U.S. patent application number 17/371935 was filed with the patent office on 2021-11-04 for 5'-cap compounds and their uses in stabilizing rna, expressing proteins and in therapy.
The applicant listed for this patent is BioNTech RNA Pharmaceuticals GmbH. Invention is credited to Stephanie Fesser, Katalin Kariko, Andreas Kuhn, Hiromi Muramatsu, Ugur Sahin.
Application Number | 20210340170 17/371935 |
Document ID | / |
Family ID | 1000005765410 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340170 |
Kind Code |
A1 |
Kuhn; Andreas ; et
al. |
November 4, 2021 |
5'-CAP COMPOUNDS AND THEIR USES IN STABILIZING RNA, EXPRESSING
PROTEINS AND IN THERAPY
Abstract
The present invention relates to 5'-cap compounds, in particular
the stabilization of RNA by such 5'-cap compounds, and provides
compositions, such as pharmaceutical compositions, and cells
comprising an RNA which is modified with such a 5'-cap compound, as
well as methods for producing a peptide or protein of interest
using the compositions or cells according to the present invention.
Furthermore, the present invention provides the RNA, compositions,
or cells for use in therapy, in particular for use in a method of
treating a disease or disorder by protein replacement therapy,
genome engineering, genetic reprogramming, and immunotherapy; a
method for increasing the stability of RNA in cells; a method for
increasing the expression of RNA in cells; and a method for
providing an RNA with a 5'-cap structure.
Inventors: |
Kuhn; Andreas; (Mainz,
DE) ; Muramatsu; Hiromi; (Mainz, DE) ; Kariko;
Katalin; (Mainz, DE) ; Fesser; Stephanie;
(Mainz, DE) ; Sahin; Ugur; (Mainz, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioNTech RNA Pharmaceuticals GmbH |
Mainz |
|
DE |
|
|
Family ID: |
1000005765410 |
Appl. No.: |
17/371935 |
Filed: |
July 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16980300 |
Sep 11, 2020 |
|
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PCT/EP2019/056502 |
Mar 14, 2019 |
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17371935 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7125 20130101;
C07H 21/02 20130101 |
International
Class: |
C07H 21/02 20060101
C07H021/02; A61K 31/7125 20060101 A61K031/7125 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2018 |
EP |
PCT/EP2018/056595 |
Claims
1. A 5'-cap compound having the 5'-cap structure according to
formula (I): ##STR00017## wherein R.sup.1 is selected from the
group consisting of optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted cycloalkyl, optionally substituted heterocyclyl,
optionally substituted aryl, and optionally substituted heteroaryl;
R.sup.2 and R.sup.3 are independently selected from the group
consisting of H, halo, OH, and optionally substituted alkoxy, or
R.sup.2 and R.sup.3 together form O--X--O, wherein X is selected
from the group consisting of optionally substituted CH.sub.2,
optionally substituted CH.sub.2CH.sub.2, optionally substituted
CH.sub.2CH.sub.2CH.sub.2, optionally substituted
CH.sub.2CH(CH.sub.3), and optionally substituted C(CH.sub.3).sub.2,
or R.sup.2 is combined with the hydrogen atom at position 4' of the
ring to which R.sup.2 is attached to form --O--CH.sub.2-- or
--CH.sub.2--O--; R.sup.4 and R.sup.6 are independently selected
from the group consisting of O, S, Se, and BH.sub.3; R.sup.5 is
selected from the group consisting of S, Se, and BH.sub.3; R.sup.7
is a mononucleotide or an oligonucleotide having 2 to 9 bases;
R.sup.8 is H, halo, or optionally substituted alkoxy; n is 1, 2, or
3; and B is a purine or pyrimidine base moiety.
2. The 5'-cap compound of claim 1, wherein R.sup.1 is selected from
the group consisting of optionally substituted C.sub.1-4 alkyl,
optionally substituted C.sub.2-4 alkenyl, and optionally
substituted aryl.
3. The 5'-cap compound of claim 1, wherein R.sup.1 is optionally
substituted C.sub.1-4 alkyl.
4. The 5'-cap compound of claim 1, wherein R.sup.1 is C.sub.1-4
alkyl.
5. The 5'-cap compound of claim 1, wherein R.sup.2 and R.sup.3 are
independently selected from the group consisting of H, F, OH,
methoxy, ethoxy, propoxy, and 2-methoxyethoxy.
6. The 5'-cap compound of claim 1, wherein R.sup.2 is OH and
R.sup.3 is methoxy or R.sup.2 is methoxy and R.sup.3 is OH.
7. The 5'-cap compound of claim 1, wherein R.sup.4 and R.sup.6 are
independently selected from the group consisting of O and S.
8. The 5'-cap compound of claim 1, wherein R.sup.4 and R.sup.6 are
O.
9. The 5'-cap compound of claim 1, wherein R.sup.5 is S.
10. The 5'-cap compound of claim 1, wherein R.sup.5 is S, n is 1 or
2, and R.sup.4 and R.sup.6 are O.
11. The 5'-cap compound of claim 10, wherein n is 1.
12. The 5'-cap compound of claim 1, wherein B is selected from the
group consisting of guanine, adenine, cytosine, thymine, uracil,
and modified forms thereof.
13. The 5'-cap compound of claim 1, wherein B is selected from the
group consisting of guanine, adenine, and modified forms
thereof.
14. The 5'-cap compound of claim 1, wherein B is guanine or
adenine.
15. The 5'-cap compound of claim 1, wherein R.sup.7 is *pGpN or
*pG.
16. The 5'-cap compound of claim 1, wherein R.sup.8 is optionally
substituted alkoxy.
17. The 5'-cap compound of claim 1, wherein R.sup.8 is methoxy.
18. The 5'-cap compound of claim 1, wherein the 5'-cap compound has
a structure according to formula (III): ##STR00018##
19. The 5'-cap compound of claim 18, wherein R.sup.1 is methyl or
ethyl.
20. The 5'-cap compound of claim 18, wherein one of R.sup.2 and
R.sup.3 is OCH.sub.3 and the other is OH.
21. The 5'-cap compound of claim 18, wherein n is 1.
22. The 5'-cap compound of claim 18, wherein B is guanine or
adenine.
23. The 5'-cap compound of claim 18, wherein R.sup.7 is a
ribomononucleotide having a free OH group at position 2'.
24. The 5'-cap compound of claim 18, wherein R.sup.8 is
methoxy.
25. The 5'-cap compound of claim 18, wherein R.sup.1 is methyl or
ethyl; one of R.sup.2 and R.sup.3 is OCH.sub.3 and the other is OH;
n is 1; R.sup.7 is a ribomononucleotide having a free OH group at
position 2'; the internucleotide linkage between the
ribomononucleotide and the ring to which R.sup.7 is attached is
phosphate; and B is guanine or adenine.
26. An RNA which is modified with a 5'-cap compound of claim 1.
27. The RNA of claim 26, wherein the RNA further comprises a
nucleotide sequence encoding a peptide or protein of interest.
28. A composition or cell comprising an RNA of claim 26.
29. The composition or cell of claim 28, wherein the RNA further
comprises a nucleotide sequence encoding a peptide or protein of
interest.
30. A method of treating a disease or disorder in a subject
comprising the step of administering to said subject: (i) an RNA
modified with a 5'-cap compound having the 5'-cap structure
according to formula (I), wherein the RNA further comprises a
nucleotide sequence encoding a peptide or protein of interest, or
(ii) a composition or cell comprising an RNA modified with a 5'-cap
compound having the 5'-cap structure according to formula (I),
wherein the RNA further comprises a nucleotide sequence encoding a
peptide or protein of interest; wherein formula (I) is:
##STR00019## wherein R.sup.1 is selected from the group consisting
of optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
and optionally substituted heteroaryl; R.sup.2 and R.sup.3 are
independently selected from the group consisting of H, halo, OH,
and optionally substituted alkoxy, or R.sup.2 and R.sup.3 together
form O--X--O, wherein X is selected from the group consisting of
optionally substituted CH.sub.2, optionally substituted
CH.sub.2CH.sub.2, optionally substituted CH.sub.2CH.sub.2CH.sub.2,
optionally substituted CH.sub.2CH(CH.sub.3), and optionally
substituted C(CH.sub.3).sub.2, or R.sup.2 is combined with the
hydrogen atom at position 4' of the ring to which R.sup.2 is
attached to form --O--CH.sub.2-- or --CH.sub.2--O--; R.sup.4 and
R.sup.6 are independently selected from the group consisting of O,
S, Se, and BH.sub.3; R.sup.5 is selected from the group consisting
of S, Se, and BH.sub.3; R.sup.7 is a mononucleotide or an
oligonucleotide having 2 to 9 bases; R.sup.8 is H, halo, or
optionally substituted alkoxy; n is 1, 2, or 3; and B is a purine
or pyrimidine base moiety.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/980,300, which was filed on Sep. 11, 2020 as a National
Stage Entry of International Application Number PCT/EP2019/056502,
which was filed on Mar. 14, 2019 and claimed priority to
International Application Number PCT/EP2018/056595, which was filed
on Mar. 15, 2018. The contents of each of the aforementioned
applications are incorporated herein by reference in their
entireties.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to 5'-cap compounds, in
particular trinucleotides or higher homologues, wherein the 5'-cap
compounds contain at least one phosphorothioate, phosphoroselenoate
and/or boranophosphate moiety in the phosphate bridge between the
first and second nucleotide, and wherein the second nucleotide is
blocked at its 2'-position. In particular, the present invention
relates to stabilization of RNA by such 5'-cap compounds, in
particular in the context of using RNA for expressing a peptide or
protein of interest, such as in the context of vaccination, and
provides compositions, such as pharmaceutical compositions, and
cells comprising an RNA which is modified with such a 5'-cap
compound, as well as methods for producing a peptide or protein of
interest using the compositions or cells according to the present
invention. Furthermore, the present invention provides the RNA,
compositions, or cells for use in therapy, in particular for use in
a method of treating a disease or disorder by protein replacement
therapy, genome engineering, genetic reprogramming, or
immunotherapy; a method for increasing the stability of RNA in
cells; a method for increasing the expression of RNA in cells; and
a method for providing an RNA with a 5'-cap structure.
BACKGROUND OF THE INVENTION
[0003] The concept of nucleic acid-encoded therapeutics was
conceived in 1990 when Wolff et al. (Science, 247: 1465-1468)
showed that direct intramuscular injection of in vitro transcribed
(IVT) mRNA or plasmid DNA (pDNA) into the skeletal muscle of mice
led to the expression of the encoded proteins in the injected
muscle. This finding was a major incentive in the field to further
investigate the applicability of nucleic acids in therapy, in
particular immunotherapy. At first, DNA based vaccines against
infectious pathogens have been studied (Cox et al., 1993, J. Virol.
67: 5664-5667; Davis et al., 1993, Hum. Mol. Genet. 2: 1847-1851;
Ulmer et al., 1993, Science 259: 1745-1749; Wang et al., 1993,
Proc. Natl. Acad. Sci. U.S.A. 90: 4156-4160). Furthermore, the
applicability of nucleic acids in gene therapy against tumors and
for induction of a specific anti-tumor immunity has been studied
(Conry et al., 1994, Cancer Res. 54: 1164-1168; Conry et al., 1995,
Gene Ther. 2: 59-65; Spooner et al., 1995, Gene Ther. 2: 173-180;
Wang et al., 1995, Hum. Gene Ther. 6: 407-418).
[0004] Nucleic acid based therapy exhibits a number of advantages.
For example, the manufacture of nucleic acid based therapeutics is
straight forward, relatively inexpensive, and DNA based
therapeutics are stable for long-term storage. However, in
particular, DNA based therapeutics exhibit a variety of potential
safety risks such as induction of anti-DNA antibodies (Gilkeson et
al., 1995, J. Clin. Invest. 95: 1398-1402) and potential
integration of the transgene into the host genome. This may lead to
the inactivation of cellular genes, an uncontrollable long term
expression of the transgene, or oncogenesis, and thus, is generally
not applicable for tumor-associated antigens with oncogenic
potential such as erb-B2 (Bargmann et al., 1986, Nature 319:
226-230) and p53 (Greenblatt et al., 1994, Cancer Res. 54:
4855-4878).
[0005] The use of RNA provides an attractive alternative to
circumvent the potential risks of DNA based therapeutics. Some of
the advantages of RNA based therapy are the transient expression
and the non-transforming character. Furthermore, RNA does not have
to be transported into the nucleus for the transgene to be
expressed, and moreover, cannot be integrated into the host
genome.
[0006] Two different strategies have been pursued for therapy with
IVT RNA, which have both been successfully tested in various animal
models. Either the RNA is directly injected into the patient by
different routes (Hoerr et al., 2000, Eur. J. Immunol. 30: 1-7) or
dendritic cells are transfected with IVT RNA using conventional
transfection methods in vitro and then the transfected dendritic
cells are administered to the patient (Heiser et al., 2000, J.
Immunol. 164: 5508-5514). It has been shown that immunization with
RNA transfected dendritic cells induces antigen-specific cytotoxic
T-lymphocytes (CTL) in vitro and in vivo (Su et al., 2003, Cancer
Res. 63: 2127-2133; Heiser et al., 2002, J. Clin. Invest. 109:
409-417). Furthermore, it has been shown that direct injection of
naked RNA into the lymph nodes of laboratory animals (intranodal
injection) leads to uptake of said RNA primarily by immature
dendritic cells, probably by a process called macropinocytosis (cf.
DE 10 2008 061 522.6). It is assumed that the RNA is translated and
the expressed protein is presented on the MHC molecules on the
surface of the antigen presenting cells to elicit an immune
response.
[0007] A major disadvantage of RNA based therapy is the instability
of the RNA in vivo. Degradation of long-chain RNA from the 5'-end
is induced in the cell by the so called "decapping" enzyme Dcp2
which cleaves m.sup.7GDP from the RNA chain. Thus, it is assumed
that the cleavage occurs between the alpha- and beta-phosphate
groups of the RNA-cap.
[0008] Eukaryotic messenger RNAs (mRNAs) carry a specific structure
at the 5'-end, the so-called cap structure. This consists of a
N.sup.7-methylated guanosine moiety, which is added to the first
transcribed nucleotide of an RNA, commonly a guanosine, via a 5'-5'
triphosphate bridge. Accordingly, this structure is often referred
to as m.sup.7GpppG. The m.sup.7GpppG structure is among others
required for translation of the mRNA into the encoded protein.
[0009] Cellular mRNAs in higher eukaryotes are further modified at
the 5'-end by methylation at the 2'-O position of the first
nucleotide after the m.sup.7Gppp moiety. This structure is called
cap1 (vs. cap0 for the non-methylated form). While this
modification was described more than 40 years ago, its function has
remained elusive until recently. Only in 2010 it was first reported
that 2'-0 methylation of the cap avoids recognition by proteins
recognizing the cap0 structure, such as IFIT proteins, especially
IFIT1. Binding of IFIT1 to cap0 mRNAs impairs binding of the
cap-binding translation initiation eIF4E, which results in
decreased translation efficiency.
[0010] Synthetic mRNAs are commonly produced by in vitro
transcription from a suitable DNA template (e.g., linearized
plasmid DNA) using a phage RNA polymerase (mostly T7 or SP6 RNA
polymerase). Capped mRNAs can be obtained by in vitro transcription
by adding an excess of a cap dinucleotide, e.g., m.sup.7GpppG, to
the reaction. However, it was reported that the cap dinucleotide
m.sup.7GpppG can be incorporated during in vitro transcription in
two orientations, from which only one is functional. Therefore,
anti-reverse cap analogs (ARCAs) have been developed that cannot be
integrated in the reverse orientation due to modifications at
either the 2'- or 3'-position of the m.sup.7guanosine.
Consequently, it was demonstrated in rabbit reticulocyte lysate and
in dendritic cells that ARCA-capped mRNAs exhibit superior
translation efficiency compared to m.sup.7GpppG-capped RNAs.
[0011] In the past decade, ARCAs were further modified in an
attempt to stabilize the mRNA against decapping enzymes and to
enhance translation efficiency by increasing the affinity for
eIF4E. Modifications include various substitutions at the bridging
and non-bridging oxygen in the phosphate bridge, extended phosphate
groups, and guanosine modifications. The task is complicated by the
fact that cap analogs being inert against the decapping enzyme
Dcp1-Dcp2 are not always good substrates for the initiation factor
and as a result only poorly translated. However, usage of
phosphorothioate modified cap analogs at the .beta.-phosphate
(beta-S-ARCA or .beta.-S-ARCA) resulted in mRNAs with both
increased translation efficiency and elongated half-life in e.g.,
dendritic cells as compared to ARCA or m.sup.7GpppG. .beta.-S-ARCA
is synthesized as a mixture of two diastereomers, referred to as D1
and D2, based on their elution pattern in HPLC, due to the
introduction of a stereogenic P center by the sulfur modification.
Interestingly, it was shown that the diastereomers have different
biological properties, in particular with respect to the resistance
against enzymatic cleavage (such as Dcp2 cleavage) and/or binding
to eIF4E. While m.sup.7GpppG was generally employed in the past,
ARCA capped mRNAs are more and more entering preclinical and now
also clinical studies.
[0012] As modification of the 2'-O position in a cap dinucleotide
inhibits incorporation by the phage RNA polymerase (as
advantageously used in ARCAs), only cap0 structures can be
co-transcriptionally added in vitro using a cap dinucleotide.
Capping of in vitro transcribed RNA can also be reached
post-transcriptionally using the corresponding enzymes, e.g., from
vaccinia virus. Here, a cap1 structure can be obtained. However,
the synthesis process then consists of two steps, transcription
followed by capping, making it more laborious. Furthermore, the
very 5' sequence of the RNAs has a strong influence on the capping
efficiency by the enzymes. Also, the method is limited to
unmodified caps due to the specificity of the enzymes. Thus, none
of the beneficial modifications as described above (e.g.,
phosphorothioate substitutions) can be incorporated in this
manner.
[0013] In summary, RNA is especially well-suited for clinical
applications. However, the use of RNA in therapy is primarily
limited by the short half-life of RNA, in particular in the
cytoplasm, and/or the recognition of the RNA by proteins
recognizing the cap0 structure, such as IFIT proteins, in
particular IFIT1 (thereby impairing the binding of the RNA to
eIF4E) both of which result in low and/or insufficient protein
expression. Thus, for RNA therapy it is of particular importance to
increase RNA stability and/or RNA expression in cells. Thus, it is
the object of the present invention to provide RNA which is
particularly suited for RNA therapy, i.e., to provide means to
particularly stabilize RNA and/or increase RNA expression in cells.
This technical problem is solved according to the present invention
by the subject-matter of the claims.
SUMMARY OF THE INVENTION
[0014] In a first aspect, the present invention provides a 5'-cap
compound having the 5'-cap structure according to formula (I):
##STR00001##
[0015] wherein R.sup.1 is selected from the group consisting of
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
and optionally substituted heteroaryl;
[0016] R.sup.2 and R.sup.3 are independently selected from the
group consisting of H, halo, OH, and optionally substituted alkoxy,
or R.sup.2 and R.sup.3 together form O--X--O, wherein X is selected
from the group consisting of optionally substituted CH.sub.2,
optionally substituted CH.sub.2CH.sub.2, optionally substituted
CH.sub.2CH.sub.2CH.sub.2, optionally substituted
CH.sub.2CH(CH.sub.3), and optionally substituted C(CH.sub.3).sub.2,
or R.sup.2 is combined with the hydrogen atom at position 4' of the
ring to which R.sup.2 is attached to form --O--CH.sub.2-- or
--CH.sub.2--O--;
[0017] R.sup.4 and R.sup.6 are independently selected from the
group consisting of O, S, Se, and BH.sub.3;
[0018] R.sup.5 is selected from the group consisting of S, Se, and
BH.sub.3;
[0019] R.sup.7 is a mononucleotide or an oligonucleotide having 2
to 9 bases;
[0020] R.sup.8 is H, halo, or optionally substituted alkoxy;
[0021] n is 1, 2, or 3; and
[0022] B is a purine or pyrimidine base moiety.
[0023] In a second aspect, the present invention provides a
composition or kit comprising a 5'-cap compound of the first
aspect. Such a kit or composition may be used to provide an RNA
with a 5'-cap structure of the present invention.
[0024] In a third aspect, the present invention provides an RNA
which is modified with a 5'-cap compound of the first aspect.
[0025] In a fourth aspect, the present invention provides a
composition or cell comprising an RNA of the third aspect.
[0026] In a particularly preferred embodiment of the third and
fourth aspects of the present invention, the RNA further comprises
a nucleotide sequence encoding a peptide or protein of
interest.
[0027] In a fifth aspect, the present invention provides a method
for producing a peptide or protein of interest comprising the step
of using the RNA of the particularly preferred embodiment of the
third aspect or the composition or cell of the particularly
preferred embodiment of the fourth aspect.
[0028] In a sixth aspect, the present invention provides a method
for expressing a peptide or protein of interest in an individual
comprising the step of administering to said individual the RNA of
the particularly preferred embodiment of the third aspect or the
composition or cell of the particularly preferred embodiment of the
fourth aspect.
[0029] In a seventh aspect, the present invention provides the RNA
of the particularly preferred embodiment of the third aspect or the
composition or cell of the particularly preferred embodiment of the
fourth aspect for use in therapy.
[0030] In an eighth aspect, the present invention provides a method
of treating a disease or disorder in a subject comprising the step
of administering to said subject the RNA of the particularly
preferred embodiment of the third aspect or the composition or cell
of the particularly preferred embodiment of the fourth aspect. The
treatment of the disease or disorder is preferably selected from
the group consisting of protein replacement therapy, genome
engineering, genetic reprogramming, and immunotherapy.
[0031] In a ninth aspect, the present invention provides the RNA of
the particularly preferred embodiment of the third aspect or the
composition or cell of the particularly preferred embodiment of the
fourth aspect for use in a method of treating a disease or disorder
in a subject. The treatment of the disease or disorder is
preferably selected from the group consisting of protein
replacement therapy, genome engineering, genetic reprogramming, and
immunotherapy.
[0032] In a tenth aspect, the present invention provides a method
of increasing the stability of an RNA in cells (such as immature
antigen presenting cells) and/or for increasing the expression of
an RNA in cells (such as immature antigen presenting cells), said
method comprising providing said RNA with the structure according
to formula (I) as defined in the first aspect; and transferring
said RNA modified with the structure according to formula (I) into
the cells.
[0033] In an eleventh aspect, the present invention provides a
method for providing an RNA with a 5'-cap structure, said method
comprising performing a transcription reaction using a template
nucleic acid in the presence of a 5'-cap compound of the first
aspect.
[0034] In further aspects, the present invention provides the
following: [0035] a method for eliciting an immune response in an
individual comprising the step of administering to said individual
the RNA of the preferred embodiment of the third aspect or the
composition (preferably in the form of a vaccine composition) or
cell (preferably an immature antigen presenting cell) of the
preferred embodiment of the fourth aspect; in one embodiment the
method is for eliciting an immune response against a virus, such as
against influenza virus (A, B, or C), cytomegalovirus (CMV), or
respiratory syncytial virus (RSV); [0036] a method of increasing a
portion of MHC molecules which present an antigen of interest on
the surface of an antigen presenting cell, said method comprising
providing an RNA comprising a nucleotide sequence encoding a
peptide or protein comprising said antigen of interest or an
antigen peptide thereof, said RNA being modified with the structure
according to formula (I) as defined in the first aspect; and
transferring said RNA modified with the structure according to
formula (I) into an immature antigen presenting cell; in one
embodiment, the antigen of interest is an antigen of a virus (such
as influenza virus (A, B, or C), CMV, or RSV) or an antigen peptide
thereof; [0037] a method for stimulating and/or activating immune
effector cells, said method comprising providing an RNA comprising
a nucleotide sequence encoding a peptide or protein comprising an
antigen of interest or an antigen peptide thereof, said RNA being
modified with the structure according to formula (I) as defined in
the first aspect; transferring said RNA modified with the structure
according to formula (I) into immature antigen presenting cells;
and contacting the antigen presenting cells with the immune
effector cells; in one embodiment, the antigen of interest is an
antigen of a virus (such as influenza virus (A, B, or C), CMV, or
RSV) or an antigen peptide thereof; [0038] a method for inducing an
immune response in an individual, said method comprising providing
an RNA comprising a nucleotide sequence encoding a peptide or
protein comprising an antigen of interest or an antigen peptide
thereof, said RNA being modified with the structure according to
formula (I) as defined in the first aspect; and administering said
RNA modified with the structure according to formula (I) to said
individual; in one embodiment, the antigen of interest is an
antigen of a virus (such as influenza virus (A, B, or C), CMV, or
RSV) or an antigen peptide thereof; and [0039] a method for
inducing an immune response in an individual, said method
comprising providing an RNA comprising a nucleotide sequence
encoding a peptide or protein comprising an antigen of interest or
an antigen peptide thereof, said RNA being modified with the
structure according to formula (I) as defined in the first aspect;
transferring said RNA modified with the structure according to
formula (I) into immature antigen presenting cells; and
administering the antigen presenting cells to said individual; in
one embodiment, the antigen of interest is an antigen of a virus
(such as influenza virus (A, B, or C), CMV, or RSV) or an antigen
peptide thereof.
[0040] Further aspects as well as advantages and novel features of
the present invention will become apparent from the following
detailed description optionally in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1: Synthesis of a P-imidazolide precursor
(Im-pm.sup.2'-OGpG) for synthesis of an exemplary 5'-cap compound
of the present invention, m.sub.2.sup.7,2'-OGppspm.sup.2'-OGpG (in
the following "Compound 1"; OR.dbd.OCH.sub.3).
[0042] FIGS. 2A-2B: FIG. 2A shows synthesis of Compound 1
(m.sub.2.sup.7,2'-OGppspm.sup.2'-OGpG; OR.dbd.OCH.sub.3) and FIG.
2B shows HRMS spectrum of Compound 1.
[0043] FIG. 3: Comparison of translatability of RNAs capped
co-transcriptionally with different 5'-cap analogs. D1 beta-S-ARCA:
D1 diastereomer of beta-S-ARCA; D2 beta-S-ARCA: D2 diastereomer of
beta-S-ARCA; D1 Compound 1: D1 diastereomer of Compound 1; D2
Compound 1: D2 diastereomer of Compound 1. Luciferase RNAs
containing the respective cap structures were electroporated into
hiDCs. Luciferase activity was recorded over 72 h.
[0044] FIGS. 4A-4C: in vivo translation of RNAs modified with
different 5'-cap analogs. D1/D2 beta-S-ARCA RNA: RNA was prepared
by IVT using either the D1 or D2 diastereomer of beta-S-ARCA; D1/D2
Compound 1 RNA: RNA was prepared by IVT using either the D1 or D2
diastereomer of Compound 1; enzymatic Cap0/Cap1 RNA: RNA was
prepared by IVT in the absence of any cap analog and then, in a
second step, enzymatically capped either by using vaccinia capping
enzyme alone (enzymatic Cap0 RNA) or by using vaccinia capping
enzyme together with methyltransferase (enzymatic Cap1 RNA). FIG.
4A shows the luciferase signal 6 hours after administration; FIG.
4B shows the luciferase signal 24 hours after administration; FIG.
4C shows the luciferase signal 48 hours after administration.
[0045] FIGS. 5A-5B: in vivo translation of murine erythropoietin
(mEPO) RNAs modified with different 5'-cap analogs having a cap0
structure (5A) or a cap1 structure (5B). ARCA G: RNA
co-transcriptionally capped with ARCA G; D1: RNA
co-transcriptionally capped with the D1 diastereomer of
beta-S-ARCA; Ecap0: RNA enzymatically capped providing a cap0
structure; ARCA G+Ecap1: RNA co-transcriptionally capped with ARCA
G, then enzymatically capped using vaccinia capping enzyme and
vaccinia methyltransferase which provide a cap1 structure;
D1+Ecap1: RNA co-transcriptionally capped with the D1 diastereomer
of beta-S-ARCA, then enzymatically capped using vaccinia capping
enzyme and vaccinia methyltransferase which provide a cap1
structure; Ecap1: RNA enzymatically capped providing a cap1
structure. mEPO mRNA (3 .mu.g) containing 1-methylpseudouridine
(m1.PSI.) were formulated in TransIT.RTM. and injected i.p. into
mice. FIGS. 5A-5B show EPO levels in plasma of mice 6 hours, 24
hours, 48 hours or 72 hours after injection.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0046] Although the present invention is further described in more
detail below, it is to be understood that this invention is not
limited to the particular methodologies, protocols and reagents
described herein as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims. Unless defined otherwise, all technical and scientific
terms used herein have the same meanings as commonly understood by
one of ordinary skill in the art.
[0047] In the following, the elements of the present invention will
be described in more detail. These elements are listed with
specific embodiments, however, it should be understood that they
may be combined in any manner and in any number to create
additional embodiments. The variously described examples and
preferred embodiments should not be construed to limit the present
invention to only the explicitly described embodiments. This
description should be understood to support and encompass
embodiments which combine the explicitly described embodiments with
any number of the disclosed and/or preferred elements. Furthermore,
any permutations and combinations of all described elements in this
application should be considered disclosed by the description of
the present application unless the context indicates otherwise. For
example, if in a preferred embodiment R.sup.1 is methyl and in
another preferred embodiment R.sup.5 is S, then in a preferred
embodiment, R.sup.1 is methyl and R.sup.5 is S. Likewise, if in a
preferred embodiment R.sup.7 is *pm.sup.2'-OGpN and in another
preferred embodiment R.sup.8 is OCH.sub.3, then in a preferred
embodiment, R.sup.7 is *pm.sup.2'-OGpN and R.sup.8 is
OCH.sub.3.
[0048] Preferably, the terms used herein are defined as described
in "A multilingual glossary of biotechnological terms: (JUPAC
Recommendations)", H. G. W. Leuenberger, B. Nagel, and H. Kolbl,
Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland,
(1995).
[0049] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, and recombinant DNA techniques which are explained in
the literature in the field (cf., e.g., Molecular Cloning: A
Laboratory Manual, 2.sup.nd Edition, J. Sambrook et al. eds., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
[0050] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated member, integer or step or group
of members, integers or steps but not the exclusion of any other
member, integer or step or group of members, integers or steps. The
term "consisting essentially of" means excluding other members,
integers or steps of any essential significance. The term
"comprising" encompasses the term "consisting essentially of"
which, in turn, encompasses the term "consisting of". Thus, at each
occurrence in the present application, the term "comprising" may be
replaced with the term "consisting essentially of" or "consisting
of". Likewise, at each occurrence in the present application, the
term "consisting essentially of" may be replaced with the term
"consisting of".
[0051] The terms "a", "an" and "the" and similar references used in
the context of describing the invention (especially in the context
of the claims) are to be construed to cover both the singular and
the plural, unless otherwise indicated herein or clearly
contradicted by the context. Recitation of ranges of values herein
is merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range.
Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by the context. The use of any and all
examples, or exemplary language (e.g., "such as"), provided herein
is intended merely to better illustrate the invention and does not
pose a limitation on the scope of the invention otherwise claimed.
No language in the specification should be construed as indicating
any non-claimed element essential to the practice of the
invention.
[0052] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0053] According to the invention, the term "nucleic acid"
comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA),
combinations thereof, and modified forms thereof. The term
comprises genomic DNA, cDNA, mRNA, recombinantly produced and
chemically synthesized molecules. According to the invention, a
nucleic acid may be present as a single-stranded or double-stranded
and linear or covalently circularly closed molecule. A nucleic acid
can, according to the invention, be isolated. The term "isolated
nucleic acid" means, according to the invention, that the nucleic
acid (i) was amplified in vitro, for example via polymerase chain
reaction (PCR) for DNA or in vitro transcription (using e.g. an RNA
polymerase) for RNA, (ii) was produced recombinantly by cloning,
(iii) was purified, for example, by cleavage and separation by gel
electrophoresis, or (iv) was synthesized, for example, by chemical
synthesis.
[0054] In the context of the present invention, the term "DNA"
relates to a molecule which comprises deoxyribonucleotide residues
and preferably is entirely or substantially composed of
deoxyribonucleotide residues. "Deoxyribonucleotide" relates to a
nucleotide which lacks a hydroxyl group at the 2'-position of a
.beta.-D-ribofuranosyl group. The term "DNA" comprises isolated DNA
such as partially or completely purified DNA, essentially pure DNA,
synthetic DNA, and recombinantly generated DNA and includes
modified DNA which differs from naturally occurring DNA by
addition, deletion, substitution and/or alteration of one or more
nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of a DNA or
internally, for example at one or more nucleotides of the DNA.
Nucleotides in DNA molecules can also comprise non-standard
nucleotides, such as non-naturally occurring nucleotides or
chemically synthesized nucleotides. These altered DNAs can be
referred to as analogs or analogs of naturally occurring DNA.
[0055] In the context of the present invention, the term "RNA"
relates to a molecule which comprises ribonucleotide residues and
preferably is entirely or substantially composed of ribonucleotide
residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl
group at the 2'-position of a .beta.-D-ribofuranosyl group. The
term "RNA" comprises isolated RNA such as partially or completely
purified RNA, essentially pure RNA, synthetic RNA, and
recombinantly generated RNA and includes modified RNA which differs
from naturally occurring RNA by addition, deletion, substitution
and/or alteration of one or more nucleotides. Such alterations can
include addition of non-nucleotide material, such as to the end(s)
of an RNA or internally, for example at one or more nucleotides of
the RNA. Nucleotides in RNA molecules can also comprise
non-standard nucleotides, such as non-naturally occurring
nucleotides or chemically synthesized nucleotides or
deoxynucleotides. These altered/modified nucleotides can be
referred to as analogs of naturally occurring nucleotides, and the
corresponding RNAs containing such altered/modified nucleotides
(i.e., altered/modified RNAs) can be referred to as analogs of
naturally occurring RNAs. A molecule is "substantially composed of
ribonucleotide residues" if the content of ribonucleotide residues
in the molecule is at least 40% (such as at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%), based on
the total number of nucleotide residues in the molecule. The total
number of nucleotide residues in a molecule is the sum of all
nucleotide residues (irrespective of whether the nucleotide
residues are standard (i.e., naturally occurring) nucleotide
residues or analogs thereof). In the context of the present
invention, the RNA, preferably the mRNA, is modified with a 5'-cap
compound of the present invention and preferably contains one or
more further modifications to further stabilize the RNA, as
described below.
[0056] According to the invention, RNA has a length of at least 20,
preferably at least 50, in particular at least 100 nucleotides,
such as 100 to 15,000, more preferably 50 to 10,000, more
preferably 100 to 5,000, in particular 200 to 1,000 nucleotides.
RNA (in particular mRNA) which encodes a peptide or protein
preferably has a length of at least 50, more preferably at least
150, in particular at least 200 nucleotides, such as 100 to 15,000,
more preferably 50 to 10,000, more preferably 100 to 5,000, in
particular 200 to 1,000 nucleotides.
