations thereof is initially expressed by the DC
and then this DC is contacted with T cells. Detection of T cells
having cytotoxic effects against the cells of interest after the
contact with DC shows that the epitope of a polypeptide or peptide
or combinations thereof has an activity of inducing the cytotoxic T
cells. Furthermore, the induced immune response can be also
examined by measuring IFN-gamma produced and released by CTL in the
presence of antigen-presenting cells that carry immobilized peptide
or combination of peptides by visualizing using anti-IFN-gamma
antibodies, such as an ELISPOT assay.
[0208] Apart from DC, peripheral blood mononuclear cells (PBMCs)
may also be used as the APC. The induction of CTL is reported to be
enhanced by culturing PBMC in the presence of GM-CSF and IL-4.
Similarly, CTL has been shown to be induced by culturing PBMC in
the presence of keyhole limpet hemocyanin (KLH) and IL-7.
[0209] The antigens confirmed to possess CTL-inducing activity by
these methods are antigens having DC activation effect and
subsequent CTL-inducing activity. Furthermore, CTLs that have
acquired cytotoxicity due to presentation of the antigen by APC can
be also used as vaccines against antigen-associated disorders.
[0210] The induction of immunity by expression of the antigen can
be further confirmed by observing the induction of antibody
production against the antigen. For example, when antibodies
against an antigen are induced in a laboratory animal immunized
with the composition encoding the antigen, and when
antigen-associated pathology is suppressed by those antibodies, the
composition is determined to induce immunity.
[0211] The induction of immunity by expression of the antigen can
be further confirmed by observing the induction of CD4+ T cells.
CD4+ T cells can also lyse target cells, but mainly supply help in
the induction of other types of immune responses, including CTL and
antibody generation. The type of CD4+ T cell help can be
characterized, as Th1, Th2, Th9, Th17, Tregulatory, or T follicular
helper (T.sub.fh) cells. Each subtype of CD4+ T cell supplies help
to certain types of immune responses. Of particular interest to
this invention, the T.sub.fh subtype provides help in the
generation of high affinity antibodies.
[0212] The therapeutic compounds or compositions of the invention
may be administered prophylactically (i.e., to prevent disease or
disorder) or therapeutically (i.e., to treat disease or disorder)
to subjects suffering from or at risk of (or susceptible to)
developing the disease or disorder. Such subjects may be identified
using standard clinical methods. In the context of the present
invention, prophylactic administration occurs prior to the
manifestation of overt clinical symptoms of disease, such that a
disease or disorder is prevented or alternatively delayed in its
progression. In the context of the field of medicine, the term
"prevent" encompasses any activity which reduces the burden of
mortality or morbidity from disease. Prevention can occur at
primary, secondary and tertiary prevention levels. While primary
prevention avoids the development of a disease, secondary and
tertiary levels of prevention encompass activities aimed at
preventing the progression of a disease and the emergence of
symptoms as well as reducing the negative impact of an already
established disease by restoring function and reducing
disease-related complications.
Nucleic Acids
[0213] In one embodiment, the invention includes a
nucleoside-modified nucleic acid molecule. In one embodiment, the
nucleoside-modified nucleic acid molecule encodes an antigen. In
one embodiment, the nucleoside-modified nucleic acid molecule
encodes a plurality of antigens. In certain embodiments, the
nucleoside-modified nucleic acid molecule encodes an antigen that
induces an adaptive immune response against the antigen. In one
embodiment, the invention includes a nucleoside-modified nucleic
acid molecule encoding an adjuvant.
[0214] The nucleotide sequences encoding an antigen or adjuvant, as
described herein, can alternatively comprise sequence variations
with respect to the original nucleotide sequences, for example,
substitutions, insertions and/or deletions of one or more
nucleotides, with the condition that the resulting polynucleotide
encodes a polypeptide according to the invention. Therefore, the
scope of the present invention includes nucleotide sequences that
are substantially homologous to the nucleotide sequences recited
herein and encode an antigen or adjuvant of interest.
[0215] In certain embodiments, the nucleotide sequence encodes an
HIV Env antigen. For example, in certain embodiments, the
nucleotide sequence encodes an HIV Env encoded by the nucleotide
sequences of SEQ ID NO: or SEQ ID NO: 2. In one embodiment, the
nucleotide sequence encodes influenza hemagglutinin (HA). For
example, in certain embodiments, the nucleotide sequence encodes HA
from PR8. For example, in one embodiment, the nucleotide sequence
encodes PR8 HA having an amino acid sequence of SEQ ID NO: 3. In
certain embodiments, the nucleotide sequence encodes PR8 HA encoded
by the nucleotide sequences of SEQ ID NO: 4 or SEQ ID NO: 5. For
example, in certain embodiments, the nucleotide sequence encodes HA
from Cal/7/2009. For example, in one embodiment, the nucleotide
sequence encodes Cal/7/2009 HA having an amino acid sequence of SEQ
ID NO: 6. In certain embodiments, the nucleotide sequence encodes
Cal/7/2009/HA encoded by the nucleotide sequences of SEQ ID NO: 7
or SEQ ID NO: 8.
[0216] As used herein, a nucleotide sequence is "substantially
homologous" to any of the nucleotide sequences described herein
when its nucleotide sequence has a degree of identity with respect
to the nucleotide sequence of at least 60%, advantageously of at
least 70%, preferably of at least 85%, and more preferably of at
least 95%. A nucleotide sequence that is substantially homologous
to a nucleotide sequence encoding an antigen can typically be
isolated from a producer organism of the antigen based on the
information contained in the nucleotide sequence by means of
introducing conservative or non-conservative substitutions, for
example. Other examples of possible modifications include the
insertion of one or more nucleotides in the sequence, the addition
of one or more nucleotides in any of the ends of the sequence, or
the deletion of one or more nucleotides in any end or inside the
sequence. The degree of identity between two polynucleotides is
determined using computer algorithms and methods that are widely
known for the persons skilled in the art.
[0217] Further, the scope of the invention includes nucleotide
sequences that encode amino acid sequences that are substantially
homologous to the amino acid sequences recited herein and preserve
the immunogenic function of the original amino acid sequence.
[0218] As used herein, an amino acid sequence is "substantially
homologous" to any of the amino acid sequences described herein
when its amino acid sequence has a degree of identity with respect
to the amino acid sequence of at least 60%, advantageously of at
least 70%, preferably of at least 85%, and more preferably of at
least 95%. The identity between two amino acid sequences is
preferably determined by using the BLASTN algorithm (BLAST Manual,
Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul,
S., et al., J. Mol. Biol. 215: 403-410 (1990)).
[0219] In one embodiment, the invention relates to a construct,
comprising a nucleotide sequence encoding an antigen. In one
embodiment, the construct comprises a plurality of nucleotide
sequences encoding a plurality of antigens. For example, in certain
embodiments, the construct encodes 1 or more, 2 or more, 5 or more,
10 or more, 15 or more, or 20 or more antigens. In one embodiment,
the invention relates to a construct, comprising a nucleotide
sequence encoding an adjuvant. In one embodiment, the construct
comprises a first nucleotide sequence encoding an antigen and a
second nucleotide sequence encoding an adjuvant.
[0220] In one embodiment, the composition comprises a plurality of
constructs, each construct encoding one or more antigens. In
certain embodiments, the composition comprises 1 or more, 2 or
more, 5 or more, 10 or more, 15 or more, or 20 or more constructs.
In one embodiment, the composition comprises a first construct,
comprising a nucleotide sequence encoding an antigen; and a second
construct, comprising a nucleotide sequence encoding an
adjuvant.
[0221] In another particular embodiment, the construct is
operatively bound to a translational control element. The construct
can incorporate an operatively bound regulatory sequence for the
expression of the nucleotide sequence of the invention, thus
forming an expression cassette.
[0222] Vectors
[0223] The nucleic acid sequences coding for the antigen or
adjuvant can be obtained using recombinant methods known in the
art, such as, for example by screening libraries from cells
expressing the gene, by deriving the gene from a vector known to
include the same, or by isolating directly from cells and tissues
containing the same, using standard techniques. Alternatively, the
gene of interest can be produced synthetically.
[0224] The nucleic acid can be cloned into a number of types of
vectors. For example, the nucleic acid can be cloned into a vector
including, but not limited to a plasmid, a phagemid, a phage
derivative, an animal virus, and a cosmid. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors, sequencing vectors and vectors optimized for in
vitro transcription.
[0225] Chemical means for introducing a polynucleotide into a host
cell include colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. An exemplary colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (e.g., an artificial
membrane vesicle).
[0226] In the case where a non-viral delivery system is utilized,
an exemplary delivery vehicle is a liposome. The use of lipid
formulations is contemplated for the introduction of the nucleic
acids into a host cell (in vitro, ex vivo or in vivo). In another
aspect, the nucleic acid may be associated with a lipid. The
nucleic acid associated with a lipid may be encapsulated in the
aqueous interior of a liposome, interspersed within the lipid
bilayer of a liposome, attached to a liposome via a linking
molecule that is associated with both the liposome and the
oligonucleotide, entrapped in a liposome, complexed with a
liposome, dispersed in a solution containing a lipid, mixed with a
lipid, combined with a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle, or otherwise associated with
a lipid. Lipid, lipid/RNA or lipid/expression vector associated
compositions are not limited to any particular structure in
solution. For example, they may be present in a bilayer structure,
as micelles, or with a "collapsed" structure. They may also simply
be interspersed in a solution, possibly forming aggregates that are
not uniform in size or shape. Lipids are fatty substances which may
be naturally occurring or synthetic lipids. For example, lipids
include the fatty droplets that naturally occur in the cytoplasm as
well as the class of compounds which contain long-chain aliphatic
hydrocarbons and their derivatives, such as fatty acids, alcohols,
amines, amino alcohols, and aldehydes.
[0227] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Chol") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform is used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology
5: 505-10). However, compositions that have different structures in
solution than the normal vesicular structure are also encompassed.
For example, the lipids may assume a micellar structure or merely
exist as nonuniform aggregates of lipid molecules. Also
contemplated are lipofectamine-nucleic acid complexes.
[0228] Regardless of the method used to introduce exogenous nucleic
acids into a host cell or otherwise expose a cell to the inhibitor
of the present invention, in order to confirm the presence of the
mRNA sequence in the host cell, a variety of assays may be
performed. Such assays include, for example, "molecular biological"
assays well known to those of skill in the art, such as Northern
blotting and RT-PCR; "biochemical" assays, such as detecting the
presence or absence of a particular peptide, e.g., by immunogenic
means (ELISAs and Western blots) or by assays described herein to
identify agents falling within the scope of the invention.
[0229] In Vitro Transcribed RNA
[0230] In one embodiment, the composition of the invention
comprises in vitro transcribed (IVT) RNA encoding an antigen. In
one embodiment, the composition of the invention comprises IVT RNA
encoding a plurality of antigens. In one embodiment, the
composition of the invention comprises IVT RNA encoding an
adjuvant. In one embodiment, the composition of the invention
comprises IVT RNA encoding one or more antigens and one or more
adjuvants.
[0231] In one embodiment, an IVT RNA can be introduced to a cell as
a form of transient transfection. The RNA is produced by in vitro
transcription using a plasmid DNA template generated synthetically.
DNA of interest from any source can be directly converted by PCR
into a template for in vitro mRNA synthesis using appropriate
primers and RNA polymerase. The source of the DNA can be, for
example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA
sequence or any other appropriate source of DNA. In one embodiment,
the desired template for in vitro transcription is an antigen
capable of inducing an adaptive immune response, including for
example an antigen associated with a pathogen or tumor, as
described elsewhere herein. In one embodiment, the desired template
for in vitro transcription is an adjuvant capable of enhancing an
adaptive immune response.
[0232] In one embodiment, the DNA to be used for PCR contains an
open reading frame. The DNA can be from a naturally occurring DNA
sequence from the genome of an organism. In one embodiment, the DNA
is a full length gene of interest of a portion of a gene. The gene
can include some or all of the 5' and/or 3' untranslated regions
(UTRs). The gene can include exons and introns. In one embodiment,
the DNA to be used for PCR is a human gene. In another embodiment,
the DNA to be used for PCR is a human gene including the 5' and 3'
UTRs. In another embodiment, the DNA to be used for PCR is a gene
from a pathogenic or commensal organism, including bacteria,
viruses, parasites, and fungi. In another embodiment, the DNA to be
used for PCR is from a pathogenic or commensal organism, including
bacteria, viruses, parasites, and fungi, including the 5' and 3'
UTRs. The DNA can alternatively be an artificial DNA sequence that
is not normally expressed in a naturally occurring organism. An
exemplary artificial DNA sequence is one that contains portions of
genes that are ligated together to form an open reading frame that
encodes a fusion protein. The portions of DNA that are ligated
together can be from a single organism or from more than one
organism.
[0233] Genes that can be used as sources of DNA for PCR include
genes that encode polypeptides that induce or enhance an adaptive
immune response in an organism. Preferred genes are genes which are
useful for a short term treatment, or where there are safety
concerns regarding dosage or the expressed gene.
[0234] In various embodiments, a plasmid is used to generate a
template for in vitro transcription of mRNA which is used for
transfection.
[0235] Chemical structures with the ability to promote stability
and/or translation efficiency may also be used. The RNA preferably
has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero
and 3000 nucleotides in length. The length of 5' and 3' UTR
sequences to be added to the coding region can be altered by
different methods, including, but not limited to, designing primers
for PCR that anneal to different regions of the UTRs. Using this
approach, one of ordinary skill in the art can modify the 5' and 3'
UTR lengths required to achieve optimal translation efficiency
following transfection of the transcribed RNA.
[0236] The 5' and 3' UTRs can be the naturally occurring,
endogenous 5' and 3' UTRs for the gene of interest. Alternatively,
UTR sequences that are not endogenous to the gene of interest can
be added by incorporating the UTR sequences into the forward and
reverse primers or by any other modifications of the template. The
use of UTR sequences that are not endogenous to the gene of
interest can be useful for modifying the stability and/or
translation efficiency of the RNA. For example, it is known that
AU-rich elements in 3' UTR sequences can decrease the stability of
mRNA. Therefore, 3' UTRs can be selected or designed to increase
the stability of the transcribed RNA based on properties of UTRs
that are well known in the art.
[0237] In one embodiment, the 5' UTR can contain the Kozak sequence
of the endogenous gene. Alternatively, when a 5' UTR that is not
endogenous to the gene of interest is being added by PCR as
described above, a consensus Kozak sequence can be redesigned by
adding the 5' UTR sequence. Kozak sequences can increase the
efficiency of translation of some RNA transcripts, but does not
appear to be required for all RNAs to enable efficient translation.
The requirement for Kozak sequences for many mRNAs is known in the
art. In other embodiments the 5' UTR can be derived from an RNA
virus whose RNA genome is stable in cells. In other embodiments
various nucleotide analogues can be used in the 3' or 5' UTR to
impede exonuclease degradation of the mRNA.
[0238] To enable synthesis of RNA from a DNA template without the
need for gene cloning, a promoter of transcription should be
attached to the DNA template upstream of the sequence to be
transcribed. When a sequence that functions as a promoter for an
RNA polymerase is added to the 5' end of the forward primer, the
RNA polymerase promoter becomes incorporated into the PCR product
upstream of the open reading frame that is to be transcribed. In
one preferred embodiment, the promoter is a T7 RNA polymerase
promoter, as described elsewhere herein. Other useful promoters
include, but are not limited to, T3 and SP6 RNA polymerase
promoters. Consensus nucleotide sequences for T7, T3 and SP6
promoters are known in the art.
[0239] In a preferred embodiment, the mRNA has both a cap on the 5'
end and a 3' poly(A) tail which determine ribosome binding,
initiation of translation and stability mRNA in the cell. On a
circular DNA template, for instance, plasmid DNA, RNA polymerase
produces a long concatameric product which is not suitable for
expression in eukaryotic cells. The transcription of plasmid DNA
linearized at the end of the 3' UTR results in normal sized mRNA
which is effective in eukaryotic transfection when it is
polyadenylated after transcription.
[0240] On a linear DNA template, phage T7 RNA polymerase can extend
the 3' end of the transcript beyond the last base of the template
(Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985);
Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65
(2003).
[0241] The conventional method of integration of polyA/T stretches
into a DNA template is molecular cloning. However polyA/T sequence
integrated into plasmid DNA can cause plasmid instability, which
can be ameliorated through the use of recombination incompetent
bacterial cells for plasmid propagation.
[0242] Poly(A) tails of RNAs can be further extended following in
vitro transcription with the use of a poly(A) polymerase, such as
E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one
embodiment, increasing the length of a poly(A) tail from 100
nucleotides to between 300 and 400 nucleotides results in about a
two-fold increase in the translation efficiency of the RNA.
Additionally, the attachment of different chemical groups to the 3'
end can increase mRNA stability. Such attachment can contain
modified/artificial nucleotides, aptamers and other compounds. For
example, ATP analogs can be incorporated into the poly(A) tail
using poly(A) polymerase. ATP analogs can further increase the
stability of the RNA.
[0243] 5' caps on also provide stability to mRNA molecules. In a
preferred embodiment, RNAs produced by the methods to include a 5'
cap1 structure. Such cap1 structure can be generated using Vaccinia
capping enzyme and 2'-O-methyltransferase enzymes (CellScript,
Madison, Wis.). Alternatively, 5' cap is provided using techniques
known in the art and described herein (Cougot, et al., Trends in
Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95
(2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966
(2005)).
[0244] RNA can be introduced into target cells using any of a
number of different methods, for instance, commercially available
methods which include, but are not limited to, electroporation
(Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM
830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser
II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg
Germany), cationic liposome mediated transfection using
lipofection, polymer encapsulation, peptide mediated transfection,
or biolistic particle delivery systems such as "gene guns" (see,
for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
In certain embodiments RNA of the invention is introduced to a cell
with a method comprising the use of TransIT.RTM.-mRNA transfection
Kit (Mirus, Madison Wis.), which, in some instances, provides high
efficiency, low toxicity, transfection.
[0245] Nucleoside-Modified RNA
[0246] In one embodiment, the composition of the present invention
comprises a nucleoside-modified nucleic acid encoding an antigen as
described herein. In one embodiment, the composition of the present
invention comprises a nucleoside-modified nucleic acid encoding a
plurality of antigens. In one embodiment, the composition of the
present invention comprises a nucleoside-modified nucleic acid
encoding an adjuvant as described herein. In one embodiment, the
composition of the present invention comprises a
nucleoside-modified nucleic acid encoding one or more antigens and
one or more adjuvants.
[0247] For example, in one embodiment, the composition comprises a
nucleoside-modified RNA. In one embodiment, the composition
comprises a nucleoside-modified mRNA. Nucleoside-modified mRNA have
particular advantages over non-modified mRNA, including for
example, increased stability, low or absent innate immunogenicity,
and enhanced translation. Nucleoside-modified mRNA useful in the
present invention is further described in U.S. Pat. No. 8,278,036,
which is incorporated by reference herein in its entirety.
[0248] In certain embodiments, nucleoside-modified mRNA does not
activate any pathophysiologic pathways, translates very efficiently
and almost immediately following delivery, and serve as templates
for continuous protein production in vivo lasting for several days
(Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012,
Mol Ther 20:948-953). The amount of mRNA required to exert a
physiological effect is small and that makes it applicable for
human therapy. For example, as described herein,
nucleoside-modified mRNA encoding an antigen has demonstrated the
ability to induce CD4+ and CD8+ T-cell and antigen-specific
antibody production. For example, in certain instances, antigen
encoded by nucleoside-modified mRNA induces greater production of
antigen-specific antibody production as compared to antigen encoded
by non-modified mRNA.
[0249] In certain instances, expressing a protein by delivering the
encoding mRNA has many benefits over methods that use protein,
plasmid DNA or viral vectors. During mRNA transfection, the coding
sequence of the desired protein is the only substance delivered to
cells, thus avoiding all the side effects associated with plasmid
backbones, viral genes, and viral proteins. More importantly,
unlike DNA- and viral-based vectors, the mRNA does not carry the
risk of being incorporated into the genome and protein production
starts immediately after mRNA delivery. For example, high levels of
circulating proteins have been measured within 15 to 30 minutes of
in vivo injection of the encoding mRNA. In certain embodiments,
using mRNA rather than the protein also has many advantages.
Half-lives of proteins in the circulation are often short, thus
protein treatment would need frequent dosing, while mRNA provides a
template for continuous protein production for several days.
Purification of proteins is problematic and they can contain
aggregates and other impurities that cause adverse effects
(Kromminga and Schellekens, 2005, Ann NY Acad Sci
1050:257-265).
[0250] In certain embodiments, the nucleoside-modified RNA
comprises the naturally occurring modified-nucleoside
pseudouridine. In certain embodiments, inclusion of pseudouridine
makes the mRNA more stable, non-immunogenic, and highly
translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson
et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al.,
2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011,
Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther
20:948-953; Kariko et al., 2005, Immunity 23:165-175).
[0251] It has been demonstrated that the presence of modified
nucleosides, including pseudouridines in RNA suppress their innate
immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Further,
protein-encoding, in vitro-transcribed RNA containing pseudouridine
can be translated more efficiently than RNA containing no or other
modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840).
Subsequently, it is shown that the presence of pseudouridine
improves the stability of RNA (Anderson et al., 2011, Nucleic Acids
Research 39:9329-9338) and abates both activation of PKR and
inhibition of translation (Anderson et al., 2010, Nucleic Acids Res
38:5884-5892). A preparative HPLC purification procedure has been
established that was critical to obtain pseudouridine-containing
RNA that has superior translational potential and no innate
immunogenicity (Kariko et al., 2011, Nucleic Acids Research
39:e142). Administering HPLC-purified, pseudourine-containing RNA
coding for erythropoietin into mice and macaques resulted in a
significant increase of serum EPO levels (Kariko et al., 2012, Mol
Ther 20:948-953), thus confirming that pseudouridine-containing
mRNA is suitable for in vivo protein therapy.
[0252] The present invention encompasses RNA, oligoribonucleotide,
and polyribonucleotide molecules comprising pseudouridine or a
modified nucleoside. In certain embodiments, the composition
comprises an isolated nucleic acid encoding an antigen, wherein the
nucleic acid comprises a pseudouridine or a modified nucleoside. In
certain embodiments, the composition comprises a vector, comprising
an isolated nucleic acid encoding an antigen, adjuvant, or
combination thereof, wherein the nucleic acid comprises a
pseudouridine or a modified nucleoside.
[0253] In one embodiment, the nucleoside-modified RNA of the
invention is IVT RNA, as described elsewhere herein. For example,
in certain embodiments, the nucleoside-modified RNA is synthesized
by T7 phage RNA polymerase. In another embodiment, the
nucleoside-modified mRNA is synthesized by SP6 phage RNA
polymerase. In another embodiment, the nucleoside-modified RNA is
synthesized by T3 phage RNA polymerase.