[0057] According to the invention, "RNA" includes mRNA, tRNA, rRNA,
snRNAs, ssRNA, dsRNAs, and inhibitory RNA, and is preferably
mRNA.
[0058] According to the invention, "dsRNA" means double-stranded
RNA and is RNA with two partially or completely complementary
strands.
[0059] According to the present invention, the term "mRNA" means
"messenger-RNA" and relates to a "transcript" which may be
generated by using a DNA template and may encode a peptide or
protein. Typically, an mRNA comprises a 5'-UTR, a peptide/protein
coding region, and a 3'-UTR. In the context of the present
invention, mRNA is preferably generated by in vitro transcription
(IVT) from a DNA template. As set forth above, the in vitro
transcription methodology is known to the skilled person, and a
variety of in vitro transcription kits is commercially
available.
[0060] mRNA is single-stranded but may contain self-complementary
sequences that allow parts of the mRNA to fold and pair with itself
to form double helices.
[0061] mRNA only possesses limited half-life in cells and in vitro.
Thus, according to the invention, the stability and/or translation
efficiency of RNA may be modified as required. For example, mRNA
may be stabilized and/or its translation increased by one or more
modifications having a stabilizing effect and/or increasing
translation efficiency of mRNA. Such modifications are described,
for example, in WO 2007/036366 the entire disclosure of which is
incorporated herein by reference. In order to increase expression
of the mRNA according to the present invention, it may be modified
within the coding region, i.e., the sequence encoding the expressed
peptide or protein, preferably without altering the sequence of the
expressed peptide or protein, e.g., to increase the GC-content to
increase mRNA stability and to perform a codon optimization and,
thus, enhance translation in cells.
[0062] RNA can be isolated from cells, can be made from a DNA
template, or can be chemically synthesized using methods known in
the art. In preferred embodiments, RNA is synthesized in vitro from
a DNA template. In one particularly preferred embodiment, RNA, in
particular mRNA, is generated by in vitro transcription from a DNA
template. The in vitro transcription methodology is known to the
skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual,
2.sup.nd Edition, J. Sambrook et al. eds., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety
of in vitro transcription kits is commercially available, e.g.,
from Thermo Fisher Scientific (such as TranscriptAid.TM. T7 kit,
MEGAscript.RTM. T7 kit, MAXIscript.RTM.), New England BioLabs Inc.
(such as HiScribe.TM. T7 kit, HiScribe.TM. T7 ARCA mRNA kit),
Promega (such as RiboMAX.TM., HeLaScribe.RTM., Riboprobe.RTM.
systems), Jena Bioscience (such as SP6 or T7 transcription kits),
and Epicentre (such as AmpliScribe.TM.). In one particularly
preferred embodiment, RNA is in vitro transcribed RNA (IVT RNA).
For providing modified RNA, correspondingly modified nucleotides,
such as modified naturally occurring nucleotides, non-naturally
occurring nucleotides and/or modified non-naturally occurring
nucleotides, can be incorporated during synthesis (preferably in
vitro transcription), or modifications can be effected in and/or
added to the RNA after transcription.
[0063] RNA according to the present invention is at least modified
with a 5'-cap compound of the present invention.
[0064] In a preferred embodiment, RNA according to the present
invention comprises a nucleic acid sequence encoding a peptide or
protein, preferably a pharmaceutically active peptide or protein,
and is capable of expressing said peptide or protein, in particular
if transferred into a cell or subject. Thus, the RNA according to
the present invention preferably contains a coding region (open
reading frame (ORF)) encoding a peptide or protein, preferably
encoding a pharmaceutically active peptide or protein. In this
respect, an "open reading frame" or "ORF" is a continuous stretch
of codons beginning with a start codon and ending with a stop
codon.
[0065] According to the invention, the term "pharmaceutically
active peptide or protein" means a peptide or protein that can be
used in the treatment of an individual where the expression of a
peptide or protein would be of benefit, e.g., in ameliorating the
symptoms of a disease or disorder. Preferably, a pharmaceutically
active peptide or protein has curative or palliative properties and
may be administered to ameliorate, relieve, alleviate, reverse,
delay onset of or lessen the severity of one or more symptoms of a
disease or disorder. Preferably, a pharmaceutically active peptide
or protein has a positive or advantageous effect on the condition
or disease state of an individual when administered to the
individual in a therapeutically effective amount. A
pharmaceutically active peptide or protein may have prophylactic
properties and may be used to delay the onset of a disease or
disorder or to lessen the severity of such disease or disorder. The
term "pharmaceutically active peptide or protein" includes entire
proteins or polypeptides, and can also refer to pharmaceutically
active fragments thereof. It can also include pharmaceutically
active analogs of a peptide or protein.
[0066] Specific examples of pharmaceutically active peptides and
proteins include, but are not limited to, cytokines, adhesion
molecules (in particular integrins), immunoglobulins (e.g.,
antibodies), immunologically active compounds (e.g., antigens),
hormones, growth factors, protease inhibitors (e.g., alpha
1-antitrypsin), enzymes (e.g., herpes simplex virus type 1
thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine
hydroxylase, pseudocholinesterase, pancreatic enzymes, and
lactase), receptors (e.g., growth factor receptors), apoptosis
regulators, transcription factors, tumor suppressor proteins,
structural proteins, reprogramming factors, genomic engineering
proteins, and blood proteins.
[0067] According to the invention, the term "cytokines" relates to
proteins which have a molecular weight of about 5 to 20 kDa and
which participate in cell signaling (e.g., paracrine, endocrine,
and/or autocrine signaling). In particular, when released,
cytokines exert an effect on the behavior of cells around the place
of their release. Examples of cytokines include lymphokines,
interleukins, chemokines, interferons, and tumor necrosis factors
(TNFs). According to the present application, cytokines do not
include hormones or growth factors. Cytokines differ from hormones
in that (i) they usually act at much more variable concentrations
than hormones and (ii) generally are made by a broad range of cells
(nearly all nucleated cells can produce cytokines). Interferons are
usually characterized by antiviral, antiproliferative and
immunomodulatory activities. Interferons are proteins that alter
and regulate the transcription of genes within a cell by binding to
interferon receptors on the regulated cell's surface, thereby
preventing viral replication within the cells. The interferons can
be grouped into two types. IFN-gamma is the sole type II
interferon; all others are type I interferons. Type I and type II
interferons differ in gene structure (type II interferon genes have
three exons; type I, one), chromosome location (in humans, type II
is located on chromosome-12; the type I interferon genes are linked
and on chromosome-9), and the types of tissues where they are
produced (type I interferons are synthesized ubiquitously, type II
by lymphocytes). Type I interferons competitively inhibit each
other binding to cellular receptors, while type II interferon has a
distinct receptor. According to the invention, the term
"interferon" or "IFN" preferably relates to type I interferons, in
particular interferon alfa and interferon beta. Particular examples
of cytokines include erythropoietin (EPO), colony stimulating
factor (CSF), granulocyte colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), tumor
necrosis factor (TNF), bone morphogenetic protein (BMP), interferon
alfa (IFN.alpha.), interferon beta (IFN.beta.), interferon gamma
(INF.gamma.), interleukin 2 (IL-2), interleukin 4 (IL-4),
interleukin 10 (IL-10), and interleukin 11 (IL-11),
[0068] According to the invention, the term "hormones" relates to a
class of signaling molecules produced by glands, wherein signaling
usually includes the following steps: (i) synthesis of a hormone in
a particular tissue; (ii) storage and secretion; (iii) transport of
the hormone to its target; (iv) binding of the hormone by a
receptor; (v) relay and amplification of the signal; and (vi)
breakdown of the hormone. Hormones differ from cytokines in that
(1) hormones usually act in less variable concentrations and (2)
generally are made by specific kinds of cells. Particular examples
of hormones include insulin, vasopressin, prolactin,
adrenocorticotropic hormone (ACTH), thyroid hormone, growth
hormones (such as human grown hormone or bovine somatotropin),
oxytocin, atrial-natriuretic peptide (ANP), glucagon, somatostatin,
cholecystokinin, gastrin, leptins, catecholamines, gonadotrophines,
trophic hormones, and dopamine. In one embodiment, a "hormone" is a
peptide or protein hormone, such as insulin, vasopressin,
prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone,
growth hormones (such as human grown hormone or bovine
somatotropin), oxytocin, atrial-natriuretic peptide (ANP),
glucagon, somatostatin, cholecystokinin, gastrin, and leptins.
[0069] According to the invention, the term "adhesion molecules"
relates to proteins which are located on the surface of a cell and
which are involved in binding of the cell with other cells or with
the extracellular matrix (ECM). Adhesion molecules are typically
transmembrane receptors and can be classified as
calcium-independent (e.g., integrins, immunoglobulin superfamily,
lymphocyte homing receptors) and calcium-dependent (cadherins and
selectins). Particular examples of adhesion molecules are
integrins, lymphocyte homing receptors, selectins (e.g.,
P-selectin), and addressins.
[0070] Integrins are also involved in signal transduction. In
particular, upon ligand binding, integrins modulate cell signaling
pathways, e.g., pathways of transmembrane protein kinases such as
receptor tyrosine kinases (RTK). Such regulation can lead to
cellular growth, division, survival, or differentiation or to
apoptosis. Particular examples of integrins include:
.alpha..sub.1.beta..sub.1, .alpha..sub.2.beta..sub.1,
.alpha..sub.3.beta..sub.1, .alpha..sub.4.beta..sub.1,
.alpha..sub.5.beta..sub.1, .alpha..sub.6.beta..sub.1,
.alpha..sub.7.beta..sub.1, .alpha..sub.L.beta..sub.2,
.alpha..sub.M.beta..sub.2, .alpha..sub.IIB.beta..sub.3,
.alpha..sub.V.beta..sub.1, .alpha..sub.V.beta..sub.3,
.alpha..sub.V.beta..sub.5, .alpha..sub.V.beta..sub.6,
.alpha..sub.V.beta..sub.8, and .alpha..sub.6.beta..sub.4.
[0071] According to the invention, the term "immunoglobulins" or
"immunoglobulin superfamily" refers to molecules which are involved
in the recognition, binding, and/or adhesion processes of cells.
Molecules belonging to this superfamily share the feature that they
contain a region known as immunoglobulin domain or fold. Members of
the immunoglobulin superfamily include antibodies (e.g., IgA, IgD,
IgE, IgG, and IgM), T cell receptors (TCRs), major
histocompatibility complex (MHC) molecules, co-receptors (e.g.,
CD4, CD8, CD19), antigen receptor accessory molecules (e.g.,
CD-3.gamma., CD3-.delta., CD-3.epsilon., CD79a, CD79b),
co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86),
and other (e.g., CD147, CD90, CD7).
[0072] According to the invention, the term "immunologically active
compound" relates to any compound altering an immune response,
preferably by inducing and/or suppressing maturation of immune
cells, inducing and/or suppressing cytokine biosynthesis, and/or
altering humoral immunity by stimulating antibody production by B
cells. Immunologically active compounds possess potent
immunostimulating activity including, but not limited to, antiviral
and antitumor activity, and can also down-regulate other aspects of
the immune response, for example shifting the immune response away
from a TH2 immune response, which is useful for treating a wide
range of TH2 mediated diseases. Immunologically active compounds
can be useful as vaccine adjuvants. Particular examples of
immunologically active compounds include interleukins, colony
stimulating factor (CSF), granulocyte colony stimulating factor
(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),
erythropoietin, tumor necrosis factor (TNF), interferons,
integrins, addressins, selectins, homing receptors, and antigens,
in particular tumor-associated antigens, pathogen-associated
antigens (such as bacterial, parasitic, or viral antigens (such as
one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens of
influenza virus (A, B, or C), CMV or RSV)), allergens, and
autoantigens.
[0073] According to the invention, the term "autoantigen" or
"self-antigen" refers to an antigen which originates from within
the body of a subject (i.e., the autoantigen can also be called
"autologous antigen") and which produces an abnormally vigorous
immune response against this normal part of the body. Such vigorous
immune reactions against autoantigens may be the cause of
"autoimmune diseases".
[0074] According to the invention, the term "allergen" refers to a
kind of antigen which originates from outside the body of a subject
(i.e., the allergen can also be called "heterologous antigen") and
which produces an abnormally vigorous immune response in which the
immune system of the subject fights off a perceived threat that
would otherwise be harmless to the subject. "Allergies" are the
diseases caused by such vigorous immune reactions against
allergens. An allergen usually is an antigen which is able to
stimulate a type-I hypersensitivity reaction in atopic individuals
through immunoglobulin E (IgE) responses. Particular examples of
allergens include allergens derived from peanut proteins (e.g., Ara
h 2.02), ovalbumin, grass pollen proteins (e.g., Phl p 5), and
proteins of dust mites (e.g., Der p 2).
[0075] According to the invention, the term "growth factors" refers
to molecules which are able to stimulate cellular growth,
proliferation, healing, and/or cellular differentiation. Typically,
growth factors act as signaling molecules between cells. The term
"growth factors" include particular cytokines and hormones which
bind to specific receptors on the surface of their target cells.
Examples of growth factors include bone morphogenetic proteins
(BMPs), fibroblast growth factors (FGFs), vascular endothelial
growth factors (VEGFs), such as VEGFA, epidermal growth factor
(EGF), insulin-like growth factor, ephrins, macrophage
colony-stimulating factor, granulocyte colony-stimulating factor,
granulocyte macrophage colony-stimulating factor, neuregulins,
neurotrophins (e.g., brain-derived neurotrophic factor (BDNF),
nerve growth factor (NGF)), placental growth factor (PGF),
platelet-derived growth factor (PDGF), renalase (RNLS)
(anti-apoptotic survival factor), T-cell growth factor (TCGF),
thrombopoietin (TPO), transforming growth factors (transforming
growth factor alpha (TGF-.alpha.), transforming growth factor beta
(TGF-.beta.)), and tumor necrosis factor-alpha (TNF-.alpha.). In
one embodiment, a "growth factor" is a peptide or protein growth
factor.
[0076] According to the invention, the term "enzymes" refers to
macromolecular biological catalysts which accelerate chemical
reactions. Like any catalyst, enzymes are not consumed in the
reaction they catalyze and do not alter the equilibrium of said
reaction. Unlike many other catalysts, enzymes are much more
specific. In one embodiment, an enzyme is essential for homeostasis
of a subject, e.g., any malfunction (in particular, decreased
activity which may be caused by any of mutation, deletion or
decreased production) of the enzyme results in a disease. Examples
of enzymes include enzymes of the biosynthesis or degradation of
cholesterol, steroidogenic enzymes, kinases, nucleases,
phosphodiesterases, methylases, de-methylases, dehydrogenases,
cellulases, proteases, lipases, phospholipases, aromatases,
cytochromes, adenylate or guanylate cyclases, and neuramidases,
such as tissue plasminogen activator, streptokinase, herpes simplex
virus type 1 thymidine kinase (HSV1-TK), hexosaminidase,
phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes
(e.g., amylase, lipase, and protease or mixtures thereof (such as
pancrelipase)), and lactase.
[0077] According to the invention, the term "receptors" refers to
protein molecules which receive signals (in particular chemical
signals called ligands) from outside a cell. The binding of a
signal (e.g., ligand) to a receptor causes some kind of response of
the cell, e.g., the intracellular activation of a kinase. Receptors
include transmembrane receptors (such as ion channel-linked
(ionotropic) receptors, G protein-linked (metabotropic) receptors,
and enzyme-linked receptors) and intracellular receptors (such as
cytoplasmic receptors and nuclear receptors). Particular examples
of receptors include steroid hormone receptors, growth factor
receptors, and peptide receptors (i.e., receptors whose ligands are
peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The
term "growth factor receptors" refers to receptors which bind to
growth factors. Growth factor receptors are the first step of the
signaling cascade for cell differentiation and proliferation.
Growth factor receptors may use the JAK/STAT, MAP kinase, and PI3
kinase pathways.
[0078] According to the invention, the term "protease inhibitors"
refers to molecules, in particular peptides or proteins, which
inhibit the function of proteases. Protease inhibitors can be
classified by the protease which is inhibited (e.g., aspartic
protease inhibitors, cysteine protease inhibitors, metalloprotease
inhibitors, serine protease inhibitors, threonine protease
inhibitors, trypsin inhibitors) or by their mechanism of action
(e.g., suicide inhibitors, such as serpins). Particular examples of
protease inhibitors include serpins, such as alpha 1-antitrypsin,
aprotinin, and bestatin.
[0079] According to the invention, the term "apoptosis regulators"
refers to molecules, in particular peptides or proteins, which
modulate apoptosis, i.e., which either activate or inhibit
apoptosis. Apoptosis regulators can be grouped into two broad
classes: those which modulate mitochondrial function and those
which regulate caspases. The first class includes proteins (e.g.,
BCL-2, BCL-xL) which act to preserve mitochondrial integrity by
preventing loss of mitochondrial membrane potential and/or release
of proapoptotic proteins such as cytochrome C into the cytosol.
Also to this first class belong proapoptotic proteins (e.g., BAX,
BAK, BIM) which promote release of cytochrome C. The second class
includes proteins such as the inhibitors of apoptosis proteins
(e.g., XIAP) or FLIP which block the activation of caspases.
Particular examples of apoptosis regulators are BAX, BCL-2, BCL-xL,
BAK, BIM, XIAP, and FLIP, in particular BAX.
[0080] According to the invention, the term "transcription factors"
relates to proteins which regulate the rate of transcription of
genetic information from DNA to messenger RNA, in particular by
binding to a specific DNA sequence. Transcription factors may
regulate cell division, cell growth, and cell death throughout
life; cell migration and organization during embryonic development;
and/or in response to signals from outside the cell, such as a
hormone. Transcription factors contain at least one DNA-binding
domain which binds to a specific DNA sequence, usually adjacent to
the genes which are regulated by the transcription factors.
Particular examples of transcription factors include hepatocyte
nuclear factors, MECP2, insulin promoter factor 1, FOXP2, FOXP3,
the STAT protein family, p53, the HOX protein family, and the SOX
proteins, such as SOX2.
[0081] According to the invention, the term "tumor suppressor
proteins" relates to molecules, in particular peptides or proteins,
which protect a cell from one step on the path to cancer.
Tumor-suppressor proteins (usually encoded by corresponding
tumor-suppressor genes) exhibit a weakening or repressive effect on
the regulation of the cell cycle and/or promote apoptosis. Their
functions may be one or more of the following: repression of genes
essential for the continuing of the cell cycle; coupling the cell
cycle to DNA damage (as long as damaged DNA is present in a cell,
no cell division should take place); initiation of apoptosis, if
the damaged DNA cannot be repaired; metastasis suppression (e.g.,
preventing tumor cells from dispersing, blocking loss of contact
inhibition, and inhibiting metastasis); and DNA repair. Particular
examples of tumor-suppressor proteins include p53, phosphatase and
tensin homolog (PTEN), SWI/SNF (SWItch/Sucrose Non-Fermentable),
von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis
coli (APC), CD95, suppression of tumorigenicity 5 (ST5),
suppression of tumorigenicity 5 (ST5), suppression of
tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3).
[0082] According to the invention, the term "structural proteins"
refers to proteins which confer stiffness and rigidity to
otherwise-fluid biological components. Structural proteins are
mostly fibrous (such as collagen and elastin) but may also be
globular (such as actin and tubulin). Usually, globular proteins
are soluble as monomers, but polymerize to form long, fibers which,
for example, may make up the cytoskeleton. Other structural
proteins are motor proteins (such as myosin, kinesin, and dynein)
which are capable of generating mechanical forces, and surfactant
proteins. Particular examples of structural proteins include
collagen, fibroin, fibrinogen, surfactant protein A, surfactant
protein B, surfactant protein C, surfactant protein D, elastin,
tubulin, actin, and myosin.
[0083] According to the invention, the term "reprogramming factors"
or "reprogramming transcription factors" relates to molecules, in
particular peptides or proteins, which, when expressed in somatic
cells optionally together with further agents such as further
reprogramming factors, lead to reprogramming or de-differentiation
of said somatic cells to cells having stem cell characteristics, in
particular pluripotency. Particular examples of reprogramming
factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.
[0084] According to the invention, the term "genomic engineering
proteins" relates to proteins which are able to insert, delete or
replace DNA in the genome of a subject. Particular examples of
genomic engineering proteins include meganucleases, zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases
(TALENs), and clustered regularly spaced short palindromic
repeat-CRISPR-associated protein 9 (CRISPR-Cas9).
[0085] According to the invention, the term "blood proteins"
relates to peptides or proteins which are present in blood plasma
of a subject, in particular blood plasma of a healthy subject.
Blood proteins have diverse functions such as transport (e.g.,
albumin, transferrin), enzymatic activity (e.g., thrombin or
ceruloplasmin), blood clotting (e.g., fibrinogen), defense against
pathogens (e.g., complement components and immunoglobulins),
protease inhibitors (e.g., alpha 1-antitrypsin), etc. Particular
examples of blood proteins include thrombin, serum albumin, Factor
VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen
activator, protein C, von Willebrand factor, antithrombin III,
glucocerebrosidase, erythropoietin, granulocyte colony stimulating
factor (G-CSF), modified Factor VIII, and anticoagulants.
[0086] According to the invention, the term "protein replacement
therapy" relates to a medical treatment which supplements or
replaces a peptide or protein which has a decreased activity in a
patient compared to a healthy subject. The decreased activity
(including zero activity which may be the case when the peptide or
protein is absent in the patient) may be the result of (i) a
decreased expression of the peptide or protein (i.e., the peptide
or protein is fully functional but the amount thereof is decreased)
or (ii) the presence of one or more mutations in the amino acid
sequence of the expressed peptide or protein (i.e., the peptide or
protein is not fully functional). For example, this decreased
activity of the peptide or protein may be the result of a gene
encoding the peptide or protein but containing one or more
mutations in such a manner that (i) the expression of said gene is
decreased or silenced thereby resulting in a decreased amount of
the peptide or protein (which may still be fully functional) and/or
(ii) the amino acid sequence of the peptide or protein encoded by
said gene contains one or more mutations thereby resulting in a
non-fully functional (or non-functional) peptide or protein.
Diseases or disorders caused by a decreased activity of a peptide
or protein in a patient may be treated by replacing or
supplementing the peptide or protein (protein replacement therapy),
e.g., by administering to a patient having such a disease or
disorder an RNA (in particular an RNA of the present invention)
comprising a nucleotide sequence encoding the peptide or protein.
The nucleotide sequence encoding the peptide or protein may be
autologous or heterologous to the patient. However, if the
decreased activity of the peptide or protein in a patient is due to
one or more mutations (i.e., resulting in a non-fully functional
(or non-functional) peptide or protein), it is preferred that the
nucleotide sequence encoding the peptide or protein is heterologous
to the patient, in particular is obtained from a healthy subject
(of the same species) expressing the peptide or protein in its
native (i.e., unmutated) form. For example, such protein
replacement therapy may comprise the step of administering to a
patient (i) an RNA (in particular an RNA of the present invention)
comprising a nucleotide sequence encoding said peptide or protein
(wherein said nucleotide sequence preferably is heterologous and
may be obtained from a healthy subject) or (ii) a composition,
e.g., a pharmaceutical composition, comprising such RNA, or
alternatively, the steps of (a) transferring an RNA (in particular
an RNA of the present invention) comprising a nucleotide sequence
encoding said peptide or protein (wherein said nucleotide sequence
preferably is heterologous and may be obtained from a healthy
subject) into a cell (wherein said cell may be autologous to the
patient) and (b) administering said transfected cell to the
patient. In alternative (i), the RNA is preferably taken up into
cells (e.g., antigen-presenting cells, such as monocytes,
macrophages, or dendritic cells, or other cells), and a translation
product of the nucleotide sequence encoding a peptide or protein is
formed (and optionally posttranslationally modified) to yield the
peptide or protein. In alternative (ii), after administration of
the transfected cells to the patient, the transfected cells
preferably express the peptide or protein.
[0087] The term "genome engineering" relates to the process in
which DNA is inserted, deleted or replaced in the genome of a
subject, preferably by using genomic engineering proteins.
Particular examples of genomic engineering proteins include
meganucleases, zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs), and clustered regularly
spaced short palindromic repeat-CRISPR-associated protein 9
(CRISPR-Cas9).
[0088] The term "genetic reprogramming" refers to the resetting of
the genetic program of a cell. A reprogrammed cell preferably
exhibits pluripotency.
[0089] In one embodiment, the pharmaceutically active peptide or
protein is a disease-associated peptide or protein, i.e., it is
causatively linked with a disease or disorder.
[0090] For example, a disease or disorder may be caused by a
decreased activity of a peptide or protein. The decreased activity
may be the result of (i) a decreased expression of the peptide or
protein (i.e., the peptide or protein is fully functional but the
amount thereof is decreased) or (ii) the presence of one or
mutations in the amino acid sequence of the expressed peptide or
protein (i.e., the peptide or protein is not fully functional). For
example, this decreased activity of the peptide or protein may be
the result of a gene encoding the peptide or protein but containing
one or mutations in such a manner that (i) the expression of said
gene is decreased or silenced thereby resulting in a decreased
amount of the peptide or protein (which may still be fully
functional) and/or (ii) the amino acid sequence of the peptide or
protein encoded by said gene contains one or more mutations thereby
resulting in a non-fully functional (or non-functional) peptide or
protein. Such diseases or disorders caused by a decreased activity
of a peptide or protein in a patient may be treated by replacing or
supplementing the peptide or protein (protein replacement therapy),
e.g., by administering to a patient having such a disease or
disorder an RNA (in particular an RNA of the present invention)
comprising a nucleotide sequence encoding the peptide or protein.
The nucleotide sequence encoding the peptide or protein may be
autologous or heterologous to the patient. However, if the
decreased activity of the peptide or protein in a patient is due to
one or more mutations (i.e., resulting in a non-fully functional
(or non-functional) peptide or protein), it is preferred that the
nucleotide sequence encoding the peptide or protein is
heterologous, in particular obtained from a healthy subject (of the
same species) expressing the peptide or protein in its native
(i.e., unmutated) form. For example, such protein replacement
therapy may comprise the step of administering to the patient (i)
an RNA (in particular an RNA of the present invention) comprising a
nucleotide sequence encoding said peptide or protein (wherein said
nucleotide sequence preferably is heterologous and may be obtained
from a healthy subject) or (ii) a composition, e.g., a
pharmaceutical composition, comprising such RNA, or alternatively,
the steps of (a) transferring an RNA (in particular an RNA of the
present invention) comprising a nucleotide sequence encoding said
peptide or protein (wherein said nucleotide sequence preferably is
heterologous and may be obtained from a healthy subject) into a
cell (wherein said cell may be autologous to the patient) and (b)
administering said transfected cell to the patient. In alternative
(i), the RNA is preferably taken up into cells (e.g.,
antigen-presenting cells, such as monocytes, macrophages, or
dendritic cells, or other cells), and a translation product of the
nucleotide sequence encoding a peptide or protein is formed (and
optionally posttranslationally modified) to yield the peptide or
protein. In alternative (ii), after administration of the
transfected cells to the patient, the transfected cells preferably
express the peptide or protein.
[0091] Alternatively, such diseases or disorders caused by a
decreased activity of a peptide or protein in a patient may be
treated by using genome engineering, e.g., by replacing the DNA
sequence encoding the peptide or protein (i.e., resulting in a
non-fully functional (or non-functional) peptide or protein) in a
patient having such a disease or disorder with a DNA sequence
encoding the peptide or protein in its native (i.e., unmutated)
form. For example, such genome engineering therapy may comprise the
step of administering to a patient (i) an RNA (in particular an RNA
of the present invention) comprising a nucleotide sequence encoding
a genomic engineering protein and (ii) a DNA comprising a
nucleotide sequence encoding the peptide or protein in its native
(i.e., unmutated) form. Upon administration, preferably, the RNA
comprising a nucleotide sequence encoding a genomic engineering
protein and the DNA comprising a nucleotide sequence encoding the
peptide or protein in its native (i.e., unmutated) form are taken
up into cells (in particular diseased cells), a translation product
of the nucleotide sequence encoding a genomic engineering protein
is formed (and optionally posttranslationally modified) to yield
the genomic engineering protein, and the genomic engineering
protein together with the DNA sequence encoding the peptide or
protein in its native (i.e., unmutated) form act to replace the
mutated DNA sequence in the genome of the cells with the DNA
sequence encoding the peptide or protein in its native (i.e.,
unmutated) form.
[0092] In a further alternative, such diseases or disorders caused
by a decreased activity of a peptide or protein in a patient may be
treated by using genetic reprogramming, e.g., by reprogramming
somatic cells (in particular autologous somatic cells) of a patient
having such as disease or disorder and administering said
reprogrammed cells to the patient. This therapeutic approach may be
particularly beneficial in patients having a disease or disorder
which causes a depletion or extinction of cells producing the
desired peptide or protein (e.g., a hormone such as insulin). For
example, such genetic reprogramming therapy may comprise the steps
of (a) introducing an RNA (in particular an RNA of the present
invention) comprising a nucleotide sequence encoding one or more
reprogramming factors into somatic cells; (b) allowing the
development of cells having stem cell characteristics; and (c)
administering the cells having stem cell characteristics to a
patient. In a preferred embodiment, the somatic cells are
autologous to the patient. Upon administration, the cells having
stem cell characteristics preferably differentiate into cells
expressing the desired peptide or protein.
[0093] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
cytokine, preferably selected from the group consisting of
erythropoietin (EPO), interleukin 4 (IL-2), and interleukin 10
(IL-11), more preferably EPO. A disease or disorder caused by a
decreased activity of a cytokine or a disease or disorder, wherein
increasing the amount of a cytokine (i) ameliorates, relieves,
alleviates, or reverses one or more symptoms of the disease or
disorder are and/or (ii) delays the onset of the disease or
disorder and/or (iii) lessens the severity of the disease or
disorder, may be treated by a corresponding protein replacement
therapy as described herein (e.g., by replacing or supplementing
the cytokine), a corresponding genome engineering therapy as
described herein, and/or a genetic reprogramming therapy as
described herein. A patient having such a disease or disorder or
being at risk of developing such a disease or disorder can be
treated accordingly.
[0094] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is an
adhesion molecule, in particular an integrin. A disease or disorder
caused by a decreased activity of an adhesion molecule or a disease
or disorder, wherein increasing the amount of an adhesion molecule
(i) ameliorates, relieves, alleviates, or reverses one or more
symptoms of the disease or disorder are and/or (ii) delays the
onset of the disease or disorder and/or (iii) lessens the severity
of the disease or disorder, may be treated by a corresponding
protein replacement therapy as described herein (e.g., by replacing
or supplementing the adhesion molecule), a corresponding genome
engineering therapy as described herein, and/or a genetic
reprogramming therapy as described herein. A patient having such a
disease or disorder or being at risk of developing such a disease
or disorder can be treated accordingly.
[0095] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
hormone, in particular vasopressin, insulin or growth hormone. A
disease or disorder caused by a decreased activity of a hormone or
a disease or disorder, wherein increasing the amount of a hormone
(i) ameliorates, relieves, alleviates, or reverses one or more
symptoms of the disease or disorder are and/or (ii) delays the
onset of the disease or disorder and/or (iii) lessens the severity
of the disease or disorder, may be treated by a corresponding
protein replacement therapy as described herein (e.g., by replacing
or supplementing the hormone), a corresponding genome engineering
therapy as described herein, and/or a genetic reprogramming therapy
as described herein. A patient having such a disease or disorder or
being at risk of developing such a disease or disorder can be
treated accordingly.
[0096] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
growth factor, in particular VEGFA. A disease or disorder caused by
a decreased activity of a growth factor or a disease or disorder,
wherein increasing the amount of a growth factor (i) ameliorates,
relieves, alleviates, or reverses one or more symptoms of the
disease or disorder are and/or (ii) delays the onset of the disease
or disorder and/or (iii) lessens the severity of the disease or
disorder, may be treated by a corresponding protein replacement
therapy as described herein (e.g., by replacing or supplementing
the growth factor), a corresponding genome engineering therapy as
described herein, and/or a genetic reprogramming therapy as
described herein. A patient having such a disease or disorder or
being at risk of developing such a disease or disorder can be
treated accordingly.
[0097] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is an
enzyme, preferably selected from the group consisting of herpes
simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase,
phenylalanine hydroxylase, pseudocholinesterase, pancreatic
enzymes, and lactase. A disease or disorder caused by a decreased
activity of an enzyme or a disease or disorder, wherein increasing
the amount of an enzyme (i) ameliorates, relieves, alleviates, or
reverses one or more symptoms of the disease or disorder are and/or
(ii) delays the onset of the disease or disorder and/or (iii)
lessens the severity of the disease or disorder, may be treated by
a corresponding protein replacement therapy as described herein
(e.g., by replacing or supplementing the enzyme), a corresponding
genome engineering therapy as described herein, and/or a genetic
reprogramming therapy as described herein. A patient having such a
disease or disorder or being at risk of developing such a disease
or disorder can be treated accordingly.
[0098] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
receptor, in particular growth factor receptors. A disease or
disorder caused by a decreased activity of a receptor or a disease
or disorder, wherein increasing the amount of a receptor (i)
ameliorates, relieves, alleviates, or reverses one or more symptoms
of the disease or disorder are and/or (ii) delays the onset of the
disease or disorder and/or (iii) lessens the severity of the
disease or disorder, may be treated by a corresponding protein
replacement therapy as described herein (e.g., by replacing or
supplementing the receptor), a corresponding genome engineering
therapy as described herein, and/or a genetic reprogramming therapy
as described herein. A patient having such a disease or disorder or
being at risk of developing such a disease or disorder can be
treated accordingly.
[0099] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is an
apoptosis regulator, in particular BAX. A disease or disorder
caused by a decreased activity of an apoptosis regulator or a
disease or disorder, wherein increasing the amount of an apoptosis
regulator (i) ameliorates, relieves, alleviates, or reverses one or
more symptoms of the disease or disorder are and/or (ii) delays the
onset of the disease or disorder and/or (iii) lessens the severity
of the disease or disorder, may be treated by a corresponding
protein replacement therapy as described herein (e.g., by replacing
or supplementing the apoptosis regulator), a corresponding genome
engineering therapy as described herein, and/or a genetic
reprogramming therapy as described herein. A patient having such a
disease or disorder or being at risk of developing such a disease
or disorder can be treated accordingly.
[0100] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
tumor suppressor protein, in particular p53. A disease or disorder
caused by a decreased activity of a tumor suppressor protein or a
disease or disorder, wherein increasing the amount of a tumor
suppressor protein (i) ameliorates, relieves, alleviates, or
reverses one or more symptoms of the disease or disorder are and/or
(ii) delays the onset of the disease or disorder and/or (iii)
lessens the severity of the disease or disorder, may be treated by
a corresponding protein replacement therapy as described herein
(e.g., by replacing or supplementing the tumor suppressor protein),
a corresponding genome engineering therapy as described herein,
and/or a genetic reprogramming therapy as described herein. A
patient having such a disease or disorder or being at risk of
developing such a disease or disorder can be treated
accordingly.