[0254] In one embodiment, the modified nucleoside is
m.sup.1acp.sup.3.PSI. (1-methyl-3-(3-amino-3-carboxypropyl)
pseudouridine. In another embodiment, the modified nucleoside is
m.sup.1.PSI. (1-methylpseudouridine). In another embodiment, the
modified nucleoside is .PSI.m (2'-O-methylpseudouridine. In another
embodiment, the modified nucleoside is m.sup.5D
(5-methyldihydrouridine). In another embodiment, the modified
nucleoside is m.sup.3' (3-methylpseudouridine). In another
embodiment, the modified nucleoside is a pseudouridine moiety that
is not further modified. In another embodiment, the modified
nucleoside is a monophosphate, diphosphate, or triphosphate of any
of the above pseudouridines. In another embodiment, the modified
nucleoside is any other pseudouridine-like nucleoside known in the
art.
[0255] In another embodiment, the nucleoside that is modified in
the nucleoside-modified RNA the present invention is uridine (U).
In another embodiment, the modified nucleoside is cytidine (C). In
another embodiment, the modified nucleoside is adenosine (A). In
another embodiment the modified nucleoside is guanosine (G).
[0256] In another embodiment, the modified nucleoside of the
present invention is m.sup.5C (5-methylcytidine). In another
embodiment, the modified nucleoside is m.sup.5U (5-methyluridine).
In another embodiment, the modified nucleoside is m.sup.6A
(N.sup.6-methyladenosine). In another embodiment, the modified
nucleoside is s.sup.2U (2-thiouridine). In another embodiment, the
modified nucleoside is .PSI. (pseudouridine). In another
embodiment, the modified nucleoside is Um (2'-O-methyluridine).
[0257] In other embodiments, the modified nucleoside is m.sup.1A
(1-methyladenosine); m.sup.2A (2-methyladenosine); Am
(2'-O-methyladenosine); ms.sup.2m.sup.6A
(2-methylthio-N.sup.6-methyladenosine); i.sup.6A
(N.sup.6-isopentenyladenosine); ms.sup.2i6A
(2-methylthio-N.sup.6isopentenyladenosine); io.sup.6A
(N.sup.6-(cis-hydroxyisopentenyl)adenosine); ms.sup.2io.sup.6A
(2-methylthio-N.sup.6-(cis-hydroxyisopentenyl) adenosine); g.sup.6A
(N.sup.6-glycinylcarbamoyladenosine); t.sup.6A
(N.sup.6-threonylcarbamoyladenosine); ms.sup.2t.sup.6A
(2-methylthio-N.sup.6-threonyl carbamoyladenosine); m.sup.6t.sup.6A
(N.sup.6-methyl-N.sup.6-threonylcarbamoyladenosine); hn.sup.6A
(N.sup.6-hydroxynorvalylcarbamoyladenosine); ms.sup.2hn.sup.6A
(2-methylthio-N.sup.6-hydroxynorvalyl carbamoyladenosine); Ar(p)
(2'-O-ribosyladenosine (phosphate)); I (inosine); m.sup.1I
(1-methylinosine); m.sup.1Im (1,2'-O-dimethylinosine); m.sup.3C
(3-methylcytidine); Cm (2'-O-methylcytidine); s.sup.2C
(2-thiocytidine); ac.sup.4C (N.sup.4-acetylcytidine); fVC
(5-formylcytidine); msCm (5,2'-O-dimethylcytidine); ac.sup.4Cm
(N.sup.4-acetyl-2'-O-methylcytidine); k.sup.2C (lysidine); m.sup.1G
(1-methylguanosine); m.sup.2G (N.sup.2-methylguanosine); m.sup.7G
(7-methylguanosine); Gm (2'-O-methylguanosine); m.sup.22G
(N.sup.2,N.sup.2-dimethylguanosine); m.sup.2Gm
(N.sup.2,2'-O-dimethylguanosine); m.sup.22Gm
(N.sup.2,N.sup.2,2'-O-trimethylguanosine); Gr(p)
(2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o.sub.2yW
(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified
hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q
(queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ
(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi
(7-aminomethyl-7-deazaguanosine); G+(archaeosine); D
(dihydrouridine); m.sup.5Um (5,2'-O-dimethyluridine); s.sup.4U
(4-thiouridine); m.sup.5s2U (5-methyl-2-thiouridine); s.sup.2Um
(2-thio-2'-O-methyluridine); acp.sup.3U
(3-(3-amino-3-carboxypropyl)uridine); ho.sup.5U (5-hydroxyuridine);
mo.sup.5U (5-methoxyuridine); cmo.sup.5U (uridine 5-oxyacetic
acid); mcmo.sup.5U (uridine 5-oxyacetic acid methyl ester);
chm.sup.5U (5-(carboxyhydroxymethyl)uridine)); mchm.sup.5U
(5-(carboxyhydroxymethyl)uridine methyl ester); mcm.sup.5U
(5-methoxycarbonylmethyluridine); mcm.sup.5Um
(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm.sup.5s.sup.2U
(5-methoxycarbonylmethyl-2-thiouridine); nm.sup.5s.sup.2U
(5-aminomethyl-2-thiouridine); mnm.sup.5U
(5-methylaminomethyluridine); mnm.sup.5s2U
(5-methylaminomethyl-2-thiouridine); mnm.sup.5se.sup.2U
(5-methylaminomethyl-2-selenouridine); nCm.sup.5U
(5-carbamoylmethyluridine); nCm.sup.5Um
(5-carbamoylmethyl-2'-O-methyluridine); cmnm.sup.5U
(5-carboxymethylaminomethyluridine); cmnm.sup.5Um
(5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm.sup.5s2U
(5-carboxymethylaminomethyl-2-thiouridine); m.sup.6.sub.2A
(N.sup.6,N.sup.6-dimethyladenosine); Im (2'-O-methylinosine);
m.sup.4C (N.sup.4-methylcytidine); m.sup.4Cm
(N.sup.4,2'-O-dimethylcytidine); hm.sup.5C
(5-hydroxymethylcytidine); m.sup.3U (3-methyluridine); cm.sup.5U
(5-carboxymethyluridine); m.sup.6Am
(N.sup.6,2'-O-dimethyladenosine); m.sup.6.sub.2Am
(N.sup.6,N.sup.6,O-2'-trimethyladenosine); m.sup.2,7G
(N.sup.2,7-dimethylguanosine); m.sup.2,2,7G
(N.sup.2,N.sup.2,7-trimethylguanosine); m.sup.3Um
(3,2'-O-dimethyluridine); m.sup.5D (5-methyldihydrouridine);
f.sup.5Cm (5-formyl-2'-O-methylcytidine); m.sup.1Gm
(1,2'-O-dimethylguanosine); m.sup.1Am (1,2'-O-dimethyladenosine);
.tau.m.sup.5U (5-taurinomethyluridine); .tau.m.sup.5s.sup.2U
(5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2
(isowyosine); or ac.sup.6A (N.sup.6-acetyladenosine).
[0258] In another embodiment, a nucleoside-modified RNA of the
present invention comprises a combination of 2 or more of the above
modifications. In another embodiment, the nucleoside-modified RNA
comprises a combination of 3 or more of the above modifications. In
another embodiment, the nucleoside-modified RNA comprises a
combination of more than 3 of the above modifications.
[0259] In another embodiment, between 0.1% and 100% of the residues
in the nucleoside-modified of the present invention are modified
(e.g. either by the presence of pseudouridine or a modified
nucleoside base). In another embodiment, 0.1% of the residues are
modified. In another embodiment, the fraction of modified residues
is 0.2%. In another embodiment, the fraction is 0.3%. In another
embodiment, the fraction is 0.4%. In another embodiment, the
fraction is 0.5%. In another embodiment, the fraction is 0.6%. In
another embodiment, the fraction is 0.8%. In another embodiment,
the fraction is 1%. In another embodiment, the fraction is 1.5%. In
another embodiment, the fraction is 2%. In another embodiment, the
fraction is 2.5%. In another embodiment, the fraction is 3%. In
another embodiment, the fraction is 4%. In another embodiment, the
fraction is 5%. In another embodiment, the fraction is 6%. In
another embodiment, the fraction is 8%. In another embodiment, the
fraction is 10%. In another embodiment, the fraction is 12%. In
another embodiment, the fraction is 14%. In another embodiment, the
fraction is 16%. In another embodiment, the fraction is 18%. In
another embodiment, the fraction is 20%. In another embodiment, the
fraction is 25%. In another embodiment, the fraction is 30%. In
another embodiment, the fraction is 35%. In another embodiment, the
fraction is 40%. In another embodiment, the fraction is 45%. In
another embodiment, the fraction is 50%. In another embodiment, the
fraction is 60%. In another embodiment, the fraction is 70%. In
another embodiment, the fraction is 80%. In another embodiment, the
fraction is 90%. In another embodiment, the fraction is 100%.
[0260] In another embodiment, the fraction is less than 5%. In
another embodiment, the fraction is less than 3%. In another
embodiment, the fraction is less than 1%. In another embodiment,
the fraction is less than 2%. In another embodiment, the fraction
is less than 4%. In another embodiment, the fraction is less than
6%. In another embodiment, the fraction is less than 8%. In another
embodiment, the fraction is less than 10%. In another embodiment,
the fraction is less than 12%. In another embodiment, the fraction
is less than 15%. In another embodiment, the fraction is less than
20%. In another embodiment, the fraction is less than 30%. In
another embodiment, the fraction is less than 40%. In another
embodiment, the fraction is less than 50%. In another embodiment,
the fraction is less than 60%. In another embodiment, the fraction
is less than 70%.
[0261] In another embodiment, 0.1% of the residues of a given
nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are
modified. In another embodiment, the fraction of the given
nucleotide that is modified is 0.2%. In another embodiment, the
fraction is 0.3%. In another embodiment, the fraction is 0.4%. In
another embodiment, the fraction is 0.5%. In another embodiment,
the fraction is 0.6%. In another embodiment, the fraction is 0.8%.
In another embodiment, the fraction is 1%. In another embodiment,
the fraction is 1.5%. In another embodiment, the fraction is 2%. In
another embodiment, the fraction is 2.5%. In another embodiment,
the fraction is 3%. In another embodiment, the fraction is 4%. In
another embodiment, the fraction is 5%. In another embodiment, the
fraction is 6%. In another embodiment, the fraction is 8%. In
another embodiment, the fraction is 10%. In another embodiment, the
fraction is 12%. In another embodiment, the fraction is 14%. In
another embodiment, the fraction is 16%. In another embodiment, the
fraction is 18%. In another embodiment, the fraction is 20%. In
another embodiment, the fraction is 25%. In another embodiment, the
fraction is 30%. In another embodiment, the fraction is 35%. In
another embodiment, the fraction is 40%. In another embodiment, the
fraction is 45%. In another embodiment, the fraction is 50%. In
another embodiment, the fraction is 60%. In another embodiment, the
fraction is 70%. In another embodiment, the fraction is 80%. In
another embodiment, the fraction is 90%. In another embodiment, the
fraction is 100%.
[0262] In another embodiment, the fraction of the given nucleotide
that is modified is less than 8%. In another embodiment, the
fraction is less than 10%. In another embodiment, the fraction is
less than 5%. In another embodiment, the fraction is less than 3%.
In another embodiment, the fraction is less than 1%. In another
embodiment, the fraction is less than 2%. In another embodiment,
the fraction is less than 4%. In another embodiment, the fraction
is less than 6%. In another embodiment, the fraction is less than
12%. In another embodiment, the fraction is less than 15%. In
another embodiment, the fraction is less than 20%. In another
embodiment, the fraction is less than 30%. In another embodiment,
the fraction is less than 40%. In another embodiment, the fraction
is less than 50%. In another embodiment, the fraction is less than
60%. In another embodiment, the fraction is less than 70%.
[0263] In another embodiment, a nucleoside-modified RNA of the
present invention is translated in the cell more efficiently than
an unmodified RNA molecule with the same sequence. In another
embodiment, the nucleoside-modified RNA exhibits enhanced ability
to be translated by a target cell. In another embodiment,
translation is enhanced by a factor of 2-fold relative to its
unmodified counterpart. In another embodiment, translation is
enhanced by a 3-fold factor. In another embodiment, translation is
enhanced by a 5-fold factor. In another embodiment, translation is
enhanced by a 7-fold factor. In another embodiment, translation is
enhanced by a 10-fold factor. In another embodiment, translation is
enhanced by a 15-fold factor. In another embodiment, translation is
enhanced by a 20-fold factor. In another embodiment, translation is
enhanced by a 50-fold factor. In another embodiment, translation is
enhanced by a 100-fold factor. In another embodiment, translation
is enhanced by a 200-fold factor. In another embodiment,
translation is enhanced by a 500-fold factor. In another
embodiment, translation is enhanced by a 1000-fold factor. In
another embodiment, translation is enhanced by a 2000-fold factor.
In another embodiment, the factor is 10-1000-fold. In another
embodiment, the factor is 10-100-fold. In another embodiment, the
factor is 10-200-fold. In another embodiment, the factor is
10-300-fold. In another embodiment, the factor is 10-500-fold. In
another embodiment, the factor is 20-1000-fold. In another
embodiment, the factor is 30-1000-fold. In another embodiment, the
factor is 50-1000-fold. In another embodiment, the factor is
100-1000-fold. In another embodiment, the factor is 200-1000-fold.
In another embodiment, translation is enhanced by any other
significant amount or range of amounts.
[0264] In another embodiment, the nucleoside-modified
antigen-encoding RNA of the present invention induces significantly
more adaptive immune response than an unmodified in
vitro-synthesized RNA molecule with the same sequence. In another
embodiment, the modified RNA molecule exhibits an adaptive immune
response that is 2-fold greater than its unmodified counterpart. In
another embodiment, the adaptive immune response is increased by a
3-fold factor. In another embodiment the adaptive immune response
is increased by a 5-fold factor. In another embodiment, the
adaptive immune response is increased by a 7-fold factor. In
another embodiment, the adaptive immune response is increased by a
10-fold factor. In another embodiment, the adaptive immune response
is increased by a 15-fold factor. In another embodiment the
adaptive immune response is increased by a 20-fold factor. In
another embodiment, the adaptive immune response is increased by a
50-fold factor. In another embodiment, the adaptive immune response
is increased by a 100-fold factor. In another embodiment, the
adaptive immune response is increased by a 200-fold factor. In
another embodiment, the adaptive immune response is increased by a
500-fold factor. In another embodiment, the adaptive immune
response is increased by a 1000-fold factor. In another embodiment,
the adaptive immune response is increased by a 2000-fold factor. In
another embodiment, the adaptive immune response is increased by
another fold difference.
[0265] In another embodiment, "induces significantly more adaptive
immune response" refers to a detectable increase in an adaptive
immune response. In another embodiment, the term refers to a fold
increase in the adaptive immune response (e.g., 1 of the fold
increases enumerated above). In another embodiment, the term refers
to an increase such that the nucleoside-modified RNA can be
administered at a lower dose or frequency than an unmodified RNA
molecule with the same species while still inducing an effective
adaptive immune response. In another embodiment, the increase is
such that the nucleoside-modified RNA can be administered using a
single dose to induce an effective adaptive immune response.
[0266] In another embodiment, the nucleoside-modified RNA of the
present invention exhibits significantly less innate immunogenicity
than an unmodified in vitro-synthesized RNA molecule with the same
sequence. In another embodiment, the modified RNA molecule exhibits
an innate immune response that is 2-fold less than its unmodified
counterpart. In another embodiment, innate immunogenicity is
reduced by a 3-fold factor. In another embodiment, innate
immunogenicity is reduced by a 5-fold factor. In another
embodiment, innate immunogenicity is reduced by a 7-fold factor. In
another embodiment, innate immunogenicity is reduced by a 10-fold
factor. In another embodiment, innate immunogenicity is reduced by
a 15-fold factor. In another embodiment, innate immunogenicity is
reduced by a 20-fold factor. In another embodiment, innate
immunogenicity is reduced by a 50-fold factor. In another
embodiment, innate immunogenicity is reduced by a 100-fold factor.
In another embodiment, innate immunogenicity is reduced by a
200-fold factor. In another embodiment, innate immunogenicity is
reduced by a 500-fold factor. In another embodiment, innate
immunogenicity is reduced by a 1000-fold factor. In another
embodiment, innate immunogenicity is reduced by a 2000-fold factor.
In another embodiment, innate immunogenicity is reduced by another
fold difference.
[0267] In another embodiment, "exhibits significantly less innate
immunogenicity" refers to a detectable decrease in innate
immunogenicity. In another embodiment, the term refers to a fold
decrease in innate immunogenicity (e.g., 1 of the fold decreases
enumerated above). In another embodiment, the term refers to a
decrease such that an effective amount of the nucleoside-modified
RNA can be administered without triggering a detectable innate
immune response. In another embodiment, the term refers to a
decrease such that the nucleoside-modified RNA can be repeatedly
administered without eliciting an innate immune response sufficient
to detectably reduce production of the recombinant protein. In
another embodiment, the decrease is such that the
nucleoside-modified RNA can be repeatedly administered without
eliciting an innate immune response sufficient to eliminate
detectable production of the recombinant protein.
Lipid Nanoparticle
[0268] In one embodiment, delivery of nucleoside-modified RNA
comprises any suitable delivery method, including exemplary RNA
transfection methods described elsewhere herein. In certain
embodiments, delivery of a nucleoside-modified RNA to a subject
comprises mixing the nucleoside-modified RNA with a transfection
reagent prior to the step of contacting. In another embodiment, a
method of present invention further comprises administering
nucleoside-modified RNA together with the transfection reagent. In
another embodiment, the transfection reagent is a cationic lipid
reagent.
[0269] In another embodiment, the transfection reagent is a
lipid-based transfection reagent. In another embodiment, the
transfection reagent is a protein-based transfection reagent. In
another embodiment, the transfection reagent is a polyethyleneimine
based transfection reagent. In another embodiment, the transfection
reagent is calcium phosphate. In another embodiment, the
transfection reagent is Lipofectin.RTM., Lipofectamine.RTM., or
TransIT.RTM.. In another embodiment, the transfection reagent is
any other transfection reagent known in the art.
[0270] In another embodiment, the transfection reagent forms a
liposome. Liposomes, in another embodiment, increase intracellular
stability, increase uptake efficiency and improve biological
activity. In another embodiment, liposomes are hollow spherical
vesicles composed of lipids arranged in a similar fashion as those
lipids which make up the cell membrane. They have, in another
embodiment, an internal aqueous space for entrapping water-soluble
compounds and range in size from 0.05 to several microns in
diameter. In another embodiment, liposomes can deliver RNA to cells
in a biologically active form.
[0271] In one embodiment, the composition comprises a lipid
nanoparticle (LNP) and one or more nucleic acid molecules described
herein. For example, in one embodiment, the composition comprises
an LNP and one or more nucleoside-modified RNA molecules encoding
one or more antigens, adjuvants, or a combination thereof.
[0272] The term "lipid nanoparticle" refers to a particle having at
least one dimension on the order of nanometers (e.g., 1-1,000 nm)
which includes one or more lipids, for example a lipid of Formula
(I), (II) or (III). In some embodiments, lipid nanoparticles are
included in a formulation comprising a nucleoside-modified RNA as
described herein. In some embodiments, such lipid nanoparticles
comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or
(III)) and one or more excipient selected from neutral lipids,
charged lipids, steroids and polymer conjugated lipids (e.g., a
pegylated lipid such as a pegylated lipid of structure (IV), such
as compound IVa). In some embodiments, the nucleoside-modified RNA
is encapsulated in the lipid portion of the lipid nanoparticle or
an aqueous space enveloped by some or all of the lipid portion of
the lipid nanoparticle, thereby protecting it from enzymatic
degradation or other undesirable effects induced by the mechanisms
of the host organism or cells e.g. an adverse immune response.
[0273] In various embodiments, the lipid nanoparticles have a mean
diameter of from about 30 nm to about 150 nm, from about 40 nm to
about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to
about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to
about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to
about 100 nm, from about 70 to about 90 nm, from about 80 nm to
about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35
nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm,
85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125
nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are
substantially non-toxic. In certain embodiments, the
nucleoside-modified RNA, when present in the lipid nanoparticles,
is resistant in aqueous solution to degradation with a
nuclease.
[0274] The LNP may comprise any lipid capable of forming a particle
to which the one or more nucleic acid molecules are attached, or in
which the one or more nucleic acid molecules are encapsulated. The
term "lipid" refers to a group of organic compounds that are
derivatives of fatty acids (e.g., esters) and are generally
characterized by being insoluble in water but soluble in many
organic solvents. Lipids are usually divided in at least three
classes: (1) "simple lipids" which include fats and oils as well as
waxes; (2) "compound lipids" which include phospholipids and
glycolipids; and (3) "derived lipids" such as steroids.
[0275] In one embodiment, the LNP comprises one or more cationic
lipids, and one or more stabilizing lipids. Stabilizing lipids
include neutral lipids and pegylated lipids.
[0276] In one embodiment, the LNP comprises a cationic lipid. As
used herein, the term "cationic lipid" refers to a lipid that is
cationic or becomes cationic (protonated) as the pH is lowered
below the pK of the ionizable group of the lipid, but is
progressively more neutral at higher pH values. At pH values below
the pK, the lipid is then able to associate with negatively charged
nucleic acids. In certain embodiments, the cationic lipid comprises
a zwitterionic lipid that assumes a positive charge on pH
decrease.
[0277] In certain embodiments, the cationic lipid comprises any of
a number of lipid species which carry a net positive charge at a
selective pH, such as physiological pH. Such lipids include, but
are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride
(DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP); 3-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
(DC-Chol),
N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl
carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane
(DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). Additionally, a number of commercial preparations
of cationic lipids are available which can be used in the present
invention. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand
Island, N.Y.); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from
Promega Corp., Madison, Wis.). The following lipids are cationic
and have a positive charge at below physiological pH: DODAP, DODMA,
DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
[0278] In one embodiment, the cationic lipid is an amino lipid.
Suitable amino lipids useful in the invention include those
described in WO 2012/016184, incorporated herein by reference in
its entirety. Representative amino lipids include, but are not
limited to, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane
(DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanediol (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), and
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA).
[0279] Suitable amino lipids include those having the formula:
##STR00015##
[0280] wherein R.sub.1 and R.sub.2 are either the same or different
and independently optionally substituted C.sub.10-C.sub.24 alkyl,
optionally substituted C.sub.10-C.sub.24 alkenyl, optionally
substituted C.sub.10-C.sub.24 alkynyl, or optionally substituted
C.sub.10-C.sub.24 acyl;
[0281] R.sub.3 and R.sub.4 are either the same or different and
independently optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, or optionally
substituted C.sub.2-C.sub.6 alkynyl or R.sub.3 and R.sub.4 may join
to form an optionally substituted heterocyclic ring of 4 to 6
carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and
oxygen;
[0282] R.sub.5 is either absent or present and when present is
hydrogen or C.sub.1-C.sub.6 alkyl;
[0283] m, n, and p are either the same or different and
independently either 0 or 1 with the proviso that m, n, and p are
not simultaneously 0;
[0284] q is 0, 1, 2, 3, or 4; and
[0285] Y and Z are either the same or different and independently
O, S, or NH.
[0286] In one embodiment, R.sub.1 and R.sub.2 are each linoleyl,
and the amino lipid is a dilinoleyl amino lipid. In one embodiment,
the amino lipid is a dilinoleyl amino lipid.
[0287] A representative useful dilinoleyl amino lipid has the
formula:
##STR00016##
[0288] wherein n is 0, 1, 2, 3, or 4.
[0289] In one embodiment, the cationic lipid is a DLin-K-DMA. In
one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA
above, wherein n is 2).
[0290] In one embodiment, the cationic lipid component of the LNPs
has the structure of Formula (I):
##STR00017##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0291] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O-- or a carbon-carbon double bond;
[0292] R.sup.1a and R.sup.1b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0293] R.sup.2a and R.sup.2b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0294] R.sup.3a and R.sup.3b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0295] R.sup.4a and R.sup.4b are, at each occurrence, independently
either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0296] R.sup.5 and R.sup.6 are each independently methyl or
cycloalkyl;
[0297] R.sup.7 is, at each occurrence, independently H or
C.sub.1-C.sub.12 alkyl;
[0298] R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12
alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to
which they are attached, form a 5, 6 or 7-membered heterocyclic
ring comprising one nitrogen atom;
[0299] a and d are each independently an integer from 0 to 24;
[0300] b and c are each independently an integer from 1 to 24;
and
[0301] e is 1 or 2.
[0302] In certain embodiments of Formula (I), at least one of
R.sup.1a, R.sup.2a, R.sup.3a or R.sup.4a is C.sub.1-C.sub.12 alkyl,
or at least one of L.sup.1 or L.sup.2 is --O(C.dbd.O)-- or
--(C.dbd.O)O--. In other embodiments, R.sup.1a and R.sup.1b are not
isopropyl when a is 6 or n-butyl when a is 8.
[0303] In still further embodiments of Formula (I), at least one of
R.sup.1a, R.sup.2a, R.sup.3a or R.sup.4a is C.sub.1-C.sub.12 alkyl,
or at least one of L.sup.1 or L.sup.2 is --O(C.dbd.O)-- or
--(C.dbd.O)O--; and
[0304] R.sup.1a and R.sup.1b are not isopropyl when a is 6 or
n-butyl when a is 8.
[0305] In other embodiments of Formula (I), R.sup.8 and R.sup.9 are
each independently unsubstituted C.sub.1-C.sub.12 alkyl; or R.sup.8
and R.sup.9, together with the nitrogen atom to which they are
attached, form a 5, 6 or 7-membered heterocyclic ring comprising
one nitrogen atom;
[0306] In certain embodiments of Formula (I), any one of L.sup.1 or
L.sup.2 may be --O(C.dbd.O)-- or a carbon-carbon double bond.
L.sup.1 and L.sup.2 may each be --O(C.dbd.O)-- or may each be a
carbon-carbon double bond.
[0307] In some embodiments of Formula (I), one of L.sup.1 or
L.sup.2 is --O(C.dbd.O)--. In other embodiments, both L.sup.1 and
L.sup.2 are --O(C.dbd.O)--.
[0308] In some embodiments of Formula (I), one of L.sup.1 or
L.sup.2 is --(C.dbd.O)O--. In other embodiments, both L.sup.1 and
L.sup.2 are --(C.dbd.O)O--.
[0309] In some other embodiments of Formula (I), one of L.sup.1 or
L.sup.2 is a carbon-carbon double bond. In other embodiments, both
L.sup.1 and L.sup.2 are a carbon-carbon double bond.
[0310] In still other embodiments of Formula (I), one of L.sup.1 or
L.sup.2 is --O(C.dbd.O)-- and the other of L.sup.1 or L.sup.2 is
--(C.dbd.O)O--. In more embodiments, one of L.sup.1 or L.sup.2 is
--O(C.dbd.O)-- and the other of L.sup.1 or L.sup.2 is a
carbon-carbon double bond. In yet more embodiments, one of L.sup.1
or L.sup.2 is --(C.dbd.O)O-- and the other of L.sup.1 or L.sup.2 is
a carbon-carbon double bond.
[0311] It is understood that "carbon-carbon" double bond, as used
throughout the specification, refers to one of the following
structures:
##STR00018##
wherein R.sup.a and R.sup.b are, at each occurrence, independently
H or a substituent. For example, in some embodiments R.sup.a and
R.sup.b are, at each occurrence, independently H, C.sub.1-C.sub.12
alkyl or cycloalkyl, for example H or C.sub.1-C.sub.12 alkyl.
[0312] In other embodiments, the lipid compounds of Formula (I)
have the following structure (Ia).
##STR00019##
[0313] In other embodiments, the lipid compounds of Formula (I)
have the following structure (Ib):
##STR00020##
[0314] In yet other embodiments, the lipid compounds of Formula (I)
have the following structure (Ic):
##STR00021##
[0315] In certain embodiments of the lipid compound of Formula (I),
a, b, c and d are each independently an integer from 2 to 12 or an
integer from 4 to 12. In other embodiments, a, b, c and d are each
independently an integer from 8 to 12 or 5 to 9. In some certain
embodiments, a is 0. In some embodiments, a is 1. In other
embodiments, a is 2. In more embodiments, a is 3. In yet other
embodiments, a is 4. In some embodiments, a is 5. In other
embodiments, a is 6. In more embodiments, a is 7. In yet other
embodiments, a is 8. In some embodiments, a is 9. In other
embodiments, a is 10. In more embodiments, a is 11. In yet other
embodiments, a is 12. In some embodiments, a is 13. In other
embodiments, a is 14. In more embodiments, a is 15. In yet other
embodiments, a is 16.
[0316] In some other embodiments of Formula (I), b is 1. In other
embodiments, b is 2. In more embodiments, b is 3. In yet other
embodiments, b is 4. In some embodiments, b is 5. In other
embodiments, b is 6. In more embodiments, b is 7. In yet other
embodiments, b is 8. In some embodiments, b is 9. In other
embodiments, b is 10. In more embodiments, b is 11. In yet other
embodiments, b is 12. In some embodiments, b is 13. In other
embodiments, b is 14. In more embodiments, b is 15. In yet other
embodiments, b is 16.
[0317] In some more embodiments of Formula (I), c is 1. In other
embodiments, c is 2. In more embodiments, c is 3. In yet other
embodiments, c is 4. In some embodiments, c is 5. In other
embodiments, c is 6. In more embodiments, c is 7. In yet other
embodiments, c is 8. In some embodiments, c is 9. In other
embodiments, c is 10. In more embodiments, c is 11. In yet other
embodiments, c is 12. In some embodiments, c is 13. In other
embodiments, c is 14. In more embodiments, c is 15. In yet other
embodiments, c is 16.
[0318] In some certain other embodiments of Formula (I), d is 0. In
some embodiments, d is 1. In other embodiments, d is 2. In more
embodiments, d is 3. In yet other embodiments, d is 4. In some
embodiments, d is 5. In other embodiments, d is 6. In more
embodiments, d is 7. In yet other embodiments, d is 8. In some
embodiments, d is 9. In other embodiments, d is 10. In more
embodiments, d is 11. In yet other embodiments, d is 12. In some
embodiments, d is 13. In other embodiments, d is 14. In more
embodiments, d is 15. In yet other embodiments, d is 16.
[0319] In some other various embodiments of Formula (I), a and d
are the same. In some other embodiments, b and c are the same. In
some other specific embodiments, a and d are the same and b and c
are the same.
[0320] The sum of a and b and the sum of c and d in Formula (I) are
factors which may be varied to obtain a lipid of Formula (I) having
the desired properties. In one embodiment, a and b are chosen such
that their sum is an integer ranging from 14 to 24. In other
embodiments, c and d are chosen such that their sum is an integer
ranging from 14 to 24. In further embodiment, the sum of a and b
and the sum of c and d are the same. For example, in some
embodiments the sum of a and b and the sum of c and d are both the
same integer which may range from 14 to 24. In still more
embodiments, a. b, c and d are selected such the sum of a and b and
the sum of c and d is 12 or greater.
[0321] In some embodiments of Formula (I), e is 1. In other
embodiments, e is 2.
[0322] The substituents at R.sup.1a, R.sup.2a, R.sup.3a and
R.sup.4a of Formula (I) are not particularly limited. In certain
embodiments R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a are H at each
occurrence. In certain other embodiments at least one of R.sup.1a,
R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.12 alkyl. In
certain other embodiments at least one of R.sup.1a, R.sup.2a,
R.sup.3a and R.sup.4a is C.sub.1-C.sub.8 alkyl. In certain other
embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and
R.sup.4a is C.sub.1-C.sub.6 alkyl. In some of the foregoing
embodiments, the C.sub.1-C.sub.5 alkyl is methyl, ethyl, n-propyl,
iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
[0323] In certain embodiments of Formula (I), R.sup.1a, R.sup.1b,
R.sup.4a and R.sup.4b are C.sub.1-C.sub.12 alkyl at each
occurrence.
[0324] In further embodiments of Formula (I), at least one of
R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b is H or R.sup.1b,
R.sup.2b, R.sup.3b and R.sup.4b are H at each occurrence.
[0325] In certain embodiments of Formula (I), R.sup.1b together
with the carbon atom to which it is bound is taken together with an
adjacent R.sup.1b and the carbon atom to which it is bound to form
a carbon-carbon double bond. In other embodiments of the foregoing
R.sup.4b together with the carbon atom to which it is bound is
taken together with an adjacent R.sup.4b and the carbon atom to
which it is bound to form a carbon-carbon double bond.
[0326] The substituents at R.sup.5 and R.sup.6 of Formula (I) are
not particularly limited in the foregoing embodiments. In certain
embodiments one or both of R.sup.5 or R.sup.6 is methyl. In certain
other embodiments one or both of R.sup.5 or R.sup.6 is cycloalkyl
for example cyclohexyl. In these embodiments the cycloalkyl may be
substituted or not substituted. In certain other embodiments the
cycloalkyl is substituted with C.sub.1-C.sub.12 alkyl, for example
tert-butyl.
[0327] The substituents at R.sup.7 are not particularly limited in
the foregoing embodiments of Formula (I). In certain embodiments at
least one R.sup.7 is H. In some other embodiments, R.sup.7 is H at
each occurrence. In certain other embodiments R.sup.7 is
C.sub.1-C.sub.12 alkyl.
[0328] In certain other of the foregoing embodiments of Formula
(I), one of R.sup.8 or R.sup.9 is methyl. In other embodiments,
both R.sup.8 and R.sup.9 are methyl.
[0329] In some different embodiments of Formula (I), R.sup.8 and
R.sup.9, together with the nitrogen atom to which they are
attached, form a 5, 6 or 7-membered heterocyclic ring. In some
embodiments of the foregoing, R.sup.8 and R.sup.9, together with
the nitrogen atom to which they are attached, form a 5-membered
heterocyclic ring, for example a pyrrolidinyl ring.
[0330] In various different embodiments, the lipid of Formula (I)
has one of the structures set forth in Table 1 below.
TABLE-US-00001 TABLE 1 Representative Lipids of Formula (I) Prep.
No. Structure Method I-1 ##STR00022## B I-2 ##STR00023## A I-3
##STR00024## A I-4 ##STR00025## B I-5 ##STR00026## B I-6
##STR00027## B I-7 ##STR00028## A I-8 ##STR00029## A I-9
##STR00030## B I-10 ##STR00031## A I-11 ##STR00032## A I-12
##STR00033## A I-13 ##STR00034## A I-14 ##STR00035## A I-15
##STR00036## A I-16 ##STR00037## A I-17 ##STR00038## A I-18
##STR00039## A I-19 ##STR00040## A I-20 ##STR00041## A I-21
##STR00042## A I-22 ##STR00043## A I-23 ##STR00044## A I-24
##STR00045## A I-25 ##STR00046## A I-26 ##STR00047## A I-27
##STR00048## A I-28 ##STR00049## A I-29 ##STR00050## A I-30
##STR00051## A I-31 ##STR00052## C I-32 ##STR00053## C I-33
##STR00054## C I-34 ##STR00055## B I-35 ##STR00056## B I-36
##STR00057## C I-37 ##STR00058## C I-38 ##STR00059## B I-39
##STR00060## B I-40 ##STR00061## B I-41 ##STR00062## B
[0331] In some embodiments, the LNPs comprise a lipid of Formula
(I), a nucleoside-modified RNA and one or more excipients selected
from neutral lipids, steroids and pegylated lipids. In some
embodiments the lipid of Formula (I) is compound I-5. In some
embodiments the lipid of Formula (I) is compound I-6.
[0332] In some other embodiments, the cationic lipid component of
the LNPs has the structure of Formula (II):
##STR00063##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0333] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--,
--C(.dbd.O)S--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a,
--OC(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)O--, or a direct
bond;
[0334] G.sup.1 is C.sub.1-C.sub.2 alkylene, --(C.dbd.O)--,
--O(C.dbd.O)--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)-- or a direct
bond;
[0335] G.sup.2 is --C(.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)S--,
--C(.dbd.O)NR.sup.a or a direct bond;
[0336] G.sup.3 is C.sub.1-C.sub.6 alkylene;
[0337] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0338] R.sup.1a and R.sup.1b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0339] R.sup.2a and R.sup.2b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0340] R.sup.3a and R.sup.3b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0341] R.sup.4a and R.sup.4b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0342] R.sup.5 and R.sup.6 are each independently H or methyl;
[0343] R.sup.7 is C.sub.4-C.sub.20 alkyl;
[0344] R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12
alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to
which they are attached, form a 5, 6 or 7-membered heterocyclic
ring;
[0345] a, b, c and d are each independently an integer from 1 to
24; and
[0346] x is 0, 1 or 2.
[0347] In some embodiments of Formula (II), L.sup.1 and L.sup.2 are
each independently --O(C.dbd.O)--, --(C.dbd.O)O-- or a direct bond.
In other embodiments, G.sup.1 and G.sup.2 are each independently
--(C.dbd.O)-- or a direct bond. In some different embodiments,
L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O-- or a direct bond; and G.sup.1 and G.sup.2 are each
independently --(C.dbd.O)-- or a direct bond.
[0348] In some different embodiments of Formula (II), L.sup.1 and
L.sup.2 are each independently --C(.dbd.O)--, --O--,
--S(O).sub.x--, --S--S--, --C(.dbd.O)S--, --SC(.dbd.O)--,
--NR.sup.a--, --NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)NR.sup.a, --OC(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)O--, --NR.sup.aS(O).sub.xNR.sup.a--,
--NR.sup.aS(O).sub.x-- or --S(O).sub.xNR.sup.a--.
[0349] In other of the foregoing embodiments of Formula (II), the
lipid compound has one of the following structures (IIA) or
(IIB).
##STR00064##
[0350] In some embodiments of Formula (II), the lipid compound has
structure (IIA). In other embodiments, the lipid compound has
structure (IIB).
[0351] In any of the foregoing embodiments of Formula (II), one of
L.sup.1 or L.sup.2 is --O(C.dbd.O)--. For example, in some
embodiments each of L.sup.1 and L.sup.2 are --O(C.dbd.O)--.
[0352] In some different embodiments of Formula (II), one of
L.sup.1 or L.sup.2 is --(C.dbd.O)O--. For example, in some
embodiments each of L.sup.1 and L.sup.2 is --(C.dbd.O)O--.
[0353] In different embodiments of Formula (II), one of L.sup.1 or
L.sup.2 is a direct bond. As used herein, a "direct bond" means the
group (e.g., L.sup.1 or L.sup.2) is absent. For example, in some
embodiments each of L.sup.1 and L.sup.2 is a direct bond.
[0354] In other different embodiments of Formula (II), for at least
one occurrence of R.sup.1a and R.sup.1b, R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0355] In still other different embodiments of Formula (II), for at
least one occurrence of R.sup.4a and R.sup.4b, R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0356] In more embodiments of Formula (II), for at least one
occurrence of R.sup.2a and R.sup.2b, R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0357] In other different embodiments of Formula (II), for at least
one occurrence of R.sup.3a and R.sup.3b, R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0358] In various other embodiments of Formula (II), the lipid
compound has one of the following structures (IIC) or (IID).
##STR00065##
wherein e, f, g and h are each independently an integer from 1 to
12.
[0359] In some embodiments of Formula (II), the lipid compound has
structure (IIC). In other embodiments, the lipid compound has
structure (IID).
[0360] In various embodiments of structures (IIC) or (IID), e, f, g
and h are each independently an integer from 4 to 10.
[0361] In certain embodiments of Formula (II), a, b, c and d are
each independently an integer from 2 to 12 or an integer from 4 to
12. In other embodiments, a, b, c and d are each independently an
integer from 8 to 12 or 5 to 9. In some certain embodiments, a is
0. In some embodiments, a is 1. In other embodiments, a is 2. In
more embodiments, a is 3. In yet other embodiments, a is 4. In some
embodiments, a is 5. In other embodiments, a is 6. In more
embodiments, a is 7. In yet other embodiments, a is 8. In some
embodiments, a is 9. In other embodiments, a is 10. In more
embodiments, a is 11. In yet other embodiments, a is 12. In some
embodiments, a is 13. In other embodiments, a is 14. In more
embodiments, a is 15. In yet other embodiments, a is 16.
[0362] In some embodiments of Formula (II), b is 1. In other
embodiments, b is 2. In more embodiments, b is 3. In yet other
embodiments, b is 4. In some embodiments, b is 5. In other
embodiments, b is 6. In more embodiments, b is 7. In yet other
embodiments, b is 8. In some embodiments, b is 9. In other
embodiments, b is 10. In more embodiments, b is 11. In yet other
embodiments, b is 12. In some embodiments, b is 13. In other
embodiments, b is 14. In more embodiments, b is 15. In yet other
embodiments, b is 16.
[0363] In some embodiments of Formula (II), c is 1. In other
embodiments, c is 2. In more embodiments, c is 3. In yet other
embodiments, c is 4. In some embodiments, c is 5. In other
embodiments, c is 6. In more embodiments, c is 7. In yet other
embodiments, c is 8. In some embodiments, c is 9. In other
embodiments, c is 10. In more embodiments, c is 11. In yet other
embodiments, c is 12. In some embodiments, c is 13. In other
embodiments, c is 14. In more embodiments, c is 15. In yet other
embodiments, c is 16.
[0364] In some certain embodiments of Formula (II), d is 0. In some
embodiments, d is 1. In other embodiments, d is 2. In more
embodiments, d is 3. In yet other embodiments, d is 4. In some
embodiments, d is 5. In other embodiments, d is 6. In more
embodiments, d is 7. In yet other embodiments, d is 8. In some
embodiments, d is 9. In other embodiments, d is 10. In more
embodiments, d is 11. In yet other embodiments, d is 12. In some
embodiments, d is 13. In other embodiments, d is 14. In more
embodiments, d is 15. In yet other embodiments, d is 16.
[0365] In some embodiments of Formula (II), e is 1. In other
embodiments, e is 2. In more embodiments, e is 3. In yet other
embodiments, e is 4. In some embodiments, e is 5. In other
embodiments, e is 6. In more embodiments, e is 7. In yet other
embodiments, e is 8. In some embodiments, e is 9. In other
embodiments, e is 10. In more embodiments, e is 11. In yet other
embodiments, e is 12.
[0366] In some embodiments of Formula (II), f is 1. In other
embodiments, f is 2. In more embodiments, f is 3. In yet other
embodiments, f is 4. In some embodiments, f is 5. In other
embodiments, f is 6. In more embodiments, f is 7. In yet other
embodiments, f is 8. In some embodiments, f is 9. In other
embodiments, f is 10. In more embodiments, f is 11. In yet other
embodiments, f is 12.
[0367] In some embodiments of Formula (II), g is 1. In other
embodiments, g is 2. In more embodiments, g is 3. In yet other
embodiments, g is 4. In some embodiments, g is 5. In other
embodiments, g is 6. In more embodiments, g is 7. In yet other
embodiments, g is 8. In some embodiments, g is 9. In other
embodiments, g is 10. In more embodiments, g is 11. In yet other
embodiments, g is 12.
[0368] In some embodiments of Formula (II), h is 1. In other
embodiments, e is 2. In more embodiments, h is 3. In yet other
embodiments, h is 4. In some embodiments, e is 5. In other
embodiments, h is 6. In more embodiments, h is 7. In yet other
embodiments, h is 8. In some embodiments, h is 9. In other
embodiments, h is 10. In more embodiments, h is 11. In yet other
embodiments, h is 12.
[0369] In some other various embodiments of Formula (II), a and d
are the same. In some other embodiments, b and c are the same. In
some other specific embodiments and a and d are the same and b and
c are the same.
[0370] The sum of a and b and the sum of c and d of Formula (II)
are factors which may be varied to obtain a lipid having the
desired properties. In one embodiment, a and b are chosen such that
their sum is an integer ranging from 14 to 24. In other
embodiments, c and d are chosen such that their sum is an integer
ranging from 14 to 24. In further embodiment, the sum of a and b
and the sum of c and d are the same. For example, in some
embodiments the sum of a and b and the sum of c and d are both the
same integer which may range from 14 to 24. In still more
embodiments, a. b, c and d are selected such that the sum of a and
b and the sum of c and d is 12 or greater.
[0371] The substituents at R.sup.1a, R.sup.2a, R.sup.3a and
R.sup.4a of Formula (II) are not particularly limited. In some
embodiments, at least one of R.sup.1a, R.sup.2a, R.sup.3a and
R.sup.4a is H. In certain embodiments R.sup.1a, R.sup.2a, R.sup.3a
and R.sup.4a are H at each occurrence. In certain other embodiments
at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is
C.sub.1-C.sub.12 alkyl. In certain other embodiments at least one
of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.8
alkyl. In certain other embodiments at least one of R.sup.1a,
R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.6 alkyl. In some
of the foregoing embodiments, the C.sub.1-C.sub.8 alkyl is methyl,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl,
n-hexyl or n-octyl.
[0372] In certain embodiments of Formula (II), R.sup.1a, R.sup.1b,
R.sup.4a and R.sup.4b are C.sub.1-C.sub.12 alkyl at each
occurrence.
[0373] In further embodiments of Formula (II), at least one of
R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b is H or R.sup.1b,
R.sup.2b, R.sup.3b and R.sup.4b are H at each occurrence.
[0374] In certain embodiments of Formula (II), R.sup.1b together
with the carbon atom to which it is bound is taken together with an
adjacent R.sup.1b and the carbon atom to which it is bound to form
a carbon-carbon double bond. In other embodiments of the foregoing
R.sup.4b together with the carbon atom to which it is bound is
taken together with an adjacent R.sup.4b and the carbon atom to
which it is bound to form a carbon-carbon double bond.
[0375] The substituents at R.sup.5 and R.sup.6 of Formula (II) are
not particularly limited in the foregoing embodiments. In certain
embodiments one of R.sup.5 or R.sup.6 is methyl. In other
embodiments each of R.sup.5 or R.sup.6 is methyl.
[0376] The substituents at R.sup.7 of Formula (II) are not
particularly limited in the foregoing embodiments. In certain
embodiments R.sup.7 is C.sub.6-C.sub.16 alkyl. In some other
embodiments, R.sup.7 is C.sub.6-C.sub.9 alkyl. In some of these
embodiments, R.sup.7 is substituted with --(C.dbd.O)OR.sup.b,
--O(C.dbd.O)R.sup.b, --C(.dbd.O)R.sup.b, --OR.sup.b,
--S(O).sub.xR.sup.b, --S--SR.sup.b, --C(.dbd.O)SR.sup.b,
--SC(.dbd.O)R.sup.b, --NR.sup.aR.sup.b, --NR.sup.aC(.dbd.O)R.sup.b,
--C(.dbd.O)NR.sup.aR.sup.b, --NR.sup.aC(.dbd.O)NR.sup.aR.sup.b,
--OC(.dbd.O)NR.sup.aR.sup.b, --NR.sup.aC(.dbd.O)OR.sup.b,
--NR.sup.aS(O).sub.xNR.sup.aR.sup.b, --NR.sup.aS(O).sub.xR.sup.b or
--S(O).sub.xNR.sup.aR.sup.b, wherein: R.sup.a is H or
C.sub.1-C.sub.12 alkyl; R.sup.b is C.sub.1-C.sub.15 alkyl; and x is
0, 1 or 2. For example, in some embodiments R.sup.7 is substituted
with --(C.dbd.O)OR.sup.b or --O(C.dbd.O)R.sup.b.