[0101] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
structural protein, in particular surfactant protein B. A disease
or disorder caused by a decreased activity of a structural protein
or a disease or disorder, wherein increasing the amount of a
structural protein (i) ameliorates, relieves, alleviates, or
reverses one or more symptoms of the disease or disorder are and/or
(ii) delays the onset of the disease or disorder and/or (iii)
lessens the severity of the disease or disorder, may be treated by
a corresponding protein replacement therapy as described herein
(e.g., by replacing or supplementing the structural protein), a
corresponding genome engineering therapy as described herein,
and/or a genetic reprogramming therapy as described herein. A
patient having such a disease or disorder or being at risk of
developing such a disease or disorder can be treated
accordingly.
[0102] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
transcription factor, in particular FOXP3. A disease or disorder
caused by a decreased activity of a transcription factor or a
disease or disorder, wherein increasing the amount of a
transcription factor (i) ameliorates, relieves, alleviates, or
reverses one or more symptoms of the disease or disorder are and/or
(ii) delays the onset of the disease or disorder and/or (iii)
lessens the severity of the disease or disorder, may be treated by
a corresponding protein replacement therapy as described herein
(e.g., by replacing or supplementing the transcription factor), a
corresponding genome engineering therapy as described herein,
and/or a genetic reprogramming therapy as described herein. A
patient having such a disease or disorder or being at risk of
developing such a disease or disorder can be treated
accordingly.
[0103] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
reprogramming factor, e.g., OCT4, SOX2, c-MYC, KLF4, LIN28 and
NANOG. A disease or disorder caused by a decreased activity of a
reprogramming factor or a disease or disorder, wherein increasing
the amount of a reprogramming factor (i) ameliorates, relieves,
alleviates, or reverses one or more symptoms of the disease or
disorder are and/or (ii) delays the onset of the disease or
disorder and/or (iii) lessens the severity of the disease or
disorder, may be treated by a corresponding protein replacement
therapy as described herein (e.g., by replacing or supplementing
the reprogramming factor), a corresponding genome engineering
therapy as described herein, and/or a genetic reprogramming therapy
as described herein. A patient having such a disease or disorder or
being at risk of developing such a disease or disorder can be
treated accordingly.
[0104] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
genomic engineering protein, in particular clustered regularly
spaced short palindromic repeat-CRISPR-associated protein 9
(CRISPR-Cas9). A disease or disorder caused by a decreased activity
of a genomic engineering protein or a disease or disorder, wherein
increasing the amount of a genomic engineering protein (i)
ameliorates, relieves, alleviates, or reverses one or more symptoms
of the disease or disorder are and/or (ii) delays the onset of the
disease or disorder and/or (iii) lessens the severity of the
disease or disorder, may be treated by a corresponding protein
replacement therapy as described herein (e.g., by replacing or
supplementing the genomic engineering protein), a corresponding
genome engineering therapy as described herein, and/or a genetic
reprogramming therapy as described herein. A patient having such a
disease or disorder or being at risk of developing such a disease
or disorder can be treated accordingly.
[0105] In one embodiment, the pharmaceutically active peptide or
protein, such as the disease-associated peptide or protein, is a
blood protein, in particular fibrinogen or alpha 1-antitrypsin. A
disease or disorder caused by a decreased activity of a blood
protein or a disease or disorder, wherein increasing the amount of
a blood protein (i) ameliorates, relieves, alleviates, or reverses
one or more symptoms of the disease or disorder are and/or (ii)
delays the onset of the disease or disorder and/or (iii) lessens
the severity of the disease or disorder, may be treated by a
corresponding protein replacement therapy as described herein
(e.g., by replacing or supplementing the blood protein), a
corresponding genome engineering therapy as described herein,
and/or a genetic reprogramming therapy as described herein. A
patient having such a disease or disorder or being at risk of
developing such a disease or disorder can be treated
accordingly.
[0106] In one embodiment, the pharmaceutically active peptide or
protein is an immunoglobulin, in particular an antibody. A disease
or disorder caused by a decreased activity of an immunoglobulin or
a disease or disorder, wherein increasing the amount of an
immunoglobulin (i) ameliorates, relieves, alleviates, or reverses
one or more symptoms of the disease or disorder are and/or (ii)
delays the onset of the disease or disorder and/or (iii) lessens
the severity of the disease or disorder, may be treated by a
corresponding protein replacement therapy as described herein
(e.g., by replacing or supplementing the immunoglobulin), a
corresponding genome engineering therapy as described herein,
and/or a genetic reprogramming therapy as described herein. A
patient having such a disease or disorder or being at risk of
developing such a disease or disorder can be treated
accordingly.
[0107] In one embodiment, the pharmaceutically active peptide or
protein is an immunologically active compound, in particular an
antigen, such as a disease-associated antigen. Thus, another
example of disease-associated peptides or proteins is a
disease-associated antigen, i.e., an antigen which is
characteristic for a disorder or disease and which is under normal
conditions, i.e., in a healthy individual, specifically expressed
in a limited number of organs and/or tissues or in specific
developmental stages (for example, the disease-associated antigen
may be under normal conditions specifically expressed in non-vital
tissue, in reproductive organs, e.g., in testis, in trophoblastic
tissue, e.g., in placenta, or in germ line cells) and is expressed
or aberrantly expressed in one or more diseased tissues. In this
context, "a limited number" preferably means not more than 3, more
preferably not more than 2 or 1. Particular examples of a
disease-associated antigen are tumor-associated antigens,
pathogen-associated antigens (e.g., antigens of a virus (such as
influenza virus (A, B, or C), CMV or RSV)) and allergens. A disease
or disorder which is characterized by a disease-associated antigen
may be treated by eliciting an immune response against said
disease-associated antigen in a patient having, or being at risk of
developing, said disease or disorder. E.g., in case the
disease-associated antigen is a tumor-associated antigen, the
immunotherapy may be considered as cancer immunotherapy; in case
the disease-associated antigen is a pathogen-associated antigen
(e.g., an antigen of a virus (such as influenza virus (A, B, or C),
CMV or RSV)), the immunotherapy can be considered as pathogen
immunotherapy; and in case the disease-associated antigen is an
allergen, the immunotherapy can be considered allergy tolerization
therapy, respectively. Thus, the RNA of the present invention may
be used to produce a disease-associated antigen which vaccinates an
individual against a malignant disease or an infectious disease or
may be used to produce an allergen which leads to allergy
tolerization.
[0108] The term "immunotherapy" relates to a treatment preferably
involving a specific immune reaction and/or immune effector
function(s).
[0109] As used herein, "de-differentiation" refers to loss of
specialization in form or function. In cells, de-differentiation
leads to a less committed cell. The term "committed" refers to
cells which are considered to be permanently committed to a
specific function. Committed cells are also referred to as
"terminally differentiated cells".
[0110] As used herein, "differentiation" refers to the adaptation
of cells for a particular form or function. In cells,
differentiation leads to a more committed cell.
[0111] A "differentiated cell" is a mature cell that has undergone
progressive developmental changes to a more specialized form or
function. Cell differentiation is the process a cell undergoes as
it matures to an overtly specialized cell type. Differentiated
cells have distinct characteristics, perform specific functions,
and are less likely to divide than their less differentiated
counterparts.
[0112] An "undifferentiated" cell, for example, an immature,
embryonic, or primitive cell, typically has a nonspecific
appearance, may perform multiple, non-specific activities, and may
perform poorly, if at all, in functions typically performed by
differentiated cells.
[0113] "Somatic cell" refers to any and all differentiated cells
and does not include stem cells, germ cells, or gametes.
Preferably, "somatic cell" as used herein refers to a terminally
differentiated cell.
[0114] A "stem cell" is a cell with the ability to self-renew, to
remain undifferentiated, and to become differentiated. A stem cell
can divide without limit, for at least the lifetime of the animal
in which it naturally resides. A stem cell is not terminally
differentiated; it is not at the end stage of a differentiation
pathway. When a stem cell divides, each daughter cell can either
remain a stem cell or embark on a course that leads toward terminal
differentiation.
[0115] The term "cells having stem cell characteristics" is used
herein to designate cells which, although they are derived from
differentiated somatic non-stem cells, exhibit one or more features
typical for stem cells, in particular embryonic stem cells. Such
features include an embryonic stem cell morphology such as compact
colonies, high nucleus to cytoplasm ratio and prominent nucleoli,
normal karyotypes, expression of telomerase activity, expression of
cell surface markers that are characteristic for embryonic stem
cells, and/or expression of genes that are characteristic for
embryonic stem cells. The cell surface markers that are
characteristic for embryonic stem cells are, for example, selected
from the group consisting of stage-specific embryonic antigen-3
(SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81,
and TRA-2-49/6E. The genes that are characteristic for embryonic
stem cells are selected, for example, from the group consisting of
endogenous OCT4, endogenous NANOG, growth and differentiation
factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth
factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1),
developmental pluripotency-associated 2 (DPPA2), DPPA4, and
telomerase reverse transcriptase (TERT). In one embodiment, the one
or more features typical for stem cells include pluripotency. In
one embodiment, the cells having stem cell characteristics exhibit
a pluripotent state. In one embodiment, the cells having stem cell
characteristics have the developmental potential to differentiate
into advanced derivatives of all three primary germ layers. In one
embodiment, the primary germ layer is endoderm and the advanced
derivative is gut-like epithelial tissue. In a further embodiment,
the primary germ layer is mesoderm and the advanced derivative is
striated muscle and/or cartilage. In an even further embodiment,
the primary germ layer is ectoderm and the advanced derivative is
neural tissue and/or epidermal tissue. In one preferred embodiment,
the cells having stem cell characteristics have the developmental
potential to differentiate into cells expressing the peptide or
protein of interest. According to the invention, generally the
terms "cells having stem cell characteristics", "cells having stem
cell properties", "reprogrammed cells" and "de-differentiated
cells" or similar terms have similar meanings and are used
interchangeably herein.
[0116] In one embodiment, RNA, in particular RNA which comprises a
nucleic acid sequence encoding a peptide or protein and which is to
be expressed in a cell, is a single stranded self-replicating RNA.
In one embodiment, the self-replicating RNA is single stranded RNA
of positive sense. In one embodiment, the self-replicating RNA is
viral RNA or RNA derived from viral RNA. In one embodiment, the
self-replicating RNA is alphaviral genomic RNA or is derived from
alphaviral genomic RNA. In one embodiment, the self-replicating RNA
is a viral gene expression vector. In one embodiment, the virus is
Semliki forest virus. In one embodiment, the self-replicating RNA
contains one or more transgenes which in one embodiment, if the RNA
is viral RNA, may partially or completely replace viral sequences
such as viral sequences encoding structural proteins.
[0117] The term "nucleoside" (abbreviated herein as "N") relates to
compounds which can be thought of as nucleotides without a
phosphate group. While a nucleoside is a nucleobase linked to a
sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a
nucleoside and one or more phosphate groups. Examples of
nucleosides include cytidine, uridine, pseudouridine, adenosine,
and guanosine.
[0118] The five standard nucleosides which usually make up
naturally occurring nucleic acids are uridine, adenosine,
thymidine, cytidine and guanosine. The five nucleosides are
commonly abbreviated to their one letter codes U, A, T, C and G,
respectively. However, thymidine is more commonly written as "dT"
("d" represents "deoxy") as it contains a 2'-deoxyribofuranose
moiety rather than the ribofuranose ring found in uridine. This is
because thymidine is found in deoxyribonucleic acid (DNA) and not
ribonucleic acid (RNA). Conversely, uridine is found in RNA and not
DNA. The remaining three nucleosides may be found in both RNA and
DNA. In RNA, they would be represented as A, C and G, whereas in
DNA they would be represented as dA, dC and dG.
[0119] A modified purine (A or G) or pyrimidine (C, T, or U) base
moiety is preferably modified by one or more alkyl groups, more
preferably one or more C.sub.1-4 alkyl groups, even more preferably
one or more methyl groups. Particular examples of modified purine
or pyrimidine base moieties include N.sup.7-alkyl-guanine,
N.sup.6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and
N(1)-alkyl-uracil, such as N.sup.7--C.sub.1-4 alkyl-guanine,
N.sup.6--C.sub.1-4 alkyl-adenine, 5-C.sub.1-4 alkyl-cytosine,
5-C.sub.1-4 alkyl-uracil, and N(1)-C.sub.1-4 alkyl-uracil,
preferably N.sup.7-methyl-guanine, N.sup.6-methyl-adenine,
5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.
[0120] The term "in vitro transcription" or "IVT" as used herein
means that the transcription (i.e., the generation of RNA) is
conducted in a cell-free manner. I.e., IVT does not use
living/cultured cells but rather the transcription machinery
extracted from cells (e.g., cell lysates or the isolated components
thereof, including an RNA polymerase (preferably T7, T3 or SP6
polymerase)).
[0121] The term "modification" in the context of modified RNA
(preferably mRNA) according to the present invention includes any
modification of an RNA (preferably mRNA) which is not naturally
present in said RNA. In particular, the term modification relates
to providing an RNA (preferably mRNA) with a 5'-cap structure of
the present invention. For example, providing an RNA (preferably
mRNA) with a 5'-cap structure of the present invention may be
achieved by in vitro transcription of a DNA template in presence of
a 5'-cap compound of the present invention, wherein said 5'-cap
structure is co-transcriptionally incorporated into the generated
RNA strand, or the RNA (preferably mRNA) may be generated, for
example, by in vitro transcription, and the 5'-cap structure may be
attached to the RNA post-transcriptionally using capping enzymes,
for example, capping enzymes of vaccinia virus.
[0122] The RNA (preferably mRNA) may comprise further modifications
in order to, e.g., increase its stability and/or decrease
immunogenicity and/or decrease cytotoxicity. For example, a further
modification of the RNA, preferably mRNA, modified with a 5'-cap
compound of the present invention may be an extension or truncation
of the naturally occurring poly(A) tail, an alteration of the 5'-
or 3'-untranslated regions (UTR) such as introduction of a UTR
which is not related to the coding region of said RNA, the
replacement of one or more naturally occurring nucleotides with
synthetic nucleotides and/or codon optimization (e.g., to alter,
preferably increase, the GC content of the RNA).
[0123] RNA (preferably mRNA) having an unmasked poly-A sequence is
translated more efficiently than RNA (preferably mRNA) having a
masked poly-A sequence. The term "poly(A) tail" or "poly-A
sequence" relates to a sequence of adenosine (in particular
adenylyl) (A) residues which typically is located on the 3'-end of
an RNA (preferably mRNA) molecule and "unmasked poly-A sequence"
means that the poly-A sequence at the 3' end of an RNA (preferably
mRNA) molecule ends with an A of the poly-A sequence and is not
followed by nucleotides other than A located at the 3' end, i.e.,
downstream, of the poly-A sequence. Furthermore, a long poly-A
sequence having a length of about 120 nucleotides results in an
optimal transcript stability and translation efficiency of an RNA
(preferably mRNA).
[0124] Therefore, in order to increase stability and/or expression
of RNA, preferably mRNA, according to the present invention, it may
be modified so as to be present in conjunction with a poly-A
sequence, preferably having a length of 10 to 500, more preferably
30 to 300, even more preferably 65 to 200 and especially 100 to 150
adenosine (in particular adenylyl) residues. In an especially
preferred embodiment the poly-A sequence has a length of
approximately 120 adenosine (in particular adenylyl) residues. To
further increase stability and/or expression of RNA, preferably of
the mRNA, according to the invention, the poly-A sequence can be
unmasked.
[0125] In addition, incorporation of a 3'-UTR into the 3'-non
translated region of an RNA (preferably mRNA) molecule can result
in an enhancement in translation efficiency. A synergistic effect
may be achieved by incorporating two or more of such 3'-UTRs (which
are preferably arranged in a head-to-tail orientation; cf., e.g.,
Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be
autologous or heterologous to the RNA (preferably mRNA) into which
they are introduced. In one particular embodiment the 3'-UTR is
derived from a globin gene or mRNA, such as a gene or mRNA of
alpha2-globin, alpha1-globin, or beta-globin, preferably
beta-globin, more preferably human beta-globin. For example, the
RNA (preferably mRNA) may be modified by the replacement of the
existing 3'-UTR with or the insertion of one or more, preferably
two copies of a 3'-UTR derived from a globin gene, such as
alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin,
more preferably human beta-globin.
[0126] The RNA (preferably mRNA) according to the invention may
have modified ribonucleotides in order to increase its stability
and/or decrease immunogenicity and/or decrease cytotoxicity. For
example, in one embodiment, in the RNA (preferably mRNA) according
to the invention 5-methylcytidine is substituted partially or
completely, preferably completely, for cytidine. Alternatively or
additionally, in one embodiment, in the RNA (preferably mRNA)
according to the invention pseudouridine or
N(1)-methylpseudouridine or 5-methyluridine is substituted
partially or completely, preferably completely, for uridine. An RNA
(preferably mRNA) which is modified by pseudouridine (substituting
partially or completely, preferably completely, for uridine) is
referred to herein as ".PSI.-modified", whereas the term
"m1.PSI.-modified" means that the RNA (preferably mRNA) contains
N(1)-methylpseudouridine (substituting partially or completely,
preferably completely, for uridine). Furthermore, the term
"m5U-modified" means that the RNA (preferably mRNA) contains
5-methyluridine (substituting partially or completely, preferably
completely, for uridine). Such .PSI.- or m1.PSI.- or m5U-modified
RNAs of the invention usually exhibit decreased immunogenicity
compared to their unmodified forms and, thus, are preferred in
applications where the induction of an immune response is to be
avoided or minimized (e.g., in protein replacement therapy, genome
engineering therapy, and genetic reprogramming therapy, as
described herein).
[0127] A combination of the above described modifications, i.e.,
incorporation of a poly-A sequence, unmasking of a poly-A sequence,
incorporation of one or more 3'-UTRs and replacing one or more
naturally occurring nucleotides with synthetic nucleotides (e.g.,
5-methylcytidine for cytidine and/or pseudouridine (.PSI.) or
N(1)-methylpseudouridine (m1.PSI.) or 5-methyluridine (m5U) for
uridine), has a synergistic influence on the stability of RNA
(preferably mRNA) and increase in translation efficiency. Thus in a
preferred embodiment, the RNA (preferably mRNA) according to the
present invention is not only modified with a 5'-cap compound of
the present invention but also contains a combination of the three
above-mentioned modifications, i.e., (i) incorporation of a poly-A
sequence, unmasking of a poly-A sequence; (ii) incorporation of one
or more 3'-UTRs; and (iii) replacing one or more naturally
occurring nucleotides with synthetic nucleotides (e.g.,
5-methylcytidine for cytidine and/or pseudouridine (.PSI.) or
N(1)-methylpseudouridine (m1.PSI.) or 5-methyluridine (m5U) for
uridine). Optionally, the codons of the RNA (preferably mRNA) of
the present invention may further be optimized, e.g., to increase
the GC content of the RNA and/or to replace codons which are rare
in the cell (or subject) in which the peptide or protein of
interest is to be expressed by codons which are synonymous frequent
codons in said cell (or subject).
[0128] The term "RNA polymerase" as used herein refers to a
DNA-dependent RNA polymerase which produces primary transcript RNA.
Examples of RNA polymerases suitable for generating IVT RNA
according to the present invention include T7, T3 and SP6 RNA
polymerases. A preferred RNA polymerase is T7 RNA polymerase.
[0129] The term "conventional 5'-cap" refers to a cap structure
found on the 5'-end of an mRNA molecule and generally consists of a
guanosine 5'-triphosphate (Gppp) which is connected via its
triphosphate moiety to the 5'-end of the next nucleotide of the
mRNA (i.e., the guanosine is connected via a 5' to 5' triphosphate
linkage to the rest of the mRNA). The guanosine may be methylated
at position N.sup.7 (resulting in the cap structure
m.sup.7Gppp).
[0130] According to the present application, the term "cap0" means
the structure "m.sup.7GpppN", wherein N is any nucleoside bearing
an OH moiety at position 2'. According to the present application,
the term "cap1" means the structure "m.sup.7GpppNm", wherein Nm is
any nucleoside bearing an OCH.sub.3 moiety at position 2'.
According to the present application, the term "cap2" means the
structure "m.sup.7GpppNmNm", wherein each Nm is independently any
nucleoside bearing an OCH.sub.3 moiety at position 2'.
[0131] In the context of the present invention, the term "5'-cap
structure of the present invention" is a 5'-cap analog that
resembles the structure of a conventional 5'-cap but is modified to
possess the ability to stabilize RNA (in particular mRNA) and/or
increase RNA expression (in particular mRNA expression), if
attached thereto, preferably in vivo or in a cell. The cell may be
any cell which can be transfected with RNA (preferably mRNA) of the
present invention and is preferably a cell obtained from a subject,
e.g., a stem cell (e.g., a mesenchymal stem cell (MSC)) or an
antigen presenting cell (e.g., an immature antigen presenting
cell), such as a dendritic cell (e.g., an immature dendritic cell).
Preferably, the 5'-cap structure of the present invention at least
comprises the structure
"1-(N.sup.7--(R.sup.1)-guanine-9-yl)-pentose-5-yl-(phosphorothioate
linkage)-N(R')--" of any one of formulas (I), (Ia), (Ib), (Ic),
(Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III), and
(IIIa) (i.e., wherein the first guanine of the 5'-cap structure is
substituted at position N.sup.7 with R.sup.1 and is connected at
N.sup.9 to C.sup.1' of the pentose bearing substituents R.sup.2 and
R.sup.3; the phosphorothioate linkage has the structure
--O--P(O)(R.sup.4)--O--P(O.sup.-)(R.sup.5)--O[--P(O.sup.-)(R.sup.6)--O].s-
ub.n; and N is any nucleoside bearing base B and being substituted
at position 2' with R.sup.8). Furthermore, if R.sup.7 in any one of
formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb),
(IIc), (IId), (IIe), (III), and (IIIa) is a ribooligonucleotide, in
which the OH group at position 2' of at least the ribose at the
5'-end of the ribooligonucleotide is replaced with a substituent
R.sup.8' selected from the group consisting of H, halo, and
optionally substituted alkoxy, and the ribose at the 3'-end of the
ribooligonucleotide has a free OH group at position 2', then the
5'-cap structure of the present invention preferably comprises in
addition to the structure
"1-(N.sup.7--(R')-guanine-9-yl)-pentose-5-yl-(phosphorothioate
linkage)-N(R.sup.8)--" of any one of formulas (I), (Ia), (Ib),
(Ic), (Id), (le), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III),
and (IIIa) also any nucleoside substituted at position 2' with
R.sup.8' (together with any internucleotide linkage between the
N(R.sup.8) moiety and the N(R.sup.8') moiety as well as any
internucleotide linkage between each pair of N(R.sup.8) moieties in
case the ribooligonucleotide contains more than one N(R.sup.8')
moiety). For example, if for providing a 5'-cap structure of the
present invention a 5'-cap compound of formula (I), wherein R.sup.7
is a ribooligonucleotide of the formula [pN(R.sup.8')].sub.2pN
(i.e., only the nucleoside at the 3'-end of the ribooligonucleotide
has a free OH group at position 2', whereas the two other
nucleosides are substituted with R.sup.8' at position 2') is used,
the 5'-cap structure of the present invention would comprises at
least the structure
"1-(N.sup.7--(R')-guanine-9-yl)-pentose-5-yl-(phosphorothioate
linkage)-N(R.sup.8)-[pN(R.sup.8')].sub.2".
[0132] According to the present application, the term "5'-capped
RNA" means RNA which contains at its 5'-end a cap structure.
[0133] Within the context of the present application, the term "RNA
which is modified with a 5'-cap compound of the present invention"
means RNA which contains at its 5'-end a 5'-cap structure of the
present invention. Similarly, the term "mRNA which is modified with
a 5'-cap compound of the present invention" means mRNA which
contains at its 5'-end a 5'-cap structure of the present invention.
Thus, in a preferred embodiment, such RNA (e.g., mRNA) modified
with a 5'-cap compound of the present invention at least comprises
at its 5'-end the structure
"1-(N.sup.7--(R')-guanine-9-yl)-pentose-5-yl-(phosphorothioate
linkage)-N(R.sup.8)--" of any one of formulas (I), (Ia), (Ib),
(Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III),
and (IIIa) (i.e., wherein the first guanine of the 5'-cap structure
is substituted at position N.sup.7 with R.sup.1 and is connected at
N.sup.9 to C.sup.1' of the pentose bearing substituents R.sup.2 and
R.sup.3; the phosphorothioate linkage has the structure
--O--P(O.sup.-)(R.sup.4)--O--P(O.sup.-)(R.sup.5)--O--[--P(O.sup.-)(R.sup.-
6)--O]; and N is any nucleoside bearing base B and being
substituted at position 2' with R'). Furthermore, if R.sup.7 in any
one of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa),
(IIb), (IIc), (IId), (IIe), (III), and (IIIa) is a
ribooligonucleotide, in which the OH group at position 2' of at
least the ribose at the 5'-end of the ribooligonucleotide is
replaced with a substituent R.sup.8' selected from the group
consisting of H, halo, and optionally substituted alkoxy, and the
ribose at the 3'-end of the ribooligonucleotide has a free OH group
at position 2', then the RNA (such as mRNA) which is modified with
a 5'-cap compound of the present invention preferably comprises at
its 5'-end in addition to the structure
"1-(N.sup.7--(R')-guanine-9-yl)-pentose-5-yl-(phosphorothioate
linkage)-N(R.sup.8)--" of any one of formulas (I), (Ia), (Ib),
(Ic), (Id), (le), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III),
and (IIIa) also any nucleoside substituted at position 2' with
R.sup.8' (together with any internucleotide linkage between the
N(R.sup.8) moiety and the N(R.sup.8') moiety as well as any
internucleotide linkage between each pair of N(R.sup.8) moieties in
case the ribooligonucleotide contains more than one N(R.sup.8')
moiety). For example, if an RNA is modified with a 5'-cap compound
of the present invention of formula (I), wherein R.sup.7 is a
ribooligonucleotide of the formula [pN(R.sup.8')].sub.2pN (i.e.,
only the nucleoside at the 3'-end of the ribooligonucleotide has a
free OH group at position 2', whereas the two other nucleosides are
substituted with R.sup.8' at position 2'), the modified RNA would
comprises at its 5'-end at least the structure
"1-(N.sup.7--(R')-guanine-9-yl)-pentose-5-yl-(phosphorothioate
linkage)-N(R')-[pN(R.sup.8')].sub.2".
[0134] The term "increasing RNA expression", preferably in
connection with an RNA modified with a 5'-cap compound of the
present invention, preferably means decreasing or even inhibiting
the recognition of the RNA by proteins recognizing a cap0
structure, e.g., IFIT proteins (in particular IFIT1).
[0135] According to the invention, a part or fragment of a peptide
or protein preferably has at least one functional property of the
peptide or protein from which it has been derived. Such functional
properties comprise a pharmacological activity, the interaction
with other peptides or proteins, an enzymatic activity, the
interaction with antibodies, and the selective binding of nucleic
acids. E.g., a pharmacological active fragment of a peptide or
protein has at least one of the pharmacological activities of the
peptide or protein from which the fragment has been derived. A part
or fragment of a peptide or protein preferably comprises a sequence
of at least 6, in particular at least 8, at least 10, at least 12,
at least 15, at least 20, at least 30 or at least 50, consecutive
amino acids of the peptide or protein. A part or fragment of a
peptide or protein preferably comprises a sequence of up to 8, in
particular up to 10, up to 12, up to 15, up to 20, up to 30 or up
to 55, consecutive amino acids of the peptide or protein.
[0136] According to the invention, an analog of a peptide or
protein is a modified form of said peptide or protein from which it
has been derived and has at least one functional property of said
peptide or protein. E.g., a pharmacological active analog of a
peptide or protein has at least one of the pharmacological
activities of the peptide or protein from which the analog has been
derived. Such modifications include any chemical modification and
comprise single or multiple substitutions, deletions and/or
additions of any molecules associated with the protein or peptide,
such as carbohydrates, lipids and/or proteins or peptides. In one
embodiment, "analogs" of proteins or peptides include those
modified forms resulting from glycosylation, acetylation,
phosphorylation, amidation, palmitoylation, myristoylation,
isoprenylation, lipidation, alkylation, derivatization,
introduction of protective/blocking groups, proteolytic cleavage or
binding to an antibody or to another cellular ligand. The term
"analog" also extends to all functional chemical equivalents of
said proteins and peptides.
[0137] In the context of the present invention, the term "vaccine
composition" relates to an antigenic preparation which comprises
RNA. The vaccine composition is administered to an individual in
order to stimulate the humoral and/or cellular immune system of the
individual against one or more antigens. In this context, the RNA
may encode the antigen, a protein or peptide comprising said
antigen or an antigen peptide. A vaccine composition in the context
of the present invention may further comprise one or more adjuvants
and/or excipients and is applied to an individual in any suitable
route in order to elicit a protective and/or therapeutic immune
reaction against the antigen.
[0138] An "antigen" according to the invention covers any substance
that will elicit an immune response and/or any substance against
which an immune response or an immune mechanism such as a cellular
response is directed. This also includes situations wherein the
antigen is processed into antigen peptides and an immune response
or an immune mechanism is directed against one or more antigen
peptides, in particular if presented in the context of MHC
molecules. In particular, an "antigen" relates to any substance,
preferably a peptide or protein, that reacts specifically with
antibodies or T-lymphocytes (T-cells). According to the present
invention, the term "antigen" comprises any molecule which
comprises at least one epitope, such as a T cell epitope.
Preferably, an antigen in the context of the present invention is a
molecule which, optionally after processing, induces an immune
reaction, which is preferably specific for the antigen (including
cells expressing the antigen).
[0139] According to the present invention, any suitable antigen may
be used, which is a candidate for an immune response, wherein the
immune response may be both a humoral as well as a cellular immune
response. In the context of some embodiments of the present
invention, the antigen is preferably presented by a cell,
preferably by an antigen presenting cell, in the context of MHC
molecules, which results in an immune response against the antigen.
An antigen is preferably a product which corresponds to or is
derived from a naturally occurring antigen. Such naturally
occurring antigens may include or may be derived from allergens,
viruses (e.g., influenza virus (A, B, or C), CMV, or RSV),
bacteria, fungi, parasites and other infectious agents and
pathogens or an antigen may also be a tumor antigen. According to
the present invention, an antigen may correspond to a naturally
occurring product, for example, a viral protein (e.g., a protein of
influenza virus A, influenza virus B, or influenza virus C, such as
PB1, PB1-F2, PB2, PA, HA, NP, NA, M1, M2, NS1, or NEP/NS2 from
influenza virus A, influenza virus B, or influenza virus C), or a
part thereof.
[0140] In a preferred embodiment, the antigen is a tumor antigen,
i.e., a part of a tumor cell which may be derived from the
cytoplasm, the cell surface or the cell nucleus, in particular
those which primarily occur intracellularly or as surface antigens
of tumor cells. For example, tumor antigens include the
carcinoembryonal antigen, al-fetoprotein, isoferritin, and fetal
sulphoglycoprotein, .alpha.2-H-ferroprotein and
.gamma.-fetoprotein, as well as various virus tumor antigens.
According to the present invention, a tumor antigen preferably
comprises any antigen which is characteristic for tumors or cancers
as well as for tumor or cancer cells with respect to type and/or
expression level. In another embodiment, the antigen is a
pathogen-associated antigen, i.e., an antigen derived from a
pathogen, e.g., from a virus (such as influenza virus (A, B, or C),
CMV, or RSV), bacterium, unicellular organism, or parasite, for
example a virus antigen such as viral ribonucleoprotein or coat
protein. In particular, the antigen should be presented by MHC
molecules which results in modulation, in particular activation of
cells of the immune system, preferably CD4.sup.+ and CD8.sup.-
lymphocytes, in particular via the modulation of the activity of a
T-cell receptor.
[0141] In some embodiments, the antigen is a tumor antigen and the
present invention involves the stimulation of an anti-tumor CTL
response against tumor cells expressing such tumor antigen and
preferably presenting such tumor antigen with class I MHC.
[0142] The term "immunogenicity" relates to the relative
effectivity of an antigen to induce an immune reaction.
[0143] The term "pathogen" relates to pathogenic microorganisms and
comprises viruses, bacteria, fungi, unicellular organisms, and
parasites. Examples for pathogenic viruses are human
immunodeficiency virus (HIV), influenza virus (e.g., influenza
virus A, influenza virus B, or influenza virus C), respiratory
syncytial virus (RSV), cytomegalovirus (CMV), herpes virus (HSV),
hepatitis A-virus (HAV), HBV, HCV, papilloma virus, and human
T-lymphotrophic virus (HTLV), such as HIV, CMV, HSV, HAV, HBV, HCV,
papilloma virus, and HTLV, preferably influenza virus, CMV, or RSV.
Unicellular organisms comprise plasmodia trypanosomes, amoeba,
etc.
[0144] Thus, in another preferred embodiment, the antigen is one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) viral antigens, i.e.,
one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens of a
virus, wherein the virus is preferably influenza virus (A, B, or
C), CMV, or RSV. Hence, in this embodiment, the present invention
preferably involves eliciting an immune response against said
virus. The one or more viral antigens are preferably selected from
the group consisting of PB1, PB1-F2, PB2, PA, HA, NP, NA, M1, M2,
NS1, and NEP/NS2 from influenza A, influenza B, or influenza C.
[0145] Examples for antigens that may be used in the present
invention are p53, ART-4, BAGE, ss-catenin/m, Bcr-abL CAMEL, CAP-1,
CASP-8, CDCl.sub.27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B,
DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu,
HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A,
preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B,
MAGE-C, MART-1/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3,
NA88-A, NF1, NY-ESO-1, NY--BR-1, p190 minor BCR-abL, Plac-1,
Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE,
SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN,
TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT, preferably
WT-1.
[0146] "A portion or fragment of an antigen" or "an antigen
peptide" according to the invention preferably is an incomplete
representation of an antigen and is capable of eliciting an immune
response against the antigen or cells characterized by expression
of the antigen and preferably by presentation of the antigen.
[0147] In this context, the invention also makes use of peptides
comprising amino acid sequences derived from antigens, also termed
"antigen peptides" herein. By "antigen peptide" or "antigen peptide
derived from an antigen" is meant an oligopeptide or polypeptide
comprising an amino acid sequence substantially corresponding to
the amino acid sequence of a fragment or peptide of an antigen. An
antigen peptide may be of any length.
[0148] Preferably, the antigen peptides are capable of stimulating
an immune response, preferably a cellular response against the
antigen or cells characterized by expression of the antigen and
preferably by presentation of the antigen. Preferably, an antigen
peptide is capable of stimulating a cellular response against a
cell characterized by presentation of an antigen with class I MHC
and preferably is capable of stimulating an antigen-responsive CTL.