[0377] In various of the foregoing embodiments of Formula (II),
R.sup.b is branched C.sub.1-C.sub.15 alkyl. For example, in some
embodiments R.sup.b has one of the following structures:
##STR00066##
[0378] In certain other of the foregoing embodiments of Formula
(II), one of R.sup.8 or R.sup.9 is methyl. In other embodiments,
both R.sup.8 and R.sup.9 are methyl.
[0379] In some different embodiments of Formula (II), R.sup.8 and
R.sup.9, together with the nitrogen atom to which they are
attached, form a 5, 6 or 7-membered heterocyclic ring. In some
embodiments of the foregoing, R.sup.8 and R.sup.9, together with
the nitrogen atom to which they are attached, form a 5-membered
heterocyclic ring, for example a pyrrolidinyl ring. In some
different embodiments of the foregoing, R.sup.8 and R.sup.9,
together with the nitrogen atom to which they are attached, form a
6-membered heterocyclic ring, for example a piperazinyl ring.
[0380] In still other embodiments of the foregoing lipids of
Formula (II), G.sup.3 is C.sub.2-C.sub.4 alkylene, for example
C.sub.3 alkylene.
[0381] In various different embodiments, the lipid compound has one
of the structures set forth in Table 2 below.
TABLE-US-00002 TABLE 2 Representative Lipids of Formula (II) Prep.
No. Structure Method II-1 ##STR00067## D II-2 ##STR00068## D II-3
##STR00069## D II-4 ##STR00070## E II-5 ##STR00071## D II-6
##STR00072## D II-7 ##STR00073## D II-8 ##STR00074## D II-9
##STR00075## D II-10 ##STR00076## D II-11 ##STR00077## D II-12
##STR00078## D II-13 ##STR00079## D II-14 ##STR00080## D II-15
##STR00081## D II-16 ##STR00082## E II-17 ##STR00083## D II-18
##STR00084## D II-19 ##STR00085## D II-20 ##STR00086## D II-21
##STR00087## D II-22 ##STR00088## D II-23 ##STR00089## D II-24
##STR00090## D II-25 ##STR00091## E II-26 ##STR00092## E II-27
##STR00093## E II-28 ##STR00094## E II-29 ##STR00095## E II-30
##STR00096## D II-31 ##STR00097## E II-32 ##STR00098## E II-33
##STR00099## E II-34 ##STR00100## E
[0382] In some embodiments, the LNPs comprise a lipid of Formula
(II), a nucleoside-modified RNA and one or more excipient selected
from neutral lipids, steroids and pegylated lipids. In some
embodiments the lipid of Formula (II) is compound II-9. In some
embodiments the lipid of Formula (II) is compound II-10. In some
embodiments the lipid of Formula (II) is compound II-11. In some
embodiments the lipid of Formula (II) is compound II-12. In some
embodiments the lipid of Formula (II) is compound II-32.
[0383] In some other embodiments, the cationic lipid component of
the LNPs has the structure of Formula (III):
##STR00101##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0384] one of L.sup.1 or L.sup.2 is --O(C.dbd.O)--, --(C.dbd.O)O--,
--C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--, --C(.dbd.O)S--,
SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O--, and the other of L.sup.1 or L.sup.2 is
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--,
--S(O).sub.x--, --S--S--, --C(.dbd.O)S--, SC(.dbd.O)--,
--NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O-- or a direct bond;
[0385] G.sup.1 and G.sup.2 are each independently unsubstituted
C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene;
[0386] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24
alkenylene, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8
cycloalkenylene;
[0387] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0388] R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24
alkyl or C.sub.6-C.sub.24 alkenyl;
[0389] R.sup.3 is H, OR.sup.5, CN, --C(.dbd.O)OR.sup.4,
--OC(.dbd.O)R.sup.4 or --NR.sup.5C(.dbd.O)R.sup.4;
[0390] R.sup.4 is C.sub.1-C.sub.12 alkyl;
[0391] R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and
[0392] x is 0, 1 or 2.
[0393] In some of the foregoing embodiments of Formula (III), the
lipid has one of the following structures (IIIA) or (IIIB):
##STR00102##
wherein:
[0394] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;
[0395] R.sup.6 is, at each occurrence, independently H, OH or
C.sub.1-C.sub.24 alkyl;
[0396] n is an integer ranging from 1 to 15.
[0397] In some of the foregoing embodiments of Formula (III), the
lipid has structure (IIIA), and in other embodiments, the lipid has
structure (IIIB).
[0398] In other embodiments of Formula (III), the lipid has one of
the following structures (IIIC) or (IIID):
##STR00103##
wherein y and z are each independently integers ranging from 1 to
12.
[0399] In any of the foregoing embodiments of Formula (III), one of
L.sup.1 or L.sup.2 is --O(C.dbd.O)--. For example, in some
embodiments each of L.sup.1 and L.sup.2 are --O(C.dbd.O)--. In some
different embodiments of any of the foregoing, L.sup.1 and L.sup.2
are each independently --(C.dbd.O)O-- or --O(C.dbd.O)--. For
example, in some embodiments each of L.sup.1 and L.sup.2 is
--(C.dbd.O)O--.
[0400] In some different embodiments of Formula (III), the lipid
has one of the following structures (IIIE) or (IIIF):
##STR00104##
[0401] In some of the foregoing embodiments of Formula (III), the
lipid has one of the following structures (IIIG), (IIIH), (IIII),
or (IIIJ):
##STR00105##
[0402] In some of the foregoing embodiments of Formula (III), n is
an integer ranging from 2 to 12, for example from 2 to 8 or from 2
to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some
embodiments, n is 3. In some embodiments, n is 4. In some
embodiments, n is 5. In some embodiments, n is 6.
[0403] In some other of the foregoing embodiments of Formula (III),
y and z are each independently an integer ranging from 2 to 10. For
example, in some embodiments, y and z are each independently an
integer ranging from 4 to 9 or from 4 to 6.
[0404] In some of the foregoing embodiments of Formula (III),
R.sup.6 is H. In other of the foregoing embodiments, R.sup.6 is
C.sub.1-C.sub.24 alkyl. In other embodiments, R.sup.6 is OH.
[0405] In some embodiments of Formula (III), G.sup.3 is
unsubstituted. In other embodiments, G3 is substituted. In various
different embodiments, G.sup.3 is linear C.sub.1-C.sub.24 alkylene
or linear C.sub.1-C.sub.24 alkenylene.
[0406] In some other foregoing embodiments of Formula (III),
R.sup.1 or R.sup.2, or both, is C.sub.6-C.sub.24 alkenyl. For
example, in some embodiments, R.sup.1 and R.sup.2 each,
independently have the following structure:
##STR00106##
wherein:
[0407] R.sup.7a and R.sup.7b are, at each occurrence, independently
H or C.sub.1-C.sub.12 alkyl; and
[0408] a is an integer from 2 to 12,
wherein R.sup.7a, R.sup.7b and a are each selected such that
R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon
atoms. For example, in some embodiments a is an integer ranging
from 5 to 9 or from 8 to 12.
[0409] In some of the foregoing embodiments of Formula (III), at
least one occurrence of R.sup.7a is H. For example, in some
embodiments, R.sup.7a is H at each occurrence. In other different
embodiments of the foregoing, at least one occurrence of R.sup.7b
is C.sub.1-C.sub.8 alkyl. For example, in some embodiments,
C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl,
n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
[0410] In different embodiments of Formula (III), R.sup.1 or
R.sup.2, or both, has one of the following structures:
##STR00107##
[0411] In some of the foregoing embodiments of Formula (III),
R.sup.3 is OH, CN, --C(.dbd.O)OR.sup.4, --OC(.dbd.O)R.sup.4 or
--NHC(.dbd.O)R.sup.4. In some embodiments, R.sup.4 is methyl or
ethyl.
[0412] In various different embodiments, the cationic lipid of
Formula (III) has one of the structures set forth in Table 3
below.
TABLE-US-00003 TABLE 3 Representative Compounds of Formula (III)
Prep. No. Structure Method III-1 ##STR00108## F III-2 ##STR00109##
F III-3 ##STR00110## F III-4 ##STR00111## F III-5 ##STR00112## F
III-6 ##STR00113## F III-7 ##STR00114## F III-8 ##STR00115## F
III-9 ##STR00116## F III-10 ##STR00117## F III-11 ##STR00118## F
III-12 ##STR00119## F III-13 ##STR00120## F III-14 ##STR00121## F
III-15 ##STR00122## F III-16 ##STR00123## F III-17 ##STR00124## F
III-18 ##STR00125## F III-19 ##STR00126## F III-20 ##STR00127## F
III-21 ##STR00128## F III-22 ##STR00129## F III-23 ##STR00130## F
III-24 ##STR00131## F III-25 ##STR00132## F III-26 ##STR00133## F
III-27 ##STR00134## F III-28 ##STR00135## F III-29 ##STR00136## F
III-30 ##STR00137## F III-31 ##STR00138## F III-32 ##STR00139## F
III-33 ##STR00140## F III-34 ##STR00141## F III-35 ##STR00142## F
III-36 ##STR00143## F
[0413] In some embodiments, the LNPs comprise a lipid of Formula
(III), a nucleoside-modified RNA and one or more excipient selected
from neutral lipids, steroids and pegylated lipids. In some
embodiments the lipid of Formula (III) is compound III-3. In some
embodiments the lipid of Formula (III) is compound III-7.
[0414] In certain embodiments, the cationic lipid is present in the
LNP in an amount from about 30 to about 95 mole percent. In one
embodiment, the cationic lipid is present in the LNP in an amount
from about 30 to about 70 mole percent. In one embodiment, the
cationic lipid is present in the LNP in an amount from about 40 to
about 60 mole percent. In one embodiment, the cationic lipid is
present in the LNP in an amount of about 50 mole percent. In one
embodiment, the LNP comprises only cationic lipids.
[0415] In certain embodiments, the LNP comprises one or more
additional lipids which stabilize the formation of particles during
their formation.
[0416] Suitable stabilizing lipids include neutral lipids and
anionic lipids.
[0417] The term "neutral lipid" refers to any one of a number of
lipid species that exist in either an uncharged or neutral
zwitterionic form at physiological pH. Representative neutral
lipids include diacylphosphatidylcholines,
diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro
sphingomyelins, cephalins, and cerebrosides.
[0418] Exemplary neutral lipids include, for example,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In one
embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
[0419] In some embodiments, the LNPs comprise a neutral lipid
selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various
embodiments, the molar ratio of the cationic lipid (e.g., lipid of
Formula (I)) to the neutral lipid ranges from about 2:1 to about
8:1.
[0420] In various embodiments, the LNPs further comprise a steroid
or steroid analogue. A "steroid" is a compound comprising the
following carbon skeleton:
##STR00144##
[0421] In certain embodiments, the steroid or steroid analogue is
cholesterol. In some of these embodiments, the molar ratio of the
cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges
from about 2:1 to 1:1.
[0422] The term "anionic lipid" refers to any lipid that is
negatively charged at physiological pH. These lipids include
phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,
diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines,
N-succinylphosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
[0423] In certain embodiments, the LNP comprises glycolipids (e.g.,
monosialoganglioside GM.sub.1). In certain embodiments, the LNP
comprises a sterol, such as cholesterol.
[0424] In some embodiments, the LNPs comprise a polymer conjugated
lipid. The term "polymer conjugated lipid" refers to a molecule
comprising both a lipid portion and a polymer portion. An example
of a polymer conjugated lipid is a pegylated lipid. The term
"pegylated lipid" refers to a molecule comprising both a lipid
portion and a polyethylene glycol portion. Pegylated lipids are
known in the art and include
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-s-DMG) and the like.
[0425] In certain embodiments, the LNP comprises an additional,
stabilizing-lipid which is a polyethylene glycol-lipid (pegylated
lipid). Suitable polyethylene glycol-lipids include PEG-modified
phosphatidylethanolamine, PEG-modified phosphatidic acid,
PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20),
PEG-modified dialkylamines, PEG-modified diacylglycerols,
PEG-modified dialkylglycerols. Representative polyethylene
glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one
embodiment, the polyethylene glycol-lipid is N-[(methoxy
poly(ethylene
glycol).sub.2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine
(PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is
PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated
diacylglycerol (PEG-DAG) such as
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG
succinate diacylglycerol (PEG-S-DAG) such as
4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(.omega.-methoxy(polyethoxy)eth-
yl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a
PEG dialkoxypropylcarbamate such as
.omega.-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate
or
2,3-di(tetradecanoxy)propyl-N-(.omega.-methoxy(polyethoxy)ethyl)carbam-
ate. In various embodiments, the molar ratio of the cationic lipid
to the pegylated lipid ranges from about 100:1 to about 25:1.
[0426] In some embodiments, the LNPs comprise a pegylated lipid
having the following structure (IV):
##STR00145##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein:
[0427] R.sup.10 and R.sup.11 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing from 10
to 30 carbon atoms, wherein the alkyl chain is optionally
interrupted by one or more ester bonds; and
[0428] z has mean value ranging from 30 to 60.
[0429] In some of the foregoing embodiments of the pegylated lipid
(IV), R.sup.10 and R.sup.11 are not both n-octadecyl when z is 42.
In some other embodiments, R.sup.10 and R.sup.11 are each
independently a straight or branched, saturated or unsaturated
alkyl chain containing from 10 to 18 carbon atoms. In some
embodiments, R.sup.10 and R.sup.11 are each independently a
straight or branched, saturated or unsaturated alkyl chain
containing from 12 to 16 carbon atoms. In some embodiments,
R.sup.10 and R.sup.11 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing 12 carbon
atoms. In some embodiments, R.sup.10 and R.sup.11 are each
independently a straight or branched, saturated or unsaturated
alkyl chain containing 14 carbon atoms. In other embodiments,
R.sup.10 and R.sup.11 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing 16 carbon
atoms. In still more embodiments, R.sup.10 and R.sup.11 are each
independently a straight or branched, saturated or unsaturated
alkyl chain containing 18 carbon atoms. In still other embodiments,
R.sup.10 is a straight or branched, saturated or unsaturated alkyl
chain containing 12 carbon atoms and R.sup.11 is a straight or
branched, saturated or unsaturated alkyl chain containing 14 carbon
atoms.
[0430] In various embodiments, z spans a range that is selected
such that the PEG portion of (II) has an average molecular weight
of about 400 to about 6000 g/mol. In some embodiments, the average
z is about 45.
[0431] In other embodiments, the pegylated lipid has one of the
following structures:
##STR00146##
wherein n is an integer selected such that the average molecular
weight of the pegylated lipid is about 2500 g/mol.
[0432] In certain embodiments, the additional lipid is present in
the LNP in an amount from about 1 to about 10 mole percent. In one
embodiment, the additional lipid is present in the LNP in an amount
from about 1 to about 5 mole percent. In one embodiment, the
additional lipid is present in the LNP in about 1 mole percent or
about 1.5 mole percent.
[0433] In some embodiments, the LNPs comprise a lipid of Formula
(I), a nucleoside-modified RNA, a neutral lipid, a steroid and a
pegylated lipid. In some embodiments the lipid of Formula (I) is
compound I-6. In different embodiments, the neutral lipid is DSPC.
In other embodiments, the steroid is cholesterol. In still
different embodiments, the pegylated lipid is compound IVa.
[0434] In certain embodiments, the LNP comprises one or more
targeting moieties which are capable of targeting the LNP to a cell
or cell population. For example, in one embodiment, the targeting
moiety is a ligand which directs the LNP to a receptor found on a
cell surface.
[0435] In certain embodiments, the LNP comprises one or more
internalization domains. For example, in one embodiment, the LNP
comprises one or more domains which bind to a cell to induce the
internalization of the LNP. For example, in one embodiment, the one
or more internalization domains bind to a receptor found on a cell
surface to induce receptor-mediated uptake of the LNP. In certain
embodiments, the LNP is capable of binding a biomolecule in vivo,
where the LNP-bound biomolecule can then be recognized by a
cell-surface receptor to induce internalization. For example, in
one embodiment, the LNP binds systemic ApoE, which leads to the
uptake of the LNP and associated cargo.
[0436] Other exemplary LNPs and their manufacture are described in
the art, for example in U.S. Patent Application Publication No.
US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176;
Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al.,
2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem
C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int
J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther
nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed
Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids.
2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam
et al., 2013, Nanomedicine, 9(5): 665-74, each of which are
incorporated by reference in their entirety.
[0437] The following Reaction Schemes illustrate methods to make
lipids of Formula (I), (II) or (III).
##STR00147##
[0438] Embodiments of the lipid of Formula (I) (e.g., compound A-5)
can be prepared according to General Reaction Scheme 1 ("Method
A"), wherein R is a saturated or unsaturated C.sub.1-C.sub.24 alkyl
or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an
integer from 1 to 24. Referring to General Reaction Scheme 1,
compounds of structure A-1 can be purchased from commercial sources
or prepared according to methods familiar to one of ordinary skill
in the art. A mixture of A-1, A-2 and DMAP is treated with DCC to
give the bromide A-3. A mixture of the bromide A-3, a base (e.g.,
N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is
heated at a temperature and time sufficient to produce A-5 after
any necessarily workup and or purification step.
##STR00148##
[0439] Other embodiments of the compound of Formula (I) (e.g.,
compound B-5) can be prepared according to General Reaction Scheme
2 ("Method B"), wherein R is a saturated or unsaturated
C.sub.1-C.sub.24 alkyl or saturated or unsaturated cycloalkyl, m is
0 or 1 and n is an integer from 1 to 24. As shown in General
Reaction Scheme 2, compounds of structure B-1 can be purchased from
commercial sources or prepared according to methods familiar to one
of ordinary skill in the art. A solution of B-1 (1 equivalent) is
treated with acid chloride B-2 (1 equivalent) and a base (e.g.,
triethylamine). The crude product is treated with an oxidizing
agent (e.g., pyridinium chlorochromate) and intermediate product
B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic
acid), and N,N-dimethylaminoamine B-4 is then treated with a
reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5
after any necessary work up and/or purification.
[0440] It should be noted that although starting materials A-1 and
B-1 are depicted above as including only saturated methylene
carbons, starting materials which include carbon-carbon double
bonds may also be employed for preparation of compounds which
include carbon-carbon double bonds.
##STR00149##
[0441] Different embodiments of the lipid of Formula (I) (e.g.,
compound C-7 or C.sub.9) can be prepared according to General
Reaction Scheme 3 ("Method C"), wherein R is a saturated or
unsaturated C.sub.1-C.sub.24 alkyl or saturated or unsaturated
cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring
to General Reaction Scheme 3, compounds of structure C-1 can be
purchased from commercial sources or prepared according to methods
familiar to one of ordinary skill in the art.
##STR00150##
[0442] Embodiments of the compound of Formula (II) (e.g., compounds
D-5 and D-7) can be prepared according to General Reaction Scheme 4
("Method D"), wherein R.sup.1a, R.sup.1b, R.sup.2a, R.sup.2b,
R.sup.3a, R.sup.3b, R.sup.4a, R.sup.4b, R.sup.6, R.sup.6, R.sup.8,
R.sup.9, L.sup.1, L.sup.2, G.sup.1, G.sup.2, G.sup.3, a, b, c and d
are as defined herein, and R.sup.7' represents R.sup.7 or a
C.sub.3-C.sub.19 alkyl. Referring to General Reaction Scheme 1,
compounds of structure D-1 and D-2 can be purchased from commercial
sources or prepared according to methods familiar to one of
ordinary skill in the art. A solution of D-1 and D-2 is treated
with a reducing agent (e.g., sodium triacetoxyborohydride) to
obtain D-3 after any necessary work up. A solution of D-3 and a
base (e.g. trimethylamine, DMAP) is treated with acyl chloride D-4
(or carboxylic acid and DCC) to obtain D-5 after any necessary work
up and/or purification. D-5 can be reduced with LiAlH4 D-6 to give
D-7 after any necessary work up and/or purification.
##STR00151##
[0443] Embodiments of the lipid of Formula (II) (e.g., compound
E-5) can be prepared according to General Reaction Scheme 5
("Method E"), wherein R.sup.1a, R.sup.1b, R.sup.2aR.sup.2b,
R.sup.3a, R.sup.3b, R.sup.4a, R.sup.4b, R.sup.6, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, L.sup.1, L.sup.2, G.sup.3, a, b, c and d are as
defined herein. Referring to General Reaction Scheme 2, compounds
of structure E-1 and E-2 can be purchased from commercial sources
or prepared according to methods familiar to one of ordinary skill
in the art. A mixture of E-1 (in excess), E-2 and a base (e.g.,
potassium carbonate) is heated to obtain E-3 after any necessary
work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP)
is treated with acyl chloride E-4 (or carboxylic acid and DCC) to
obtain E-5 after any necessary work up and/or purification.
##STR00152##
[0444] General Reaction Scheme 6 provides an exemplary method
(Method F) for preparation of Lipids of Formula (III). G.sup.1,
G.sup.3, R.sup.1 and R.sup.3 in General Reaction Scheme 6 are as
defined herein for Formula (III), and G1' refers to a one-carbon
shorter homologue of G1. Compounds of structure F-1 are purchased
or prepared according to methods known in the art. Reaction of F-1
with diol F-2 under appropriate condensation conditions (e.g., DCC)
yields ester/alcohol F-3, which can then be oxidized (e.g., PCC) to
aldehyde F-4. Reaction of F-4 with amine F-5 under reductive
amination conditions yields a lipid of Formula (III).
[0445] It should be noted that various alternative strategies for
preparation of lipids of Formula (III) are available to those of
ordinary skill in the art. For example, other lipids of Formula
(III) wherein L.sup.1 and L.sup.2 are other than ester can be
prepared according to analogous methods using the appropriate
starting material. Further, General Reaction Scheme 6 depicts
preparation of a lipids of Formula (III), wherein G.sup.1 and
G.sup.2 are the same; however, this is not a required aspect of the
invention and modifications to the above reaction scheme are
possible to yield compounds wherein G.sup.1 and G.sup.2 are
different.
[0446] It will be appreciated by those skilled in the art that in
the process described herein the functional groups of intermediate
compounds may need to be protected by suitable protecting groups.
Such functional groups include hydroxy, amino, mercapto and
carboxylic acid. Suitable protecting groups for hydroxy include
trialkylsilyl or diarylalkylsilyl (for example,
t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),
tetrahydropyranyl, benzyl, and the like. Suitable protecting groups
for amino, amidino and guanidino include t-butoxycarbonyl,
benzyloxycarbonyl, and the like. Suitable protecting groups for
mercapto include --C(O)--R'' (where R'' is alkyl, aryl or
arylalkyl), p-methoxybenzyl, trityl and the like. Suitable
protecting groups for carboxylic acid include alkyl, aryl or
arylalkyl esters. Protecting groups may be added or removed in
accordance with standard techniques, which are known to one skilled
in the art and as described herein. The use of protecting groups is
described in detail in Green, T. W. and P. G. M. Wutz, Protective
Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill
in the art would appreciate, the protecting group may also be a
polymer resin such as a Wang resin, Rink resin or a
2-chlorotrityl-chloride resin.
Antigen
[0447] The present invention provides a composition that induces an
adaptive immune response in a subject. In one embodiment, the
composition comprises an antigen. In one embodiment, the
composition comprises a nucleic acid sequence which encodes an
antigen. For example, in certain embodiments, the composition
comprises a nucleoside-modified RNA encoding an antigen. The
antigen may be any molecule or compound, including but not limited
to a polypeptide, peptide or protein that induces an adaptive
immune response in a subject.
[0448] In one embodiment, the antigen comprises a polypeptide or
peptide associated with a pathogen, such that the antigen induces
an adaptive immune response against the antigen, and therefore the
pathogen. In one embodiment, the antigen comprises a fragment of a
polypeptide or peptide associated with a pathogen, such that the
antigen induces an adaptive immune response against the
pathogen.
[0449] In certain embodiments, the antigen comprises an amino acid
sequence that is substantially homologous to the amino acid
sequence of an antigen described herein and retains the immunogenic
function of the original amino acid sequence. For example, in
certain embodiments, the amino acid sequence of the antigen has a
degree of identity with respect to the original amino acid sequence
of at least 60%, advantageously of at least 70%, preferably of at
least 85%, and more preferably of at least 95%.
[0450] In one embodiment, the antigen is encoded by a nucleic acid
sequence of a nucleic acid molecule. In certain embodiments, the
nucleic acid sequence comprises DNA, RNA, cDNA, a variant thereof,
a fragment thereof, or a combination thereof. In one embodiment,
the nucleic acid sequence comprises a modified nucleic acid
sequence. For example, in one embodiment the antigen-encoding
nucleic acid sequence comprises nucleoside-modified RNA, as
described in detail elsewhere herein. In certain instances, the
nucleic acid sequence comprises include additional sequences that
encode linker or tag sequences that are linked to the antigen by a
peptide bond.
[0451] In certain embodiments, the antigen, encoded by the
nucleoside-modified nucleic acid molecule, comprises a protein,
peptide, a fragment thereof, or a variant thereof, or a combination
thereof from any number of organisms, for example, a virus, a
parasite, a bacterium, a fungus, or a mammal. For example, in
certain embodiments, the antigen is associated with an autoimmune
disease, allergy, or asthma. In other embodiments, the antigen is
associated with cancer, herpes, influenza, hepatitis B, hepatitis
C, human papilloma virus (HPV), ebola, pneumococcus, Haemophilus
influenza, meningococcus, dengue, tuberculosis, malaria, norovirus
or human immunodeficiency virus (HIV). In certain embodiments, the
antigen comprises a consensus sequence based on the amino acid
sequence of two or more different organisms. In certain
embodiments, the nucleic acid sequence encoding the antigen is
optimized for effective translation in the organism in which the
composition is delivered.
[0452] In one embodiment, the antigen comprises a tumor-specific
antigen or tumor-associated antigen, such that the antigen induces
an adaptive immune response against the tumor. In one embodiment,
the antigen comprises a fragment of a tumor-specific antigen or
tumor-associated antigen, such that the antigen induces an adaptive
immune response against the tumor. In certain embodiment, the
tumor-specific antigen or tumor-associated antigen is a mutation
variant of a host protein.
[0453] Viral Antigens
[0454] In one embodiment, the antigen comprises a viral antigen, or
fragment thereof, or variant thereof. In certain embodiments, the
viral antigen is from a virus from one of the following families:
Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae,
Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae,
Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae,
Picornaviridae, Poxviridae, Reoviridae, Retroviridae,
Rhabdoviridae, or Togaviridae. In certain embodiments, the viral
antigen is from papilloma viruses, for example, human papilloma
virus (HPV), human immunodeficiency virus (HIV), polio virus,
hepatitis B virus, hepatitis C virus, smallpox virus (Variola major
and minor), vaccinia virus, influenza virus, rhinoviruses, dengue
fever virus, equine encephalitis viruses, rubella virus, yellow
fever virus, Norwalk virus, hepatitis A virus, human T-cell
leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II),
California encephalitis virus, Hanta virus (hemorrhagic fever),
rabies virus, Ebola fever virus, Marburg virus, measles virus,
mumps virus, respiratory syncytial virus (RSV), herpes simplex 1
(oral herpes), herpes simplex 2 (genital herpes), herpes zoster
(varicella-zoster, a.k.a., chickenpox), cytomegalovirus (CMV), for
example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and
mouth disease virus, chikungunya virus, lassa virus, arenavirus, or
cancer causing virus.
[0455] Hepatitis Antigen
[0456] In one embodiment, the antigen comprises a hepatitis virus
antigen (i.e., hepatitis antigen), or fragment thereof, or variant
thereof. In certain embodiments, the hepatitis antigen comprises an
antigen or immunogen from hepatitis A virus (HAV), hepatitis B
virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV),
and/or hepatitis E virus (HEV). In certain embodiments, the
hepatitis antigen is full-length or immunogenic fragments of
full-length proteins.
[0457] In one embodiment, the hepatitis antigen comprises an
antigen from HAV. For example, in certain embodiments, the
hepatitis antigen comprises a HAV capsid protein, a HAV
non-structural protein, a fragment thereof, a variant thereof, or a
combination thereof.
[0458] In one embodiment, the hepatitis antigen comprises an
antigen from HCV. For example, in certain embodiments, the
hepatitis antigen comprises a HCV nucleocapsid protein (i.e., core
protein), a HCV envelope protein (e.g., E1 and E2), a HCV
non-structural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and
NS5b), a fragment thereof, a variant thereof, or a combination
thereof.
[0459] In one embodiment, the hepatitis antigen comprises an
antigen from HDV. For example, in certain embodiments, the
hepatitis antigen comprises a HDV delta antigen, fragment thereof,
or variant thereof.
[0460] In one embodiment, the hepatitis antigen comprises an
antigen from HEV. For example, in certain embodiments, the
hepatitis antigen comprises a HEV capsid protein, fragment thereof,
or variant thereof.
[0461] In one embodiment, the hepatitis antigen comprises an
antigen from HBV. For example, in certain embodiments, the
hepatitis antigen comprises a HBV core protein, a HBV surface
protein, a HBV DNA polymerase, a HBV protein encoded by gene X,
fragment thereof, variant thereof, or combination thereof. In
certain embodiments, the hepatitis antigen comprises a HBV genotype
A core protein, a HBV genotype B core protein, a HBV genotype C
core protein, a HBV genotype D core protein, a HBV genotype E core
protein, a HBV genotype F core protein, a HBV genotype G core
protein, a HBV genotype H core protein, a HBV genotype A surface
protein, a HBV genotype B surface protein, a HBV genotype C surface
protein, a HBV genotype D surface protein, a HBV genotype E surface
protein, a HBV genotype F surface protein, a HBV genotype G surface
protein, a HBV genotype H surface protein, fragment thereof,
variant thereof, or combination thereof.
[0462] Human Papilloma Virus (HPV) Antigen
[0463] In one embodiment, the antigen comprises a human papilloma
virus (HPV) antigen, or fragment thereof, or variant thereof. For
example, in certain embodiments, the antigen comprises an antigen
from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause
cervical cancer, rectal cancer, and/or other cancers. In one
embodiment, the antigen comprises an antigen from HPV types 6 and
11, which cause genital warts, and are known to be causes of head
and neck cancer. For example, in certain embodiments, the HPV
antigen comprises a HPV E6 or E7 domain, or fragments, or variant
thereof from any HPV type.
[0464] RSV Antigen
[0465] In one embodiment, the antigen comprises an RSV antigen or
fragment thereof, or variant thereof. For example, in certain
embodiments, the RSV antigen comprises a human RSV fusion protein
(also referred to herein as "RSV F", "RSV F protein" and "F
protein"), or fragment or variant thereof. In one embodiment, the
human RSV fusion protein is conserved between RSV subtypes A and B.
In certain embodiments, the RSV antigen comprises a RSV F protein,
or fragment or variant thereof, from the RSV Long strain (GenBank
AAX23994.1). In one embodiment, the RSV antigen comprises a RSV F
protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment
or variant thereof. In certain embodiments, the RSV antigen is a
monomer, a dimer or trimer of the RSV F protein, or a fragment or
variant thereof. According to the invention, in certain
embodiments, the RSV F protein is in a prefusion form or a
postfusion form.
[0466] In one embodiment, the RSV antigen comprises a human RSV
attachment glycoprotein (also referred to herein as "RSV G", "RSV G
protein" and "G protein"), or fragment or variant thereof. The
human RSV G protein differs between RSV subtypes A and B. In one
embodiment, the antigen comprises a RSV G protein, or fragment or
variant thereof, from the RSV Long strain (GenBank AAX23993). In
one embodiment, the RSV antigen comprises RSV G protein from: the
RSV subtype B isolate H5601, the RSV subtype B isolate H1068, the
RSV subtype B isolate H5598, the RSV subtype B isolate H1123, or a
fragment or variant thereof.
[0467] In other embodiments, the RSV antigen comprises a human RSV
non-structural protein 1 ("NS1 protein"), or fragment or variant
thereof. For example, in one embodiment, the RSV antigen comprises
RSV NS1 protein, or fragment or variant thereof, from the RSV Long
strain (GenBank AAX23987.1). In one embodiment, the RSV antigen
comprises RSV non-structural protein 2 ("NS2 protein"), or fragment
or variant thereof. For example, in one embodiment, the RSV antigen
comprises RSV NS2 protein, or fragment or variant thereof, from the
RSV Long strain (GenBank AAX23988.1). In one embodiment, the RSV
antigen comprises human RSV nucleocapsid ("N") protein, or fragment
or variant thereof. For example, in one embodiment, the RSV antigen
is RSV N protein, or fragment or variant thereof, from the RSV Long
strain (GenBank AAX23989.1). In one embodiment, the RSV antigen
comprises human RSV Phosphoprotein ("P") protein, or fragment or
variant thereof. For example, in one embodiment, the RSV antigen
comprises RSV P protein, or fragment or variant thereof, from the
RSV Long strain (GenBank AAX23990.1). In one embodiment, the RSV
antigen comprises human RSV Matrix protein ("M") protein, or
fragment or variant thereof. For example, in one embodiment, the
RSV antigen comprises RSV M protein, or fragment or variant
thereof, from the RSV Long strain (GenBank AAX23991.1).
[0468] In still other embodiments, the RSV antigen comprises human
RSV small hydrophobic ("SH") protein, or fragment or variant
thereof. For example, in one embodiment, the RSV antigen comprises
RSV SH protein, or fragment or variant thereof, from the RSV Long
strain (GenBank AAX23992.1). In one embodiment, the RSV antigen
comprises human RSV Matrix protein2-1 ("M2-1") protein, or fragment
or variant thereof. For example, in one embodiment, the RSV antigen
comprises RSV M2-1 protein, or fragment or variant thereof, from
the RSV Long strain (GenBank AAX23995.1). In one embodiment, the
RSV antigen comprises RSV Matrix protein 2-2 ("M2-2") protein, or
fragment or variant thereof. For example, in one embodiment, the
RSV antigen comprises RSV M2-2 protein, or fragment or variant
thereof, from the RSV Long strain (GenBank AAX23997.1). In one
embodiment, the RSV antigen comprises RSV Polymerase L ("L")
protein, or fragment or variant thereof. For example, in one
embodiment, the RSV antigen comprises RSV L protein, or fragment or
variant thereof, from the RSV Long strain (GenBank AAX23996.1).
[0469] Influenza Antigen
[0470] In one embodiment, the antigen comprises an influenza
antigen or fragment thereof, or variant thereof. The influenza
antigens are those capable of eliciting an adaptive immune response
in a mammal against one or more influenza serotypes. In certain
embodiments, the antigen comprises the full length translation
product Hemagglutinin (HA)0, subunit HA1, subunit HA2, a variant
thereof, a fragment thereof or a combination thereof. In certain
embodiments, the influenza hemagglutinin antigen is derived from
one or more strains of influenza A serotype H1, influenza A
serotype H2, or influenza B.
[0471] In one embodiment, the influenza antigen contains at least
one antigenic epitope that can be effective against particular
influenza immunogens against which an immune response can be
induced. In certain embodiments, the antigen may provide an entire
repertoire of immunogenic sites and epitopes present in an intact
influenza virus.
[0472] In some embodiments, the influenza antigen comprises H1 HA,
H2 HA, H3 HA, H5 HA, or a BHA antigen. In certain embodiments, the
influenza antigen comprises neuraminidase (NA), matrix protein,
nucleoprotein, M2 ectodomain-nucleoprotein (M2e-NP), a variant
thereof, a fragment thereof, or combinations thereof.
[0473] Human Immunodeficiency Virus (HIV) Antigen
[0474] In one embodiment, the antigen comprises an HIV antigen or
fragment thereof, or variant thereof.
[0475] In certain embodiments, the HIV antigen comprises an
envelope (Env) protein or fragment or variant thereof. For example,
in certain embodiments, the HIV antigen comprises an Env protein
selected from gp120, gp41, or a combination thereof.
[0476] In certain embodiments, the HIV antigen comprises at least
one of nef, gag, pol, vif, vpr, vpu, tat, rev, or a fragment of
variant thereof.
[0477] The HIV antigen may be derived from any strain of HIV. For
example, in certain embodiments the HIV antigen comprises an
antigen from HIV groups M, N, O, and P, and subtype A, HIV subtype
B, HIV subtype C, HIV subtype D, subtype E, subtype F, subtype G,
subtype H, subtype J, or subtype K. In one embodiment, the HIV
antigen comprises Env or fragment or variant thereof, from the
HIV-R3A strain (R3A-Env).
[0478] Parasite Antigens
[0479] In certain embodiments, the antigen comprises a parasite
antigen or fragment or variant thereof. In certain embodiments, the
parasite is a protozoa, helminth, or ectoparasite. In certain
embodiments, the helminth (i.e., worm) is a flatworm (e.g., flukes
and tapeworms), a thorny-headed worm, or a round worm (e.g.,
pinworms). In certain embodiments, the ectoparasite is lice, fleas,
ticks, and mites.
[0480] In certain embodiments, the parasite is any parasite causing
the following diseases: Acanthamoeba keratitis, Amoebiasis,
Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas
disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis,
Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis,
Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis,
Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama
fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis,
Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis,
Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis,
Toxoplasmosis, Trichinosis, and Trichuriasis.
[0481] In certain embodiments, the parasite is Acanthamoeba,
Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug,
Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba
histolytica, Fasciola hepatica, Giardia lamblia, Hookworm,
Leishmania, Linguatula serrata, Liver fluke, Loa loa,
Paragonimus--lung fluke, Pinworm, Plasmodium falciparum,
Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma
gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.
[0482] Malaria Antigen
[0483] In one embodiment, the antigen comprises a malaria antigen
(i.e., PF antigen or PF immunogen), or fragment thereof, or variant
thereof. For example, in one embodiment, the antigen comprises an
antigen from a parasite causing malaria. In one embodiment, the
malaria causing parasite is Plasmodium falciparum.
[0484] In some embodiments, the malaria antigen comprises one or
more of P. falciparum immunogens CS; LSA1; TRAP; CelTOS; and Amal.
The immunogens may be full length or immunogenic fragments of full
length proteins.
[0485] Bacterial Antigens
[0486] In one embodiment, the antigen comprises a bacterial antigen
or fragment or variant thereof. In certain embodiments, the
bacterium is from any one of the following phyla: Acidobacteria,
Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae,
Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria,
Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia,
Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes,
Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria,
Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria,
Thermotogae, and Verrucomicrobia.
[0487] In certain embodiments, the bacterium is a gram positive
bacterium or a gram negative bacterium. In certain embodiments, the
bacterium is an aerobic bacterium or an anaerobic bacterium. In
certain embodiments, the bacterium is an autotrophic bacterium or a
heterotrophic bacterium. In certain embodiments, the bacterium is a
mesophile, a neutrophile, an extremophile, an acidophile, an
alkaliphile, a thermophile, psychrophile, halophile, or an
osmophile.
[0488] In certain embodiments, the bacterium is an anthrax
bacterium, an antibiotic resistant bacterium, a disease causing
bacterium, a food poisoning bacterium, an infectious bacterium,
Salmonella bacterium, Staphylococcus bacterium, Streptococcus
bacterium, or tetanus bacterium. In certain embodiments, bacterium
is a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus
anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or
Clostridium difficile.
[0489] Mycobacterium tuberculosis Antigens
[0490] In one embodiment, the antigen comprises a Mycobacterium
tuberculosis antigen (i.e., TB antigen or TB immunogen), or
fragment thereof, or variant thereof. The TB antigen can be from
the Ag85 family of TB antigens, for example, Ag85A and Ag85B. The
TB antigen can be from the Esx family of TB antigens, for example,
EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS,
EsxT, EsxU, EsxV, and EsxW.
[0491] Fungal Antigens
[0492] In one embodiment, the antigen comprises a fungal antigen or
fragment or variant thereof. In certain embodiments, the fungus is
Aspergillus species, Blastomyces dermatitidis, Candida yeasts
(e.g., Candida albicans), Coccidioides, Cryptococcus neoformans,
Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma
capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix
schenckii, Exserohilum, or Cladosporium.
[0493] Tumor Antigens
[0494] In certain embodiments, the antigen comprises a tumor
antigen, including for example a tumor-associated antigen or a
tumor-specific antigen. In the context of the present invention,
"tumor antigen" or "hyperporoliferative disorder antigen" or
"antigen associated with a hyperproliferative disorder" refer to
antigens that are common to specific hyperproliferative disorders.
In certain aspects, the hyperproliferative disorder antigens of the
present invention are derived from cancers including, but not
limited to, primary or metastatic melanoma, mesothelioma, thymoma,
lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's
lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, cervical
cancer, bladder cancer, kidney cancer and adenocarcinomas such as
breast cancer, prostate cancer, ovarian cancer, pancreatic cancer,
and the like.
[0495] Tumor antigens are proteins that are produced by tumor cells
that elicit an immune response, particularly T-cell mediated immune
responses. In one embodiment, the tumor antigen of the present
invention comprises one or more antigenic cancer epitopes
immunogenically recognized by tumor infiltrating lymphocytes (TIL)
derived from a cancer tumor of a mammal. The selection of the
antigen will depend on the particular type of cancer to be treated
or prevented by way of the composition of the invention.
[0496] Tumor antigens are well known in the art and include, for
example, a glioma-associated antigen, carcinoembryonic antigen
(CEA), .beta.-human chorionic gonadotropin, alphafetoprotein (AFP),
lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human
telomerase reverse transcriptase, RU1, RU2 (AS), intestinal
carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific
antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA,
Her2/neu, survivin and telomerase, prostate-carcinoma tumor
antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2,
CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and
mesothelin.
[0497] In one embodiment, the tumor antigen comprises one or more
antigenic cancer epitopes associated with a malignant tumor.
Malignant tumors express a number of proteins that can serve as
target antigens for an immune attack. These molecules include but
are not limited to tissue-specific antigens such as MART-1,
tyrosinase and GP 100 in melanoma and prostatic acid phosphatase
(PAP) and prostate-specific antigen (PSA) in prostate cancer. Other
target molecules belong to the group of transformation-related
molecules such as the oncogene HER-2/Neu/ErbB-2. Yet another group
of target antigens are onco-fetal antigens such as carcinoembryonic
antigen (CEA). In B-cell lymphoma the tumor-specific idiotype
immunoglobulin constitutes a truly tumor-specific immunoglobulin
antigen that is unique to the individual tumor. B-cell
differentiation antigens such as CD19, CD20 and CD37 are other
candidates for target antigens in B-cell lymphoma. Some of these
antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as
targets for passive immunotherapy with monoclonal antibodies with
limited success.
[0498] The type of tumor antigen referred to in the invention may
also be a tumor-specific antigen (TSA) or a tumor-associated
antigen (TAA). A TSA is unique to tumor cells and does not occur on
other cells in the body. A TAA associated antigen is not unique to
a tumor cell and instead is also expressed on a normal cell under
conditions that fail to induce a state of immunologic tolerance to
the antigen. The expression of the antigen on the tumor may occur
under conditions that enable the immune system to respond to the
antigen. TAAs may be antigens that are expressed on normal cells
during fetal development when the immune system is immature and
unable to respond or they may be antigens that are normally present
at extremely low levels on normal cells but which are expressed at
much higher levels on tumor cells.
[0499] Non-limiting examples of TSA or TAA antigens include the
following: Differentiation antigens such as MART-1/MelanA (MART-I),
gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific
multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,
p15; overexpressed embryonic antigens such as CEA; overexpressed
oncogenes and mutated tumor-suppressor genes such as p53, Ras,
HER-2/neu; unique tumor antigens resulting from chromosomal
translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR;
and viral antigens, such as the Epstein Barr virus antigens EBVA
and the human papillomavirus (HPV) antigens E6 and E7. Other large,
protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6,
RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72,
CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1,
p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG,
BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50,
CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344,
MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2
binding protein\cyclophilin C-associated protein, TAAL6, TAG72,
TLP, and TPS.
[0500] In a preferred embodiment, the antigen includes but is not
limited to CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met,
PSMA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR,
and the like.
Adjuvant
[0501] In one embodiment, the composition comprises an adjuvant. In
one embodiment, the composition comprises a nucleic acid molecule
encoding an adjuvant. In one embodiment, the adjuvant-encoding
nucleic acid molecule is IVT RNA. In one embodiment, the
adjuvant-encoding nucleic acid molecule is nucleoside-modified
RNA.
[0502] Exemplary adjuvants include, but is not limited to,
alpha-interferon, gamma-interferon, platelet derived growth factor
(PDGF), TNF.alpha., TNF.beta., GM-CSF, epidermal growth factor
(EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial
thymus-expressed chemokine (TECK), mucosae-associated epithelial
chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15
having the signal sequence deleted and optionally including the
signal peptide from IgE. Other genes which may be useful adjuvants
include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES,
L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1,
LFA-I, VLA-I, Mac-1, p150.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2,
LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L,
vascular growth factor, fibroblast growth factor, IL-7, nerve
growth factor, vascular endothelial growth factor, Fas, TNF
receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD,
NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos,
c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB,
Inactive NIK, SAP K, SAP-I, INK, interferon response genes, NFkB,
Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK
LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C,
NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig,
anti-TIM3-Ig and functional fragments thereof.
Pharmaceutical Compositions
[0503] The 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 a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0504] Although the description of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical 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
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans and other primates, mammals
including commercially relevant mammals such as non-human primates,
cattle, pigs, horses, sheep, cats, and dogs.
[0505] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for ophthalmic, oral, rectal, vaginal, parenteral,
topical, pulmonary, intranasal, buccal, intravenous,
intracerebroventricular, intradermal, intramuscular, or another
route of administration. Other contemplated formulations include
projected nanoparticles, liposomal preparations, resealed
erythrocytes containing the active ingredient, and
immunogenic-based formulations.