Preferably, according to the invention, the antigen peptides are
MHC class I and/or class II presented peptides or can be processed
to produce MHC class I and/or class II presented peptides.
Preferably, the antigen peptides comprise an amino acid sequence
substantially corresponding to the amino acid sequence of a
fragment of an antigen. Preferably, said fragment of an antigen is
an MHC class I and/or class II presented peptide. Preferably, an
antigen peptide according to the invention comprises an amino acid
sequence substantially corresponding to the amino acid sequence of
such fragment and is processed to produce such fragment, i.e., an
MHC class I and/or class II presented peptide derived from an
antigen.
[0149] If an antigen peptide is to be presented directly, i.e.,
without processing, in particular without cleavage, it has a length
which is suitable for binding to an MHC molecule, in particular a
class I MHC molecule, and preferably is 7-20 amino acids in length,
more preferably 7-12 amino acids in length, more preferably 8-11
amino acids in length, in particular 9 or 10 amino acids in length.
Preferably the sequence of an antigen peptide which is to be
presented directly is derived from the amino acid sequence of an
antigen, i.e., its sequence substantially corresponds and is
preferably completely identical to a fragment of an antigen.
[0150] If an antigen peptide is to be presented following
processing, in particular following cleavage, the peptide produced
by processing has a length which is suitable for binding to an MHC
molecule, in particular a class I MHC molecule, and preferably is
7-20 amino acids in length, more preferably 7-12 amino acids in
length, more preferably 8-11 amino acids in length, in particular 9
or 10 amino acids in length. Preferably, the sequence of the
peptide which is to be presented following processing is derived
from the amino acid sequence of an antigen, i.e., its sequence
substantially corresponds and is preferably completely identical to
a fragment of an antigen. Thus, an antigen peptide according to the
invention in one embodiment comprises a sequence of 7-20 amino
acids in length, more preferably 7-12 amino acids in length, more
preferably 8-11 amino acids in length, in particular 9 or 10 amino
acids in length which substantially corresponds and is preferably
completely identical to a fragment of an antigen and following
processing of the antigen peptide makes up the presented peptide.
However, the antigen peptide may also comprise a sequence which
substantially corresponds and preferably is completely identical to
a fragment of an antigen which is even longer than the above stated
sequence. In one embodiment, an antigen peptide may comprise the
entire sequence of an antigen.
[0151] Peptides having amino acid sequences substantially
corresponding to a sequence of a peptide which is presented by the
class I MHC may differ at one or more residues that are not
essential for TCR recognition of the peptide as presented by the
class I MHC, or for peptide binding to MHC. Such substantially
corresponding peptides are also capable of stimulating an
antigen-responsive CTL. Peptides having amino acid sequences
differing from a presented peptide at residues that do not affect
TCR recognition but improve the stability of binding to MHC may
improve the immunogenicity of the antigen peptide, and may be
referred to herein as "optimized peptide". Using existing knowledge
about which of these residues may be more likely to affect binding
either to the MHC or to the TCR, a rational approach to the design
of substantially corresponding peptides may be employed. Resulting
peptides that are functional are contemplated as antigen
peptides.
[0152] In one embodiment, an antigen peptide when presented in the
context of MHC such as MHC of antigen presenting cells is
recognized by a T cell receptor. The antigen peptide if recognized
by a T cell receptor may be able to induce in the presence of
appropriate co-stimulatory signals, clonal expansion of the T cell
carrying the T cell receptor specifically recognizing the antigen
peptide. Preferably, antigen peptides, in particular if presented
in the context of MHC molecules, are capable of stimulating an
immune response, preferably a cellular response against the antigen
from which they are derived or cells characterized by expression of
the antigen and preferably characterized by presentation of the
antigen.
[0153] The term "epitope" refers to an antigenic determinant in a
molecule such as an antigen, i.e., to a part in or fragment of the
molecule that is recognized by the immune system, for example, that
is recognized by a T cell, in particular when presented in the
context of MHC molecules. An epitope of a protein preferably
comprises a continuous or discontinuous portion of said protein and
is preferably between 5 and 100, preferably between 5 and 50, more
preferably between 8 and 30, most preferably between 10 and 25
amino acids in length, for example, the epitope may be preferably
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or
25 amino acids in length. It is particularly preferred that the
epitope in the context of the present invention is a T cell
epitope.
[0154] Terms such as "epitope", "T cell epitope", "fragment of an
antigen", "immunogenic peptide" and "antigen peptide" are used
interchangeably herein and preferably relate to an incomplete
representation of an antigen which is preferably capable of
eliciting an immune response against the antigen or a cell
expressing or comprising and preferably presenting the antigen.
Preferably, the terms relate to an immunogenic portion of an
antigen. Preferably, it is a portion of an antigen that is
recognized (i.e., specifically bound) by a T cell receptor, in
particular if presented in the context of MHC molecules. Certain
preferred immunogenic portions bind to an MHC class I or class II
molecule.
[0155] The term "target" shall mean an agent such as a cell or
tissue which is a target for an immune response such as a cellular
immune response. Targets include cells that present an antigen or
an antigen epitope, i.e. a peptide fragment derived from an
antigen. In one embodiment, the target cell is a cell expressing an
antigen and preferably presenting said antigen with class I
MHC.
[0156] The term "portion" refers to a fraction. With respect to a
particular structure such as an amino acid sequence or protein the
term "portion" thereof may designate a continuous or a
discontinuous fraction of said structure.
[0157] The terms "part" and "fragment" are used interchangeably
herein and refer to a continuous element. For example, a part of a
structure such as an amino acid sequence or protein refers to a
continuous element of said structure.
[0158] "Antigen processing" refers to the degradation of an antigen
into processing products which are fragments of said antigen (e.g.,
the degradation of a protein into peptides) and the association of
one or more of these fragments (e.g., via binding) with MHC
molecules for presentation by cells, preferably antigen-presenting
cells to specific T-cells.
[0159] By "antigen-responsive CTL" is meant a CD8+ T-cell that is
responsive to an antigen or a peptide derived from said antigen,
which is presented with class I MHC on the surface of antigen
presenting cells.
[0160] According to the invention, CTL responsiveness may include
sustained calcium flux, cell division, production of cytokines such
as IFN-.gamma. and TNF-.alpha., up-regulation of activation markers
such as CD44 and CD69, and specific cytolytic killing of tumor
antigen expressing target cells. CTL responsiveness may also be
determined using an artificial reporter that accurately indicates
CTL responsiveness.
[0161] The terms "immune response" and "immune reaction" are used
herein interchangeably in their conventional meaning and refer to
an integrated bodily response to an antigen and preferably refers
to a cellular immune response, a humoral immune response, or both.
According to the invention, the term "immune response to" or
"immune response against" with respect to an agent such as an
antigen, cell or tissue, relates to an immune response such as a
cellular response directed against the agent. An immune response
may comprise one or more reactions selected from the group
consisting of developing antibodies against one or more antigens
and expansion of antigen-specific T-lymphocytes, preferably
CD4.sup.+ and CD8.sup.+ T-lymphocytes, more preferably CD8.sup.+
T-lymphocytes, which may be detected in various proliferation or
cytokine production tests in vitro.
[0162] The terms "inducing an immune response" and "eliciting an
immune response" and similar terms in the context of the present
invention refer to the induction of an immune response, preferably
the induction of a cellular immune response, a humoral immune
response, or both. The immune response may be
protective/preventive/prophylactic and/or therapeutic. The immune
response may be directed against any immunogen or antigen or
antigen peptide, preferably against a tumor-associated antigen or a
pathogen-associated antigen (e.g., an antigen of a virus (such as
influenza virus (A, B, or C), CMV or RSV)). "Inducing" in this
context may mean that there was no immune response against a
particular antigen or pathogen before induction, but it may also
mean that there was a certain level of immune response against a
particular antigen or pathogen before induction and after induction
said immune response is enhanced. Thus, "inducing the immune
response" in this context also includes "enhancing the immune
response". Preferably, after inducing an immune response in an
individual, said individual is protected from developing a disease
such as an infectious disease or a cancerous disease or the disease
condition is ameliorated by inducing an immune response.
[0163] The terms "cellular immune response", "cellular response",
"cell-mediated immunity" or similar terms are meant to include a
cellular response directed to cells characterized by expression of
an antigen and/or presentation of an antigen with class I or class
II MHC. The cellular response relates to cells called T cells or T
lymphocytes which act as either "helpers" or "killers". The helper
T cells (also termed CD4.sup.+ T cells) play a central role by
regulating the immune response and the killer cells (also termed
cytotoxic T cells, cytolytic T cells, CD8.sup.+ T cells or CTLs)
kill cells such as diseased cells.
[0164] The term "humoral immune response" refers to a process in
living organisms wherein antibodies are produced in response to
agents and organisms, which they ultimately neutralize and/or
eliminate. The specificity of the antibody response is mediated by
T and/or B cells through membrane-associated receptors that bind
antigen of a single specificity. Following binding of an
appropriate antigen and receipt of various other activating
signals, B lymphocytes divide, which produces memory B cells as
well as antibody secreting plasma cell clones, each producing
antibodies that recognize the identical antigenic epitope as was
recognized by its antigen receptor. Memory B lymphocytes remain
dormant until they are subsequently activated by their specific
antigen. These lymphocytes provide the cellular basis of memory and
the resulting escalation in antibody response when re-exposed to a
specific antigen.
[0165] The term "antibody" as used herein, refers to an
immunoglobulin molecule, which is able to specifically bind to an
epitope on an antigen. In particular, the term "antibody" refers to
a glycoprotein comprising at least two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. The term
"antibody" includes monoclonal antibodies, recombinant antibodies,
human antibodies, humanized antibodies, chimeric antibodies and
combinations of any of the foregoing. Each heavy chain is comprised
of a heavy chain variable region (VH) and a heavy chain constant
region (CH). Each light chain is comprised of a light chain
variable region (VL) and a light chain constant region (CL). The
variable regions and constant regions are also referred to herein
as variable domains and constant domains, respectively. The VH and
VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions
(CDRs), interspersed with regions that are more conserved, termed
framework regions (FRs). Each VH and VL is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs
of a VH are termed HCDR1, HCDR2 and HCDR3, the CDRs of a VL are
termed LCDR1, LCDR2 and LCDR3. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of an antibody comprise the heavy
chain constant region (CH) and the light chain constant region
(CL), wherein CH can be further subdivided into constant domain
CH1, a hinge region, and constant domains CH2 and CH3 (arranged
from amino-terminus to carboxy-terminus in the following order:
CH1, CH2, CH3). The constant regions of the antibodies may mediate
the binding of the immunoglobulin to host tissues or factors,
including various cells of the immune system (e.g., effector cells)
and the first component (C1q) of the classical complement system.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoactive
portions of intact immunoglobulins. Antibodies are typically
tetramers of immunoglobulin molecules. Antibodies 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.
[0166] The term "immunoglobulin" relates to proteins of the
immunoglobulin superfamily, preferably to antigen receptors such as
antibodies or the B cell receptor (BCR). The immunoglobulins are
characterized by a structural domain, i.e., the immunoglobulin
domain, having a characteristic immunoglobulin (Ig) fold. The term
encompasses membrane bound immunoglobulins as well as soluble
immunoglobulins. Membrane bound immunoglobulins are also termed
surface immunoglobulins or membrane immunoglobulins, which are
generally part of the BCR. Soluble immunoglobulins are generally
termed antibodies. Immunoglobulins generally comprise several
chains, typically two identical heavy chains and two identical
light chains which are linked via disulfide bonds. These chains are
primarily composed of immunoglobulin domains, such as the V.sub.L
(variable light chain) domain, C.sub.L (constant light chain)
domain, V.sub.H (variable heavy chain) domain, and the C.sub.H
(constant heavy chain) domains C.sub.H1, C.sub.H2, C.sub.H3, and
C.sub.H4. There are five types of mammalian immunoglobulin heavy
chains, i.e., .alpha., .delta., .epsilon., .gamma., and .mu. which
account for the different classes of antibodies, i.e., IgA, IgD,
IgE, IgG, and IgM. As opposed to the heavy chains of soluble
immunoglobulins, the heavy chains of membrane or surface
immunoglobulins comprise a transmembrane domain and a short
cytoplasmic domain at their carboxy-terminus. In mammals there are
two types of light chains, i.e., lambda and kappa. The
immunoglobulin chains comprise a variable region and a constant
region. The constant region is essentially conserved within the
different isotypes of the immunoglobulins, wherein the variable
part is highly divers and accounts for antigen recognition.
[0167] The terms "vaccination" and "immunization" describe the
process of treating an individual for therapeutic or prophylactic
reasons and relate to the procedure of administering one or more
immunogen(s) or antigen(s) or derivatives thereof, in particular in
the form of RNA coding therefor, as described herein to an
individual and stimulating an immune response against said one or
more immunogen(s) or antigen(s) or cells characterized by
presentation of said one or more immunogen(s) or antigen(s).
[0168] By "cell characterized by presentation of an antigen" or
"cell presenting an antigen" or "MHC molecules which present an
antigen on the surface of an antigen presenting cell" or similar
expressions is meant a cell such as a diseased cell, in particular
a tumor cell, or an antigen presenting cell presenting the antigen
or an antigen peptide, either directly or following processing, in
the context of MHC molecules, preferably MHC class I and/or MHC
class II molecules, most preferably MHC class I molecules.
[0169] In the context of the present invention, terms such as
"protect", "prevent", "prophylactic", "preventive", or "protective"
relate to the prevention or treatment or both of the occurrence
and/or the propagation of a disease in an individual and, in
particular, to minimizing the chance that an individual will
develop a disease or to delaying the development of a disease. For
example, a person at risk for a disease would be a candidate for
therapy to prevent a disease. A prophylactic administration of an
agent (e.g., RNA) or composition (such as a pharmaceutical
composition, e.g., a vaccine composition) described herein can
protect the recipient from the development of a disease, e.g., from
an infection by a pathogen (e.g., a virus, such as influenza virus
(A, B, or C), CMV or RSV) or from the dissemination or metastasis
of existing tumors. A therapeutic administration of an agent (e.g.,
RNA) or composition (such as a pharmaceutical composition)
described herein may lead to the inhibition of the progress/growth
of the disease. This comprises the deceleration of the
progress/growth of the disease, in particular a disruption of the
progression of the disease, which preferably leads to elimination
of the disease.
[0170] The term "adjuvant" relates to compounds which when
administered in combination with an antigen, an antigen peptide, or
a nucleic acid (such as RNA, preferably mRNA) encoding said antigen
or antigen peptide to an individual prolongs or enhances or
accelerates the immune response. In the context of the present
invention, RNA (preferably mRNA) may be administered with any
adjuvants. It is assumed that adjuvants exert their biological
activity by one or more mechanisms, including an increase of the
surface of the antigen, a prolongation of the retention of the
antigen in the body, a retardation of the antigen release,
targeting of the antigen to macrophages, increase of the uptake of
the antigen, enhancement of antigen processing, stimulation of
cytokine release, stimulation and activation of immune cells such
as B-cells, macrophages, dendritic cells, T-cells and unspecific
activation of immune cells. For example, compounds which allow the
maturation of the DCs, e.g. lipopolysaccharides or CD40 ligand,
form a class of suitable adjuvants. Generally, any agent which
influences the immune system of the type of a "danger signal" (LPS,
GP96, dsRNA etc.) or cytokines, such as GM-CSF, can be used as an
adjuvant which enables an immune response to be intensified and/or
influenced in a controlled manner. CpG oligodeoxynucleotides (Krieg
et al., 1995, Nature 374: 546-549) can optionally also be used in
this context. Further types of adjuvants include oil emulsions
(e.g., Freund's adjuvants), mineral compounds (such as alum),
bacterial products (such as Bordetella pertussis toxin), liposomes,
immune-stimulating complexes, cytokines (e.g., monokines,
lymphokines, interleukins or chemokines, such as IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-.alpha.,
IFN-.gamma., GM-CSF, LT-.alpha., or growth factors, e.g. hGH),
lipopeptides (e.g., Pam3Cys). In case the RNA (preferably mRNA) of
the invention in one embodiment may encode an immunostimulating
agent and said immunostimulating agent encoded by said RNA is to
act as the primary immunostimulant, however, only a relatively
small amount of CpG DNA is necessary (compared with
immunostimulation with only CpG DNA). Examples for adjuvants are
monophosphoryl-lipid-A (MPL SmithKline Beecham). Saponins such as
QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739),
QS7, QS17, QS18, and QS-L1 (So et al., 1997, Mol. Cells 7:
178-186), incomplete Freund's adjuvants, complete Freund's
adjuvants, vitamin E, montanid, alum, CpG oligonucleotides, and
various water-in-oil emulsions which are prepared from biologically
degradable oils such as squalene and/or tocopherol. Particularly
preferred adjuvants are cytokines, such as monokines, lymphokines,
interleukins or chemokines, e.g. IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-.alpha., IFN-.gamma.,
GM-CSF, LT-.alpha., growth factors, e.g. hGH or lipopeptides, such
as Pam3Cys, all of which are suitable for use as adjuvants in the
pharmaceutical compositions of the present invention or when RNA of
the present invention is used in therapy.
[0171] Terms such as "increasing", "enhancing", or "prolonging"
preferably relate to an increase, enhancement, or prolongation by
about at least 10%, preferably at least 20%, preferably at least
30%, more preferably at least 40%, more preferably at least 50%,
even more preferably at least 80%, and most preferably at least
100%. These terms may also relate to an increase, enhancement, or
prolongation from zero or a non-measurable or non-detectable level
to a level of more than zero or a level which is measurable or
detectable.
[0172] Terms such as "decreasing", "reducing" or "inhibiting"
relate to the ability to cause an overall decrease, preferably of
5% or greater, 10% or greater, 20% or greater, more preferably of
50% or greater, and most preferably of 75% or greater, in the
level. This also includes a complete or essentially complete
decrease, i.e. a decrease to zero or essentially to zero.
[0173] Terms such as "transferring", "transfecting" or "introducing
into cells" are used interchangeably herein and relate to the
introduction of nucleic acids, in particular exogenous or
heterologous nucleic acids, preferably RNA (such as mRNA) into a
cell. According to the present invention, the cell can form part of
an organ, a tissue and/or an organism. The introduction of nucleic
acids, in particular exogenous or heterologous nucleic acids,
preferably RNA (such as mRNA) into a cell can be performed in vivo
or in vitro.
[0174] "Antigen-presenting cells" (APCs) are cells which display
antigen, in particular peptide fragments of protein antigens, in
association with MHC molecules on their cell surface. T cells may
recognize this complex using their T cell receptor (TCR).
Antigen-presenting cells process antigens and present them to T
cells. An antigen presenting cell includes, but is not limited to,
monocytes/macrophages, B cells and dendritic cells (DCs). In a
preferred embodiment, the APCs according to the present invention
are mammalian, preferably human, mouse, or rat.
[0175] Non-professional antigen-presenting cells do not
constitutively express the MHC class II proteins required for
interaction with naive T cells; these are expressed only upon
stimulation of the non-professional antigen-presenting cells by
certain cytokines such as IFN.gamma..
[0176] Professional antigen-presenting cells are very efficient at
internalizing antigen, either by phagocytosis or by
receptor-mediated endocytosis, and then displaying a fragment of
the antigen, bound to a class II MHC molecule, on their membrane.
The T cell recognizes and interacts with the antigen-class II MHC
molecule complex on the membrane of the antigen-presenting cell. An
additional co-stimulatory signal is then produced by the
antigen-presenting cell, leading to activation of the T cell. The
expression of co-stimulatory molecules is a defining feature of
professional antigen-presenting cells.
[0177] The main types of professional antigen-presenting cells are
dendritic cells, which have the broadest range of antigen
presentation, and are probably the most important
antigen-presenting cells, macrophages, B-cells, and certain
activated epithelial cells.
[0178] Dendritic cells (DCs) are leukocyte populations that present
antigens captured in peripheral tissues to T cells via both MHC
class II and I antigen presentation pathways. It is well known that
dendritic cells are potent inducers of immune responses and the
activation of these cells is a critical step for the induction of
immunity.
[0179] Dendritic cells are conveniently categorized as "immature"
and "mature" cells, which can be used as a simple way to
discriminate between two well characterized phenotypes. However,
this nomenclature should not be construed to exclude all possible
intermediate stages of differentiation.
[0180] Immature dendritic cells are characterized as antigen
presenting cells with a high capacity for antigen uptake and
processing, which correlates with the high expression of Fc.gamma.
receptor and mannose receptor. The mature phenotype is typically
characterized by a lower expression of these markers, but a high
expression of cell surface molecules responsible for T cell
activation such as class I and class II MHC, adhesion molecules (e.
g. CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,
CD86 and 4-1 BB).
[0181] Dendritic cell maturation is referred to as the status of
dendritic cell activation at which such antigen-presenting
dendritic cells lead to T cell priming, while presentation by
immature dendritic cells results in tolerance. Dendritic cell
maturation is chiefly caused by biomolecules with microbial
features detected by innate receptors (bacterial DNA, viral RNA,
endotoxin, etc.), pro-inflammatory cytokines (TNF, IL-1, IFNs),
ligation of CD40 on the dendritic cell surface by CD40L, and
substances released from cells undergoing stressful cell death. The
dendritic cells can be derived by culturing bone marrow cells in
vitro with cytokines, such as granulocyte-macrophage
colony-stimulating factor (GM-CSF) and tumor necrosis factor
alpha.
[0182] The term "immunoreactive cell" or "effector cell" in the
context of the present invention relates to a cell which exerts
effector functions during an immune reaction. An "immunoreactive
cell" preferably is capable of binding an antigen or a cell
characterized by expression and/or presentation of an antigen or an
antigen peptide derived from an antigen and mediating an immune
response. For example, such cells secrete cytokines and/or
chemokines, kill microbes, secrete antibodies, recognize infected
or cancerous cells, and optionally eliminate such cells. For
example, immunoreactive cells comprise T cells (cytotoxic T cells,
helper T cells, tumor infiltrating T cells), B cells, natural
killer cells, neutrophils, macrophages, and dendritic cells.
Preferably, in the context of the present invention,
"immunoreactive cells" are T cells, preferably CD4.sup.+ and/or
CD8.sup.+ T cells.
[0183] Preferably, an "immunoreactive cell" recognizes an antigen
or an antigen peptide derived from an antigen with some degree of
specificity, in particular if presented in the context of MHC
molecules such as on the surface of antigen presenting cells or
diseased cells such as tumor cells. Preferably, said recognition
enables the cell that recognizes an antigen or an antigen peptide
derived from said antigen to be responsive or reactive. If the cell
is a helper T cell (CD4.sup.+ T cell) bearing receptors that
recognize an antigen or an antigen peptide derived from an antigen
in the context of MHC class II molecules such responsiveness or
reactivity may involve the release of cytokines and/or the
activation of CD8.sup.+ lymphocytes (CTLs) and/or B-cells. If the
cell is a CTL such responsiveness or reactivity may involve the
elimination of cells presented in the context of MHC class I
molecules, i.e., cells characterized by presentation of an antigen
with class I MHC, for example, via apoptosis or perforin-mediated
cell lysis. According to the invention, CTL responsiveness may
include sustained calcium flux, cell division, production of
cytokines such as IFN-.gamma. and TNF-.alpha., up-regulation of
activation markers such as CD44 and CD69, and specific cytolytic
killing of antigen expressing target cells. CTL responsiveness may
also be determined using an artificial reporter that accurately
indicates CTL responsiveness. Such CTL that recognizes an antigen
or an antigen peptide derived from an antigen and are responsive or
reactive are also termed "antigen-responsive CTL" herein. If the
cell is a B cell such responsiveness may involve the release of
immunoglobulins.
[0184] The term "T cell" or "T lymphocyte" relates to
thymus-derived cells that participate in a variety of cell-mediated
immune reactions and includes T helper cells (CD4+ T cells) and
cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T
cells.
[0185] T cells belong to a group of white blood cells known as
lymphocytes, and play a central role in cell-mediated immunity.
They can be distinguished from other lymphocyte types, such as B
cells and natural killer cells by the presence of a special
receptor on their cell surface called T cell receptor (TCR). The
thymus is the principal organ responsible for the maturation of T
cells. Several different subsets of T cells have been discovered,
each with a distinct function.
[0186] T helper cells assist other white blood cells in immunologic
processes, including maturation of B cells into plasma cells and
activation of cytotoxic T cells and macrophages, among other
functions. These cells are also known as CD4+ T cells because they
express the CD4 protein on their surface. Helper T cells become
activated when they are presented with peptide antigens by MHC
class II molecules that are expressed on the surface of
antigen-presenting cells (APCs). Once activated, they divide
rapidly and secrete small proteins called cytokines that regulate
or assist in the active immune response.
[0187] Cytotoxic T cells destroy virally infected cells and tumor
cells, and are also implicated in transplant rejection. These cells
are also known as CD8+ T cells since they express the CD8
glycoprotein at their surface. These cells recognize their targets
by binding to antigen associated with MHC class I, which is present
on the surface of nearly every cell of the body.
[0188] A majority of T cells have a T cell receptor (TCR) existing
as a complex of several proteins. The actual T cell receptor is
composed of two separate peptide chains, which are produced from
the independent T cell receptor alpha and beta (TCR.alpha. and
TCR.beta.) genes and are called .alpha.- and .beta.-TCR chains.
.gamma..delta. T cells (gamma delta T cells) represent a small
subset of T cells that possess a distinct T cell receptor (TCR) on
their surface. However, in .gamma..delta. T cells, the TCR is made
up of one .gamma.-chain and one 6-chain. This group of T cells is
much less common (2% of total T cells) than the up T cells.
[0189] The structure of the T cell receptor is very similar to
immunoglobulin Fab fragments, which are regions defined as the
combined light and heavy chain of an antibody arm. Each chain of
the TCR is a member of the immunoglobulin superfamily and possesses
one N-terminal immunoglobulin (Ig)-variable (V) domain, one
Ig-constant (C) domain, a transmembrane/cell membrane-spanning
region, and a short cytoplasmic tail at the C-terminal end. The
variable domain of both the TCR .alpha.-chain and .beta.-chain have
three hypervariable or complementarity determining regions (CDRs),
whereas the variable region of the .beta.-chain has an additional
area of hypervariability (HV4) that does not normally contact
antigen and therefore is not considered a CDR. CDR3 is the main CDR
responsible for recognizing processed antigen, although CDR1 of the
.alpha.-chain has also been shown to interact with the N-terminal
part of the antigenic peptide, whereas CDR1 of the .beta.-chain
interacts with the C-terminal part of the peptide. CDR2 is thought
to recognize the MHC. CDR4 of the .beta.-chain is not thought to
participate in antigen recognition, but has been shown to interact
with superantigens. The constant domain of the TCR domain consists
of short connecting sequences in which a cysteine residue forms
disulfide bonds, which forms a link between the two chains.
[0190] The term "peripheral blood mononuclear cell" or "PBMC"
relates to a peripheral blood cell having a round nucleus. These
cells consist of lymphocytes (T cells, B cells, NK cells) and
monocytes, whereas erythrocytes and platelets have no nuclei, and
granulocytes (neutrophils, basophils, and eosinophils) have
multi-lobed nuclei. These cells can be extracted from whole blood
using ficoll and gradient centrifugation, which will separate the
blood into a top layer of plasma, followed by a layer of PBMCs and
a bottom fraction of polymorphonuclear cells (such as neutrophils
and eosinophils) and erythrocytes.
[0191] The term "major histocompatibility complex" and the
abbreviation "MHC" include MHC class I and MHC class II molecules
and relate to a complex of genes which occurs in all vertebrates.
MHC proteins or molecules are important for signaling between
lymphocytes and antigen presenting cells or diseased cells in
immune reactions, wherein the MHC proteins or molecules bind
peptides and present them for recognition by T cell receptors. The
proteins encoded by the MHC are expressed on the surface of cells,
and display both self antigens (peptide fragments from the cell
itself) and nonself antigens (e.g., fragments of invading
microorganisms) to a T cell.
[0192] The MHC region is divided into three subgroups, class I,
class II, and class III. MHC class I proteins contain an
.alpha.-chain and $2-microglobulin (not part of the MHC encoded by
chromosome 15). They present antigen fragments to cytotoxic T
cells. On most immune system cells, specifically on
antigen-presenting cells, MHC class II proteins contain .alpha.-
and .beta.-chains and they present antigen fragments to T-helper
cells. MHC class III region encodes for other immune components,
such as complement components and some that encode cytokines.
[0193] In humans, genes in the MHC region that encode
antigen-presenting proteins on the cell surface are referred to as
human leukocyte antigen (HLA) genes. However the abbreviation MHC
is often used to refer to HLA gene products. HLA genes include the
nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1,
HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.
[0194] In one preferred embodiment of all aspects of the invention
relating to immunotherapy or immune responses, an MHC molecule is
an HLA molecule.
[0195] The term "immune effector functions" or "effector functions"
in the context of the present invention includes any functions
mediated by components of the immune system that result, for
example, in the killing of cells. Preferably, the immune effector
functions in the context of the present invention are T cell
mediated effector functions. Such functions comprise in the case of
a helper T cell (CD4.sup.+ T cell) the recognition of an antigen or
an antigen peptide derived from an antigen in the context of MHC
class II molecules by T cell receptors, the release of cytokines
and/or the activation of CD8.sup.+ lymphocytes (CTLs) and/or
B-cells, and in the case of CTL the recognition of an antigen or an
antigen peptide derived from an antigen in the context of MHC class
I molecules by T cell receptors, the elimination of cells presented
in the context of MHC class I molecules, i.e., cells characterized
by presentation of an antigen with class I MHC, for example, via
apoptosis or perforin-mediated cell lysis, production of cytokines
such as IFN-.gamma. and TNF-.alpha., and specific cytolytic killing
of antigen expressing target cells.
[0196] The term "immune effector cells" in the context of the
present invention relates to cells which exert effector functions
during an immune reaction. "Immune effector cells" preferably are
capable of binding an antigen or a cell characterized by
presentation of an antigen and mediating an immune response. For
example, such cells secrete cytokines and/or chemokines, kill
microbes, secrete antibodies, recognize infected or cancerous
cells, and optionally eliminate such cells. For example, immune
effector cells comprise T-cells (cytotoxic T-cells, helper T-cells,
tumor infiltrating T-cells), B-cells, natural killer cells,
neutrophils, macrophages, and dendritic cells. Preferably, in the
context of the present invention, "immune effector cells" are
T-cells, preferably CD4.sup.+ and/or CD8.sup.+ cells.
[0197] Preferably, an "immune effector cell" recognizes an antigen
or an antigen peptide derived from said antigen with some degree of
specificity, in particular if presented in the context of MHC
molecules such as on the surface of antigen presenting cells or
diseased cells such as tumor cells. Preferably, said recognition
enables the cell that recognizes an antigen or an antigen peptide
derived from said antigen to be responsive. If the cell is a helper
T-cell (CD4.sup.+ T-cell) bearing receptors that recognize an
antigen or an antigen peptide derived from said antigen in the
context of MHC class II molecules such responsiveness may involve
the release of cytokines and/or the activation of CD8.sup.+
lymphocytes (CTLs) and/or B-cells. If the cell is a CTL such
responsiveness may involve the elimination of cells presented in
the context of MHC class I molecules, i.e., cells characterized by
presentation of an antigen with class I MHC, for example, via
apoptosis or perform-mediated cell lysis. Such CTL that recognizes
an antigen or an antigen peptide derived from said antigen and are
responsive are also termed "antigen-responsive CTL" herein. If the
cell is a B-cell such responsiveness may involve the release of
immunoglobulins.
[0198] The term "half-life" relates to the period of time which is
needed to eliminate half of the activity, amount, or number of
molecules. In the context of the present invention, the half-life
of an RNA (preferably mRNA) is indicative for the stability of said
RNA. The half-life of RNA may influence the "duration of
expression" of the RNA. It can be expected that RNA having a long
half-life will be expressed for an extended time period.
[0199] Of course, if according to the present invention it is
desired to decrease stability and/or translation efficiency of RNA,
it is possible to modify RNA so as to interfere with the function
of elements as described above increasing the stability and/or
translation efficiency of RNA.
[0200] The terms "patient", "individual", "subject", or "animal"
are used interchangeably and relate to vertebrates. For example,
vertebrates in the context of the present invention are mammals,
birds (e.g., poultry), reptiles, amphibians, bony fishes, and
cartilaginous fishes, in particular domesticated animals of any of
the foregoing as well as animals in captivity such as animals of
zoos, and are preferably mammals. Mammals in the context of the
present invention include, but are not limited to, humans,
non-human primates, domesticated mammals, such as dogs, cats,
sheep, cattle, goats, pigs, horses etc., laboratory mammals such as
mice, rats, rabbits, guinea pigs, etc. as well as mammals in
captivity such as mammals of zoos. The term "animal" as used herein
also includes humans. The term "subject" may also include a
patient, i.e., an animal, preferably a human having a disease,
preferably a disease as described herein.
[0201] According to the invention, the term "chronic patient" or
"long-term patient" refers to a patient having a chronic disease or
disorder. A "chronic disease or disorder" is a disease or disorder
which is persistent and/or whose effects (e.g., symptoms) are
persistent for at least 3 weeks, such as at least 4 weeks, at least
1 month, at least 2 months, at least 3 months, at least 4 months,
at least 5 months, at least 6 months, at least 12 months, at least
2 years, at least 3 years, at least 4 years, at least 5 years, or
at least 10 years, e.g., up to 4 weeks, up to 1 month, up to 2
months, up to 3 months, up to 4 months, up to 5 months, up to 6
months, up to 12 months, up to 2 years, up to 3 years, or up to 4
years, up to 5 years, up to 10 years, or the entire life of the
patient.
[0202] According to the invention, the term "tumor" or "tumor
disease" refers to a swelling or lesion formed by an abnormal
growth of cells (called neoplastic cells or tumor cells). By "tumor
cell" is meant an abnormal cell that grows by a rapid, uncontrolled
cellular proliferation and continues to grow after the stimuli that
initiated the new growth cease. Tumors show partial or complete
lack of structural organization and functional coordination with
the normal tissue, and usually form a distinct mass of tissue,
which may be either benign, pre-malignant, or malignant.
[0203] Preferably, a tumor disease according to the invention is a
cancer disease, i.e., a malignant disease, and a tumor cell is a
cancer cell. Preferably, a tumor disease is characterized by cells
in which an antigen, i.e., a tumor antigen, is expressed or
abnormally expressed. Preferably, a tumor disease or a tumor cell
is characterized by presentation of a tumor antigen with class I
MHC.
[0204] "Abnormal expression" means according to the invention that
expression is altered, preferably increased, compared to the state
in a healthy individual. An increase in expression refers to an
increase by at least 10%, in particular at least 20%, at least 50%
or at least 100%. In one embodiment, expression is only found in a
diseases tissue, while expression in a healthy tissue is
repressed.
[0205] Preferably, a tumor disease according to the invention is
cancer, wherein the term "cancer" according to the invention
comprises leukemias, seminomas, melanomas, teratomas, lymphomas,
neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney
cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer,
cancer of the brain, cervical cancer, intestinal cancer, liver
cancer, colon cancer, stomach cancer, intestine cancer, head and
neck cancer, gastrointestinal cancer, lymph node cancer, esophagus
cancer, colorectal cancer, pancreas cancer, ear, nose and throat
(ENT) cancer, breast cancer, prostate cancer, cancer of the uterus,
ovarian cancer and lung cancer and the metastases thereof. Examples
thereof are lung carcinomas, mamma carcinomas, prostate carcinomas,
colon carcinomas, renal cell carcinomas, cervical carcinomas, or
metastases of the cancer types or tumors described above. The term
"cancer" according to the invention also comprises cancer
metastases.
[0206] In one embodiment, the RNA according to the invention is
(modified) RNA, in particular (modified) mRNA, encoding a peptide
or protein. According to the invention, the term "RNA encoding a
peptide or protein" means that the RNA, if present in the
appropriate environment, preferably within a cell, can direct the
assembly of amino acids to produce, i.e., express, the peptide or
protein during the process of translation. Preferably, RNA (such as
mRNA) according to the invention is able to interact with the
cellular translation machinery allowing translation of the peptide
or protein.
[0207] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a nucleic acid to serve as templates
for synthesis of other polymers and macromolecules in biological
processes having either a defined sequence of nucleotides or a
defined sequence of amino acids. Thus, a nucleic acid encodes a
protein if expression (translation and optionally transcription) of
the nucleic acid produces the protein in a cell or other biological
system.
[0208] The term "expression" is used according to the invention in
its most general meaning and comprises the production of RNA and/or
peptides or proteins, e.g., by transcription and/or translation.
With respect to RNA, the term "expression" or "translation" relates
in particular to the production of peptides or proteins. It also
comprises partial expression of nucleic acids. Moreover, expression
can be transient or stable.
[0209] In the context of the present invention, the term
"transcription" relates to a process, wherein the genetic code in a
DNA sequence is transcribed into RNA. Subsequently, the RNA may be
translated into protein.
[0210] According to the present invention, the term "transcription"
comprises "in vitro transcription", wherein the term "in vitro
transcription" relates to a process wherein RNA, in particular
mRNA, is in vitro synthesized in a cell-free system, preferably
using appropriate cell extracts. Preferably, cloning vectors are
applied for the generation of transcripts. These cloning vectors
are generally designated as transcription vectors and are according
to the present invention encompassed by the term "vector".
According to the present invention, the RNA used in the present
invention may be obtained by in vitro transcription of an
appropriate DNA template. The promoter for controlling
transcription can be any promoter for any RNA polymerase.
Particular examples of RNA polymerases are the T7, T3, and SP6 RNA
polymerases. Preferably, the in vitro transcription according to
the invention is controlled by a T7 or SP6 promoter. A DNA template
for in vitro transcription may be obtained by cloning of a nucleic
acid, in particular cDNA, and introducing it into an appropriate
vector for in vitro transcription. The cDNA may be obtained by
reverse transcription of RNA.
[0211] The cDNA containing vector template may comprise vectors
carrying different cDNA inserts which following transcription
results in a population of different RNA molecules optionally
capable of expressing different peptides or proteins or may
comprise vectors carrying only one species of cDNA insert which
following transcription only results in a population of one RNA
species capable of expressing only one peptide or protein. Thus, it
is possible to produce RNA capable of expressing a single peptide
or protein only or to produce compositions of different RNAs such
as RNA libraries and whole-cell RNA capable of expressing more than
one peptide or protein, e.g., a composition of peptides or
proteins. The present invention envisions the introduction of all
such RNA into cells.
[0212] The term "vector" as used herein includes any vectors known
to the skilled person including plasmid vectors, cosmid vectors,
phage vectors such as lambda phage, viral vectors such as
adenoviral or baculoviral vectors, retro- or lentiviral vectors,
transposons or artificial chromosome vectors such as bacterial
artificial chromosomes (BAC), yeast artificial chromosomes (YAC),
or P1 artificial chromosomes (PAC). Said vectors include expression
as well as cloning vectors. Expression vectors comprise plasmids as
well as viral vectors and generally contain a desired coding
sequence and appropriate DNA sequences necessary for the expression
of the operably linked coding sequence in a particular host
organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in
vitro expression systems. Cloning vectors are generally used to
engineer and amplify a certain desired DNA fragment and may lack
functional sequences needed for expression of the desired DNA
fragments.
[0213] The nucleic acid encoding a peptide or protein can be cloned
into a number of types of vectors. However, the present invention
should not be construed to be limited to any particular vector.
Instead, the present invention should be construed to encompass a
wide plethora of vectors which are readily available and well-known
in the art. In specific embodiments, the vector is selected from
the group consisting of a viral vector, a bacterial vector, and a
mammalian cell vector. Many such systems are commercially and
widely available.
[0214] The vector may be provided to a cell in the form of a viral
vector. Viral vector technology is well known in the art. Viruses,
which are useful as vectors include, but are not limited to,
retroviruses, adenoviruses, adeno-associated viruses, herpes
viruses, and lentiviruses. Preferably, the virus is
helper-dependent adenovirus (HD-Ad). In general, a suitable vector
contains an origin of replication functional in at least one
organism, a promoter sequence, convenient restriction endonuclease
sites, and one or more selectable markers.
[0215] Those of skill in the art of molecular biology generally
know how to use promoters, enhancers, and cell type combinations
for protein expression. The promoters employed may be constitutive,
tissue-specific, inducible, and/or useful under the appropriate
conditions to direct high level expression of the introduced
nucleic acid segment encoding a peptide or protein. The promoter
may be heterologous or endogenous. Constitutive promoter sequences
which may be used according to the invention, include, but are not
limited to the immediate early cytomegalovirus (CMV) promoter
sequence, the simian virus 40 (SV40) early promoter, mouse mammary
tumor virus (MMTV), human immunodeficiency virus (HIV) long
terminal repeat (LTR) promoter, Moloney virus promoter, the avian
leukemia virus promoter, Epstein-Barr virus immediate early
promoter, Rous sarcoma virus promoter, as well as human gene
promoters such as, but not limited to, the actin promoter, the
myosin promoter, the hemoglobin promoter, and the muscle creatine
promoter. Further, the invention should not be limited to the use
of constitutive promoters. Inducible promoters are also
contemplated as part of the invention. The use of an inducible
promoter in the invention provides a molecular switch capable of
turning on expression of the polynucleotide sequence which it is
operatively linked when such expression is desired, or turning off
the expression when expression is not desired. Examples of
inducible promoters include, but are not limited to a
metallothionine promoter, a glucocorticoid promoter, a progesterone
promoter, and a tetracycline promoter. Further, the invention
includes the use of a tissue specific promoter, which promoter is
active only in a desired tissue. Tissue specific promoters are well
known in the art and include, but are not limited to, the HER-2
promoter and the PSA associated promoter sequences.
[0216] In order to assess the expression of a peptide or protein,
the expression vector to be introduced into a cell can also contain
either a selectable marker gene or a reporter gene or both to
facilitate identification and selection of expressing cells from
the population of cells sought to be transfected or infected
through viral vectors. In other embodiments, the selectable marker
may be carried on a separate piece of DNA and used in a
co-transfection procedure. Both selectable markers and reporter
genes may be flanked with appropriate regulatory sequences to
enable expression in the cells. Useful selectable markers are known
in the art and include, for example, antibiotic-resistance genes,
such as neo and the like. Reporter genes are used for identifying
potentially transfected cells and for evaluating the functionality
of regulatory sequences. Reporter genes that encode for easily
assayable proteins are well known in the art. In general, a
reporter gene is a gene that is not present in or expressed by the
recipient organism or tissue and that encodes a protein whose
expression is manifested by some easily detectable property, e.g.,
enzymatic activity. Expression of the reporter gene is assayed at a
suitable time after the nucleic acid has been introduced into the
recipient cells. Suitable reporter genes may include genes encoding
luciferase, beta-galactosidase, chloramphenicol acetyl transferase,
secreted alkaline phosphatase, or the green fluorescent protein
gene.
[0217] The vector can be readily introduced into a cell by any
method in the art. For example, the expression vector can be
transferred into a cell by physical, chemical or biological means.
Physical methods for introducing a nucleic acid into a cell include
calcium phosphate precipitation, lipofection, particle bombardment,
microinjection, electroporation, and the like. Methods for
producing cells comprising vectors and/or exogenous nucleic acids
are well-known in the art. See, for example, Sambrook et al. (2001,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York), and Ausubel et al. (1997, Current Protocols
in Molecular Biology, John Wiley & Sons, New York).
[0218] Biological methods for introducing a nucleic acid of
interest into a cell include the use of DNA and RNA vectors. Viral
vectors, and especially retroviral vectors, have become the most
widely used method for inserting genes into mammalian, e.g., human
cells. Other viral vectors can be derived from lentivirus,
poxviruses, herpes simplex virus I, adenoviruses and
adeno-associated viruses, and the like.
[0219] Chemical means for introducing a nucleic acid into a 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. A preferred colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art.
[0220] Regardless of the method used to introduce exogenous nucleic
acids into a cell or otherwise increase the cellular level of a
peptide or protein in a cell, in order to confirm the presence
and/or amount of the peptide or protein or its encoding nucleic
acid in the cell, a variety of assays may be performed. Such assays
include, for example, Southern and Northern blotting, RT-PCR and
PCR and assays for detecting the presence or absence of a
particular peptide, e.g., by immunological means (ELISAs and
Western blots).
[0221] The term "cell" means any cell that can be transfected with
RNA (preferably mRNA), wherein the RNA to be transfected is
preferably exogenous or heterologous RNA. A cell may be obtained
from any subject and in one embodiment may be obtained from a
patient having a disorder or disease. If the cell is obtained from
a patient having a disorder or disease, the cell may contain
genetic material which is homologous to the RNA to be introduced
but which results in a peptide or protein having decreased
activity. The decreased activity may be the result of (i) a
decreased expression of the peptide or protein (i.e., the peptide
or protein is fully functional but the amount thereof is decreased)
or (ii) the presence of one or more mutations in the amino acid
sequence of the expressed peptide or protein (i.e., the peptide or
protein is not fully functional). For example, such homologous
genetic material which results in a peptide or protein having
decreased activity may be a gene containing one or more mutations
in such a manner that (i) the expression of said gene containing
one or more mutations is decreased or silenced thereby resulting in
a decreased amount of the fully functional peptide or protein
and/or (ii) the amino acid sequence of the peptide or protein
encoded by said gene contains one or more mutations thereby
resulting in a not fully functional (or non-functional) peptide or
protein.
[0222] In case the fully functional peptide or protein is expressed
in the cell or patient but in an amount too low to maintain the
functions of the cell or patient (e.g., leading to the development
of a disease or disorder in the patient in which the cell is
contained), a therapy to replace or supplement said peptide or
protein (protein replacement therapy) would be beneficial. Such
protein replacement therapy may comprise the step of administering
an RNA comprising a nucleotide sequence encoding said peptide or
protein (or a composition, such as a pharmaceutical composition,
comprising such RNA) to the patient, or alternatively, the steps of
(a) transferring an RNA comprising a nucleotide sequence encoding
said peptide or protein into a cell (which may be obtained from the
patient) and (b) administering said transfected cell to the
patient.
[0223] In case the decreased activity of a peptide or protein in a
patient (and thus, the development of a disease or disorder) is due
to the presence of one or more mutations in the amino acid sequence
of said peptide or protein (i.e., the peptide or protein is not
fully functional), a genome engineering therapy would be
beneficial. Such genome engineering therapy may comprise the step
of administering to the patient (i) an RNA (in particular an RNA of
the present invention) comprising a nucleotide sequence encoding a
genomic engineering protein and (ii) a DNA comprising a nucleotide
sequence encoding the peptide or protein in its native (i.e.,
unmutated) form.
[0224] Alternatively, a genetic reprogramming therapy would be
beneficial, in particular with patients having a disease or
disorder which causes a depletion or extinction of cells producing
the desired peptide or protein (e.g., a hormone such as insulin).
For example, such genetic reprogramming therapy may comprise the
steps of (a) introducing an RNA (in particular an RNA of the
present invention) comprising a nucleotide sequence encoding one or
more reprogramming factors into somatic cells; (b) allowing the
development of cells having stem cell characteristics; and (c)
administering the cells having stem cell characteristics to a
patient. In a preferred embodiment, the somatic cells are
autologous to the patient.
[0225] The term "translation" according to the invention relates to
the process in the ribosomes of a cell by which a strand of mRNA
directs the assembly of a sequence of amino acids to make a peptide
or protein. The translation may be performed in vivo (e.g., in a
cell, tissue, or organism) or in vitro (e.g., using a cell-free
system).
[0226] Expression control sequences or regulatory sequences, which
according to the invention may be linked functionally with a
nucleic acid, can be homologous or heterologous with respect to the
nucleic acid. A coding sequence and a regulatory sequence are
linked together "functionally" if they are bound together
covalently, so that the transcription or translation of the coding
sequence is under the control or under the influence of the
regulatory sequence. If the coding sequence is to be translated
into a functional protein, with functional linkage of a regulatory
sequence with the coding sequence, induction of the regulatory
sequence leads to a transcription of the coding sequence, without
causing a reading frame shift in the coding sequence or inability
of the coding sequence to be translated into the desired protein or
peptide.
[0227] The term "expression control sequence" or "regulatory
sequence" comprises, according to the invention, promoters,
ribosome-binding sequences and other control elements, which
control the transcription of a nucleic acid or the translation of
the derived RNA. In certain embodiments of the invention, the
regulatory sequences can be controlled. The precise structure of
regulatory sequences can vary depending on the species or depending
on the cell type, but generally comprises 5'-untranscribed and 5'-
and 3-untranslated sequences, which are involved in the initiation
of transcription or translation, such as TATA-box,
capping-sequence, CAAT-sequence and the like. In particular,
5'-untranscribed regulatory sequences comprise a promoter region
that includes a promoter sequence for transcriptional control of
the functionally bound gene. Regulatory sequences can also comprise
enhancer sequences or upstream activator sequences.
[0228] According to the invention it is preferred that a nucleic
acid such as RNA (preferably mRNA) encoding a peptide or protein
once taken up by or introduced, i.e. transfected or transduced,
into a cell which cell may be present in vitro or in a subject
results in expression of said peptide or protein. The cell may
express the encoded peptide or protein intracellularly (e.g. in the
cytoplasm and/or in the nucleus), may secrete the encoded peptide
or protein, or may express it on the surface.
[0229] According to the invention, terms such as "nucleic acid
expressing" and "nucleic acid encoding" or similar terms are used
interchangeably herein and with respect to a particular peptide or
polypeptide mean that the nucleic acid, if present in the
appropriate environment, preferably within a cell, can be expressed
to produce said peptide or polypeptide.
[0230] According to the invention, RNA is to be transferred into
cells either in vitro or in vivo, e.g., by administration of RNA
intraperitoneally, intramuscularly, or intradermally or, in case
the cells are immature antigen presenting cells, into the lymphatic
system (such as into the lymph nodes). According to the present
invention, any technique which is suitable to transfer RNA into
cells may be used to introduce RNA into cells. Preferably, the RNA
is transfected into cells by standard techniques. Such techniques
comprise transfection of nucleic acid calcium phosphate
precipitates, transfection of nucleic acids which are associated
with DEAE, the transfection or infection with viruses which carry
the nucleic acids of interest, electroporation, lipofection, and
microinjection. According to the present invention, the
administration of a nucleic acid is either achieved as naked
nucleic acid or in combination with an administration reagent.
Preferably, administration of nucleic acids is in the form of naked
nucleic acids. Preferably, the RNA is administered in combination
with stabilizing substances such as RNase inhibitors. In a
particularly preferred embodiment, the RNA and/or the compositions
of the present invention are administered as naked RNA preferably
intraperitoneally, intramuscularly, by intranodal injection or
transdermal administration. In case of antigen-presenting cells
(such as immature antigen-presenting cells), preferably dendritic
cells (such as immature dendritic cells), a conventional
transfection technique is not absolutely necessary to introduce
naked RNA into said cells, since in particular immature
antigen-presenting cells such as immature dendritic cells are
capable of taking up naked RNA by macropinocytosis. Preferably, the
introduction of RNA which encodes a peptide or protein of interest
into a cell results in expression of said peptide or protein of
interest in the cell. In particular embodiments, the targeting of
the nucleic acids to particular cells is preferred. In such
embodiments, a carrier which is applied for the administration of
the nucleic acid to a cell (for example, a retrovirus or a
liposome), exhibits a targeting molecule. For example, a molecule
such as an antibody which is specific for a surface membrane
protein on the target cell or a ligand for a receptor on the target
cell may be incorporated into the nucleic acid carrier or may be
bound thereto. In case the nucleic acid is administered by
liposomes, proteins which bind to a surface membrane protein which
is associated with endocytosis may be incorporated into the
liposome formulation in order to enable targeting and/or uptake.
Such proteins encompass capsid proteins of fragments thereof which
are specific for a particular cell type, antibodies against
proteins which are internalized, proteins which target an
intracellular location etc.
[0231] The term "peptide" as used herein comprises oligo- and
polypeptides and refers to substances comprising two or more,
preferably 3 or more, preferably 4 or more, preferably 6 or more,
preferably 8 or more, preferably 10 or more, preferably 13 or more,
preferably 16 more, preferably 21 or more and up to preferably 8,
10, 20, 30, 40 or 50, in particular 100 amino acids joined
covalently by peptide bonds. The term "protein" preferentially
refers to large peptides, preferably to peptides with more than 100
amino acid residues, but in general the terms "peptide" and
"protein" are synonyms and are used interchangeably herein.
[0232] The term "immunologically active compound" relates to any
compound altering an immune response, preferably by inducing and/or
suppressing maturation of immune cells, inducing and/or suppressing
cytokine biosynthesis, and/or altering humoral immunity by
stimulating antibody production by B cells. Immunologically active
compounds possess potent immunostimulating activity including, but
not limited to, antiviral and antitumor activity, and can also
down-regulate other aspects of the immune response, for example
shifting the immune response away from a TH2 immune response, which
is useful for treating a wide range of TH2 mediated diseases.
Immunologically active compounds can be useful as vaccine
adjuvants.
[0233] In one embodiment, RNA (such as mRNA) that codes for an
antigen such a disease-associated antigen is administered to a
mammal, in particular if treating a mammal having a disease
involving or expressing the antigen (disease-associated antigen) is
desired. The RNA is preferably taken up into the mammal's
antigen-presenting cells (monocytes, macrophages, dendritic cells
or other cells). An antigenic translation product of the RNA is
formed and the product is displayed on the surface of the cells for
recognition by T cells. In one embodiment, the antigen or a product
produced by optional procession thereof is displayed on the cell
surface in the context of MHC molecules for recognition by T cells
through their T cell receptor leading to their activation.
[0234] The term "portion of MHC molecules which present an antigen
of interest" refers to the fraction of MHC molecules on the surface
of an antigen presenting cell which are loaded with, i.e., are
bound to, a particular antigen or an antigen peptide derived from
said antigen relative to the total amount of MHC molecules on the
surface of the cell. In a preferred embodiment, the RNA modified
with a 5'-cap compound of the present invention is capable of
increasing the portion of MHC molecules which present an antigen of
interest on the surface of an antigen presenting cell into which
the RNA was transferred. This is in comparison to an RNA which does
not carry the 5'-cap structure of the 5'-cap compound of the
present invention, in particular, an RNA which carries a
conventional RNA cap.
[0235] According to the invention, the terms "disease", "disorder",
and "condition" are used herein interchangeably and refer to any
pathological state, including infectious diseases (i.e., diseases
caused by a pathogen), tumor diseases, and undesirable
inflammation.
[0236] By "being at risk" is meant an individual, i.e., a patient,
that is identified as having a higher than normal chance of
developing a disease compared to the general population. In
addition, an individual who has had, or who currently has, a
disease is a subject who has an increased risk for developing a
disease, as such a subject may continue to develop a disease.
[0237] The term "in vivo" relates to the situation in a
subject.
[0238] The term "autologous" is used to describe anything that is
derived from the same subject. For example, "autologous cell"
refers to a cell derived from the same subject. Such procedures are
advantageous because they overcome the immunological barrier which
otherwise results in rejection.
[0239] The term "heterologous" is used to describe something
consisting of multiple different elements. As an example, the
transfer of one individual's bone marrow into a different
individual constitutes a heterologous transplant. A heterologous
gene is a gene derived from a source other than the subject.
[0240] The term "non-nucleotidic linker" as used herein means any
linker of two nucleosides which is not phosphate or a phosphate
derivate (such as phosphorothioate, boranophosphate,
imidophosphate, alkylene phosphate, phosphorodithioate,
alkylphosphonate, phosphotriester, or phosphoroamidite).
Preferably, a non-nucleotide linker is a peptide, an amine, an
aliphatic hydrocarbon (e.g. alkyl), or an aromatic hydrocarbon,
wherein the hydrocarbons optionally can include one or more
functional groups including, but not limited to, hydroxy, amino,
thiol, thioether, ether, amide, thioamide, ester, urea, or
thiourea. A particular example of such non-nucleotidic linkers
includes, but is not limited to, an alkyl linker. The alkyl linker
may be branched or unbranched, cyclic or acyclic, substituted or
unsubstituted, saturated or unsaturated, chiral, achiral or racemic
mixture. The alkyl linkers can have from 2 to 18 carbon atoms, such
as from 3 to 9 carbon atoms. Some alkyl linkers include one or more
functional groups including, but not limited to, hydroxy, amino,
thiol, thioether, ether, amide, thioamide, ester, urea, and
thioether. Such alkyl linkers can include, but are not limited to,
1-propanol, 1,2-propanediol, 1,3-propanediol, 1,2,3-propanetriol,
triethylene glycol, hexaethylene glycol, polyethylene glycol
linkers (e.g. [--O--CH.sub.2CH.sub.2-]c, (c=1, 2, 3, 4, 5, 6, 7, 8,
or 9)), methyl linkers, ethyl linkers, propyl linkers, butyl
linkers, or hexyl linkers. In some embodiments, the non-nucleotidic
linker is glycerol or a glycerol homolog of the formula
HO--(CH.sub.2).sub.o--CH(OH)--(CH.sub.2).sub.p--OH, wherein o and p
independently are integers from 1 to 6, e.g., from 1 to 4, or from
1 to 3. In some other embodiments, the non-nucleotidic linker is a
derivative of 1,3-diamino-2-hydroxypropane, such as those having
the formula
HO--(CH.sub.2).sub.m--C(O)NH--CH.sub.2--CH(OH)--CH.sub.2--NHC(OMCH.sub.2)-
.sub.m--OH, wherein each m is independently an integer from 0 to
10, e.g., from 0 to 6, from 2 to 6, or from 2 to 4.
[0241] The term "alkyl" refers to a monoradical of a saturated
straight or branched hydrocarbon. Preferably, the alkyl group
comprises from 1 to 20 carbon atoms, such as from 1 to 12 or from 1
to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon
atoms, more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to
4 carbon atoms. Exemplary alkyl groups include methyl, ethyl,
propyl, iso-propyl, butyl (e.g., n-butyl, iso-butyl, tert-butyl),
pentyl (e.g., n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl),
1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl,
2,2-dimethylbutyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl,
n-nonyl, n-decyl, and the like. A "substituted alkyl" means that
one or more (such as 1 to the maximum number of hydrogen atoms
bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to
10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen
atoms of the alkyl group are replaced with a substituent other than
hydrogen (when more than one hydrogen atom is replaced the
substituents may be the same or different). Preferably, the
substituent other than hydrogen is a 1.sup.st level substituent, a
2.sup.nd level substituent, or a 3.sup.rd level substituent as
specified herein, such as halogen or optionally substituted aryl.
Examples of a substituted alkyl include trifluoromethyl,
2,2,2-trichloroethyl, arylalkyl (also called "aralkyl", e.g.,
benzyl, chloro(phenyl)methyl, 4-methylphenylmethyl,
(2,4-dimethylphenyl)methyl, o-fluorophenylmethyl, 2-phenylpropyl,
2-, 3-, or 4-carboxyphenylalkyl), or heteroarylalkyl (also called
"heteroaralkyl").
[0242] The term "alkenyl" refers to a monoradical of an unsaturated
straight or branched hydrocarbon having at least one carbon-carbon
double bond. Generally, the maximum number of carbon-carbon double
bonds in the alkenyl group can be equal to the integer which is
calculated by dividing the number of carbon atoms in the alkenyl
group by 2 and, if the number of carbon atoms in the alkenyl group
is uneven, rounding the result of the division down to the next
integer. For example, for an alkenyl group having 9 carbon atoms,
the maximum number of carbon-carbon double bonds is 4. Preferably,
the alkenyl group has 1 to 4, i.e., 1, 2, 3, or 4, carbon-carbon
double bonds. Preferably, the alkenyl group comprises from 2 to 20
carbon atoms, such as from 2 to 12 or from 2 to 10 carbon atoms,
i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2
to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon
atoms. Thus, in a preferred embodiment, the alkenyl group comprises
from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double
bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2,
3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and
1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1
or 2 carbon-carbon double bonds. The carbon-carbon double bond(s)
may be in cis (Z) or trans (E) configuration. Exemplary alkenyl
groups include ethenyl (i.e., vinyl), 1-propenyl, 2-propenyl (i.e.,
allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,
3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,
5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl,
5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl,
5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl,
4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl,
2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl,
8-decenyl, 9-decenyl, and the like. If an alkenyl group is attached
to a nitrogen atom, the double bond cannot be alpha to the nitrogen
atom. A "substituted alkenyl" means that one or more (such as 1 to
the maximum number of hydrogen atoms bound to an alkenyl group,
e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to
5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl
group are replaced with a substituent other than hydrogen (when
more than one hydrogen atom is replaced the substituents may be the
same or different). Preferably, the substituent other than hydrogen
is a 1.sup.st level substituent, a 2.sup.nd level substituent, or a
3.sup.rd level substituent as specified herein, such as halogen or
optionally substituted aryl. An example of a substituted alkenyl is
styryl (i.e., 2-phenylvinyl).
[0243] The term "alkynyl" refers to a monoradical of an unsaturated
straight or branched hydrocarbon having at least one carbon-carbon
triple bond. Generally, the maximum number of carbon-carbon triple
bonds in the alkynyl group can be equal to the integer which is
calculated by dividing the number of carbon atoms in the alkynyl
group by 2 and, if the number of carbon atoms in the alkynyl group
is uneven, rounding the result of the division down to the next
integer. For example, for an alkynyl group having 9 carbon atoms,
the maximum number of carbon-carbon triple bonds is 4. Preferably,
the alkynyl group has 1 to 4, i.e., 1, 2, 3, or 4, more preferably
1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group
comprises from 2 to 20 carbon atoms, such as from 2 to 12 or from 2
to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon
atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon
atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the
alkynyl group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4,
or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more
preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4
(preferably 1 or 2) carbon-carbon triple bonds, such as 2 to 6
carbon atoms and 1, 2 or 3 carbon-carbon triple bonds or 2 to 4
carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary
alkynyl groups include ethynyl, 1-propynyl (i.e.,
--C.ident.CCH.sub.3), 2-propynyl (i.e., --CH.sub.2C.ident.CH or
propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,
2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl,
3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl,
3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octynyl,
2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl,
1-nonylyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl, 6-nonynyl,
7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl,
5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, and the
like. If an alkynyl group is attached to a nitrogen atom, the
triple bond cannot be alpha to the nitrogen atom. A "substituted
alkynyl" means that one or more (such as 1 to the maximum number of
hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6,
7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or
1 or 2) hydrogen atoms of the alkynyl group are replaced with a
substituent other than hydrogen (when more than one hydrogen atom
is replaced the substituents may be the same or different).
Preferably, the substituent other than hydrogen is a 1.sup.st level
substituent, a 2.sup.nd level substituent, or a 3.sup.rd level
substituent as specified herein, such as halogen or optionally
substituted aryl.
[0244] The term "aryl" or "aromatic ring" refers to a monoradical
of an aromatic cyclic hydrocarbon. Preferably, the aryl group
contains 3 to 14 (e.g., 5 to 10, such as 5, 6, or 10) carbon atoms
which can be arranged in one ring (e.g., phenyl) or two or more
condensed rings (e.g., naphthyl). Exemplary aryl groups include
cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl,
azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl"
refers to a monocyclic ring containing 6 carbon atoms or an
aromatic bicyclic ring system containing 10 carbon atoms. Preferred
examples are phenyl and naphthyl. A "substituted aryl" means that
one or more (such as 1 to the maximum number of hydrogen atoms
bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to
10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen
atoms of the aryl group are replaced with a substituent other than
hydrogen (when more than one hydrogen atom is replaced the
substituents may be the same or different). Preferably, the
substituent other than hydrogen is a 1.sup.st level substituent, a
2.sup.nd level substituent, or a 3.sup.rd level substituent as
specified herein, such as halogen, --CN, nitro, --OR.sup.11 (e.g.,
--OH), --SR.sup.11 (e.g., --SH), --N(R.sup.12)(R.sup.13) (e.g.,
--NH.sub.2), .dbd.Z (e.g., .dbd.O, .dbd.S, or .dbd.NH), alkyl
(e.g., C.sub.1-6 alkyl), alkenyl (e.g., C.sub.2-6 alkenyl), and
alkynyl (e.g., C.sub.2-6 alkynyl). Examples of a substituted aryl
include biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl,
4-hydroxyphenyl, methoxyphenyl (i.e., 2-, 3-, or 4-methoxyphenyl),
and 4-ethoxyphenyl.
[0245] The term "heteroaryl" or "heteroaromatic ring" means an aryl
group as defined above in which one or more carbon atoms in the
aryl group are replaced by heteroatoms of O, S, or N. Preferably,
heteroaryl refers to a five or six-membered aromatic monocyclic
ring wherein 1, 2, or 3 carbon atoms are replaced by the same or
different heteroatoms of O, N, or S. Alternatively, it means an
aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5
carbon atoms are replaced with the same or different heteroatoms of
O, N, or S. Preferably, in each ring of the heteroaryl group the
maximum number of O atoms is 1, the maximum number of S atoms is 1,
and the maximum total number of O and S atoms is 2. Exemplary
heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl,
oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl,
tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,
pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl,
isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl,
benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl,
benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl,
benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl,
pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl,
phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl,
pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl,
phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl,
phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and
phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include
furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl,
imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl,
thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl),
pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted
heteroaryl" means that one or more (such as 1 to the maximum number
of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3,
or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with
a substituent other than hydrogen (when more than one hydrogen atom
is replaced the substituents may be the same or different).
Preferably, the substituent other than hydrogen is a 1.sup.st level
substituent, a 2.sup.nd level substituent, or a 3.sup.rd level
substituent as specified herein, such as halogen, --CN, nitro,
--OR.sup.11 (e.g., --OH), --SR.sup.11 (e.g., --SH),
--N(R.sup.12)(R.sup.13) (e.g., --NH.sub.2), .dbd.Z (e.g., .dbd.O,
.dbd.S, or .dbd.NH), alkyl (e.g., C.sub.1-6 alkyl), alkenyl (e.g.,
C.sub.2-6 alkenyl), and alkynyl (e.g., C.sub.2-6 alkynyl). Examples
of a substituted heteroaryl include 3-phenylpyrrolyl, 2,3'-bifuryl,
4-methylpyridyl, 2-, or 3-ethylindolyl.
[0246] The term "cycloalkyl" or "cycloaliphatic" represents cyclic
non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to
14 carbon atoms, such as 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7,
8, 9, or 10 carbon atoms, more preferably 3 to 7 carbon atoms. In
one embodiment, the cycloalkyl group has 1, 2, or more (preferably
1 or 2) double bonds. Exemplary cycloalkyl groups include
cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl,
cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl,
cylcodecyl, cylcodecenyl, and adamantyl. The term "cycloalkyl" is
also meant to include bicyclic and tricyclic versions thereof. If
bicyclic rings are formed it is preferred that the respective rings
are connected to each other at two adjacent carbon atoms, however,
alternatively the two rings are connected via the same carbon atom,
i.e., they form a spiro ring system or they form "bridged" ring
systems. Preferred examples of cycloalkyl include
C.sub.3-C.sub.5-cycloalkyl, in particular cyclopropyl, cyclobutyl,
cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,
cyclohexadienyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl,
spiro[3,4]octyl, spiro[4,3]octyl, spiro[4,5]decanyl,
bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl
(i.e., norbornyl), bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl,
bicyclo[4.2.0]octyl, bicyclo[4.3.0]nonyl,
1,2,3,4-tetrahydronaphthyl (i.e., tetralinyl), and
bicyclo[4.4.0]decanyl (i.e., decalinyl). A "substituted cycloalkyl"
means that one or more (such as 1 to the maximum number of hydrogen
atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2)
hydrogen atoms of the cycloalkyl group are replaced with a
substituent other than hydrogen (when more than one hydrogen atom
is replaced the substituents may be the same or different).
Preferably, the substituent other than hydrogen is a 1.sup.st level
substituent, a 2.sup.nd level substituent, or a 3.sup.rd level
substituent as specified herein, such as halogen, --CN, nitro,
--OR.sup.11 (e.g., --OH), --SR.sup.11 (e.g., --SH),
--N(R.sup.12)(R.sup.13) (e.g., --NH.sub.2), .dbd.Z (e.g., .dbd.O,
.dbd.S, or .dbd.NH), alkyl (e.g., C.sub.1-6 alkyl), alkenyl (e.g.,
C.sub.2-6 alkenyl), and alkynyl (e.g., C.sub.2-6 alkynyl). Examples
of a substituted cycloalkyl include oxocyclohexyl, oxocyclopentyl,
fluorocyclohexyl, and oxocyclohexenyl.
[0247] The term "heterocyclyl" or "heterocyclic ring" means a
cycloalkyl group as defined above in which from 1, 2, 3, or 4
carbon atoms in the cycloalkyl group are replaced by heteroatoms of
oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur,
preferably O, S, or N. A heterocyclyl group has preferably 1 or 2
rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring
atoms. Preferably, in each ring of the heterocyclyl group the
maximum number of O atoms is 1, the maximum number of S atoms is 1,
and the maximum total number of O and S atoms is 2. The term
"heterocyclyl" is also meant to encompass partially or completely
hydrogenated forms (such as dihydro, tetrahydro or perhydro forms)
of the above-mentioned heteroaryl groups. Exemplary heterocyclyl
groups include morpholinyl, pyrrolidinyl, imidazolidinyl,
pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl,
di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and
tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides,
and cyclic anhydrides. A "substituted heterocyclyl" means that one
or more (such as 1 to the maximum number of hydrogen atoms bound to
a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10,
such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen
atoms of the heterocyclyl group are replaced with a substituent
other than hydrogen (when more than one hydrogen atom is replaced
the substituents may be the same or different). Preferably, the
substituent other than hydrogen is a 1.sup.st level substituent, a
2.sup.nd level substituent, or a 3.sup.rd level substituent as
specified herein, such as halogen, --CN, nitro, --OR.sup.11 (e.g.,
--OH), --SR.sup.11 (e.g., --SH), --N(R.sup.12)(R.sup.13) (e.g.,
--NH.sub.2), .dbd.Z (e.g., .dbd.O, .dbd.S, or .dbd.NH), alkyl
(e.g., C.sub.1-6 alkyl), alkenyl (e.g., C.sub.2-6 alkenyl), and
alkynyl (e.g., C.sub.2-6 alkynyl).