[0506] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, 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 or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0507] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and 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.
[0508] In addition to the active ingredient, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents.
[0509] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0510] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, intraocular, intravitreal, subcutaneous,
intraperitoneal, intramuscular, intradermal, intrasternal
injection, intratumoral, intravenous, intracerebroventricular and
kidney dialytic infusion techniques.
[0511] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e. powder or granular) form for reconstitution
with a suitable vehicle (e.g. sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0512] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0513] A pharmaceutical composition of the invention may be
prepared, packaged, 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 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. 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
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a sealed container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
More preferably, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[0514] 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 the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0515] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e., powder or granular) form for reconstitution
with a suitable vehicle (e.g., sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0516] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations that are useful include those
that comprise the active ingredient in microcrystalline form, in a
liposomal preparation, or as a component of a biodegradable polymer
system. Compositions for sustained release or implantation may
comprise pharmaceutically acceptable polymeric or hydrophobic
materials such as an emulsion, an ion exchange resin, a sparingly
soluble polymer, or a sparingly soluble salt.
[0517] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Remington's
Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co.,
Easton, Pa.), which is incorporated herein by reference.
Treatment Methods
[0518] The present invention provides methods of inducing an
adaptive immune response in a subject comprising administering an
effective amount of a composition comprising one or more isolated
nucleic acids encoding one or more antigens, one or more adjuvants,
or a combination thereof.
[0519] In one embodiment, the method provides immunity in the
subject to an infection, disease, or disorder associated with an
antigen. The present invention thus provides a method of treating
or preventing the infection, disease, or disorder associated with
the antigen. For example, the method may be used to treat or
prevent a viral infection, bacterial infection, fungal infection,
parasitic infection, or cancer, depending upon the type of antigen
of the administered composition. Exemplary antigens and associated
infections, diseases, and tumors are described elsewhere
herein.
[0520] In one embodiment, the composition is administered to a
subject having an infection, disease, or cancer associated with the
antigen. In one embodiment, the composition is administered to a
subject at risk for developing the infection, disease, or cancer
associated with the antigen. For example, the composition may be
administered to a subject who is at risk for being in contact with
a virus, bacteria, fungus, parasite, or the like. In one
embodiment, the composition is administered to a subject who has
increased likelihood, though genetic factors, environmental
factors, or the like, of developing cancer.
[0521] In one embodiment, the method comprises administering a
composition comprising one or more nucleoside-modified nucleic acid
molecules encoding one or more antigens and one or more adjuvant.
In one embodiment, the method comprises administering a composition
comprising a first nucleoside-modified nucleic acid molecule
encoding one or more antigens and a second nucleoside-modified
nucleic acid molecule encoding one or more adjuvants. In one
embodiment, the method comprises administering a first composition
comprising one or more nucleoside-modified nucleic acid molecules
encoding one or more antigens and administering a second
composition comprising one or more nucleoside-modified nucleic acid
molecules encoding one or more adjuvants.
[0522] In certain embodiments, the method comprises administering
to subject a plurality of nucleoside-modified nucleic acid
molecules encoding a plurality of antigens, adjuvants, or a
combination thereof.
[0523] In certain embodiments, the method of the invention allows
for sustained expression of the antigen or adjuvant, described
herein, for at least several days following administration.
However, the method, in certain embodiments, also provides for
transient expression, as in certain embodiments, the nucleic acid
is not integrated into the subject genome.
[0524] In certain embodiments, the method comprises administering
nucleoside-modified RNA which provides stable expression of the
antigen or adjuvant described herein. In some embodiments,
administration of nucleoside-modified RNA results in little to no
innate immune response, while inducing an effective adaptive immune
response.
[0525] Administration of the compositions of the invention in a
method of treatment can be achieved in a number of different ways,
using methods known in the art. In one embodiment, the method of
the invention comprises systemic administration of the subject,
including for example enteral or parenteral administration. In
certain embodiments, the method comprises intradermal delivery of
the composition. In another embodiment, the method comprises
intravenous delivery of the composition. In some embodiments, the
method comprises intramuscular delivery of the composition. In one
embodiment, the method comprises subcutaneous delivery of the
composition. In one embodiment, the method comprises inhalation of
the composition. In one embodiment, the method comprises intranasal
delivery of the composition.
[0526] It will be appreciated that the composition of the invention
may be administered to a subject either alone, or in conjunction
with another agent.
[0527] The therapeutic and prophylactic methods of the invention
thus encompass the use of pharmaceutical compositions encoding an
antigen, adjuvant, or a combination thereof, described herein to
practice the methods of the invention. The pharmaceutical
compositions useful for practicing the invention may be
administered to deliver a dose of from ng/kg/day and 100 mg/kg/day.
In one embodiment, the invention envisions administration of a dose
which results in a concentration of the compound of the present
invention from 10 nM and 10 .mu.M in a mammal.
[0528] Typically, dosages which may be administered in a method of
the invention to a mammal, preferably a human, range in amount from
0.01 .mu.g to about 50 mg per kilogram of body weight of the
mammal, while the precise dosage administered will vary depending
upon any number of factors, including but not limited to, the type
of mammal and type of disease state being treated, the age of the
mammal and the route of administration. Preferably, the dosage of
the compound will vary from about 0.1 .mu.g to about 10 mg per
kilogram of body weight of the mammal. More preferably, the dosage
will vary from about 1 .mu.g to about 1 mg per kilogram of body
weight of the mammal.
[0529] The composition may be administered to a mammal as
frequently as several times daily, or it may be administered less
frequently, such as once a day, once a week, once every two weeks,
once a month, or even less frequently, such as once every several
months or even once a year or less. The frequency of the dose will
be readily apparent to the skilled artisan and will depend upon any
number of factors, such as, but not limited to, the type and
severity of the disease being treated, the type and age of the
mammal, etc.
[0530] In certain embodiments, administration of an immunogenic
composition or vaccine of the present invention may be performed by
single administration or boosted by multiple administrations.
[0531] In one embodiment, the invention includes a method
comprising administering one or more compositions encoding one or
more antigens or adjuvants described herein. In certain
embodiments, the method has an additive effect, wherein the overall
effect of the administering the combination is approximately equal
to the sum of the effects of administering each antigen or
adjuvant. In other embodiments, the method has a synergistic
effect, wherein the overall effect of administering the combination
is greater than the sum of the effects of administering each
antigen or adjuvant.
EXPERIMENTAL EXAMPLES
[0532] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0533] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out the preferred
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure.
Example 1: Induction of Adaptive Immune Response by Modified RNA
Encoding HIV Env Protein
[0534] Experiments were conducted to investigate the ability of
modified RNA encoding HIV Env protein to induce adaptive immunity
in a mouse model. In a first set of experiments animals received
two intradermal injections of 3 .mu.h, 10 .mu.g (E10) or 30 .mu.g
(E30) HIV-1 CD4-independent R3A envelope encoding mRNA encapsulated
into lipid nanoparticles (ENV-LNP). The lipid nanoparticles of
Examples 1-4 comprised mRNA, cationic lipid (compound I-6), DSPC,
cholesterol and pegylated lipid (compound 14-6), and were prepared
according to Example 15. The lipid nanoparticles of Example 5
comprised mRNA, the indicated cationic lipid, DSPC, cholesterol and
pegylated lipid (compound 14-6), and were also prepared according
to Example 15. The resulting ENV-LNP had a mean diameter of 76 nm
and polydispersity index of 0.007. Encapsulation efficiency was
determined to be 95% using Quant-IT Ribogreen (Thermo-Fisher) to
assay free mRNA in an LNP sample vs. total mRNA in a corresponding
sample containing 2% v/v Triton TX100 surfactant to disrupt the
LNPs and expose the total mRNA. Control mice were injected with 30
.mu.g firefly luciferase encoding mRNA complexed into lipid
nanoparticles (LUC). These control LNPs had a mean diameter of 74
nm with polydispersity index of 0.007 and encapsulation efficiency
of 92%. There was a 4-week interval between mRNA-LNP injections and
animals were sacrificed 14 days after the second injection (FIG.
1). Cells and serum were analyzed from the immunized animals.
[0535] Multicolor flow cytometry was used to measure intracellular
cytokine production in cells after stimulation with peptide pools
of 15-mers overlapping by 11 amino acids of the complete envelope
sequence.
[0536] It was observed that the immunization with ENV-LNPs induces
IFN-.gamma., TNF-.alpha., and IL-2 production (FIG. 2 and FIG. 3)
by antigen specific CD4+ T cells. Further analysis was conducted to
evaluate the distribution of mono,- bi,- and trifunctional antigen
specific CD4+ T cells in vaccinated animals. It was observed that
Env-treated animals produced a higher percentage of CD4+ cells
producing all three of IFN-.gamma., TNF-.alpha., IL-2; both
IFN-.gamma. and TNF-.alpha.; and both TNF-.alpha. and IL-2 (FIG.
4). Collectively, this data demonstrates that immunization with
ENV-LNPs elicit robust and high quality CD4+ T cell responses.
[0537] It was also observed that immunization with ENV-LNP results
in significant increase in T follicular helper (Tfh) cell numbers
that are critical for the generation of high affinity antibody
responses. CD4, CXCR5 and PD-1 markers were used to determine Tfh
cells (FIG. 5). The same ratios of T.sub.fh cells are obtained for
CXCR5+ICOS+CD4+ and PD-1+ICOS+CD4+ T cells.
[0538] Cytokine production of antigen specific CD8+ T cells was
also examined. It was observed that the immunization with ENV-LNPs
induces IFN-.gamma., TNF-.alpha., IL-2 and CD107a production (FIG.
6 and FIG. 7) by antigen specific CD8+ T cells. Further analysis
was conducted to evaluate the distribution of mono,- bi,- and
trifunctional antigen specific CD8+ T cells in vaccinated animals.
It was observed that E10 treated animals produced a higher
percentage of CD8+ cells producing all three of IFN-.gamma.,
TNF-.alpha., and CD107a; both IFN-.gamma. and CD107a; both
TNF-.alpha. and CD107a, and CD107a alone (FIG. 8). Further, E30
treated animals produced a higher percentage of CD8+ cells
producing all three of IFN-.gamma., TNF-.alpha., and CD107a.
Collectively, this data demonstrates that immunization with
ENV-LNPs elicits robust CD8+ T cell responses.
[0539] ELISA assays were performed to investigate antigen specific
B cell responses to the HIV envelope immunogen in mice immunized
with 3 .mu.g, 10 .mu.g, or 30 .mu.g of HIV-1 CD4-independent R3A
envelope encoding mRNA encapsulated into lipid nanoparticles.
Specifically, HIV-1g120 specific 1gG titers were measured after two
injections of mRNA-LNP. Titers were measured by a gp120 specific
ELISA assay. It was observed that ENV-LNP treated mice exhibited an
antigen specific B cell response, as measured by the increased
level of gp120-specific IgG compared to control (FIG. 9A). Further,
it was observed that similar amounts of Env-specific IgG1 and IgG2a
are produced 2 weeks after 2 immunizations.
[0540] Experiments were conducted to examine the functional
activity of anti-ENV antibodies produced after 2 immunizations with
HIV envelope iR3A encoding modified mRNA-LNP complexes.
Neutralization titers to an easy to neutralize tier 1 strain of HIV
called MN and a difficult to neutralize tier 2 strain of HIV called
X2278_C2_B6 were determined and control neutralization titers to
moloney leukemia virus (MLV) were subtracted. It was observed that
high levels of tier 1 neutralization (FIG. 10) and tier 2
neutralization (FIG. 11) was induced by immunization with
intradermal iR3A modified mRNA-LNPs.
[0541] Another set of experiments were conducted where mice
received a single intradermal injection of 30 .mu.g HIV-1
CD4-independent R3A envelope encoding mRNA encapsulated into lipid
nanoparticles (ENV). Control mice were injected with 30 .mu.g
firefly luciferase encoding mRNA complexed into lipid nanoparticles
(LUC). Animals were sacrificed 14 days after mRNA administration
(FIG. 12).
[0542] Cytokine production of antigen specific CD4+ cells was
measured in the animals treated with a single intradermal dose of
30 .mu.g ENV-LNP. It was observed that the immunization with a
single injection of ENV-LNPs induces IFN-.gamma., TNF-.alpha., IL-2
and CD107a production (FIG. 13 and FIG. 14) by antigen specific
CD4+ T cells. Further analysis was conducted to evaluate the
distribution of mono,- bi,- and trifunctional antigen specific CD4+
T cells in vaccinated animals. It was observed ENV-LNP treated
animals produced a higher percentage of CD4+ cells producing both
TNF-.alpha. and IL-2 (FIG. 15). Collectively, this data
demonstrates that immunization with a single dose of ENV-LNPs
elicit robust CD4+ T cell responses.
[0543] It was also observed that immunization with ENV-LNP results
in significant increase in Tfh cell numbers. CD4, CXCR5 and PD-1
markers were used to determine total Tfh cells (FIG. 16).
[0544] Cytokine production by antigen specific Tfh cells was then
examined. It was observed that immunization with a single injection
of ENV-LNPs induces IFN-.gamma., TNF-.alpha., and IL-2 production
(FIG. 17) by antigen specific Tfh cells.
[0545] Further analysis was conducted to evaluate the distribution
of mono,- bi,- and trifunctional antigen specific Tfh cells in
vaccinated animals. It was observed that animals treated with a
single dose of ENV-LNP produced a higher percentage of Tfh cells
producing all three of IFN-.gamma., TNF-.alpha., IL2 (FIG. 18).
Collectively, this data demonstrates that immunization with a
single dose of ENV-LNPs elicits robust Tfh cell immune
response.
[0546] ELISA assays were performed to investigate antigen specific
B cell responses in mice immunized with a single dose of ENV-LNP.
Specifically, HIV-1g120 specific IgG titers were measured after a
single injection of mRNA-LNP. Titers were measured by a gp120
specific ELISA assay. It was observed that the single dose of
ENV-LNP induced a robust antigen specific B cell response, as
measured by the increased level of gp120-specific IgG compared to
control and naive animals (FIG. 19).
[0547] Experiments were conducted to compare the adaptive immune
response induced by LNP-complexed nucleoside-modified RNA versus
nucleoside-modified RNA delivered alone. Mice were immunized 2
times with 10 .mu.g of unmodified, 1-methyl-pseudouridine modified,
or 1-methyl-pseudouridine modified and LNP complexed mRNA (all
encoding iR3A antigen) by the intradermal route at 1 month
intervals.
[0548] Spleen cells were analyzed by a 6 hour stimulation with
envelope overlapping peptides and analyzed for expression of CD107A
or intracellular IFN-.gamma., TNF-.alpha., and IL-2 versus CD3+,
CD8+ T cells (FIG. 20) or for expression of intracellular
IFN-.gamma., TNF-.alpha., and IL-2 versus CD3+, CD4+ T cells (FIG.
21). Nucleoside-modified RNA-LNP responses in CD8+ and CD4+ T-cells
were significantly greater (p<0.01) than uncomplexed modified or
unmodified mRNA or control (luciferase modified mRNA) treated mice
(FIG. 20 and FIG. 21) demonstrating the superiority of LNP
complexing.
[0549] Experiments were also conducted to examine envelope-specific
antibody responses induced by immunization with uncomplexed or
complexed nucleoside-modified RNA. Mice were immunized 2 times with
10 g of uncomplexed 1-methyl-pseudouridine modified mRNA encoding
HIV envelope iR3A (naked iR3A), 1-methyl-pseudouridine-LNP
complexed mRNA encoding luciferase (luc-LNP), or
1-methyl-pseudouridine-LNP complexed iR3A encoding mRNA (iR3A-LNP)
by the intradermal route at 1 month intervals. Serum was analyzed
for envelope (gp120) specific responses by ELISA. Serum was diluted
1:1000 and analyzed. A monoclonal antibody specific for gp120 was
used to determine concentration in serum. It was observed that
immunization with iR3A-LNP resulted in increased gp120-specific
antibody response.
Example 2: Induction of Adaptive Immune Response by Modified RNA
Encoding Influenza Antigen
[0550] Experiments presented herein demonstrate that
nucleoside-modified RNA which encodes an influenza antigen (i.e.
hemagglutinin (HA)) induces an influenza-specific adaptive immune
response in a subject. In these studies HA from PR8 and
A/Cal/7/2009 influenza strains were used. The amino acid sequence,
nucleotide sequence, and codon optimized sequences for the PR8 and
A/Cal/7/2009 HA are provided below
[0551] Experiments were conducted using m1.psi.-modified mRNA.
Initial experiments were conducted to examine cytokine production
in CD4+ T cells, 10 days after a single administration of PR8
HA-encoding modified mRNA-LNP (30 .mu.g). It was observed that a
single intradermal administration of 30 .mu.g PR8 HA-encoding
modified mRNA-LNP induced increased production of IFN-.gamma.,
TNF-.alpha., and IL-2, as compared to luciferase-encoding mRNA and
split virus (FIG. 23). Split virus is used in standard
intramuscular flu vaccines. The virus is initially grown in
chick-embryo allantoic fluid. The fluid is harvested, clarified,
concentrated and purified to eliminate almost all the egg protein.
The virus is then disrupted with chemicals that inactivate it and
break it into components to generate split virus.
[0552] Further, a polyfunctional CD4+ T cell response after the
single intradermal administration of 30 .mu.g PR8 HA-encoding
modified mRNA-LNP was observed, where PR8 HA-encoding modified
mRNA-LNP induced the expression of all 3 measured cytokines in a
significantly greater percentage of cells, as compared to control
luciferase-encoding RNA and intramuscular injection with 1000 HAU
of inactivated PR8 virus (FIG. 24).
[0553] Experiments were conducted to examine cytokine production in
CD8+ T cells, 10 days after a single intradermal administration of
PR8 HA-encoding modified mRNA-LNP (30 .mu.g). It was observed that
a single administration of 30 .mu.g PR8 HA-encoding modified
mRNA-LNP induced increased production of IFN-.gamma. and
TNF-.alpha. as compared to luciferase-encoding mRNA and split virus
(FIG. 25).
[0554] Further experiments were conducted to detect the level of
neutralization, as measured by HI titer, induced 14 days or 28 days
post-intradermal injection of either 10 .mu.g or 30 .mu.g of PR8
HA-encoding modified mRNA-LNP. Neutralization titers were measured
by the standard hemaglutinin inhibition assay, where turkey red
blood cells were coated with PR8 hemagglutinin. Serum at 2-fold
increasing dilutions was added to the RBCs and the titer where
hemagglutination was lost was measured. It was observed that both
administration of 10 .mu.g and 30 .mu.g of PR8 HA-encoding modified
mRNA-LNP resulted in increased titer as compared to luciferase
encoding mRNA (FIG. 26). Further, the level of neutralization
induced by acute infection with PR8 influenza was lower than that
induced by modified mRNA-LNP (Wolf et al., 2011, J Clin Invest,
121: 3954-3964).
[0555] It was further observed that a single administration of PR8
HA-encoding modified mRNA-LNP induces the production of germinal
center B cells (FIG. 27) as measured by being IgD.sup.-,
B220.sup.+, CD138.sup.-, CD19.sup.+, IgM.sup.-, CD3.sup.- and
CD14.sup.- Additionally, a single intradermal administration of PR8
HA-encoding modified mRNA-LNP induces an increase in total memory B
cells in the spleen, as measured by the number of CD11c.sup.+
T-BET.sup.+ cells (FIG. 28).
[0556] Additional studies were conducted to examine the effect of
administration of HA-encoding modified mRNA on T follicular helper
(Tfh) cells, which are critical in driving B cell response and
memory. It was observed that a single administration of PR8
HA-encoding modified mRNA-LNP induces an increase in total Tfh
cells in the spleen (FIG. 29). Further, administration of 30 .mu.g
PR8 HA-encoding modified mRNA-LNP resulted in an increase in the
production of IFN-.gamma. and IL-2 in CD4+ Tfh cells, as compared
to treatment with luciferase-encoding mRNA and live virus control
when spleen cells were stimulated with overlapping HA peptides and
Tfh cells were defined as Bcl6+(FIG. 30).
[0557] The cytokine expression of IL-4, IL-21, and IFN-.gamma. was
measured in Tfh cells purified from the spleens of mice immunized
with PR8 HA-encoding modified mRNA-LNP (FIG. 31). Spleen cells 10
days after PRB modified mRNA-LNP immunization were isolated. T
cells were selected by either positive selection with CD3 or
negative selection with CD14, CD19, CD16, CD56. Total T cells were
either directly analyzed (all T cells) or further purified by
selection of CXCR5+ and PD-1+ cells, T follicular helper cells.
Levels of IL-4, IL-21, and IFN-g were measured by real time PCR
using GAPDH as a control. Data are expressed as fold difference
compared to a universal standard mRNA. Further, it was observed
that administration of the PR8 HA-encoding modified mRNA-LNP does
not increase the percentage of Tfh regulatory cells (FIG. 32).
[0558] Experiments were also conducted to examine the effects of
influenza challenge on mice that were immunized with either 10
.mu.g or 30 .mu.g of PR8 HA-encoding modified mRNA-LNP. It was
observed that that challenged mice which were immunized
intradermally with either 10 .mu.g or 30 .mu.g of PR8 HA-encoding
modified mRNA-LNP maintained their weight throughout the 15 days
post-infection study, while control animals exhibited reduced
weight (FIG. 33) and significant mortality.
[0559] Experiments were conducted to examine the influenza stalk
response to evaluate the potential to induce universal protection
across influenza strains, and to measure affinity maturation driven
by Tfh cells. Current influenza vaccines do not induce stalk
responses.
[0560] Experiments were conducted to evaluate HA binding ability of
sera of animals treated with a single intradermal administration of
either 10 .mu.g or 30 .mu.g of PR8 HA-encoding modified mRNA-LNP.
It was observed that binding to H1 HA (H1-head/H1-stalk) was
increased 4 weeks after administration, as compared to 2 weeks
after administration (FIG. 34). Further, it was observed that
administration of either 10 .mu.g or 30 .mu.g of PR8 HA-encoding
modified mRNA-LNP induced IgG specific for the stalk, as
demonstrated by the ability to bind to H5-head/H1-stalk hybrid HA
(FIG. 35). It was also observed that whole HA binding and HA stalk
binding increases over time up to 63 days after a single
intradermal immunization (FIG. 36).
[0561] The persistence of the adaptive immune response induced by
the PR8 HA-encoding modified mRNA-LNP was then evaluated. It was
observed that the antibody response after single intradermal
administration of PR8 HA-encoding modified mRNA-LNP remains
unchanged 6 months after administration (FIG. 37).
[0562] Additional experiments were conducted using m1.PSI. modified
RNA-LNP, where the m1.PSI. modified RNA encodes A/California/7/2009
HA (hereinafter "CA09 HA"). This is a different influenza HA that
was used in the 2015-2016 vaccine and is clinically significant. It
was observed that a single intradermal administration of 30 .mu.g
CA09 HA-encoding mRNA-LNP induced an antigen-specific adaptive
immune response, as measured by HA inhibition titer 2 weeks after
the single intradermal administration (FIG. 38).