[0248] The term "aromatic" as used in the context of hydrocarbons
means that the whole molecule has to be aromatic. For example, if a
monocyclic aryl is hydrogenated (either partially or completely)
the resulting hydrogenated cyclic structure is classified as
cycloalkyl for the purposes of the present invention. Likewise, if
a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the
resulting hydrogenated bi- or polycyclic structure (such as
1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes
of the present invention (even if one ring, such as in
1,2-dihydronaphthyl, is still aromatic). A similar distinction is
made within the present application between heteroaryl and
heterocyclyl. For example, indolinyl, i.e., a dihydro variant of
indolyl, is classified as heterocyclyl for the purposes of the
present invention, since only one ring of the bicyclic structure is
aromatic and one of the ring atoms is a heteroatom.
[0249] The term "halogen" or "halo" means fluoro, chloro, bromo, or
iodo, preferably fluoro. The term "hydroxy" means OH. The term
"alkoxy" means O-alkyl, wherein alkyl is as defined above, and
includes methoxy, ethoxy, propoxy, butoxy, iso-butoxy, sec-butoxy,
pentoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, and decyloxy. The
term "substituted alkoxy" means O-(substituted alkyl), wherein
substituted alkyl is as defined above, and includes
2-methoxyethoxy. The term "nitro" means NO.sub.2. The term "cyano"
means the group --CN. The term "isocyano" means the group --NC. The
term "cyanato" means the group --OCN. The term "isocyanato" means
the group --NCO. The term "thiocyanato" means the group --SCN. The
term "isothiocyanato" means the group --NCS. The term "azido" means
N.sub.3.
[0250] The term "amino" includes unsubstituted amino (i.e., the
group --NH.sub.2) and substituted amino (i.e., mono- or
disubstituted amino, wherein one or two of the hydrogen atoms have
been replaced with a group other than hydrogen). Thus, the term
"amino" means the group --N(R.sup.12)(R.sup.13), wherein R.sup.12
and R.sup.13 are, in each case, independently selected from the
group consisting of --H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, and heterocyclyl, or R.sup.12 and R.sup.13 may join
together with the nitrogen atom to which they are attached to form
the group --N.dbd.CR.sup.15R.sup.16, wherein each of the alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl
groups is optionally substituted with one or more (such as 1 to the
maximum number of hydrogen atoms bound to the alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g.,
1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1
to 4, or 1 to 3, or 1 or 2) independently selected R.sup.31;
R.sup.15 and R.sup.16 are independently selected from the group
consisting of --H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, heterocyclyl, and --NH.sub.yR.sup.20.sub.2-y, or
R.sup.15 and R.sup.16 may join together with the atom to which they
are attached to form a ring which is optionally substituted with
one or more (such as 1 to the maximum number of hydrogen atoms
bound to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10,
such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently
selected R.sup.31, wherein each of the alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally
substituted with one or more (such as 1 to the maximum number of
hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl,
aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1
or 2) independently selected R.sup.30; y is an integer from 0 to 2;
R.sup.20 is selected from the group consisting of alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein
each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,
and heterocyclyl groups is optionally substituted with one or more
(such as 1 to the maximum number of hydrogen atoms bound to the
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or
heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10,
such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently
selected R.sup.30; and R.sup.30 is a 1.sup.st (or 2.sup.nd or
3.sup.rd) level substituent.
[0251] The term "imino" means the group --N(R.sup.14)--, wherein
both free valences of the nitrogen atom may bind to one other atom
(e.g., C) resulting in a double bond (e.g., C.dbd.N(R.sup.14)) or
to different atoms (e.g., two C atoms) resulting two single bonds
(e.g., C--N(R.sup.14)--C). In each case, R.sup.14 is selected from
the group consisting of --H, alkyl, alkenyl, alkynyl, cycloalkyl,
aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl
groups is optionally substituted with one or more (such as 1 to the
maximum number of hydrogen atoms bound to the alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g.,
1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1
to 4, or 1 to 3, or 1 or 2) independently selected R.sup.30; and
R.sup.30 is a 1.sup.st (or 2.sup.nd or 3.sup.rd) level
substituent.
[0252] The term "optionally substituted" indicates that one or more
(such as 1 to the maximum number of hydrogen atoms bound to a
moiety, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as
between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atom(s) may
be replaced with a group/substituent (i.e., a 1.sup.st level
substituent) different from hydrogen such as alkyl (preferably,
C.sub.1-6 alkyl), alkenyl (preferably, C.sub.2-6 alkenyl), alkynyl
(preferably, C.sub.2-6 alkynyl), aryl (preferably, 3- to
14-membered aryl), heteroaryl (preferably, 3- to 14-membered
heteroaryl), cycloalkyl (preferably, 3- to 14-membered cycloalkyl),
heterocyclyl (preferably, 3- to 14-membered heterocyclyl), halogen,
--CN, --NC, --NCO, --CNO, --SCN, --NCS, --N.sub.3, --NO.sub.2,
--OR.sup.71, --N(R.sup.72)(R.sup.73), --ON(R.sup.72)(R.sup.73),
--N*(--O)(R.sup.72)(R.sup.73), --S(O).sub.0-2R.sup.71 (i.e.,
--SR.sup.71, --S(O)R.sup.71, or --S(O).sub.2R.sup.71),
--S(O).sub.0-2OR.sup.71 (e.g., --S(O).sub.1-2OR.sup.71),
--OS(O).sub.0-2OR.sup.71 (e.g., --OS(O).sub.1-2OR.sup.71),
--S(O).sub.0-2N(R.sup.72)(R.sup.73) (e.g.,
--S(O).sub.1-2N(R.sup.72)(R.sup.73)),
--OS(O).sub.0-2N(R.sup.72)(R.sup.73) (e.g.,
--OS(O).sub.1-2N(R.sup.72)(R.sup.73)),
--N(R.sup.71)S(O).sub.0-2R.sup.71 (e.g.,
--N(R.sup.71)S(O).sub.1-2R.sup.71),
--NR.sup.71S(O).sub.0-2OR.sup.71 (e.g.,
--NR.sup.71S(O).sub.1-2OR.sup.71),
--NR.sup.71S(O).sub.0-2N(R.sup.72)(R.sup.73) (e.g.,
--NR.sup.71S(O).sub.1-2N(R.sup.72)(R.sup.73)),
--C(.dbd.Z.sup.1)R.sup.71, --C(.dbd.Z.sup.1)Z.sup.1R.sup.71,
--Z.sup.1C(.dbd.Z.sup.1)R.sup.71, and
--Z.sup.1C(.dbd.Z.sup.1)Z.sup.1R.sup.71, and/or any two 1.sup.st
level substituents which are bound to the same carbon atom of a
cycloalkyl or heterocyclyl group may join together to form
.dbd.Z.sup.1, wherein each of the alkyl, alkenyl, alkynyl, aryl,
heteroaryl, cycloalkyl, and heterocyclyl groups of the 1.sup.st
level substituent may themselves be substituted by one or more
(e.g., one, two or three) substituents (i.e., 2.sup.nd level
substituents) selected from the group consisting of C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, 3- to 14-membered
aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl,
3- to 14-membered heterocyclyl, halogen, --CF.sub.3, --CN, --NC,
--NCO, --CNO, --SCN, --NCS, --N.sub.3, --NO.sub.2, --OR.sup.81,
--N(R.sup.82)(R.sup.83), --ON(R.sup.82)(R.sup.83),
--N*(--O.sup.-)(R.sup.82)(R.sup.83), --S(O).sub.0-2R.sup.81 (i.e.,
--SR.sup.81, --S(O)R.sup.81, or --S(O).sub.2R.sup.81),
--S(O).sub.0-2OR.sup.81 (e.g., --S(O).sub.1-2OR.sup.81),
--OS(O).sub.0-2R.sup.81 (e.g., --OS(O).sub.1-2R.sup.81),
--OS(O).sub.0-2OR.sup.81 (e.g., --OS(O).sub.1-20R.sup.81),
--S(O).sub.0-2N(R.sup.82)(R.sup.83) (e.g.,
--S(O).sub.1-2N(R.sup.82)(R.sup.83)),
--OS(O).sub.0-2N(R.sup.82)(R.sup.83) (e.g.,
--OS(O).sub.1-2N(R.sup.82)(R.sup.83)),
--N(R.sup.81)S(O).sub.0-2R.sup.81 (e.g.,
--N(R.sup.81)S(O).sub.1-2R.sup.81),
--NR.sup.81S(O).sub.0-2OR.sup.81 (e.g.,
--NR.sup.81S(O).sub.1-2OR.sup.81),
--NR.sup.81S(O).sub.0-2N(R.sup.82)(R.sup.83) (e.g.,
--NR.sup.81S(O).sub.1-2N(R.sup.82)(R.sup.83)),
--C(.dbd.Z.sup.2)R.sup.81, --C(.dbd.Z.sup.2)Z.sup.2R.sup.81,
--Z.sup.2C(.dbd.Z.sup.2)R.sup.81, and
--Z.sup.2C(.dbd.Z.sup.2)Z.sup.2R.sup.81, and/or any two 2.sup.nd
level substituents which are bound to the same carbon atom of a
cycloalkyl or heterocyclyl group may join together to form
.dbd.Z.sup.2, wherein each of the C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, 3- to 14-membered aryl, 3- to
14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to
14-membered heterocyclyl groups of the 2.sup.rd level substituent
is optionally substituted with one or more (e.g., one, two or
three) substituents (i.e., 3.sup.rd level substituents)
independently selected from the group consisting of C.sub.1-3
alkyl, halogen, --CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS,
--N.sub.3, --NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3,
--S(C.sub.1-3 alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl),
--N(C.sub.1-3 alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl), --C(.dbd.O)(C.sub.1-3
alkyl), --C(.dbd.O)OH, --C(.dbd.O)O(C.sub.1-3 alkyl),
--C(.dbd.O)NH.sub.2-z (C.sub.1-3 alkyl), --OC(.dbd.O)(C.sub.1-3
alkyl), --OC(.dbd.O)O(C.sub.1-3 alkyl),
--OC(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--NHC(.dbd.O)(C.sub.1-3 alkyl), --NHC(.dbd.O)NH.sub.z-2(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and
--N(C.sub.1-3 alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
wherein z is 0, 1, or 2 and C.sub.1-3 alkyl is methyl, ethyl,
propyl or isopropyl, and/or any two 3.sup.rd level substituents
which are bound to the same carbon atom of a cycloalkyl or
heterocyclyl group may join together to form .dbd.O, .dbd.S,
.dbd.NH, or .dbd.N(C.sub.1-3 alkyl);
[0253] wherein
[0254] R.sup.71, R.sup.72, and R.sup.73 are independently selected
from the group consisting of --H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, 3- to 7-membered cycloalkyl, 5- or
6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered
heterocyclyl, wherein each of the C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, 3- to 7-membered cycloalkyl, 5- or
6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered
heterocyclyl groups is optionally substituted with one, two or
three substituents selected from the group consisting of C.sub.1-3
alkyl, halogen, --CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS,
--N.sub.3, --NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3,
--S(C.sub.1-3 alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl),
--N(C.sub.1-3 alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--C(.dbd.O)(C.sub.1-3 alkyl), --C(.dbd.O)OH, --C(.dbd.O)O(C.sub.1-3
alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--OC(.dbd.O)(C.sub.1-3 alkyl), --OC(.dbd.O)O(C.sub.1-3 alkyl),
--OC(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--NHC(.dbd.O)(C.sub.1-3 alkyl), --NHC(.dbd.O)NH.sub.z-2(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and
--N(C.sub.1-3 alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
wherein z is 0, 1, or 2 and C.sub.1-3 alkyl is methyl, ethyl,
propyl or isopropyl,
[0255] or R.sup.72 and R.sup.73 may join together with the nitrogen
atom to which they are attached to form a 5- or 6-membered ring,
which is optionally substituted with one, two or three substituents
selected from the group consisting of C.sub.1-3 alkyl, halogen,
--CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS, --N.sub.3,
--NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3, --S(C.sub.1-3
alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl), --N(C.sub.1-3
alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--C(.dbd.O)(C.sub.1-3 alkyl), --C(.dbd.O)OH, --C(.dbd.O)O(C.sub.1-3
alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--OC(.dbd.O)(C.sub.1-3 alkyl), --OC(.dbd.O)O(C.sub.1-3 alkyl),
--OC(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--NHC(.dbd.O)(C.sub.1-3 alkyl), --NHC(.dbd.O)NH.sub.z-2(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and
--N(C.sub.1-3 alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
wherein z is 0, 1, or 2 and C.sub.1-3 alkyl is methyl, ethyl,
propyl or isopropyl;
[0256] R.sup.81, R.sup.82, and R.sup.83 are independently selected
from the group consisting of --H, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, 3- to 6-membered cycloalkyl, 5- or
6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered
heterocyclyl, wherein each of the C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, 3- to 6-membered cycloalkyl, 5- or
6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered
heterocyclyl groups is optionally substituted with one, two or
three substituents selected from the group consisting of C.sub.1-3
alkyl, halogen, --CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS,
--N.sub.3, --NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3,
--S(C.sub.1-3 alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl),
--N(C.sub.1-3 alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--C(.dbd.O)(C.sub.1-3 alkyl), --C(.dbd.O)OH, --C(.dbd.O)O(C.sub.1-3
alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--OC(.dbd.O)(C.sub.1-3 alkyl), --OC(.dbd.O)O(C.sub.1-3 alkyl),
--OC(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--NHC(.dbd.O)(C.sub.1-3 alkyl), --NHC(.dbd.O)NH.sub.z-2(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and
--N(C.sub.1-3 alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
wherein z is 0, 1, or 2 and C.sub.1-3 alkyl is methyl, ethyl,
propyl or isopropyl,
[0257] or R.sup.82 and R.sup.83 may join together with the nitrogen
atom to which they are attached to form a 5- or 6-membered ring,
which is optionally substituted with one, two or three substituents
selected from the group consisting of C.sub.1-3 alkyl, halogen,
--CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS, --N.sub.3,
--NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3, --S(C.sub.1-3
alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl), --N(C.sub.1-3
alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--C(.dbd.O)(C.sub.1-3 alkyl), --C(.dbd.O)OH, --C(.dbd.O)O(C.sub.1-3
alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--OC(.dbd.O)(C.sub.1-3 alkyl), --OC(.dbd.O)O(C.sub.1-3 alkyl),
--OC(.dbd.O)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
--NHC(.dbd.O)(C.sub.1-3 alkyl), --NHC(.dbd.O)NH.sub.z-2(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and
--N(C.sub.1-3 alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z,
wherein z is 0, 1, or 2 and C.sub.1-3 alkyl is methyl, ethyl,
propyl or isopropyl;
[0258] Z.sup.1 and Z.sup.2 are independently selected from O, S,
and N(R.sup.84), wherein R.sup.14 is --H or C.sub.1-3 alkyl.
[0259] Typical 1.sup.st level substituents are preferably selected
from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, 3- to 14-membered (such as 5- or 6-membered)
aryl, 3- to 14-membered (such as 5- or 6-membered) heteroaryl, 3-
to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to
14-membered (such as 3- to 7-membered) heterocyclyl, halogen, --CN,
--NC, --NCO, --CNO, --SCN, --NCS, --N.sub.3, --NO.sub.2,
--OR.sup.71, --N(R.sup.72)(R.sup.73), --S(O).sub.0-2R.sup.71,
--S(O).sub.0-2OR.sup.71, --OS(O).sub.0-2R.sup.71,
--OS(O).sub.0-2OR.sup.71, --S(O).sub.0-2N(R.sup.72)(R.sup.73),
--OS(O).sub.0-2N(R.sup.72)(R.sup.73),
--N(R.sup.71)S(O).sub.0-2R.sup.71,
--NR.sup.71S(O).sub.0-2OR.sup.71, --C(.dbd.Z.sup.1)R.sup.71,
--C(.dbd.Z.sup.1)Z.sup.1R.sup.71, --Z.sup.1C(.dbd.Z.sup.1)R.sup.71,
and --Z.sup.1C(.dbd.Z.sup.1)Z.sup.1R.sup.71, such as C.sub.1-4
alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, 5- or 6-membered aryl,
5- or 6-membered heteroaryl, 3- to 7-membered cycloalkyl, 3- to
7-membered heterocyclyl, halogen, --CF.sub.3, --CN, --NC, --NCO,
--CNO, --SCN, --NCS, --N.sub.3, --NO.sub.2, --OH, --O(C.sub.1-3
alkyl), --S(C.sub.1-3 alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl),
--N(C.sub.1-3 alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z, --C(.dbd.O)OH,
--C(.dbd.O)O(C.sub.1-3 alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.O)(C.sub.1-3 alkyl),
--NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and --N(C.sub.1-3
alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z, wherein z is 0,
1, or 2 and C.sub.1-3 alkyl is methyl, ethyl, propyl or isopropyl;
Z.sup.1 is independently selected from O, S, NH and N(CH.sub.3);
and R.sup.71, R.sup.72, and R.sup.73 are as defined above or,
preferably, are independently selected from the group consisting of
--H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, 5- or
6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered
heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and
heterocyclyl groups is optionally substituted with one, two or
three substituents selected from the group consisting of C.sub.1-3
alkyl, halogen, --CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS,
--N.sub.3, --NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3,
--S(C.sub.1-3 alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl),
--N(C.sub.1-3 alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z, --C(.dbd.O)OH,
--C(.dbd.O)O(C.sub.1-3 alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.O)(C.sub.1-3 alkyl),
--NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and --N(C.sub.1-3
alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z, wherein z is 0,
1, or 2 and C.sub.1-3 alkyl is methyl, ethyl, propyl or isopropyl;
or R.sup.72 and R.sup.73 may join together with the nitrogen atom
to which they are attached to form a 5- or 6-membered ring, which
is optionally substituted with one, two or three substituents
selected from the group consisting of C.sub.1-3 alkyl, halogen,
--CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN, --NCS, --N.sub.3,
--NO.sub.2, --OH, --O(C.sub.1-3 alkyl), --OCF.sub.3, --S(C.sub.1-3
alkyl), --NH.sub.2, --NH(C.sub.1-3 alkyl), --N(C.sub.1-3
alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3 alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z, --C(.dbd.O)OH,
--C(.dbd.O)O(C.sub.1-3 alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.O)(C.sub.1-3 alkyl),
--NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl).sub.z, and --N(C.sub.1-3
alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z, wherein z is 0,
1, or 2 and C.sub.1-3 alkyl is methyl, ethyl, propyl or
isopropyl.
[0260] Typical 2.sup.nd level substituents are preferably selected
from the group consisting of C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, 5- or 6-membered aryl, 5- or 6-membered
heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered
heterocyclyl, halogen, --CF.sub.3, --CN, --NC, --NCO, --CNO, --SCN,
--NCS, --N.sub.3, --NO.sub.2, --OH, --O(C.sub.1-3 alkyl),
--OCF.sub.3, --S(C.sub.1-3 alkyl), --NH.sub.2, --NH(C.sub.1-3
alkyl), --N(C.sub.1-3 alkyl).sub.2, --NHS(O).sub.2(C.sub.1-3alkyl),
--S(O).sub.2NH.sub.2-z(C.sub.1-3 alkyl).sub.z, --C(.dbd.O)OH,
--C(.dbd.O)O(C.sub.1-3 alkyl), --C(.dbd.O)NH.sub.2-z(C.sub.1-3
alkyl).sub.z, --NHC(.dbd.O)(C.sub.1-3 alkyl),
--NHC(.dbd.NH)NH.sub.z-2(C.sub.1-3 alkyl), and --N(C.sub.1-3
alkyl)C(.dbd.NH)NH.sub.2-z(C.sub.1-3 alkyl).sub.z, wherein z is 0,
1, or 2 and C.sub.1-3 alkyl is methyl, ethyl, propyl or isopropyl.
Particularly preferred 2.sup.nd level substituents include
4-morpholinyl, homomorpholinyl, 4-piperidinyl, homopiperidinyl
(i.e., azepanyl, in particular 4-azepanyl), 4-piperazinyl,
homopiperazinyl (i.e., diazepanyl, in particular 2,4-diazepanyl),
N-methyl-piperazin-4-yl, N-methyl-homopiperazinyl,
--CH.sub.2CH.sub.2OCH.sub.3, --OCH.sub.2CH.sub.2OCH.sub.3,
--CH.sub.2CH.sub.2NH.sub.2-z(CH.sub.3).sub.z,
--OCH.sub.2CH.sub.2NH.sub.2-z(CH.sub.3).sub.z, --CF.sub.3, and
--OCF.sub.3.
[0261] Typical 3.sup.rd level substituents are preferably selected
from the group consisting of phenyl, furanyl, pyrrolyl, thienyl,
imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyrazinyl,
pyrimidinyl, pyridazinyl, partially and completely hydrogenated
forms of the forgoing groups, morpholino, C.sub.1-3 alkyl, halogen,
--NC, --NCO, --CNO, --SCN, --NCS, --N.sub.3, --CF.sub.3, --OH,
--OCH.sub.3, --OCF.sub.3, --SCH.sub.3,
--NH.sub.2-z(CH.sub.3).sub.z, --C(.dbd.O)OH, and
--C(.dbd.O)OCH.sub.3, wherein z is 0, 1, or 2.
[0262] The term "optional" or "optionally" as used herein means
that the subsequently described event, circumstance or condition
may or may not occur, and that the description includes instances
where said event, circumstance, or condition occurs and instances
in which it does not occur.
[0263] "Isomers" are compounds having the same molecular formula
but differ in structure ("structural isomers") or in the
geometrical positioning of the functional groups and/or atoms
("stereoisomers"). "Enantiomers" are a pair of stereoisomers which
are non-superimposable mirror-images of each other. A "racemic
mixture" or "racemate" contains a pair of enantiomers in equal
amounts and is denoted by the prefix (+). "Diastereomers" are
stereoisomers which are not enantiomers. "Tautomers" are structural
isomers of the same chemical substance that spontaneously
interconvert with each other, even when pure.
[0264] The 5'-cap compound of the present invention or an RNA
modified with a 5'-cap compound of the present invention may be
isotopically labeled, i.e., one or more atoms are replaced by a
corresponding atom having the same number of protons but differing
in the number of neutrons. For example, a hydrogen atom may be
replaced by a deuterium atom. Exemplary isotopes which can be used
in the 5'-cap compound of the present invention or an RNA modified
with a 5'-cap compound of the present invention include deuterium,
.sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.18F, .sup.32S,
.sup.36Cl, and .sup.125I. The term "isotopically enriched" means
that the occurrence of the isotope is beyond the natural abundance.
A 5'-cap compound of the present invention which is isotopically
labeled or RNAs modified with such an isotopically labeled 5'-cap
compound of the present application can be produced by using
correspondingly isotopically labeled nucleotides during the in
vitro transcription or by adding such correspondingly isotopically
labeled nucleotides after transcription.
[0265] The phrase "the stereochemical configuration at the P atom
comprising the substituent R.sup.5 corresponds to that at the
P.sub..beta. atom of the D1 diastereomer of beta-S-ARCA" means that
a phosphorous atom comprising the substituent R.sup.5 and having a
chiral center, and therefore capable of existing in either of two
stereochemical configurations, is present in predominately one
desired stereochemical configuration, i.e., that at the
P.sub..beta. atom of the D1 diastereomer of beta-S-ARCA. As the
case may be for the P.sub..beta. atom of the D1 diastereomer of
beta-S-ARCA this could either be the (R) configuration or the (S)
configuration. Preferably, greater than 50% of the group of
interest has the desired stereochemical configuration, preferably
at least 75% of the group of interest has the desired
stereochemical configuration, more preferably at least 90% of the
group of interest has the desired stereochemical configuration,
even more preferably at least 95% of the group of interest has the
desired stereochemical configuration, and most preferably at least
99% of the group of interest has the desired stereochemical
configuration.
[0266] The D1 diastereomer of beta-S-ARCA (3-S-ARCA) has the
following structure:
##STR00002##
[0267] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is
the diastereomer of beta-S-ARCA which elutes first on an HPLC
column compared to the D2 diastereomer of beta-S-ARCA
(beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The
HPLC preferably is an analytical HPLC. In one embodiment, a
Supelcosil LC-18-T RP column, preferably of the format: 5 m,
4.6.times.250 mm is used for separation, whereby a flow rate of 1.3
ml/min can be applied. In one embodiment, a gradient of methanol in
ammonium acetate, for example, a 0-25% linear gradient of methanol
in 0.05 M ammonium acetate, pH=5.9, within 15 min is used.
UV-detection (VWD) can be performed at 260 nm and fluorescence
detection (FLD) can be performed with excitation at 280 nm and
detection at 337 nm.
[0268] The term "naturally occurring", as used herein in context
with an object, refers to the fact that an object can be found in
nature. For example, a protein, amino acid or nucleic acid that is
present in an organism (including viruses), that can be isolated
from a source in nature and that has not been intentionally
modified by man in the laboratory is naturally occurring.
[0269] The present invention relates to modification of RNA,
preferably mRNA, to increase the stability and/or expression of
said RNA, preferably in immune cells, more preferably in immature
immune cells, even more preferably in immature antigen presenting
cells, and most preferably in immature dendritic cells.
[0270] The modified RNA described in the present invention is
particularly useful for RNA vaccination.
[0271] 5'-Cap Compound
[0272] In a first aspect, the present application provides a 5'-cap
compound having the 5'-cap structure according to formula (I):
##STR00003##
[0273] wherein R.sup.1 is selected from the group consisting of
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
and optionally substituted heteroaryl;
[0274] R.sup.2 and R.sup.3 are independently selected from the
group consisting of H, halo, OH, and optionally substituted alkoxy,
or R.sup.2 and R.sup.3 together form O--X--O, wherein X is selected
from the group consisting of optionally substituted CH.sub.2,
optionally substituted CH.sub.2CH.sub.2, optionally substituted
CH.sub.2CH.sub.2CH.sub.2, optionally substituted
CH.sub.2CH(CH.sub.3), and optionally substituted C(CH.sub.3).sub.2,
or R.sup.2 is combined with the hydrogen atom at position 4' of the
ring to which R.sup.2 is attached to form --O--CH.sub.2-- or
--CH.sub.2--O--;
[0275] R.sup.4 and R.sup.6 are independently selected from the
group consisting of O, S, Se, and BH.sub.3;
[0276] R.sup.5 is selected from the group consisting of S, Se, and
BH.sub.3;
[0277] R.sup.7 is a mononucleotide or an oligonucleotide having 2,
3, 4, 5, 6, 7, 8, or 9 (such as 2, 3, 4, 5, or 6) bases;
[0278] R.sup.8 is H, halo, or optionally substituted alkoxy;
[0279] n is 1, 2, or 3; and
[0280] B is a purine or pyrimidine base moiety.
[0281] In one embodiment, the 5'-cap compound has the formula
(Ia)
##STR00004##
[0282] wherein R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, n, and B are as defined above or below and
R.sup.1 is selected such that the 5'-cap compound does not inhibit
translation of the RNA comprising said 5'-cap compound. In one
embodiment of the 5'-cap compound of formula (Ia), R.sup.1 is
selected such that the capped RNA, in particular the 5'-cap
structure of the capped RNA is recognized by the translation
initiation machinery, preferably in vivo and in vitro, preferably
the capped RNA, in particular the 5'-cap structure of the capped
RNA is recognized by the eukaryotic translation initiation
machinery. For example, the skilled person may determine whether a
capped RNA or the 5'-cap structure of the capped RNA is recognized
by the eukaryotic translation initiation machinery by determining
the affinity of the eukaryotic translation initiation factor eIF4E
for said capped RNA or said 5'-cap structure.
[0283] In one embodiment of the 5'-cap compound of formula (Ia),
R.sup.1 is selected from the group consisting of optionally
substituted C.sub.1-C.sub.4 alkyl (e.g., methyl, ethyl, propyl,
butyl, benzyl, phenylethyl, and naphthylmethyl, any of which may be
optionally substituted); optionally substituted C.sub.2-C.sub.4
alkenyl (e.g., ethenyl, propenyl, or butenyl, any of which may be
optionally substituted), and optionally substituted aryl.
[0284] In a preferred embodiment of the 5'-cap compound of formula
(Ia), R.sup.1 is selected from the group consisting of
C.sub.1-C.sub.4 alkyl and optionally substituted aryl. In a
preferred embodiment of the 5'-cap compound of formula (Ia),
R.sup.1 is selected from the group consisting of methyl, ethyl,
optionally substituted benzyl, optionally substituted phenylethyl,
and optionally substituted naphthylmethyl. In a preferred
embodiment of the 5'-cap compound of formula (Ia), R.sup.1 is
methyl or ethyl, more preferably methyl.
[0285] In one embodiment, the 5'-cap compound has the formula
(Ib)
##STR00005##
[0286] wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, n, and B are as defined above (in particular with respect
to one or more of formulas (I) and (Ta)) or below and the
configuration of R.sup.2 and R.sup.3 is such that the 5'-cap
compound can only be incorporated into an RNA chain in one
orientation. Pasquinelli et al. (1995, RNA J. 1: 957-967) have
demonstrated that during in vitro transcription bacteriophage RNA
polymerases use the 7-methylguanosine unit for initiation of
transcription whereby around 40-50% of the transcripts with cap
possess the cap-dinucleotide in a reverse orientation (i.e., the
initial reaction product is Gpppm.sup.7GpN).
[0287] Compared to the RNAs containing a cap structure in the
correct orientation RNAs containing a cap structure in reverse
orientation (also called RNAs with a reverse cap) are not
functional with respect to translation of the encoded proteins.
Thus, it is desirable to incorporate the cap in the correct
orientation, i.e., resulting in an RNA with a cap structure
essentially corresponding to m.sup.7GpppGpN etc. It has been shown
that the reverse integration of the cap-dinucleotide is inhibited
by the substitution of either the 2'- or the 3'-OH group of the
methylated guanosine unit (Stepinski et al., 2001; RNA J.
7:1486-1495; Peng et al., 2002; Org. Lett. 24:161-164). RNAs which
are synthesized in presence of such "anti reverse cap analogs",
i.e., ARCAs, are translated more efficiently than RNAs which have
been in vitro transcribed in presence of the conventional 5'-cap
m.sup.7GpppG. Furthermore, Kore et al. (J. Am. Chem. Soc. 2009,
131:6364-6365) found that locked nucleic acid (LNA)-modified
dinucleotide mRNA cap analogues are also not incorporated in the
reverse orientation into an RNA strand.
[0288] Consequently, in a preferred embodiment of the 5'-cap
compound of formula (Ib), R.sup.1 is selected such that the
eukaryotic translation initiation machinery is capable of
recognizing the RNA capped with the 5'-cap compound of the present
invention and at least one (or both of) R.sup.2 and R.sup.3 is
(are) selected such that the 5'-cap compound cannot be incorporated
in reverse orientation into an RNA strand.
[0289] In one embodiment of the 5'-cap compound of formula (Ib),
R.sup.2 and R.sup.3 are independently selected from the group
consisting of H, F, OH, methoxy, ethoxy, propoxy, and
2-methoxyethoxy. In a preferred embodiment of the 5'-cap compound
of formula (Ib), one of R.sup.2 and R.sup.3 is OH, and the other is
not OH. In another preferred embodiment of the 5'-cap compound of
formula (Ib), at least one of R.sup.2 and R.sup.3 is not OH. For
example, in one embodiment of the 5'-cap compound of formula (Ib),
R.sup.2 is selected from the group consisting of H, F, methoxy,
ethoxy, propoxy and 2-methoxyethoxy, preferably from the group
consisting of H, F, methoxy, ethoxy, and propoxy.
[0290] In any one of the embodiments of the 5'-cap compound of
formula (Ib) described above, the ring structure comprising the
substituents R.sup.2 and R.sup.3 may have the stereochemical
configuration of ribose. In this embodiment, it is preferred that
at least one of R.sup.2 and R.sup.3 is not OH.
[0291] In those of the above embodiments, where R.sup.2 (or
R.sup.3) is not OH it is preferably selected from the group
consisting of H, halo, and optionally substituted C.sub.1-C.sub.10
alkoxy, more preferably from the group consisting of H, F, methoxy,
ethoxy, propoxy, and 2-methoxyethoxy, more preferably from the
group consisting of H, F, methoxy, ethoxy, and propoxy. More
preferably, it is methoxy.
[0292] In a preferred embodiment of the 5'-cap compound of formula
(Ib), in particular when the ring structure comprising the
substituents R.sup.2 and R.sup.3 has the stereochemical
configuration of ribose, R.sup.2 is OH and R.sup.3 is methoxy or
R.sup.2 is methoxy and R.sup.3 is OH.
[0293] In one embodiment of the 5'-cap compound of formula (Ib),
R.sup.2 and R.sup.3 together form O--X--O, wherein X is selected
from the group consisting of CH.sub.2 and C(CH.sub.3).sub.2, both
of which may be optionally substituted.
[0294] In one embodiment of the 5'-cap compound of formula (Ib),
the stereochemical configuration of the ring structure comprising
the substituents R.sup.2 and R.sup.3 does not correspond to the
stereochemical configuration of ribose. For example, the
stereochemical configuration of the ring structure comprising the
substituents R.sup.2 and R.sup.3 may correspond to the
stereochemical configuration of arabinose, xylose, or lyxose, in
particular when the stereochemical configuration of said ring
structure corresponds to that of arabinose. In these embodiments,
it is preferred that R.sup.2 and R.sup.3 are both OH. However, in
these embodiments, it is also possible that R.sup.2 and R.sup.3 are
selected as specified above.