[0563] Further, it was observed that a single administration of 30
.mu.g of CA09 HA-encoding mRNA-LNP induced increased IFN-.gamma.,
TNF-.alpha., and IL-2 in CD4+ T cells, measured 2 weeks after the
single administration, as compared to poly(C) control (FIG. 39). It
was also observed that that single administration of 30 .mu.g of
CA09 HA-encoding mRNA-LNP resulted in increased percentage of Tfh
cells, measured 2 weeks after single administration (FIG. 40).
[0564] An experiment was conducted to examine the effectiveness of
intramuscular delivery of CA09 HA-encoding mRNA-LNP. Subjects were
administered either 10 .mu.g, 30 .mu.g, or 90 .mu.g of CA09
HA-encoding mRNA-LNPs, administered by intramuscular injection, or
with 3 .mu.g, 10 .mu.g, or 30 g of CA09 HA-encoding mRNA-LNPs
administered by intradermal injection. It was observed that
intramuscular injection resulted in a similar immune response as
compared to intradermal injection but required 3 times as much mRNA
(FIG. 41).
[0565] The data presented herein demonstrate the clear superiority
of the modified mRNA-LNP vaccine for influenza. Importantly, it is
shown that only a single immunization in a naive host is needed for
complete protection against influenza. It is understood that this
is the first reported demonstration that a single immunization with
a non-replicating vaccine is capable of inducing a high titer IgG
response and over a quarter of the response is directed at the
stalk. While not wishing to be bound by any particular theory, the
potent antibody response is likely due to the Tfh response that
makes up half of the CD4 helper response. Further, it is
demonstrated the mRNA-LNP vaccine can be effective following
delivery via different routes, which greatly expands the utility of
the mRNA-LNP vaccine.
Example 3: Mechanism of Modified mRNA Induction of Potent Tfh
Responses
[0566] Nucleoside modified mRNA in LNPs does not induce an innate
immune response. It was examined whether it is the lack of adjuvant
effect that results in the potent Tfh response. To investigate
this, PR8 HA mRNA was manufactured that only differs by the lack of
nucleoside modification but contains modification of the nucleoside
sequence. This results in similar levels of translation but the
unmodified mRNA induces an innate immune response.
[0567] It was observed that codon-optimized FPLC-purified, but
unmodified HA mRNA induces type 1 interferon production,
demonstrating that the unmodified HA mRNA induces an innate immune
response. However, the m1.PSI.-modified mRNA did not induce an
innate immune response (FIG. 42). Further, it was observed that
intravenous injection of HPLC purified, nucleoside modified
mRNA-LNP does not induce the production of proinflammatory
cytokines IL-6, IFN-.alpha., or TNF-.alpha. (FIG. 43) demonstrating
a lack of innate immune activation.
[0568] Experiments were conducted to compare m1.psi. modified mRNA
with unmodified codon optimized mRNA in their ability to induce a
CD4+ T cell response. It was observed that the nucleoside modified
HA-encoding mRNA (does not induce innate immune response) induces a
better CD4+ T cell response, as measured by the increased
production of IFN-.gamma., TNF-.alpha., and IL-2, as compared to
unmodified HA-encoding mRNA (which does induce innate immune
response) (FIG. 44).
[0569] Experiments were conducted to examine the adaptive immune
response generated by intradermal administration of 30 .mu.g of
m1.PSI.-modified HA-encoding mRNA compared to 30 .mu.g of
unmodified codon-optimized PR8 HA-encoding mRNA. It was observed
that m1.PSI.-modified HA-encoding mRNA produced increased levels of
Tfh cells in the spleen, as compared to unmodified HA-encoding mRNA
and to controls (FIG. 45). Additionally, administration of
m1.PSI.-modified HA-encoding mRNA resulted in a greater antigen
specific Tfh cell response, as measured by percentage of
CD4+Bcl6+IFN-.gamma.+ T cells, as compared to unmodified
HA-encoding mRNA (FIG. 46). Finally, m1.PSI.-modified HA-encoding
mRNA induced greater HA-specific antibody response, as measured by
HA inhibition titers 10 days after single administration (FIG.
47).
[0570] These experiments demonstrate that HA-encoding mRNAs that
only differed in containing m1.PSI. versus unmodified mRNA and the
ability to activate RNA sensors (only unmodified), but had similar
levels of translation, have a differential ability in inducing an
adaptive immune response. The m1.PSI. modified mRNA was observed to
induce a greater antigen-specific immune response. Most
importantly, the lack of innate immune activation or adjuvant
activity or induction of IFN-.alpha. induced the induction of
potent Tfh cells.
Example 4: mRNA Delivery
[0571] Experiments were conducted to visualize the expression of
the m1.PSI.modified mRNA. Mice were injected with 0.1 .mu.g, 1
.mu.g, or 5 .mu.g of naked or LNP complexed m1.PSI. luciferase
encoding mRNA and imaged by In Vivo Imaging (IVIS). mRNA
translation was observed in all conditions (FIG. 48). LNP
complexing increases the level and duration of mRNA translation
(FIG. 48).
Example 5: Comparison of LNP Formulations
[0572] Experiments were conducted to examine the effectiveness of
various LNP formulations, as measured by the effective translation
of the encapsulated mRNA. LNPs comprising Luciferase encoding m-RNA
were prepared as described in Example 15. The tested LNPs comprised
cationic lipid I-5, I-6, II-9, II-10, II-11, II-12, II-32, III-3 or
III-7. Other components were as described in Example 15. Six week
old BALB/c mice were intradermally injected with 3 .mu.g of
Luciferase encoding mRNA-LNPs. The expression of luciferase was
measured by IVIS. The data show that mRNA can be effectively
delivered using a variety of LNPs (FIG. 49). Accordingly, the data
provide evidence that a wide variety of LNPs can be used to deliver
nucleoside-modified RNA encoding at least one antigen
Example 6: Synthesis of Compound I-5
[0573] Compound I-5 was prepared according to method B as
follows:
[0574] A solution of hexan-1,6-diol (10 g) in methylene chloride
(40 mL) and tetrahydrofuran (20 mL) was treated with
2-hexyldecanoyl chloride (10 g) and triethylamine (10 mL). The
solution was stirred for an hour and the solvent removed. The
reaction mixture was suspended in hexane, filtered and the filtrate
washed with water. The solvent was removed and the residue passed
down a silica gel (50 g) column using hexane, followed by methylene
chloride, as the eluent, yielding 6-(2'-hexyldecanoyloxy)hexan-1-ol
as an oil (7.4 g).
[0575] The purified product (7.4 g) was dissolved in methylene
chloride (50 mL) and treated with pyridinium chlorochromate (5.2 g)
for two hours. Diethyl ether (200 mL) as added and the supernatant
filtered through a silica gel bed. The solvent was removed from the
filtrate and resultant oil passed down a silica gel (50 g) column
using a ethyl acetate/hexane (0-5%) gradient.
6-(2'-hexyldecanoyloxy)dodecanal (5.4 g) was recovered as an
oil.
[0576] A solution of the product (4.9 g), acetic acid (0.33 g) and
2-N,N-dimethylaminoethylamine (0.40 g) in methylene chloride (20
mL) was treated with sodium triacetoxyborohydride (2.1 g) for two
hours. The solution was washed with aqueous sodium hydroxide. The
organic phase was dried over anhydrous magnesium sulfate, filtered
and the solvent removed. The residue was passed down a silica gel
(50 g) column using a methanol/methylene chloride (0-8%) gradient
to yield the desired product (1.4 g) as a colorless oil.
Example 7: Synthesis of Compound I-6
[0577] Compound I-6 was prepared according to method B as
follows:
[0578] A solution of nonan-1,9-diol (12.6 g) in methylene chloride
(80 mL) was treated with 2-hexyldecanoic acid (10.0 g), DCC (8.7 g)
and DMAP (5.7 g). The solution was stirred for two hours. The
reaction mixture was filtered and the solvent removed. The residue
was dissolved in warmed hexane (250 mL) and allowed to crystallize.
The solution was filtered and the solvent removed. The residue was
dissolved in methylene chloride and washed with dilute hydrochloric
acid. The organic fraction was dried over anhydrous magnesium
sulfate, filtered and the solvent removed. The residue was passed
down a silica gel column (75 g) using 0-12% ethyl acetate/hexane as
the eluent, yielding 9-(2'-hexyldecanoyloxy)nonan-1-ol (9.5 g) as
an oil.
[0579] The product was dissolved in methylene chloride (60 mL) and
treated with pyridinium chlorochromate (6.4 g) for two hours.
Diethyl ether (200 mL) was added and the supernatant filtered
through a silica gel bed. The solvent was removed from the filtrate
and resultant oil passed down a silica gel (75 g) column using a
ethyl acetate/hexane (0-12%) gradient, yielding
9-(2'-ethylhexanoyloxy)nonanal (6.1 g) as an oil.
[0580] A solution of the crude product (6.1 g), acetic acid (0.34
g) and 2-N,N-dimethylaminoethylamine (0.46 g) in methylene chloride
(20 mL) was treated with sodium triacetoxyborohydride (2.9 g) for
two hours. The solution was diluted with methylene chloride washed
with aqueous sodium hydroxide, followed by water. The organic phase
was dried over anhydrous magnesium sulfate, filtered and the
solvent removed. The residue was passed down a silica gel (75 g)
column using a methanol/methylene chloride (0-8%) gradient,
followed by a second column (20 g) using a methylene
chloride/acetic acid/methanol gradient. The purified fractions were
dissolved in methylene chloride, washed with dilute aqueous sodium
hydroxide solution, dried over anhydrous magnesium sulfate,
filtered and the solvent removed, to yield the desired product (1.6
g) as a colorless oil.
Example 8: Synthesis of Compound II-9
##STR00153##
[0582] Compound II-9 was prepared according to method D as
follows:
Step 1
[0583] 3-dimethylamine-1-propylamine (1 eq. 1.3 mmol, 133 mg, 163
uL; MW102.18, d 0.812) and the ketone 9a (1 eq., 0.885 g, 1.3 mmol)
were mixed in DCE (8 mL) and then treated with sodium
triacetoxyborohydride (1.4 eq., 1.82 mmol, 386 mg; MW211.94) and
AcOH (1 eq., 1.3 mmol, 78 mg, 74 uL, MW 60.05, d 1.06). The mixture
was stirred at RT under an Ar atmosphere for 2 days. The reaction
mixture was diluted with hexanes-EtOAc (9:1) and quenched by adding
0.1 N NaOH (20 mL). The organic phase was separated, washed with
sat NaHCO.sub.3, brine, dried over sodium sulfate, decanted and
concentrated to give the desired product 9b as a slightly yellow
cloudy oil (1.07 g, 1.398 mmol).
Step 2
[0584] A solution of nonanoyl chloride (1.3 eq., 1.27 mmol, 225 mg)
in benzene (10 mL) was added via syringe to a solution of the
compound 9b from step 1 (0.75 g, 0.98 mmol) and triethylamine (5
eq, 4.90 mmol, 0.68 mL) and DMAP (20 mg) in benzene (10 mL) at RT
in 10 min. After addition, the mixture was stirred at RT overnight.
Methanol (5.5 mL) was added to remove excess acyl chloride. After 3
h, the mixture was filtered through a pad of silica gel (1.2 cm).
Concentration gave a colorless oil (0.70 g).
[0585] The crude product (0.70 g) was purified by flash dry column
chromatography on silica gel (0 to 4% MeOH in chloroform). This
yielded 457 mg of colorless oil, 0.50 mmol, 51%. 1HNMR (400 MHz,
CDCl3) .delta.: 4.54-4.36 (very br., estimated 0.3H, due to slow
isomerization about amide bond), 3.977, 3.973 (two sets of
doublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09
(m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two sets of singlet, 6H),
1.76-1.56 (m, 10H), 1.49-1.39 (m, 4H), 1.37-1.11 (62H), 0.92-0.86
(m, 15H).
Example 9: Synthesis of Compound II-10
[0586] Compound II-10 was prepared according to the general
procedure D to yield 245 mg of colorless oil, 0.27 mmol, total
yield 53% for 2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.87
(quintet-like, 6.3 Hz, 2H), 4.54-4.36 (very br., estimated 0.3H,
due to slow isomerization about amide bond), 3.63 (quintet-like,
6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22
(two sets of singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m, 12H),
1.37-1.11 (60H), 0.92-0.86 (m, 15H).
Example 10: Synthesis of Compound II-11
[0587] Compound II-11 was prepared according to the general
procedure D to yield 239 mg of colorless oil, 0.26 mmol, total
yield 52% for 2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.87
(quintet-like, 6.3 Hz, 2H), 4.54-4.36 (very br., estimated 0.3H,
due to slow isomerization about amide bond), 3.63 (quintet-like,
6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22
(two sets of singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m, 12H),
1.37-1.11 (62H), 0.92-0.86 (m, 15H).
Example 11: Synthesis of Compound II-12
[0588] Compound II-12 was prepared according to the general
procedure D to yield 198 mg of colorless oil, 0.20 mmol, total
yield 46% for 2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.:
4.54-4.36 (very br., estimated 0.3H, due to slow isomerization
about amide bond), 3.974, 3.971 (two sets of doublets, 5.8 Hz, 4H),
3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m,
8H), 2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 10H),
1.49-1.39 (m, 4H), 1.37-1.11 (76H), 0.92-0.86 (m, 15H).
Example 12: Synthesis of Compound III-3
[0589] A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g),
acetic acid (0.33 g) and 4-aminobutan-1-ol (0.23 g) in methylene
chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3
g) for two hours. The solution was washed with aqueous sodium
bicarbonate solution. The organic phase was dried over anhydrous
magnesium sulfate, filtered and the solvent removed. The residue
was passed down a silica gel column using a methanol/methylene
chloride (0-8/100-92%) gradient, yielding compound 3 as a colorless
oil (0.4 g).
Example 13: Synthesis of Compound III-7
[0590] A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g),
acetic acid (0.14 g) and 5-aminopentan-1-ol (0.24 g) in methylene
chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3
g) for two hours. The solution was washed with aqueous sodium
hydrogen carbonate solution. The organic phase was dried over
anhydrous magnesium sulfate, filtered and the solvent removed. The
residue was passed down a silica gel column using a
methanol/methylene chloride (0-8/100-92%) gradient, yielding
compound 7 as a colorless oil (0.5 g)
Example 14: Synthesis of a Representative PEG Lipid
##STR00154##
[0592] Pegylated lipid 14-6 ("PEG-DMA") was prepared according to
the above reaction scheme, wherein n approximates the center of the
range of ethylene oxide repeating units in the pegylated lipid.
Synthesis of 14-1 and 14-2
[0593] To a solution of myristic acid (6 g, 26 mmol) in toluene (50
mL) was added oxalyl chloride (39 mmol, 1.5 eq. 5 g) at RT. After
the resulting mixture was heated at 70.degree. C. for 2h, the
mixture was concentrated. The residue was taken up in toluene and
concentrated again. The residual oil was added via a syringe to a
concentrated ammonia solution (20 mL) at 10.degree. C. The reaction
mixture was filtered and washed with water. The white solid was
dried in vacuo. The desired product was obtained as a white solid
(3.47 g, 15 mmol, 58.7%).
Synthesis of 14-3
[0594] To suspension of 20-2 (3.47 g, 15 mmol) in THE (70 mL) was
added in portions of lithium aluminium hydride (1.14 g, 30 mmol) at
RT during 30 min period of time. Then the mixture was heated to
reflux gently (oil bath at 65.degree. C.) overnight. The mixture
was cooled to 5.degree. C. and sodium sulphate 9 hydrate was added.
The mixture was stirred for 2h, filtered through a layer of celite,
washed with 15% of MeOH in DCM (200 mL). The filtrate and washings
were combined and concentrated. The residual solid was dried in
vacuo. The desired product was obtained as a white solid (2.86 13.4
mmol, 89.5%).
Synthesis of 14-4
[0595] To a solution of myristic acid (3.86 g, 16.9 mmol) in
benzene (40 mL) and DMF (1 drop) was added oxalyl chloride (25.35
mmol, 1.5 eq. 3.22 g) at RT. The mixture was stirred at RT for 1.5
h. Heated at 60.degree. C. for 30 min. The mixture was
concentrated. The residue was taken up in toluene and concentrated
again. The residual oil (light yellow) was taken in 20 mL of
benzene and added via syringe to a solution of 20-3 (2.86 13.4
mmol) and triethylamine (3.53 mL, 1.5 eq) in benzene (40 mL) at
10.degree. C. After addition, the resulting mixture was stirred at
RT overnight. The reaction mixture was diluted with water and was
adjusted to pH 6-7 with 20% H.sub.2SO.sub.4. The mixture was
filtered and washed with water. A pale solid was obtained. The
crude product was recrystallized from methanol. This gave the
desired product as an off-white solid (5.65 g, 13 mmol, 100%).
Synthesis of 14-5
[0596] To suspension of 20-4 (5.65 g, 13 mmol) in THE (60 mL) was
added in portions lithium aluminium hydride (0.99 g, 26 mmol) at RT
during 30 min period of time. Then the mixture was heated to reflux
gently overnight. The mixture was cooled to 0.degree. C. and sodium
sulphate 9 hydrate. The mixture was stirred for 2h, then filtered
through a pad of celite and silica gel and washed with ether first.
The filtrate turned cloudy and precipitation formed. Filtration
gave a white solid. The solid was recrystallized from MeOH and a
colorless crystalline solid (2.43 g).
[0597] The pad of celite and silica gel was then washed 5% of MeOH
in DCM (400 mL) and then 10% of MeOH in DCM with 1% of
triethylamine (300 mL). The fractions containing the desired
product were combined and concentrated. A white solid was obtained.
The solid was recrystallized from MeOH and a colorless crystalline
solid (0.79 g). The above two solids (2.43 g and 0.79 g) were
combined and dried in vacuo (3.20 g, 60%). 1HNMR (CDCl3 at 7.27
ppm) .delta.: 2.58 (t-like, 7.2 Hz, 4H), 1.52-1.44 (m, 4H),
1.33-1.24 (m, 44H), 0.89 (t-like, 6.6 Hz, 6H), 2.1-1.3 (very broad,
1H).
Synthesis of 14-6
[0598] To a solution of 20-5 (7 mmol, 2.87 g) and triethylamine (30
mmol, 4.18 mL) in DCM (100 mL) was added a solution of mPEG-NHS
(from NOF, 5.0 mmol, 9.97 g, PEG MW approx. 2,000, n=about 45) in
DCM (120 mL,). After 24 h the reaction solution was washed with
water (300 mL). The aqueous phase was extracted twice with DCM (100
mL.times.2). DCM extracts were combined, washed with brine (100
mL). The organic phase was dried over sodium sulfate, filtered,
concentrated partially. The concentrated solution (ca 300 mL) was
cooled at ca -15 C. Filtration gave a white solid (1.030 g, the
unreacted starting amine). To the filtration was added Et.sub.3N
(1.6 mmol, 0.222 mL, 4 eq) and acetic anhydride (1.6 mmol, 164 mg).
The mixture was stirred at RT for 3h and then concentrated to a
solid. The residual solid was purified by column chromatography on
silica gel (0-8% methanol in DCM). This gave the desired product as
a white solid (9.211 g). 1HNMR (CDCl3 at 7.27 ppm) .delta.: 4.19
(s, 2H), 3.83-3.45 (m, 180-200H), 3.38 (s, 3H), 3.28 (t-like, 7.6
Hz, 2H, CH2N), 3.18 (t-like, 7.8 Hz, 2H, CH2N), 1.89 (s, 6.6H,
water), 1.58-1.48 (m, 4H), 1.36-1.21 (m, 48-50H), 0.88 (t-like, 6.6
Hz, 6H).
Example 15: Preparation of Lipid Nanoparticle Compositions
[0599] LNPs were prepared as follows. Cationic lipid, DSPC,
cholesterol and PEG-lipid (compound 14-6) were solubilized in
ethanol at a molar ratio of approximately 50:10:38.5:1.5. LNPs for
Examples 1, 2, 3 and 4 included cationic lipid compound I-6 and the
foregoing components. LNPs of Example 5 included the indicated
cationic lipid and the foregoing components. Lipid nanoparticles
(LNP) were prepared at a total lipid to mRNA weight ratio of
approximately 10:1 to 30:1. Briefly, the mRNA was diluted to 0.05
to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps
were used to mix the ethanolic lipid solution with the mRNA aqueous
solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow
rates above 15 ml/min. The ethanol was then removed and the
external buffer replaced with PBS by dialysis. Finally, the lipid
nanoparticles were filtered through a 0.2 m pore sterile filter.
Lipid nanoparticle particle size was 70-90 nm diameter as
determined by quasi-elastic light scattering using a Malvern
Zetasizer Nano (Malvern, UK).
Example 16: Sequences
[0600] Table 4 below provides sequence identifiers and description
of nucleic acid and amino acid sequences described herein.