[0295] In one embodiment, the 5'-cap compound has the formula
(Ic)
##STR00006##
[0296] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.6,
R.sup.7, R.sup.8, n, and B are as defined above (in particular with
respect to one or more of formulas (I), (Ia), and (Ib)) or below
and R.sup.5 is S or Se, preferably S. In one embodiment of the
5'-cap compound of formula (Ic), R.sup.5 is S or Se, preferably S,
and n is 1 or 2. In one embodiment of the 5'-cap compound of
formula (Ic), R.sup.5 is S and n is 1 or 2, preferably 1. In any of
the above embodiments of the 5'-cap compound of formula (Ic), it is
preferred that R.sup.4 and R.sup.6 are independently selected from
the group consisting of O, Se, and S, more preferably from the
group consisting of 0 and S. In any of the above embodiments,
wherein n is 2 or 3, it is to be understood that R.sup.6 may
independently be selected for each [R.sup.6PO.sub.2] moiety. For
example, if n is 2, the 5'-cap compound contains two
[R.sup.6PO.sub.2] moieties, wherein the two R.sup.6 residues may be
the same (e.g., R.sup.6 in both [R.sup.6PO.sub.2] moieties is O) or
different (e.g., R.sup.6 in one [R.sup.6PO.sub.2] moiety is O,
whereas R.sup.6 in the other [R.sup.6PO.sub.2] moiety is S). In one
embodiment of the 5'-cap compound of formula (Ic), R.sup.5 is S or
Se, preferably S, n is 1 or 2, preferably 1, and R.sup.4 and
R.sup.6 are independently selected from the group consisting of O
and S, more preferably R.sup.4 and R.sup.6 are O. In one embodiment
of the 5'-cap compound of formula (Ic), R.sup.5 is S, n is 1 or 2,
preferably 1, and R.sup.4 and R.sup.6 are O.
[0297] In one embodiment of the 5'-cap compound of formula (Ic),
the stereochemical configuration at the P atom comprising the
substituent R.sup.5 corresponds to that at the P.sub..beta. atom of
the D1 diastereomer of beta-S-ARCA.
[0298] In one embodiment, the 5'-cap compound has the formula
(Id)
##STR00007##
[0299] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.8, n, and B are as defined above (in particular with
respect to one or more of formulas (I), (Ia), (Ib), and (Ic)) or
below and R.sup.7 is bonded via its 5'-end to the ring to which
R.sup.8 is attached. In one embodiment of the 5'-cap compound of
formula (Id), R.sup.7 is a ribomononucleotide or
ribooligonucleotide. In one preferred embodiment of the 5'-cap
compound of formula (Id), R.sup.7 is a ribonucleotide having a free
OH group at position 2'. In another preferred embodiment of the
5'-cap compound of formula (Id), R.sup.7 is a ribooligonucleotide,
wherein both the ribose moiety at the 3'-end of the
ribooligonucleotide and the ribose moiety at the 5'-end of the
ribooligonucleotide have a free OH group at position 2'. In another
preferred embodiment of the 5'-cap compound of formula (Id),
R.sup.7 is a ribooligonucleotide, wherein the OH group at position
2' of at least the ribose at the 5'-end of the ribooligonucleotide
is replaced with a substituent selected from the group consisting
of H, halo, and optionally substituted alkoxy (such as H, F,
methoxy, ethoxy, propoxy, or 2-methoxyethoxy, preferably H, F,
methoxy, ethoxy, or propoxy, most preferably methoxy), and the
ribose at the 3'-end of the ribooligonucleotide has a free OH group
at position 2'. In any of the above embodiments of formula (Id), it
is preferred that the internucleotide linkage between the
mononucleotide or oligonucleotide and the ring to which R.sup.7 is
attached is selected from the group consisting of phosphate,
phosphorothioate, boranophosphate, imidophosphate, alkylene
phosphate, phosphorodithioate, alkylphosphonate, phosphotriester,
phosphoroamidite, and non-nucleotide linker, preferably from the
group consisting of phosphate, phosphorothioate, and
phosphorodithioate (in one embodiment the internucleotide linkage
between the mononucleotide or oligonucleotide and the ring to which
R.sup.7 is attached is phosphate). In any of the above embodiments
of formula (Id), where R.sup.7 is an oligonucleotide, in particular
a ribooligonucleotide, it is preferred that the internucleotide
linkage(s) between the nucleotides in the oligonucleotide is(are)
selected from the group consisting of phosphate, phosphorothioate,
boranophosphate, imidophosphate, alkylene phosphate,
phosphorodithioate, alkylphosphonate, phosphotriester,
phosphoroamidite, and non-nucleotide linker, preferably from the
group consisting of phosphate, phosphorothioate, and
phosphorodithioate (in one embodiment the internucleotide
linkage(s) between the nucleotides in the oligonucleotide is(are)
phosphate). In one embodiment of the 5'-cap compound of formula
(Id), R.sup.7 is *[pN(R.sup.8')].sub.a[pN].sub.b, wherein *
indicates the attachment point of R.sup.7 to the ring to which
R.sup.7 is attached; each N(R.sup.8') is a nucleoside (preferably
adenosine, guanosine, uridine, 5-methyluridine, or cytidine) which
is substituted with R.sup.8' (being selected from the group
consisting of H, halo, and optionally substituted alkoxy,
preferably from the group consisting of H, F, methoxy, ethoxy,
propoxy and 2-methoxyethoxy, more preferably from the group
consisting of H, F, methoxy, ethoxy, and propoxy, most preferably
methoxy) at position 2'; each N is a ribonucleoside (preferably
adenosine, guanosine, uridine, 5-methyluridine, or cytidine) having
a free OH group at position 2'; each p is a phosphate moiety; a is
0, 1, 2, 3, 4, 5, 6, 7, or 8; b is 1, 2, 3, 4, 5, 6, 7, 8, or 9;
and a+b is 1, 2, 3, 4, 5, 6, 7, 8, or 9 (preferably a is 0, 1, or
2; b is 1, 2, 3, 4, 5, or 6; and a+b is 1, 2, 3, 4, 5, or 6). In
one embodiment of the 5'-cap compound of formula (Id), R.sup.7 is
*pGpN or *pG, wherein N is adenosine, guanosine, uridine,
5-methyluridine, or cytidine and wherein * indicates the attachment
point of R.sup.7 to the ring to which R.sup.7 is attached. In one
embodiment of the 5'-cap compound of formula (Id), R.sup.7 is
*pm.sup.2'-OGpN, wherein N is adenosine, guanosine, uridine,
5-methyluridine, or cytidine and wherein * indicates the attachment
point of R.sup.7 to the ring to which R.sup.7 is attached.
[0300] In one embodiment, the 5'-cap compound has the formula
(Ie)
##STR00008##
[0301] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, and n are as defined above (in
particular with respect to one or more of formulas (I), (Ia), (Ib),
(Ic), and (Id)) or below and B is a naturally occurring purine or
pyrimidine base moiety or a modified form thereof. In one
embodiment of the 5'-cap compound of formula (Ie), B is selected
from the group consisting of guanine, adenine, cytosine, thymine,
uracil, and modified forms thereof, preferably from the group
consisting of guanine, adenine, cytosine, uracil, and modified
forms thereof, more preferably from the group consisting of
guanine, adenine, cytosine, and modified forms thereof, more
preferably from the group consisting of guanine, adenine, and
modified forms thereof. In one embodiment of the 5'-cap compound of
formula (Je), the modified purine or pyrimidine base moiety is
modified by one or more alkyl groups, preferably one or more
C.sub.1-4alkyl groups, more preferably one or more methyl groups.
In a preferred embodiment of the 5'-cap compound of formula (Ie),
the modified purine or pyrimidine base moiety is selected from the
group consisting of N.sup.7-alkyl-guanine, N.sup.6-alkyl-adenine,
5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, preferably
from the group consisting of N.sup.7--C.sub.1-4alkyl-guanine,
N.sup.6--C.sub.1-4alkyl-adenine, 5-C.sub.1-4alkyl-cytosine,
5-C.sub.1-4alkyl-uracil, and N(1)-C.sub.1-4alkyl-uracil, more
preferably from the group consisting of N.sup.7-methyl-guanine,
N.sup.6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and
N(1)-methyl-uracil. In a preferred embodiment of the 5'-cap
compound of formula (Ie), the naturally occurring purine or
pyrimidine base moiety is G or A, preferably G. In a more preferred
embodiment of the 5'-cap compound of formula (Ie), B is G or A,
preferably G.
[0302] In any of the above embodiments of the 5'-cap compound of
any one of formulas (I), (Ia), (Ib), (Ic), (Id), and (Ie), it is
preferred that R.sup.8 is selected from the group consisting of H,
F, methoxy, ethoxy, propoxy, and 2-methoxyethoxy, more preferably
from the group consisting of H, F, methoxy, ethoxy, and propoxy.
Most preferably, in any of the above embodiments of the 5'-cap
compound any one of formulas (I), (Ia), (Ib), (Ic), (Id), and (Ie),
R.sup.8 is methoxy.
[0303] In one embodiment, the 5'-cap compound has the formula
(II)
##STR00009##
[0304] wherein R.sup.1 is selected from the group consisting of
optionally substituted C.sub.1-C.sub.4 alkyl and optionally
substituted aryl;
[0305] R.sup.2 and R.sup.3 are independently selected from the
group consisting of H, F, OH, methoxy, ethoxy, propoxy, and
2-methoxyethoxy;
[0306] R.sup.4 and R.sup.6 are independently selected from the
group consisting of O and S;
[0307] R.sup.5 is S or Se;
[0308] R.sup.7 is a ribomononucleotide or a ribooligonucleotide
having 2, 3, 4, 5, or 6 (such as 2 or 3) bases;
[0309] R.sup.8 is selected from the group consisting of H, F,
methoxy, ethoxy, propoxy, and 2-methoxyethoxy;
[0310] n is 1, 2, or 3; and
[0311] B is a purine or pyrimidine base moiety.
[0312] In one embodiment, the 5'-cap compound has the formula
(IIa)
##STR00010##
[0313] wherein R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, n, and B are as defined above (in particular with
respect to one or more of formulas (I), (Ia), (Ib), (Ic), (Id),
(Ie), and (TI)) or below and R.sup.1 is selected from the group
consisting of methyl, ethyl, benzyl, phenylethyl, and
naphthylmethyl, more preferably from the group consisting of methyl
and ethyl. In a preferred embodiment of the 5'-cap compound of
formula (IIa), R.sup.1 is methyl or ethyl, more preferably
methyl.
[0314] In one embodiment, the 5'-cap compound has the formula
(IIb)
##STR00011##
[0315] wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, n, and B are as defined above (in particular with respect
to one or more of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (TI),
and (IIa)) or below and at least one of R.sup.2 and R.sup.3 is not
OH. In one embodiment of the 5'-cap compound of formula (IIb), one
of R.sup.2 and R.sup.3 is OH, and the other is not OH. In one
embodiment of the 5'-cap compound of formula (IIb), the ring
structure comprising the substituents R.sup.2 and R.sup.3 has the
stereochemical configuration of ribose. In this embodiment, it is
preferred that at least one of R.sup.2 and R.sup.3 is not OH. In
those of the above embodiments, where R.sup.2 (or R.sup.3) is not
OH it is preferably selected from the group consisting of H, F,
methoxy, ethoxy, and propoxy. More preferably, it is methoxy.
[0316] In a preferred embodiment of the 5'-cap compound of formula
(IIb), in particular when the ring structure comprising the
substituents R.sup.2 and R.sup.3 has the stereochemical
configuration of ribose, R.sup.2 is OH and R.sup.3 is methoxy or
R.sup.2 is methoxy and R.sup.3 is OH.
[0317] In one embodiment of the 5'-cap compound of formula (IIb),
the stereochemical configuration of the ring structure comprising
the substituents R.sup.2 and R.sup.3 does not correspond to the
stereochemical configuration of ribose. For example, the
stereochemical configuration of the ring structure comprising the
substituents R.sup.2 and R.sup.3 may correspond to the
stereochemical configuration of arabinose, xylose, or lyxose, in
particular when the stereochemical configuration of said ring
structure corresponds to that of arabinose. In these embodiments,
it is preferred that R.sup.2 and R.sup.3 are both OH. However, in
these embodiments, it is also possible that R.sup.2 and R.sup.3 are
selected as specified above.
[0318] In one embodiment, the 5'-cap compound has the formula
(IIc)
##STR00012##
[0319] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.6,
R.sup.7, R.sup.8, n, and B are as defined above (in particular with
respect to one or more of formulas (I), (Ia), (Tb), (Ic), (Id),
(Ie), (II), (IIa), and (IIb)) or below. In one embodiment of the
5'-cap compound of formula (IIc), n is 1 or 2. In any of the above
embodiments, wherein n is 2 or 3, it is to be understood that
R.sup.6 may independently selected for each [R.sup.6PO.sub.2]
moiety. For example, if n is 2, the 5'-cap compound contains two
[R.sup.6PO.sub.2] moieties, wherein the two R.sup.6 residues may be
the same (e.g., R.sup.6 in both [R.sup.6PO.sub.2] moieties is O) or
different (e.g., R.sup.6 in one [R.sup.6PO.sub.2] moiety is O,
whereas R.sup.6 in the other [R.sup.6PO.sub.2] moiety is S). In one
embodiment of the 5'-cap compound of formula (IIc), R.sup.4 and
R.sup.6 are O . In one embodiment of the 5'-cap compound of formula
(IIc), n is 1 or 2, preferably 1, and R.sup.4 and R.sup.6 are
O.
[0320] In one embodiment, the 5'-cap compound has the formula
(IId)
##STR00013##
[0321] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.8, n, and B are as defined above (in particular with
respect to one or more of formulas (I), (Ia), (Ib), (Ic), (Id),
(Ie), (II), (IIa), (IIb), and (IIc)) or below and R.sup.7 is bonded
via its 5'-end to the ring to which R.sup.8 is attached. In one
preferred embodiment of the 5'-cap compound of formula (IId),
R.sup.7 is a ribonucleotide having a free OH group at position 2'.
In another preferred embodiment of the 5'-cap compound of formula
(IId), R.sup.7 is a ribooligonucleotide, wherein both the ribose
moiety at the 3'-end of the ribooligonucleotide and the ribose
moiety at the 5'-end of the ribooligonucleotide have a free OH
group at position 2'. In another preferred embodiment of the 5'-cap
compound of formula (IId), R.sup.7 is a ribooligonucleotide,
wherein the OH group at position 2' of at least the ribose at the
5'-end of the ribooligonucleotide is replaced with a substituent
selected from the group consisting of H, halo, and optionally
substituted alkoxy (such as H, F, methoxy, ethoxy, propoxy, or
2-methoxyethoxy, preferably H, F, methoxy, ethoxy, or propoxy, most
preferably methoxy), and the ribose at the 3'-end of the
ribooligonucleotide has a free OH group at position 2'. In any of
the above embodiments of formula (IId), it is preferred that the
internucleotide linkage between the mononucleotide or
oligonucleotide and the ring to which R.sup.7 is attached is
selected from the group consisting of phosphate, phosphorothioate,
boranophosphate, imidophosphate, alkylene phosphate,
phosphorodithioate, alkylphosphonate, phosphotriester,
phosphoroamidite, and non-nucleotide linker, more preferably the
internucleotide linkage between the mononucleotide or
oligonucleotide and the ring to which R.sup.7 is attached is
phosphate. In any of the above embodiments of formula (IId), where
R.sup.7 is an oligonucleotide, in particular a ribooligonucleotide,
it is preferred that the internucleotide linkage(s) between the
nucleotides in the oligonucleotide is(are) selected from the group
consisting of phosphate, phosphorothioate, boranophosphate,
imidophosphate, alkylene phosphate, phosphorodithioate,
alkylphosphonate, phosphotriester, phosphoroamidite, and
non-nucleotide linker, more preferably the internucleotide
linkage(s) between the nucleotides in the oligonucleotide is(are)
phosphate. In one embodiment of the 5'-cap compound of formula
(IId), R.sup.7 is *[pN(R.sup.8')].sub.a[pN].sub.b, wherein *
indicates the attachment point of R.sup.7 to the ring to which
R.sup.7 is attached; each N(R.sup.8') is a nucleoside (preferably
adenosine, guanosine, uridine, 5-methyluridine, or cytidine) which
is substituted with R.sup.8' (being selected from the group
consisting of H, halo, and optionally substituted alkoxy,
preferably from the group consisting of H, F, methoxy, ethoxy,
propoxy and 2-methoxyethoxy, more preferably from the group
consisting of H, F, methoxy, ethoxy, and propoxy, most preferably
methoxy) at position 2'; each N is a ribonucleoside (preferably
adenosine, guanosine, uridine, 5-methyluridine, or cytidine) having
a free OH group at position 2'; each p is a phosphate moiety; a is
0, 1, 2, 3, 4, 5, 6, 7, or 8; b is 1, 2, 3, 4, 5, 6, 7, 8, or 9;
and a+b is 1, 2, 3, 4, 5, 6, 7, 8, or 9 (preferably a is 0, 1, or
2; b is 1, 2, 3, 4, 5, or 6; and a+b is 1, 2, 3, 4, 5, or 6). In
one embodiment of the 5'-cap compound of formula (IId), R.sup.7 is
*pGpN or *pG, wherein N is adenosine, guanosine, uridine,
5-methyluridine, or cytidine and wherein * indicates the attachment
point of R.sup.7 to the ring to which R.sup.7 is attached. In one
embodiment of the 5'-cap compound of formula (IId), R.sup.7 is
*pm.sup.2'-OGpN, wherein N is adenosine, guanosine, uridine,
5-methyluridine, or cytidine and wherein * indicates the attachment
point of R.sup.7 to the ring to which R.sup.7 is attached.
[0322] In one embodiment, the 5'-cap compound has the formula
(IIe)
##STR00014##
[0323] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, and n are as defined above (in
particular with respect to one or more of formulas (I), (Ia), (Ib),
(Ic), (Id), (le), (II), (IIa), (IIb), (IIc), and (IId)) or below
and B is a naturally occurring purine or pyrimidine base moiety or
a modified form thereof. In one embodiment of the 5'-cap compound
of formula (IIe), B is selected from the group consisting of
guanine, adenine, cytosine, thymine, uracil, and modified forms
thereof, preferably from the group consisting of guanine, adenine,
cytosine, uracil, and modified forms thereof, more preferably from
the group consisting of guanine, adenine, cytosine, and modified
forms thereof, more preferably from the group consisting of
guanine, adenine, and modified forms thereof. In one embodiment of
the 5'-cap compound of formula (IIe), the modified purine or
pyrimidine base moiety is modified by one or more alkyl groups,
preferably one or more C.sub.1-4alkyl groups, more preferably one
or more methyl groups. In a preferred embodiment of the 5'-cap
compound of formula (IIe), the modified purine or pyrimidine base
moiety is selected from the group consisting of
N.sup.7-alkyl-guanine, N.sup.6-alkyl-adenine, 5-alkyl-cytosine,
5-alkyl-uracil, and N(1)-alkyl-uracil, preferably from the group
consisting of N.sup.7--C.sub.1-4alkyl-guanine,
N.sup.6--C.sub.1-4alkyl-adenine, 5-C.sub.1-4alkyl-cytosine,
5-C.sub.1-4alkyl-uracil, and N(1)-C.sub.1-4alkyl-uracil, more
preferably from the group consisting of N.sup.7-methyl-guanine,
N.sup.6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and
N(1)-methyl-uracil. In a preferred embodiment of the 5'-cap
compound of formula (IIe), the naturally occurring purine or
pyrimidine base moiety is G or A, preferably G. In a more preferred
embodiment of the 5'-cap compound of formula (IIe), B is G or A,
preferably G.
[0324] In any of the above embodiments of the 5'-cap compound of
any one of formulas (II), (IIa), (IIb), (IIc), (IId), and (IIe), it
is preferred that R is selected from the group consisting of H, F,
methoxy, ethoxy, and propoxy. Most preferably, in any of the above
embodiments of the 5'-cap compound of any one of formulas (II),
(IIa), (IIb), (IIc), (IId), and (IIe), R.sup.8 is methoxy.
[0325] In one embodiment, the 5'-cap compound has the formula
(III)
##STR00015##
[0326] wherein R.sup.1 is methyl, ethyl, benzyl, phenylethyl, or
naphthylmethyl, more preferably methyl or ethyl; R.sup.2 and
R.sup.3 are independently selected from the group consisting of H,
F, OH, and methoxy, wherein preferably at least one of R.sup.2 and
R.sup.3 is not OH;
[0327] R.sup.7 is a ribomononucleotide, ribodinucleotide or a
ribotrinucleotide bonded via its 5'-end to the ring to which
R.sup.8 is attached, wherein the internucleotide linkage between
the ribomononucleotide, ribodinucleotide or ribotrinucleotide and
the ring to which R.sup.7 is attached is selected from the group
consisting of phosphate, phosphorothioate, and phosphorodithioate,
and wherein if R.sup.7 is a ribodinucleotide or a
ribotrinucleotide, the internucleotide linkage(s) between the
nucleotides in the ribodinucleotide or ribotrinucleotide is(are)
selected from the group consisting of phosphate, phosphorothioate,
and phosphorodithioate;
[0328] R.sup.8 is selected from the group consisting of H, F, and
methoxy;
[0329] n is 1 or 2; and
[0330] B is selected from the group consisting of guanine, adenine,
cytosine, thymine, and uracil, preferably from the group consisting
of guanine, adenine, cytosine, and uracil, more preferably from the
group consisting of guanine, adenine, and cytosine, more preferably
from the group consisting of guanine and adenine.
[0331] In one embodiment, the 5'-cap compound has the formula
(IIIa)
##STR00016##
[0332] wherein R.sup.1 is methyl or ethyl; one of R.sup.2 and
R.sup.3 is OCH.sub.3 and the other is OH (e.g., R.sup.2 is
OCH.sub.3 and R.sup.3 is OH or R.sup.2 is OH and R.sup.3 is
OCH.sub.3); R.sup.8 methoxy; n is 1; the internucleotide linkage
between the ribomononucleotide, ribodinucleotide or
ribotrinucleotide and the ring to which R.sup.7 is attached is
phosphate or phosphorothioate, preferably phosphate, and wherein if
R.sup.7 is a ribodinucleotide or a ribotrinucleotide, the
internucleotide linkage(s) between the nucleotides in the
ribodinucleotide or ribotrinucleotide is(are) phosphate or
phosphorothioate, preferably phosphate; and B is guanine or
adenine, preferably guanine. In one embodiment of the 5'-cap
compound of formula (IIIa), R.sup.7 is a ribomononucleotide having
a free OH group at position 2'. In another preferred embodiment of
the 5'-cap compound of formula (IIIa), R.sup.7 is a ribodi- or
ribotrinucleotide, wherein both the ribose moiety at the 3'-end of
the ribodi- or ribotrinucleotide and the ribose moiety at the
5'-end of the ribodi- or ribotrinucleotide have a free OH group at
position 2'. In another preferred embodiment of the 5'-cap compound
of formula (IIIa), R.sup.7 is a ribodi- or ribotrinucleotide,
wherein the OH group at position 2' of at least the ribose at the
5'-end of the ribodi- or ribotrinucleotide is replaced with a
substituent selected from the group consisting of H, F, methoxy,
ethoxy, propoxy, and 2-methoxyethoxy (preferably from the group
consisting of H, F, methoxy, ethoxy, and propoxy, most preferably
said substituent is methoxy), and the ribose at the 3'-end of the
ribodi- or ribotrinucleotide has a free OH group at position 2'. In
one embodiment of the 5'-cap compound of formula (IIIa), R.sup.7 is
*pGpN or *pG, wherein N is adenosine, guanosine, uridine,
5-methyluridine, or cytidine and wherein * indicates the attachment
point of R.sup.7 to the ring to which R.sup.7 is attached. In one
embodiment of the 5'-cap compound of formula (IIIa), R.sup.7 is
*pm.sup.2'-OGpN, wherein N is adenosine, guanosine, uridine,
5-methyluridine, or cytidine and wherein * indicates the attachment
point of R.sup.7 to the ring to which R.sup.7 is attached.
[0333] 5'-cap compounds of the present invention can be synthesized
starting from commercially available compounds (such as
(pN).sub.2-4) using standard procedures. These oligonucleotides can
be converted into the corresponding P-imidazolide derivatives by
reacting them with imidazole in the presence of an activation
system (e.g., 2,2'-dithiodipyridine/triphenylphosphine; cf. FIG. 1
and Mukaiyama and Hashimoto 1971 (Bull. Chem. Soc. Jpn. 44, 2284
(1971))). The nucleotide subunit bearing a modified phosphate
bridge (e.g., m.sub.2.sup.7,2'OMeGDP.beta.S) may be synthesized as
described in Kowalska et al. 2008 (RNA 14, 1119-1131 (2008)). Then,
the two precursors may be coupled to yield the final 5'-cap
compound of the invention; cf. e.g., FIG. 2. Diastereoisomers may
be separated by RP HPLC (e.g., using a Discovery Amide RP C16
column).
[0334] Preferably, when the 5'-cap compound of the present
invention is used to prepare a correspondingly 5'-capped RNA, the
5'-cap structure upon transfer of the 5'-capped RNA into cells is
capable of increasing the stability of the RNA, decreasing or
inhibiting the recognition of the RNA by proteins recognizing the
cap0 structure, e.g., IFIT proteins (in particular IFIT1),
increasing translation efficiency of the RNA, prolonging
translation of the RNA, increasing total protein expression of the
RNA, and/or, if RNA comprises a nucleotide sequence encoding an
antigen, increasing the immune response against said antigen when
compared to the same RNA without the 5'-cap structure. If RNA
comprises a nucleotide sequence encoding an antigen, it is
preferred that the cells are immature antigen presenting cells,
such as immature dendritic cells. The skilled person may readily
determine whether the 5'-cap structure of the 5'-capped RNA is
capable of exerting the above functions, for example, by generating
two RNAs, e.g., by in vitro transcription, which only differ in the
5'-cap structure, wherein one of the RNA carries a 5'-cap structure
according to any one of the formulas (I), (Ia), (Ib), (Ic), (Id),
(Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III), and (IIIa)
and the other RNA (reference RNA) (i) does not comprise a 5'-cap
structure, (ii) carries a conventional mRNA 5'-cap, i.e., a
methyl-7-guanosine cap, or (iii) carries any other cap with which
the function of the 5'-cap structure according to any one of the
formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb),
(IIc), (IId), (IIe), (III), and (IIIa) should be compared. For
example, the reference RNA may carry a 5'-cap structure which
corresponds to the D2 diastereomer of beta-S-ARCA. It is
particularly preferred that the 5'-cap structure of the 5'-capped
RNA upon transfer of the modified RNA into cells is capable of
increasing the stability of the RNA, decreasing or inhibiting the
recognition of the RNA by proteins recognizing the cap0 structure
e.g., IFIT proteins (in particular IFIT1), increasing translation
efficiency of the RNA, prolonging translation of the RNA,
increasing total protein expression of the RNA, and/or, if RNA
comprises a nucleotide sequence encoding an antigen, increasing the
immune response against said antigen when compared to a reference
RNA, such as the same RNA having a conventional mRNA 5'-cap.
[0335] Preferably, the stability and translation efficiency of RNA
modified with a 5'-cap compound of the present invention (in
particular a 5'-cap compound according to any one of the formulas
(I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc),
(IId), (IIe), (III), and (IIIa)) may be further modified as
required. For example, the RNA may be stabilized and its
translation increased by one or more modifications having a
stabilizing and/or translation efficiency increasing effect. Such
modifications are, for example, described in WO 2007/036366
incorporated herein by reference.
[0336] For example, RNA having an unmasked poly-A sequence
(unmasked poly-A tail) is translated more efficiently than RNA
having a masked poly-A sequence. The term "poly-A sequence" relates
to a sequence of adenyl (A) residues which typically is located at
the 3'-end of an RNA molecule and "unmasked poly-A sequence" means
that the poly-A sequence at the 3'-end of an RNA molecule ends with
an A of the poly-A sequence and is not followed by nucleotides
other than A located at the 3'-end, i.e., down-stream, of the
poly-A sequence. Furthermore, a long poly-A sequence of about 120
nucleotides results in optimal transcript stability and translation
efficiency.
[0337] Thus, the RNA, preferably the mRNA, modified with a 5'-cap
compound of the present invention (in particular a 5'-cap compound
according to any one of the formulas (I), (Ia), (Ib), (Ic), (Id),
(le), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III), and (IIIa))
may preferably further comprise a poly-A tail having a length of 10
to 500, preferably having a length of 30 to 300, more preferably
having a length of 65 to 200, more preferably having a length of
100 to 150 nucleotides, e.g., 100, 110, 120, 130, 140, or 150
nucleotides, preferably 120 nucleotides. Preferably, said poly-A
sequence is an unmasked poly-A sequence. Thus, preferably, the RNA,
preferably the mRNA, modified with a 5'-cap compound of the present
invention (in particular a 5'-cap compound according to any one of
the formulas (I), (Ia), (Ib), (Ic), (Id), (le), (II), (IIa), (IIb),
(IIc), (IId), (IIe), (III), and (IIIa)) comprises an unmasked
poly-A tail having a length of 10 to 500, preferably having a
length of 30 to 300, more preferably having a length of 65 to 200,
more preferably having a length of 100 to 150 nucleotides, e.g.,
100, 110, 120, 130, 140, or 150 nucleotides, preferably 120
nucleotides.
[0338] In addition, incorporation of a 3'-untranslated region (UTR)
into the 3'-untranslated region of an RNA molecule can result in an
enhancement in translation efficiency. A synergistic effect may be
achieved by incorporating two or more of such 3'-UTRs. The 3'-UTRs
may be autologous or heterologous to the RNA into which they are
introduced, for example, it may be the 3'-UTR of the beta-globin
mRNA. Thus, preferably, the RNA, preferably the mRNA, modified with
a 5'-cap compound of the present invention (in particular a 5'-cap
compound according to any one of the formulas (I), (Ia), (Ib),
(Ic), (Id), (le), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III),
and (IIIa)) may further comprise one or more copies, preferably two
copies of the 3'-untranslated region (3'-UTR) of the beta-globin
gene, preferably of the human beta-globin gene.
[0339] In addition, the replacement of uridine with pseudouridine
or N(1)-methylpseudouridine or 5-methyl-uridine (m5U) resulting in
T- or m1T- or m5U-modified RNAs can decrease the immunogenicity of
the thus modified RNAs. Therefore, preferably, in the RNA,
preferably mRNA, modified with a 5'-cap compound of the present
invention (in particular a 5'-cap compound according to any one of
the formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb),
(IIc), (IId), (IIe), (III), and (IIIa)) pseudouridine or
N(1)-methylpseudouridine or 5-methyluridine (m5U) is substituted
partially or completely, preferably completely, for uridine. I.e.,
in one preferred embodiment, the RNA of the invention is .PSI.- or
m1.PSI.- or m5U-modified or any combination thereof (e.g., .PSI.-
and m1.PSI.-modified or T- and m5U-modified or m1.PSI.- and
m5U-modified or .PSI.- and m1.PSI.- and m5U-modified).
[0340] In some embodiments, the modified nucleoside replacing one
or more uridine in the RNA may be any one or more of
3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine,
6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U),
4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine,
5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine
(e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid
(cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U),
5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine,
5-carboxyhydroxymethyl-uridine (chm5U),
5-carboxyhydroxymethyl-uridine methyl ester (mchm5U),
5-methoxycarbonylmethyl-uridine (mcm5U),
5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U),
5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine
(mmu5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine
(mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U),
5-carbamoylmethyl-uridine (ncm5U),
5-carboxymethylaminomethyl-uridine (cmmu5U),
5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U),
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyl-uridine (.TM.5U), 1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine (.tau.m5s2U),
1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine
(m5s2U), 1-methyl-4-thio-pseudouridine (m1s4.PSI.),
4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3.PSI.),
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),
dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine
(m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine,
2-methoxy-uridine, 2-methoxy-4-thio-uridine,
4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,
N.sup.1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine
(acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3
.PSI.), 5-(isopentenylaminomethyl)uridine (imu5U),
5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U),
.alpha.-thio-uridine, 2'-O-methyl-uridine (Um),
5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (.PSI.m),
2-thio-2'-O-methyl-uridine (s2Um),
5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um),
5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um),
5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um),
3,2'-O-dimethyl-uridine (m3Um),
5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um),
1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine,
2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine,
5-[3-(1-E-propenylamino)uridine, or any other modified uridine
known in the art.
[0341] It is particularly preferred that the RNA, preferably the
mRNA, modified with a 5'-cap compound of the present invention (in
particular a 5'-cap compound according to any one of the formulas
(I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc),
(IId), (IIe), (III), and (IIIa)) is modified by a combination of
the above described modifications, i.e., by at least two (e.g., at
least 3 or by all 4) of the following modifications: incorporation
of a poly-A sequence, unmasking of a poly-A sequence, incorporation
of one or more 3'-UTRs, and replacement of uridine with
pseudouridine or N(1)-methylpseudouridine or 5-methyluridine or a
combination thereof.
[0342] In a particularly preferred embodiment, the RNA, preferably
the mRNA, modified with a 5'-cap compound of the present invention
(in particular a 5'-cap compound according to any one of the
formulas (I), (Ia), (Ib), (Ic), (Id), (le), (II), (IIa), (IIb),
(IIc), (IId), (IIe), (III), and (IIIa)) encodes a pharmaceutically
active peptide or protein, e.g., selected from the group consisting
of cytokines, such as erythropoietin; adhesion molecules, such as
an integrin; immunoglobulins; immunologically active compounds,
e.g., antigens, such as tumor-associated antigens,
pathogen-associated antigens (e.g., one or more (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10) antigens of a virus, such as one or more
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens of influenza
virus (A, B, or C), CMV, or RSV), allergens, or autoantigens;
hormones, such as vasopressin, insulin or growth hormone; growth
factors, such as VEGFA; enzymes, such as herpes simplex virus type
1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine
hydroxylase, pseudocholinesterase, pancreatic enzymes, or lactase;
receptors, such as growth factor receptors; protease inhibitors,
such as alpha 1-antitrypsin; apoptosis regulators, such as BAX;
transcription factors, such as FOXP3; tumor suppressor proteins,
such as p53; structural proteins, such as surfactant proteins;
reprogramming factors, such as OCT4, SOX2, c-MYC, KLF4, LIN28, or
NANOG; genomic engineering proteins, such as clustered regularly
spaced short palindromic repeat-CRISPR-associated protein 9
(CRISPR-Cas9); and blood proteins, such as fibrinogen. For example,
the pharmaceutically active peptide or protein may be a peptide or
protein comprising an immunogen, antigen or antigen peptide,
wherein the peptide or protein may be processed after expression to
provide said immunogen, antigen or antigen peptide. Alternatively,
the peptide or protein itself may be the immunogen, antigen or
antigen peptide.
[0343] Compositions and Kits
[0344] In a further aspect, the present invention provides a
composition or kit comprising a 5'-cap compound of the present
invention. Such composition or kit may be used for providing an RNA
with a 5'-cap structure of the present invention and/or for
increasing the stability of an RNA, e.g., in the corresponding
methods disclosed herein. In one embodiment of this aspect, the kit
may further comprise reagents typically used in in vitro
transcription reactions (e.g., NTPs, an RNA polymerase, one or more
buffers, and/or a DNA template) and/or instructions for use.