TABLE-US-00004 TABLE 4 Sequences Sequence Description Number
Nucleic acid sequence encoding Env SEQ ID NO: 1 Entire mRNA
encoding Env (with UTRs SEQ ID NO: 2 and poly(A) tail) PR8 HA amino
acid sequence SEQ ID NO: 3 Native nucleoside sequence encoding PR8
SEQ ID NO: 4 HA Codon optimized used in mRNA SEQ ID NO: 5 encoding
PR8 HA Ca1/7/2009 HA amino acid sequence SEQ ID NO: 6 Native
nucleoside sequence encoding SEQ ID NO: 7 Ca1/7/2009 HA Codon
optimized sequence used in SEQ ID NO: 8 mRNA encoding Ca1/7/2009
HA
[0601] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
Sequence CWU 1
1
812544DNAHuman immunodeficiency virusmisc_feature(1507)..(1509)n is
a, c, g, t or u 1atgagagtga agggcatccg gcgcaactgc cagcggtggt
ggaagtgggg aatcatgctg 60ctgggcatcc tgatgatctg caacgccgag cagctgtggg
tcaccgtgta ctacggcgtg 120cccgtgtgga aagaggccac caccaccctg
ttctgcgcca gcgacgccaa gggccacgac 180accgaggccc acaacgtgtg
ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gaaatcgtgc
tggaaaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggaa
300cagatgcacg aggacgtgat cagcctgtgg gaccagagcc tgaagccctg
cgtgaagctg 360acccccttct gcgtgaccct gaactgcacc gacgtgatga
acaacgtgaa caccaccaca 420aacagcagcg agcggatgat caagaagggc
gagatcaaga actgcagctt caacatcaac 480accaacatgc ggaacaaggt
gcagaagaag cacgccctgt tctacaagct ggacgtggtg 540cccatcgaca
acaccagcta cagactgatc agctgcaaca ccagcgtgat cacccaggcc
600tgccccaagg tgtccttcga gcccatcccc atccactact gcgcccccgc
cggcttcgcc 660atcctgaagt gccgggacaa gaagttcaac ggcaccggcc
cctgcaccaa cgtgagcacc 720gtgcagtgca cccacggcat cagacccgtg
gtgagcaccc agctgctgtt caacggcagc 780ctggccgagg aggacgtcgt
gatcaagagc gccaacttca gcgacaacgc caagaccatc 840ctggtgcagc
tgaacgagac agtcgtgatc aactgcacca gacccggcaa caacacccgg
900aaaagagtga cactgggccc cggccgggtg tactacacca ccggccagat
catcggcgac 960atccggaagg cccactgcaa cctgagcaga gccggctgga
acaacaccct ggaacggatc 1020gccatcaagc tgagagagca gttccagaac
aagacaatcg ccttcaacca gagcagcgga 1080ggcgaccccg agatcaccaa
gatcagcttc aactgcggcg gcgagttctt ctactgcaac 1140agcacacagc
tgttcaacgg aacctggaac ggcacatggc tggacgtgaa gcagggcgac
1200ggcaccatca ccctgccctg cagaatcaag cagatcatca acctgtggca
ggaagtgggc 1260aaggccatgt acgccccccc catcagcgga cagatccggt
gcagcagcaa catcaccggc 1320ctgctgctga ccagagacgg cggcaccagc
aacgagacaa ccaccaccga gacattccgg 1380cccggaggag gaaacatgaa
ggacaactgg cgcagcgagc tgtaccggta caaagtgatc 1440aagatcgagc
ccctgggcgt ggcccccaca aaggcccgga gaagggtggt gcagcgcgag
1500aaaagannng ccgtgggaat cggcgccgtg ttcctgggct tcctgggagc
cgccggaagc 1560acaatgggcg ccgccagcat gaccctgacc gtgcaggcca
gacagctgct gagcggcatc 1620gtgcagcagc agaccaacct gctgagagcc
atcgaggcac agcagcagct gctgaaactg 1680accgtgtggg gcatcaagca
gctgcagaca agagtgctgg ccgtggaaag atacctgaag 1740gaccagcagc
tgctgggaat ctggggctgc agcggcaaac tgatctgcac caccaacgtg
1800ccctggaaca ccagctggag caacaagagc atgcaccaga tttgggacaa
catgacctgg 1860atgcagtggg agagagagat cgacaactac acaggcctga
tctacagcct gatcgaggaa 1920agccagaacc agcaggaaaa gaacgaacag
gaactgctgg ccctggacga gtgggccagc 1980ctgtggaact ggttcgacat
caccaagtgg ctgcggtaca tcaagatatt catcatcatc 2040gtgggcggcc
tgatcggcct gcggatcgtg ttcaccgtgc tgagcatcgt gaacagagtg
2100cggaagggct acagccccct gagcttccag accagactgc ccacacccag
aggccccgac 2160agacccggcg gcatcgagga ggaaggcggc gacagagaca
gggacggctc cggccccctc 2220gtgaacggct tcctggccat catctgggtg
gacctgcgga gcctgtgcct gttcagctac 2280cacagactgc gggacctgct
gctgatcgtg gccagaatcg tggaactgct gggcagaagg 2340ggctgggagg
ccctgaagta ctggtggaac ctgctgcagt actggtccca ggaactgaag
2400aacagcgccg tgtccctgct gaacgccatc gccatcgccg tggccgaggg
caccgacaga 2460gtgatcgaag tgctgcagag agccggacgg gccatcctgc
acatccccag aagaatccgg 2520cagggcctgg aaagggccct gctg
254422960DNAHuman immunodeficiency virusmisc_feature(1653)..(1655)n
is a, c, g, t or u 2ggaataaaag tctcaacaca acatatacaa aacaaacgaa
tctcaagcaa tcaagcattc 60tacttctatt gcagcaattt aaatcatttc ttttaaagca
aaagcaattt tctgaaaatt 120ttcaccattt acgaacgata gcgctgatga
gagtgaaggg catccggcgc aactgccagc 180ggtggtggaa gtggggaatc
atgctgctgg gcatcctgat gatctgcaac gccgagcagc 240tgtgggtcac
cgtgtactac ggcgtgcccg tgtggaaaga ggccaccacc accctgttct
300gcgccagcga cgccaagggc cacgacaccg aggcccacaa cgtgtgggcc
acccacgcct 360gcgtgcccac cgaccccaac ccccaggaaa tcgtgctgga
aaacgtgacc gagaacttca 420acatgtggaa gaacaacatg gtggaacaga
tgcacgagga cgtgatcagc ctgtgggacc 480agagcctgaa gccctgcgtg
aagctgaccc ccttctgcgt gaccctgaac tgcaccgacg 540tgatgaacaa
cgtgaacacc accacaaaca gcagcgagcg gatgatcaag aagggcgaga
600tcaagaactg cagcttcaac atcaacacca acatgcggaa caaggtgcag
aagaagcacg 660ccctgttcta caagctggac gtggtgccca tcgacaacac
cagctacaga ctgatcagct 720gcaacaccag cgtgatcacc caggcctgcc
ccaaggtgtc cttcgagccc atccccatcc 780actactgcgc ccccgccggc
ttcgccatcc tgaagtgccg ggacaagaag ttcaacggca 840ccggcccctg
caccaacgtg agcaccgtgc agtgcaccca cggcatcaga cccgtggtga
900gcacccagct gctgttcaac ggcagcctgg ccgaggagga cgtcgtgatc
aagagcgcca 960acttcagcga caacgccaag accatcctgg tgcagctgaa
cgagacagtc gtgatcaact 1020gcaccagacc cggcaacaac acccggaaaa
gagtgacact gggccccggc cgggtgtact 1080acaccaccgg ccagatcatc
ggcgacatcc ggaaggccca ctgcaacctg agcagagccg 1140gctggaacaa
caccctggaa cggatcgcca tcaagctgag agagcagttc cagaacaaga
1200caatcgcctt caaccagagc agcggaggcg accccgagat caccaagatc
agcttcaact 1260gcggcggcga gttcttctac tgcaacagca cacagctgtt
caacggaacc tggaacggca 1320catggctgga cgtgaagcag ggcgacggca
ccatcaccct gccctgcaga atcaagcaga 1380tcatcaacct gtggcaggaa
gtgggcaagg ccatgtacgc cccccccatc agcggacaga 1440tccggtgcag
cagcaacatc accggcctgc tgctgaccag agacggcggc accagcaacg
1500agacaaccac caccgagaca ttccggcccg gaggaggaaa catgaaggac
aactggcgca 1560gcgagctgta ccggtacaaa gtgatcaaga tcgagcccct
gggcgtggcc cccacaaagg 1620cccggagaag ggtggtgcag cgcgagaaaa
gannngccgt gggaatcggc gccgtgttcc 1680tgggcttcct gggagccgcc
ggaagcacaa tgggcgccgc cagcatgacc ctgaccgtgc 1740aggccagaca
gctgctgagc ggcatcgtgc agcagcagac caacctgctg agagccatcg
1800aggcacagca gcagctgctg aaactgaccg tgtggggcat caagcagctg
cagacaagag 1860tgctggccgt ggaaagatac ctgaaggacc agcagctgct
gggaatctgg ggctgcagcg 1920gcaaactgat ctgcaccacc aacgtgccct
ggaacaccag ctggagcaac aagagcatgc 1980accagatttg ggacaacatg
acctggatgc agtgggagag agagatcgac aactacacag 2040gcctgatcta
cagcctgatc gaggaaagcc agaaccagca ggaaaagaac gaacaggaac
2100tgctggccct ggacgagtgg gccagcctgt ggaactggtt cgacatcacc
aagtggctgc 2160ggtacatcaa gatattcatc atcatcgtgg gcggcctgat
cggcctgcgg atcgtgttca 2220ccgtgctgag catcgtgaac agagtgcgga
agggctacag ccccctgagc ttccagacca 2280gactgcccac acccagaggc
cccgacagac ccggcggcat cgaggaggaa ggcggcgaca 2340gagacaggga
cggctccggc cccctcgtga acggcttcct ggccatcatc tgggtggacc
2400tgcggagcct gtgcctgttc agctaccaca gactgcggga cctgctgctg
atcgtggcca 2460gaatcgtgga actgctgggc agaaggggct gggaggccct
gaagtactgg tggaacctgc 2520tgcagtactg gtcccaggaa ctgaagaaca
gcgccgtgtc cctgctgaac gccatcgcca 2580tcgccgtggc cgagggcacc
gacagagtga tcgaagtgct gcagagagcc ggacgggcca 2640tcctgcacat
ccccagaaga atccggcagg gcctggaaag ggccctgctg taaactagta
2700gtgactgact aggatctggt taccactaaa ccagcctcaa gaacacccga
atggagtctc 2760taagctacat aataccaact tacacttaca aaatgttgtc
ccccaaaatg tagccattcg 2820tatctgctcc taataaaaag aaagtttctt
cacattctaa aaaaaaaaaa aaaaaaaaaa 2880aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940aaaaaaaaaa
aaaaaaaaac 29603565PRTInfluenza virus 3Met Lys Ala Asn Leu Leu Val
Leu Leu Ser Ala Leu Ala Ala Ala Asp1 5 10 15Ala Asp Thr Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30Val Asp Thr Val Leu
Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45Leu Leu Glu Asp
Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60Ala Pro Leu
Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly65 70 75 80Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe145 150 155 160Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Leu Tyr 195 200 205Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln
Ala225 230 235 240Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro
Gly Asp Thr Ile 245 250 255Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala
Pro Met Tyr Ala Phe Ala 260 265 270Leu Ser Arg Gly Phe Gly Ser Gly
Ile Ile Thr Ser Asn Ala Ser Met 275 280 285His Glu Cys Asn Thr Lys
Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300Ser Leu Pro Tyr
Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro305 310 315 320Lys
Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn 325 330
335Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
340 345 350Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly
Tyr His 355 360 365His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
Gln Lys Ser Thr 370 375 380Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys
Val Asn Thr Val Ile Glu385 390 395 400Lys Met Asn Ile Gln Phe Thr
Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415Glu Lys Arg Met Glu
Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430Asp Ile Trp
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445Arg
Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455
460Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly
Cys465 470 475 480Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
Glu Ser Val Arg 485 490 495Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
Glu Glu Ser Lys Leu Asn 500 505 510Arg Glu Lys Val Asp Gly Val Lys
Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525Ile Leu Ala Ile Tyr Ser
Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540Ser Leu Gly Ala
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln545 550 555 560Cys
Arg Ile Cys Ile 56541698DNAInfluenza virus 4atgaaggcaa acctactggt
cctgttaagt gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa
caattcaacc gacactgttg acacagtact cgagaagaat 120gtgacagtga
cacactctgt taacctgctc gaagacagcc acaacggaaa actatgtaga
180ttaaaaggaa tagccccact acaattgggg aaatgtaaca tcgccggatg
gctcttggga 240aacccagaat gcgacccact gcttccagtg agatcatggt
cctacattgt agaaacacca 300aactctgaga atggaatatg ttatccagga
gatttcatcg actatgagga gctgagggag 360caattgagct cagtgtcatc
attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca
acacaaacgg agtaacggca gcatgctccc atgaggggaa aagcagtttt
480tacagaaatt tgctatggct gacggagaag gagggctcat acccaaagct
gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg
gtattcatca cccgcctaac 600agtaaggaac aacagaatct ctatcagaat
gaaaatgctt atgtctctgt agtgacttca 660aattataaca ggagatttac
cccggaaata gcagaaagac ccaaagtaag agatcaagct 720gggaggatga
actattactg gaccttgcta aaacccggag acacaataat atttgaggca
780aatggaaatc taatagcacc aatgtatgct ttcgcactga gtagaggctt
tgggtccggc 840atcatcacct caaacgcatc aatgcatgag tgtaacacga
agtgtcaaac acccctggga 900gctataaaca gcagtctccc ttaccagaat
atacacccag tcacaatagg agagtgccca 960aaatacgtca ggagtgccaa
attgaggatg gttacaggac taaggaacac tccgtccatt 1020caatccagag
gtctatttgg agccattgcc ggttttattg aagggggatg gactggaatg
1080atagatggat ggtatggtta tcatcatcag aatgaacagg gatcaggcta
tgcagcggat 1140caaaaaagca cacaaaatgc cattaacggg attacaaaca
aggtgaacac tgttatcgag 1200aaaatgaaca ttcaattcac agctgtgggt
aaagaattca acaaattaga aaaaaggatg 1260gaaaatttaa ataaaaaagt
tgatgatgga tttctggaca tttggacata taatgcagaa 1320ttgttagttc
tactggaaaa tgaaaggact ctggatttcc atgactcaaa tgtgaagaat
1380ctgtatgaga aagtaaaaag ccaattaaag aataatgcca aagaaatcgg
aaatggatgt 1440tttgagttct accacaagtg tgacaatgaa tgcatggaaa
gtgtaagaaa tgggacttat 1500gattatccca aatattcaga agagtcaaag
ttgaacaggg aaaaggtaga tggagtgaaa 1560ttggaatcaa tggggatcta
tcagattctg gcgatctact caactgtcgc cagttcactg 1620gtgcttttgg
tctccctggg ggcaatcagt ttctggatgt gttctaatgg atctttgcag
1680tgcagaatat gcatctga 169851698DNAArtificial SequenceChemically
synthesized 5atgaaggcga acctgctggt cctgctgagc gcgctggcgg cggcggacgc
ggacacgatc 60tgcatcggct accacgcgaa caacagcacc gacacggtcg acacggtcct
cgagaagaac 120gtgaccgtga cccacagcgt caacctgctc gaggacagcc
acaacgggaa gctgtgcagg 180ctcaagggca tcgccccgct gcagctgggg
aagtgcaaca tcgccggctg gctcttgggg 240aaccccgagt gcgacccgct
gctcccggtg aggagctggt cctacatcgt ggagaccccg 300aactcggaga
acgggatctg ctacccgggg gacttcatcg actacgagga gctgagggag
360cagttgagct cggtgtcgtc cttcgagagg ttcgagatct tccccaagga
gagctcgtgg 420cccaaccaca acaccaacgg ggtcacggcc gcgtgctccc
acgaggggaa gagcagcttc 480tacaggaact tgctgtggct gacggagaag
gagggctcgt acccgaagct gaagaactcg 540tacgtgaaca agaaggggaa
ggaggtcctc gtactgtggg gcatccacca cccgccgaac 600agcaaggagc
agcagaacct ctaccagaac gagaacgcgt acgtctccgt ggtgacctcg
660aactacaaca ggaggttcac cccggagatc gcggagaggc ccaaggtcag
ggaccaggcc 720gggaggatga actactactg gaccttgctg aagcccggcg
acaccatcat cttcgaggcg 780aacgggaacc tgatcgcacc gatgtacgcg
ttcgcgctga gcaggggctt cgggtccggc 840atcatcacct cgaacgcgtc
catgcacgag tgcaacacga agtgccagac gcccctgggc 900gcgatcaaca
gcagcctccc gtaccagaac atccacccgg tcacgatcgg ggagtgcccc
960aagtacgtca ggagcgccaa gttgaggatg gtgaccgggc tcaggaacac
gccgtccatc 1020cagtccaggg gcctgttcgg ggccatcgcc gggttcatcg
aggggggctg gaccggcatg 1080atcgacgggt ggtacgggta ccaccaccag
aacgagcagg ggtcgggcta cgcggcggac 1140cagaagagca cgcagaacgc
catcaacggg atcacgaaca aggtgaacac ggtcatcgag 1200aagatgaaca
tccagttcac ggccgtgggg aaggagttca acaagttgga gaagaggatg
1260gagaacttga acaagaaggt cgacgacggg ttcctggaca tctggacgta
caacgcggag 1320ttgttggtgc tgctggagaa cgagaggacg ctggacttcc
acgactcgaa cgtgaagaac 1380ctgtacgaga aggtgaagag ccagttgaag
aacaacgcca aggagatcgg caacgggtgc 1440ttcgagttct accacaagtg
cgacaacgag tgcatggaga gcgtgaggaa cgggacgtac 1500gactacccca
agtactccga agagtcgaag ttgaacaggg agaaggtgga cggggtgaag
1560ttggagtcga tggggatcta ccagatcctg gcgatctact cgacggtcgc
cagctccctg 1620gtgctgttgg tctccctggg ggcgatcagc ttctggatgt
gctccaacgg gtcgttgcag 1680tgcaggatct gcatctga 16986566PRTInfluenza
virus 6Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala
Asn1 5 10 15Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Asp Thr 20 25 30Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
Leu Arg Gly Val 50 55 60Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala
Gly Trp Ile Leu Gly65 70 75 80Asn Pro Glu Cys Glu Ser Leu Ser Thr
Ala Ser Ser Trp Ser Tyr Ile 85 90 95Val Glu Thr Pro Ser Ser Asp Asn
Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110Ile Asp Tyr Glu Glu Leu
Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125Glu Arg Phe Glu
Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135 140Ser Asn
Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser145 150 155
160Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro
165 170 175Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val
Leu Val 180 185 190Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp
Gln Gln Ser Leu 195 200 205Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val
Gly Ser Ser Arg Tyr Ser 210 215 220Lys Lys Phe Lys Pro Glu Ile Ala
Ile Arg Pro Lys Val Arg Gly Gln225 230 235 240Glu Gly Arg Met Asn
Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255Ile Thr Phe
Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270Ala
Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280
285Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn
290 295 300Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly
Lys Cys305 310 315 320Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu
Ala Thr Gly Leu Arg 325 330 335Asn Ile Pro Ser Ile Gln Ser Arg Gly
Leu Phe Gly Ala Ile Ala Gly 340 345 350Phe Ile Glu Gly Gly Trp Thr
Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365His His Gln Asn Glu
Gln Gly
Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380Thr Gln Asn Ala Ile
Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile385 390 395 400Glu Lys
Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His 405 410
415Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe
420 425 430Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu
Glu Asn 435 440 445Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
Asn Leu Tyr Glu 450 455 460Lys Val Arg Ser Gln Leu Lys Asn Asn Ala
Lys Glu Ile Gly Asn Gly465 470 475 480Cys Phe Glu Phe Tyr His Lys
Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495Lys Asn Gly Thr Tyr
Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510Asn Arg Glu
Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr 515 520 525Gln
Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val 530 535
540Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser
Leu545 550 555 560Gln Cys Arg Ile Cys Ile 56571701DNAInfluenza
virus 7atgaaagcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc
agacacatta 60tgtataggtt atcatgcgaa caattcaaca gacactgtag acacagtact
agaaaagaat 120gtaacagtaa cacactctgt taaccttcta gaagacaagc
ataacgggaa actatgcaaa 180ctaagagggg tagccccatt gcatttgggt
aaatgtaaca ttgctggctg gatcctggga 240aatccagagt gtgaatcact
ctccacagca agctcatggt cctacattgt ggaaacacct 300agttcagaca
atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag
360caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac
aagttcatgg 420cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc
ctcatgctgg agcaaaaagc 480ttctacaaaa atttaatatg gctagttaaa
aaaggaaatt catacccaaa gctcagcaaa 540tcctacatta atgataaagg
gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600actagtgctg
accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggtca
660tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt
gaggggtcaa 720gaagggagaa tgaactatta ctggacacta gtagagccgg
gagacaaaat aacattcgaa 780gcaactggaa atctagtggt accgagatat
gcattcgcaa tggaaagaaa tgctggatct 840ggtattatca tttcagatac
accagtccac gattgcaata caacttgtca aacacccaag 900ggtgctataa
acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt
960ccaaaatatg taaaaagcac aaaattgaga ctggccacag gattgaggaa
tatcccgtct 1020attcaatcta gaggcctatt tggggccatt gccggtttca
ttgaaggggg gtggacaggg 1080atggtagatg gatggtacgg ttatcaccat
caaaatgagc aggggtcagg atatgcagcc 1140gacctgaaga gcacacagaa
tgccattgac gagattacta acaaagtaaa ttctgttatt 1200gaaaagatga
atacacagtt cacagcagta ggtaaagagt tcaaccacct ggaaaaaaga
1260atagagaatt taaataaaaa agttgatgat ggtttcctgg acatttggac
ttacaatgcc 1320gaactgttgg ttctattgga aaatgaaaga actttggact
accacgattc aaatgtgaag 1380aacttatatg aaaaggtaag aagccagcta
aaaaacaatg ccaaggaaat tggaaacggc 1440tgctttgaat tttaccacaa
atgcgataac acgtgcatgg aaagtgtcaa aaatgggact 1500tatgactacc
caaaatactc agaggaagca aaattaaaca gagaagaaat agatggggta
1560aagctggaat caacaaggat ttaccagatt ttggcgatct attcaactgt
cgccagttca 1620ttggtactgg tagtctccct gggggcaatc agtttctgga
tgtgctctaa tgggtctcta 1680cagtgtagaa tatgtattta a
170181701DNAArtificial SequenceChemically synthisized 8atgaaagcaa
tactagtagt actgctatac acattcgcaa ccgcaaacgc agacacatta 60tgcataggct
accacgcgaa caactcaaca gacaccgtag acacagtact agaaaagaac
120gtaacagtaa cacactccgt caacctccta gaagacaagc acaacgggaa
actatgcaaa 180ctaagagggg tagccccatt gcacttgggc aaatgcaaca
tcgctggctg gatcctggga 240aacccagagt gcgaatcact ctccacagca
agctcatggt cctacatcgt ggaaacaccg 300agctcagaca acggaacgtg
ctacccagga gacttcatcg actacgagga gctaagagag 360caattgagct
cagtgtcatc attcgaaagg ttcgagatat tccccaagac aagctcatgg
420cccaaccacg actcgaacaa aggcgtaacg gcagcatgcc cgcacgccgg
agcaaaaagc 480ttctacaaaa acttaatatg gctagtgaaa aaaggaaact
catacccaaa gctcagcaaa 540tcctacatca acgacaaagg gaaagaagtc
ctcgtgctat ggggcatcca ccacccatcg 600accagcgccg accaacaaag
cctctaccag aacgcagacg catacgtgtt cgtggggtca 660tcaagataca
gcaagaagtt caagccggaa atagcaataa gacccaaagt gaggggccaa
720gaagggagaa tgaactacta ctggacacta gtagagccgg gagacaaaat
aacattcgaa 780gcaaccggaa acctagtggt accgagatac gcattcgcaa
tggaaagaaa cgccggatcc 840ggcatcatca tatcagacac accagtccac
gactgcaaca caacctgcca aacacccaag 900ggcgcgataa acaccagcct
cccattccag aacatacacc cgatcacaat cggaaaatgc 960ccaaaatacg
taaaaagcac aaaattgaga ctggccacag gattgaggaa tatcccgtcc
1020atccaatcca gaggcctatt cggggccatc gccggcttca tcgaaggggg
gtggacaggg 1080atggtagatg gatggtacgg ttaccaccac caaaacgagc
aggggtcagg atacgcagcc 1140gacctgaaga gcacacagaa cgccatcgac
gagatcacga acaaagtaaa ctccgtcatc 1200gaaaagatga acacacagtt
cacagcagta ggcaaagagt tcaaccacct ggaaaaaaga 1260atagagaact
taaacaaaaa agtcgacgac ggcttcctgg acatctggac ctacaacgcc
1320gaactgttgg tcctattgga aaacgaaaga accttggact accacgactc
aaacgtgaag 1380aacttatacg aaaaggtaag aagccagcta aaaaacaacg
ccaaggaaat cggaaacggc 1440tgcttcgaat tctaccacaa atgcgacaac
acgtgcatgg aaagcgtcaa aaacgggacg 1500tacgactacc caaaatactc
agaggaagca aaattaaaca gagaagaaat agacggggta 1560aagctggaat
caacaaggat ctaccagatc ttggcgatct actcaaccgt cgccagctca
1620ttggtactgg tagtctccct gggggcaatc agtttctgga tgtgctccaa
cgggtcccta 1680cagtgcagaa tatgcatcta a 1701
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