[0345] In a further aspect, the present invention provides a
composition, preferably a pharmaceutical composition, comprising an
RNA (preferably mRNA) modified with a 5'-cap compound of the
present invention (such composition comprising an RNA of the
invention is also referred to herein as RNA composition of the
invention). The composition, in particular pharmaceutical
composition, of this aspect may comprise the RNA (preferably mRNA)
modified with a 5'-cap compound of the present invention in
combination with and one or more pharmaceutically acceptable
excipients. In one embodiment, the pharmaceutical composition
comprises an RNA (preferably mRNA) modified with a 5'-cap compound
of the present invention, one or more pharmaceutically acceptable
excipients and one or more additional/supplementary active
compounds.
[0346] In a further aspect, the present application provides a
pharmaceutical composition as specified herein for use in
therapy.
[0347] For example, in particular in those embodiments where the
RNA modified with a 5'-cap compound of the present invention
comprises a nucleotide sequence encoding a peptide or protein, the
pharmaceutical compositions of the present invention may be used in
protein replacement therapy, genome engineering therapy, genomic
reprogramming therapy, or immunotherapy.
[0348] Illustrative applications of protein replacement therapy for
the RNA or pharmaceutical compositions of the present invention
include the treatment (including prophylactic treatment) of a
condition, disorder or disease caused by a decreased activity of a
peptide or protein, e.g., anemia (replacement protein: e.g.,
erythropoietin), diabetes (replacement protein: e.g., vasopressin),
congential lung disease (replacement protein: e.g., surfactant
protein B), asthma (replacement protein: e.g., FOXP3), myocardial
infarction (replacement protein: e.g., VEGFA), melanoma
(replacement protein: e.g., BAX), autoimmune diabetes (replacement
protein: e.g., IL-4), autoimmune myocarditis (replacement protein:
e.g., IL-10), inflammation (replacement proteins: e.g., P-selectin
glycoprotein ligand-1 (PSGL-1), Sialyl-Lewisx (SLeX), and IL-10)),
factor VII deficiency (replacement protein: e.g., factor VIIa),
hemophilia A (replacement protein: e.g., factor VIII), hemophilia B
(replacement protein: e.g., factor IX), factor X deficiency
(replacement protein: e.g., factor X), factor XI deficiency
(replacement protein: e.g., factor XI), factor XIII deficiency
(replacement protein: e.g., factor XIII), von Willebrand disease
(replacement protein: e.g., von Willebrand factor), protein C
deficiency (replacement protein: e.g., protein C), antithrombin
deficiency (replacement protein: e.g., antithrombin III),
fibrinogen deficiency (replacement protein: e.g., fibrinogen),
hereditary angioedema (replacement protein: e.g., C1-esterase
inhibitor), al-PI deficiency (replacement protein: e.g., alpha-1
proteinase inhibitor), Gaucher disease (replacement protein: e.g.,
glucocerebrosidase), mucopolysaccharidosis I (replacement protein:
e.g., alpha-L-iduronidase), mucopolysaccharidosis II (replacement
protein: e.g., iduronate sulfatase), mucopolysaccharidosis VI
(replacement protein: e.g., N-acetylgalactosamine-4-sulfatase),
mucopolysaccharidosis IVA (replacement protein: e.g.,
N-acetylgalactosamine-6-sulfatase), mucopolysaccharidosis IIIA
(replacement protein: e.g., heparan sulfate sulfatase), Fabry
disease (replacement protein: e.g., alpha-galactosidase A), Pompe
disease (replacement protein: e.g., alpha-glucosidase),
Niemann-Pick type B disease (replacement protein: e.g., acid
sphingomyelinase), alpha-mannosidosis, (replacement protein: e.g.,
alpha-mannosidase), metachromatic leukodystrophy (replacement
protein: e.g., arylsulphatase A), LAL deficiency (replacement
protein: e.g., lysosomal acid lipase (LAL)), sucraseisomaltase
deficiency (replacement protein: e.g., sucrose-isomaltase), ADA
deficiency (replacement protein: e.g., adenosine deaminase (ADA)),
primary IGF-1 deficiency (replacement protein: e.g., insulin-like
growth factor 1 (IGF-1)), hypophosphatasia (replacement protein:
e.g., alkaline phosphatase), and acute intermittent porphyria
(replacement protein: e.g., porphobilinogen deaminase).
[0349] Illustrative applications of genome engineering therapy for
the RNA or pharmaceutical compositions of the present invention
include the treatment (including prophylactic treatment) of a
condition, disorder or disease selected from the group consisting
of X-linked severe combined immunodeficiency (X-SCID) (correction
with DNA encoding the interleukin-2 receptor common gamma chain
(IL-2Ry)), Xeroderma pigmentosum (correction with native, i.e.,
unmutated DNA), and the conditions, disorders and diseases
specified above with respect to illustrative applications of
protein replacement therapy. A further genome engineering therapy
for the RNA or pharmaceutical compositions of the present invention
includes genome editing making use of, e.g., CRISPR/CAS.
[0350] Illustrative applications of genetic reprogramming therapy
for the RNA or pharmaceutical compositions of the present invention
include the treatment (including prophylactic treatment) of any of
the conditions, disorders and diseases specified above with respect
to illustrative applications of protein replacement therapy and/or
illustrative applications of genome engineering therapy.
[0351] Illustrative immunotherapeutic applications for the
pharmaceutical compositions of the present invention include the
treatment (including prophylactic treatment) of a condition,
disorder or disease selected from the group consisting of
infectious diseases (e.g., those caused by a pathogen such as
viruses (such as influenza virus (A, B, or C), CMV, or RSV),
bacteria, fungi or other microorganisms); an undesirable
inflammation (such as an immune disorder); and cancer.
[0352] Cancer (medical term: malignant neoplasm) is a class of
diseases in which a group of cells display uncontrolled growth
(division beyond the normal limits), invasion (intrusion on and
destruction of adjacent tissues), and sometimes metastasis (spread
to other locations in the body via lymph or blood). These three
malignant properties of cancers differentiate them from benign
tumors, which are self-limited, and do not invade or metastasize.
Most cancers form a tumor, i.e., a swelling or lesion formed by an
abnormal growth of cells (called neoplastic cells or tumor cells),
but some, like leukemia, do not. The term "cancer" according to the
invention comprises leukemias, seminomas, melanomas, teratomas,
lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial
cancer, kidney cancer, adrenal cancer, thyroid cancer, blood
cancer, skin cancer, cancer of the brain, cervical cancer,
intestinal cancer, liver cancer, colon cancer, stomach cancer,
intestine cancer, head and neck cancer, gastrointestinal cancer,
lymph node cancer, esophagus cancer, colorectal cancer, pancreas
cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate
cancer, cancer of the uterus, ovarian cancer and lung cancer and
the metastases thereof. Examples thereof are lung carcinomas, mamma
carcinomas, prostate carcinomas, colon carcinomas, renal cell
carcinomas, cervical carcinomas, or metastases of the cancer types
or tumors described above. The term cancer according to the
invention also comprises cancer metastases.
[0353] Examples of cancers treatable with the RNA and
pharmaceutical compositions of the present invention include
malignant melanoma, all types of carcinoma (colon, renal cell,
bladder, prostate, non-small cell and small cell lung carcinoma,
etc.), lymphomas, sarcomas, blastomas, gliomas, etc.
[0354] Malignant melanoma is a serious type of skin cancer. It is
due to uncontrolled growth of pigment cells, called
melanocytes.
[0355] According to the invention, a "carcinoma" is a malignant
tumor derived from epithelial cells. This group represents the most
common cancers, including the common forms of breast, prostate,
lung and colon cancer.
[0356] Lymphoma and leukemia are malignancies derived from
hematopoietic (blood-forming) cells.
[0357] A sarcoma is a cancer that arises from transformed cells in
one of a number of tissues that develop from embryonic mesoderm.
Thus, sarcomas include tumors of bone, cartilage, fat, muscle,
vascular, and hematopoietic tissues.
[0358] Blastic tumor or blastoma is a tumor (usually malignant)
which resembles an immature or embryonic tissue. Many of these
tumors are most common in children.
[0359] A glioma is a type of tumor that starts in the brain or
spine. It is called a glioma because it arises from glial cells.
The most common site of gliomas is the brain.
[0360] By "metastasis" is meant the spread of cancer cells from its
original site to another part of the body. The formation of
metastasis is a very complex process and depends on detachment of
malignant cells from the primary tumor, invasion of the
extracellular matrix, penetration of the endothelial basement
membranes to enter the body cavity and vessels, and then, after
being transported by the blood, infiltration of target organs.
Finally, the growth of a new tumor, i.e., a secondary tumor or
metastatic tumor, at the target site depends on angiogenesis. Tumor
metastasis often occurs even after the removal of the primary tumor
because tumor cells or components may remain and develop metastatic
potential. In one embodiment, the term "metastasis" according to
the invention relates to "distant metastasis" which relates to a
metastasis which is remote from the primary tumor and the regional
lymph node system.
[0361] Exemplary immune disorders include, but are not limited to,
autoimmune diseases (for example, diabetes mellitus, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis and psoriatic arthritis), multiple sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosus,
autoimmune thyroiditis, dermatitis (including atopic dermatitis and
eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's
disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
sepsis and septic shock, inflammatory bowel disorder, cutaneous
lupus erythematosus, scleroderma, vaginitis, proctitis, drug
eruptions, leprosy reversal reactions, erythema nodosum leprosum,
autoimmune uveitis, allergic encephalomyelitis, acute necrotizing
hemorrhagic encephalopathy, idiopathic bilateral progressive
sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
glomerulonephritis, idiopathic sprue, lichen planus, Graves'
disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior,
and interstitial lung fibrosis), graft-versus-host disease, cases
of transplantation, and allergy such as, atopic allergy.
[0362] Exemplary viruses include, but are not limited to, are human
immunodeficiency virus (HIV), Epstein-Barr virus (EBV),
cytomegalovirus (CMV) (e.g., CMV5), human herpesviruses (HHV)
(e.g., HHV6, 7 or 8), herpes simplex viruses (HSV), bovine herpes
virus (BHV) (e.g., BHV4), equine herpes virus (EHV) (e.g., EHV2),
human T-Cell leukemia viruses (HTLV)5, Varicella-Zoster virus
(VZV), measles virus, papovaviruses (JC and BK), hepatitis viruses
(e.g., HBV or HCV), myxoma virus, adenovirus, parvoviruses, polyoma
virus, influenza viruses (e.g., influenza virus A, influenza virus
B, or influenza virus C), respiratory syncytial virus (RSV),
papillomaviruses and poxviruses such as vaccinia virus, and
molluscum contagiosum virus (MCV), and lyssaviruses. Such virus may
or may not express an apoptosis inhibitor. Exemplary diseases
caused by viral infection include, but are not limited to, chicken
pox, Cytomegalovirus infections, genital herpes, Hepatitis B and C,
influenza, and shingles, and rabies.
[0363] Exemplary bacteria include, but are not limited to,
Campylobacter jejuni, Enterobacter species, Enterococcus faecium,
Enterococcus faecalis, Escherichia coli (e.g., F. coli O157:H7),
Group A streptococci, Haemophilus influenzae, Helicobacter pylori,
listeria, Mycobacterium tuberculosis, Pseudomonas aeruginosa, S.
pneumoniae, Salmonella, Shigella, Staphylococcus aureus, and
Staphylococcus epidermidis, and Borrelia and Rickettsia. Exemplary
diseases caused by bacterial infection include, but are not limited
to, anthrax, cholera, diphtheria, foodborne illnesses, leprosy,
meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock,
syphilis, tetanus, tuberculosis, typhoid fever, and urinary tract
infection, and Lyme disease and Rocky Mountain spotted fever.
[0364] Particular examples of infectious diseases treatable with
the RNA or pharmaceutical compositions of the present invention
include viral infectious diseases, such as AIDS (HIV), hepatitis A,
B or C, herpes, herpes zoster (chicken-pox), German measles
(rubella virus), yellow fever, dengue fever; infectious diseases
caused by flaviviruses; influenza; infectious diseases caused by
RSV; infectious diseases caused by CMV; hemorrhagic infectious
diseases (Marburg or Ebola viruses); bacterial infectious diseases
(such as Legionnaire's disease (Legionella), gastric ulcer
(Helicobacter), cholera (Vibrio), infections by E. coli,
Staphylococci, Salmonella or Streptococci (tetanus); infections by
protozoan pathogens such as malaria, sleeping sickness,
leishmaniasis, toxoplasmosis, i.e. infections by Plasmodium,
Trypanosoma, Leishmania and Toxoplasma; or fungal infections, which
are caused, e.g., by Cryptococcus neoformans, Histoplasma
capsulatum, Coccidioides immitis, Blastomyces dermatitidis or
Candida albicans.
[0365] For administration according to the invention, in
particular, in the form of a pharmaceutical composition (e.g.,
vaccine composition), RNA may be naked RNA or may be incorporated
in a carrier, for example, liposomes or other particles for gene
transfer, and is preferably in the form of naked RNA.
[0366] The RNA (preferably mRNA) modified with a 5'-cap compound of
the present invention or the pharmaceutical compositions of the
present invention can be used alone or in conjunction with one or
more additional/supplementary active compounds which can be
administered prior to, simultaneously with or after administration
of the RNA or pharmaceutical composition of the present invention.
Such one or more additional/supplementary active compounds include
immunosuppressants (e.g., for applications where the induction of
an immune response is to be avoided or minimized (e.g., in protein
replacement therapies, genome engineering therapies, and genetic
reprogramming therapies, as described herein)), nucleic acids
(e.g., plasmids) comprising a nucleotide sequence encoding a
peptide or protein (in particular in genome engineering therapies,
where, for example, said nucleotide sequence is to be inserted into
the genome of a patient, e.g., in order to replace the
corresponding mutated nucleotide sequence in the genome of the
patient), compounds for cell differentiation (e.g., compounds which
induce the differentiation of cells having stem cell
characteristics into cells expressing a peptide or protein (in
particular a pharmaceutically active peptide or protein), in
particular in genetic reprogramming therapies), chemotherapeutic
drugs for cancer patients (e.g. gemcitabine, etopophos, cis-platin,
carbo-platin), antiviral agents, anti-parasite agents,
anti-bacterial agents, immunotherapeutic agents (e.g., antigens or
fragments thereof (in particular immunogenic fragments thereof)),
and adjuvants, and, if administered simultaneously with the RNA of
the present invention, may be present in a pharmaceutical
composition of the present invention.
[0367] In particular in case of a vaccine composition, the one or
more additional/supplementary active compounds can comprise an
immunotherapeutic agent, preferably an immunotherapeutic agent
inducing or effecting a targeted, i.e., specific, immune reaction.
Thus, in one embodiment, the RNA and pharmaceutical compositions of
the present invention can be used in conjunction with an
immunotherapeutic agent, preferably an immunotherapeutic agent
inducing or effecting a targeted, i.e., specific, immune reaction.
Such immunotherapeutic agents include agents directed against a
disease-associated antigen such as therapeutic antibodies or agents
inducing an immune response directed against a disease-associated
antigen or cells expressing a disease-associated antigen. Useful
immunotherapeutic agents include proteins or peptides inducing a B
cell or T cell response against the disease-associated antigen or
cells expressing the disease-associated antigen. These proteins or
peptides may comprise a sequence essentially corresponding to or
being identical to the sequence of the disease-associated antigen
or one or more fragments thereof. In one embodiment, the protein or
peptide comprises the sequence of an MHC presented peptide derived
from the disease-associated antigen. Instead of administering the
protein or peptide it is also possible to administer nucleic acid,
preferably RNA such as mRNA, encoding the protein or peptide. The
RNA encoding the protein or peptide may be the RNA (preferably
mRNA) modified with a 5'-cap compound of the present invention.
Alternatively or additionally, the RNA encoding the protein or
peptide may be a different RNA not according to the present
invention which RNA may be administered simultaneously with (in
this case the RNA may form part of a pharmaceutical composition of
the invention) and/or prior to and/or after administration of a
pharmaceutical composition of the invention. Accordingly, the
pharmaceutical composition of the present invention may be used in
genetic vaccination, wherein an immune response is stimulated by
introduction into an individual a suitable nucleic acid molecule
(DNA or mRNA) which codes for an antigen or a fragment thereof.
[0368] In one embodiment, a disease-associated antigen is a
tumor-associated antigen. In this embodiment, the RNA (preferably
mRNA) modified with a 5'-cap compound of the present invention and
the pharmaceutical compositions of the present invention may be
useful in treating cancer or cancer metastasis. Preferably, the
diseased organ or tissue is characterized by diseased cells such as
cancer cells expressing a disease-associated antigen and/or being
characterized by association of a disease-associated antigen with
their surface. Immunization with intact or substantially intact
tumor-associated antigen or fragments thereof such as MHC class I
and class II peptides or nucleic acids, in particular mRNA,
encoding such antigen or fragment makes it possible to elicit a MHC
class I and/or a class II type response and thus, stimulate T cells
such as CD8+ cytotoxic T lymphocytes which are capable of lysing
cancer cells and/or CD4+ T cells. Such immunization may also elicit
a humoral immune response (B cell response) resulting in the
production of antibodies against the tumor-associated antigen.
Furthermore, antigen presenting cells (APC) such as dendritic cells
(DCs) can be loaded with MHC class I-presented peptides directly or
by transfection with nucleic acids encoding tumor antigens or tumor
antigen peptides in vitro and administered to a patient.
[0369] According to the present invention, a tumor-associated
antigen preferably comprises any antigen which is characteristic
for tumors or cancers as well as for tumor or cancer cells with
respect to type and/or expression level. In one embodiment, the
term "tumor-associated antigen" relates to proteins that are under
normal conditions, i.e., in a healthy individual, specifically
expressed in a limited number of organs and/or tissues or in
specific developmental stages, for example, the tumor-associated
antigen may be under normal conditions specifically expressed in
stomach tissue, preferably in the gastric mucosa, in reproductive
organs, e.g., in testis, in trophoblastic tissue, e.g., in
placenta, or in germ line cells, and are expressed or aberrantly
expressed in one or more tumor or cancer tissues. In this context,
"a limited number" preferably means not more than 3, more
preferably not more than 2 or 1. The tumor-associated antigens in
the context of the present invention include, for example,
differentiation antigens, preferably cell type specific
differentiation antigens, i.e., proteins that are under normal
conditions specifically expressed in a certain cell type at a
certain differentiation stage, cancer/testis antigens, i.e.,
proteins that are under normal conditions specifically expressed in
testis and sometimes in placenta, and germ line specific antigens.
In the context of the present invention, the tumor-associated
antigen is preferably associated with the cell surface of a cancer
cell and is preferably not or only rarely expressed in normal
tissues. Preferably, the tumor-associated antigen or the aberrant
expression of the tumor-associated antigen identifies cancer cells.
In the context of the present invention, the tumor-associated
antigen that is expressed by a cancer cell in an individual, e.g.,
a patient suffering from a cancer disease, is preferably a
self-protein in said individual. In preferred embodiments, the
tumor-associated antigen in the context of the present invention is
expressed under normal conditions specifically in a tissue or organ
that is non-essential, i.e., tissues or organs which when damaged
by the immune system do not lead to death of the individual, or in
organs or structures of the body which are not or only hardly
accessible by the immune system. In one embodiment, the amino acid
sequence of the tumor-associated antigen is identical between the
tumor-associated antigen which is expressed in normal tissues and
the tumor-associated antigen which is expressed in cancer tissues.
Preferably, a tumor-associated antigen is presented in the context
of MHC molecules by a cancer cell in which it is expressed.
[0370] Examples for differentiation antigens which ideally fulfill
the criteria for tumor-associated antigens as contemplated by the
present invention as target structures in tumor immunotherapy, in
particular, in tumor vaccination are the cell surface proteins of
the claudin family, such as CLDN6 and CLDN18.2. These
differentiation antigens are expressed in tumors of various
origins, and are particularly suited as target structures in
connection with antibody-mediated cancer immunotherapy due to their
selective expression (no expression in a toxicity relevant normal
tissue) and localization to the plasma membrane.
[0371] Particular examples for antigens that may be useful in the
present invention are those explicitly specified herein including
p53 and WT-1.
[0372] The RNA or pharmaceutical compositions according to the
present invention are generally applied in "pharmaceutically
acceptable amounts" and in "pharmaceutically acceptable
preparations". The term "pharmaceutically acceptable" refers to the
non-toxicity of a material which does not interact with the action
of the active agent(s) of the pharmaceutical composition.
[0373] A "therapeutically effective amount" relates to an amount
which--alone or in combination with further dosages--results in a
desired reaction or a desired effect. In the case of the therapy of
a particular disease or a particular condition, the desired
reaction relates to the inhibition of the progress of the disease.
This comprises the deceleration of the progress of the disease, in
particular a disruption of the progression of the disease. The
desired reaction for a therapy of a disease or a condition may also
be the retardation of the occurrence or the inhibition of the
occurrence of the disease or the condition. An effective amount of
the composition according to the present invention is dependent on
the condition or disease, the severity of the disease, the
individual parameters of the patient, including age, physiological
condition, height and weight, the duration of the treatment, the
type of an optionally accompanying therapy, the specific
administration route, and similar factors. In case the reaction of
a patient is insufficient with an initial dosage, higher dosages
(or higher effective dosages which may be achieved by a more
localized administration route) may be applied. In general, for a
treatment or for an induction or increase of an immune reaction in
a human preferably dosages of the RNA in the range of 1 ng to 700
.mu.g, 1 ng to 500 .mu.g, 1 ng to 300 .mu.g, 1 ng to 200 .mu.g, or
1 ng to 100 .mu.g are formulated and administered.
[0374] According to the present invention, the administration of an
RNA (such as mRNA) is either achieved as naked nucleic acid or in
combination with one or more pharmaceutically acceptable
excipients. Preferably, administration of nucleic acids is in the
form of naked nucleic acids. Preferably, the RNA is administered in
combination with stabilizing substances such as RNase inhibitors.
The present invention also envisions the repeated introduction of
nucleic acids into cells to allow sustained expression for extended
time periods. However, due to the presence of the 5'-cap structure
of the present invention and optionally other stabilizing
modifications, the RNAs of the present invention preferably exhibit
the advantage that they can be administered less frequently than
RNAs not containing the 5'-cap structure of the present invention.
Thus, using the RNAs of the present invention preferably provides
the benefit to the patient that, for example with respect to the
protein replacement therapy, less administrations (such as
injections) of RNA (or pharmaceutical compositions) of the
invention are required to achieve the desired effect (e.g., an
expression of the desired peptide or protein in an amount
sufficient to maintain the functions of the patient (e.g., to
maintain the homeostasis of the patient)). Thus, in one embodiment
the RNA of the invention (such as the RNA composition or
pharmaceutical composition of the invention) is administered to a
patient (e.g., by injection, such as intraperitoneal,
intramuscular, or intradermal injection) at most once per day
(i.e., the time period between two administrations is at least 24
h, such as at least 30 h, at least 36 h, or at least 42),
preferably at most once per two days (i.e., the time period between
two administrations is at least 48 h, such as at least 54 h, at
least 60, or at least 66 h), preferably at most once per three days
(i.e., the time period between two administrations is at least 72
h, such as at least 78 h, at least 84 h, or at least 90 h) or at
most once per four days (i.e., the time period between two
administrations is at least 96 h, such as at least 102 h, at least
108 h, or at least 114 h). Accordingly, the present invention is
particularly beneficial for chronic patients and/or long-term
patients, e.g., patients who are treated over an extended period of
time, e.g., who receive the RNA of the invention (such as the RNA
composition or pharmaceutical composition of the invention) over an
extended period of time, wherein the extended period of time
preferably is at least 1 week, such as at least 2 weeks, at least 3
weeks, at least 4 weeks, at least 1 month, at least 2 months, at
least 3 months, at least 4 months, at least 5 months, at least 6
months, at least 12 months, at least 2 years, at least 3 years, at
least 4 years, at least 5 years, or at least 10 years, e.g., up to
2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2
months, up to 3 months, up to 4 months, up to 5 months, up to 6
months, up to 12 months, up to 2 years, up to 3 years, or up to 4
years, up to 5 years, up to 10 years, or the entire life of the
patient. Thus, in one embodiment the RNA of the invention (such as
the RNA composition or pharmaceutical composition of the invention)
is administered to a chronic patient or long-term patient (e.g., by
injection, such as intraperitoneal, intramuscular, or intradermal
injection) at most once per day (i.e., the time period between two
administrations is at least 24 h, such as at least 30 h, at least
36 h, or at least 42), preferably at most once per two days (i.e.,
the time period between two administrations is at least 48 h, such
as at least 54 h, at least 60, or at least 66 h), preferably at
most once per three days (i.e., the time period between two
administrations is at least 72 h, such as at least 78 h, at least
84 h, or at least 90 h) or at most once per four days (i.e., the
time period between two administrations is at least 96 h, such as
at least 102 h, at least 108 h, or at least 114 h) for an extended
time period, in particular, at least 1 week, such as at least 2
weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at
least 2 months, at least 3 months, at least 4 months, at least 5
months, at least 6 months, at least 12 months, at least 2 years, at
least 3 years, at least 4 years, at least 5 years, or at least 10
years, e.g., up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1
month, up to 2 months, up to 3 months, up to 4 months, up to 5
months, up to 6 months, up to 12 months, up to 2 years, up to 3
years, or up to 4 years, up to 5 years, up to 10 years, or the
entire life of the chronic or long-term patient.
[0375] Cells can be transfected with any excipients (in particular
carriers) with which RNA can be associated, e.g., by forming
complexes with the RNA or forming vesicles in which the RNA is
enclosed or encapsulated, resulting in increased stability of the
RNA compared to naked RNA. Excipients (in particular carriers)
useful according to the invention include, for example,
lipid-containing carriers such as cationic lipids, liposomes, in
particular cationic liposomes, and micelles, and nanoparticles.
Cationic lipids may form complexes with negatively charged nucleic
acids. Any cationic lipid may be used according to the invention.
Furthermore, cells can be taken from an individual, the cells can
be transfected with RNA or a pharmaceutical composition of the
invention, and the transfected cells can be inserted into the
individual.
[0376] Preferably, the introduction of RNA which encodes a peptide
or polypeptide into a cell, in particular into a cell present in
vivo, results in expression of said peptide or polypeptide in the
cell. In particular embodiments, the targeting of the nucleic acids
to particular cells is preferred. In such embodiments, a carrier
which is applied for the administration of the nucleic acid to a
cell (for example, a retrovirus or a liposome), exhibits a
targeting molecule. For example, a molecule such as an antibody
which is specific for a surface membrane protein on the target cell
or a ligand for a receptor on the target cell may be incorporated
into the nucleic acid carrier or may be bound thereto. In case the
nucleic acid is administered by liposomes, proteins which bind to a
surface membrane protein which is associated with endocytosis may
be incorporated into the liposome formulation in order to enable
targeting and/or uptake. Such proteins encompass capsid proteins or
fragments thereof which are specific for a particular cell type,
antibodies against proteins which are internalized, proteins which
target an intracellular location, etc.
[0377] In certain embodiments of the present disclosure, the capped
RNA described herein may be present in RNA lipoplex particles. The
RNA lipoplex particles and compositions comprising RNA lipoplex
particles described herein are useful for delivery of the capped
RNA described herein to a target tissue after parenteral
administration, in particular after intravenous administration. The
RNA lipoplex particles may be prepared using liposomes that may be
obtained by injecting a solution of the lipids in ethanol into
water or a suitable aqueous phase. In one embodiment, the aqueous
phase has an acidic pH. In one embodiment, the aqueous phase
comprises acetic acid, e.g., in an amount of about 5 mM. In one
embodiment, the liposomes and RNA lipoplex particles comprise at
least one cationic lipid and at least one additional lipid. In one
embodiment, the at least one cationic lipid comprises
1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one
embodiment, the at least one additional lipid comprises
1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE),
cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC). In one embodiment, the at least one cationic lipid
comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)
and the at least one additional lipid comprises
1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).
In one embodiment, the liposomes and RNA lipoplex particles
comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)
and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine
(DOPE). Liposomes may be used for preparing RNA lipoplex particles
by mixing the liposomes with RNA. Specific spleen targeting RNA
lipoplex particles are described in WO 2013/143683, herein
incorporated by reference. It has been found that RNA lipoplex
particles having a net negative charge may be used to
preferentially target spleen tissue or spleen cells such as
antigen-presenting cells, in particular dendritic cells.
Accordingly, following administration of the RNA lipoplex
particles, RNA accumulation and/or RNA expression in the spleen
occurs. Thus, RNA lipoplex particles of the disclosure may be used
for expressing RNA in the spleen. In an embodiment, after
administration of the RNA lipoplex particles, no or essentially no
RNA accumulation and/or RNA expression in the lung and/or liver
occurs. In one embodiment, after administration of the RNA lipoplex
particles, RNA accumulation and/or RNA expression in antigen
presenting cells, such as professional antigen presenting cells in
the spleen occurs. Thus, RNA lipoplex particles of the disclosure
may be used for expressing RNA in such antigen presenting cells. In
one embodiment, the antigen presenting cells are dendritic cells
and/or macrophages.
[0378] The term "excipient" when used herein is intended to
indicate all substances in a pharmaceutical composition which are
not active agents (e.g., which are therapeutically inactive
ingredients that do not exhibit any therapeutic effect in the
amount/concentration used), such as, e.g., salts, carriers,
binders, lubricants, thickeners, surface active agents, dispersing
agents, preservatives, emulsifiers, buffering agents, wetting
agents, flavoring agents, colorants, stabilizing agents (such as
RNase inhibitors) or antioxidants all of which are preferably
pharmaceutically acceptable.
[0379] "Pharmaceutically acceptable salts" comprise, for example,
acid addition salts which may, for example, be formed by using a
pharmaceutically acceptable acid such as hydrochloric acid,
sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic
acid, benzoic acid, citric acid, tartaric acid, carbonic acid or
phosphoric acid. Furthermore, suitable pharmaceutically acceptable
salts may include alkali metal salts (e.g., sodium or potassium
salts); alkaline earth metal salts (e.g., calcium or magnesium
salts); ammonium (NH.sub.4.sup.+); and salts formed with suitable
organic ligands (e.g., quaternary ammonium and amine cations formed
using counteranions such as halide, hydroxide, carboxylate,
sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate).
Illustrative examples of pharmaceutically acceptable salts include,
but are not limited to, acetate, adipate, alginate, arginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate,
bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate,
camphorate, camphorsulfonate, camsylate, carbonate, chloride,
citrate, clavulanate, cyclopentanepropionate, digluconate,
dihydrochloride, dodecylsulfate, edetate, edisylate, estolate,
esylate, ethanesulfonate, formate, fumarate, galactate,
galacturonate, gluceptate, glucoheptonate, gluconate, glutamate,
glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,
hexanoate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate,
hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate,
lactobionate, laurate, lauryl sulfate, malate, maleate, malonate,
mandelate, mesylate, methanesulfonate, methylsulfate, mucate,
2-naphthalenesulfonate, napsylate, nicotinate, nitrate,
N-methylglucamine ammonium salt, oleate, oxalate, pamoate
(embonate), palmitate, pantothenate, pectinate, persulfate,
3-phenylpropionate, phosphate/diphosphate, phthalate, picrate,
pivalate, polygalacturonate, propionate, salicylate, stearate,
sulfate, suberate, succinate, tannate, tartrate, teoclate,
tosylate, triethiodide, undecanoate, valerate, and the like (see,
for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm.
Sci., 66, pp. 1-19 (1977)). Salts which are not pharmaceutically
acceptable may be used for preparing pharmaceutically acceptable
salts and are included in the invention.
[0380] The compositions according to the present invention may
comprise a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
The "pharmaceutically acceptable carrier" may be in the form of a
solid, semisolid, liquid, or combinations thereof.
[0381] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions, sterile non-aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. The use of such
media and agents for pharmaceutically active agents is known in the
art. Except insofar as any conventional media or agent is
incompatible with the active agent, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Exemplary pharmaceutically acceptable carriers for an injectable
formulation include water, an isotonic buffered saline solution
(e.g., Ringer or Ringer lactate), ethanol, polyols (e.g.,
glycerol), polyalkylene glycols (e.g., propylene glycol and liquid
polyethylene glycol), hydrogenated naphthalenes, and, in
particular, biocompatible lactide polymers (e.g., lactide/glycolide
copolymers or polyoxyethylene/polyoxy-propylene copolymers).
[0382] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0383] Suitable buffering agents for use in the pharmaceutical
compositions of the invention include acetic acid in a salt, citric
acid in a salt, boric acid in a salt and phosphoric acid in a
salt.
[0384] Suitable preservatives for use in the pharmaceutical
compositions of the invention include various antibacterial and
antifungal agents, such as benzalkonium chloride, chlorobutanol,
paraben, sorbic acid, and thimerosal. Prevention of the presence of
microorganisms may also be ensured by sterilization procedures
(e.g., sterilization filtration, in particular sterilization
microfiltration).
[0385] The pharmaceutical composition of the invention may be
administered to an individual by any route, preferably
parenterally. The expressions "parenteral administration" and
"administered parenterally" as used herein mean modes of
administration other than enteral administration ("enteral
administration" and "administered enterally" as used herein mean
that the drug administered is taken up by the stomach and/or the
intestine). Parenteral administration is usually by injection
and/or infusion and includes, without limitation, intravenous,
intramuscular, intraarterial, intrathecal, intracapsular,
intraosseous, intraorbital, intracardiac, intranodal, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, intracerebral,
intracerebroventricular, subarachnoid, intraspinal, epidural
intrasternal, and topical administration. For applications other
than immunotherapy (e.g., for protein replacement therapy, genome
engineering therapy, or genetic reprogramming therapy), it is
preferred that the pharmaceutical composition of the invention is
administered intraperitoneally, intramuscularly, or intradermally.
For immunotherapeutical applications, it is preferred that the
pharmaceutical composition of the invention is administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intralymphaticly, intradermally or intranodally, more preferably
intradermally or intranodally, e.g., by intranodal injection.
[0386] The pharmaceutical composition of the present invention can
be administered by a variety of methods known in the art. As will
be appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
[0387] The active agents (i.e., the RNA of the invention and
optionally one or more additional/supplementary active compounds)
can be prepared with carriers that will protect the compounds
against rapid release, such as a controlled release formulation,
including implants, transdermal patches, and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
the preparation of such formulations are generally known to those
skilled in the art. See, e.g., Sustained and Controlled Release
Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,
New York, 1978.
[0388] To administer the active agent (i.e., the RNA of the
invention and optionally one or more additional/supplementary
active compounds) by certain routes of administration, it may be
necessary to coat the active agent with, or co-administer the
compound with, a material to prevent its inactivation and/or to
increase the effectiveness of the active agent (in particular the
RNA of the invention) to be translated. For example, the active
agent may be administered to an individual in an appropriate
carrier, for example, lipid-containing carriers (in particular
cationic lipids), liposomes (such as water-in-oil-in-water CGF
emulsions as well as conventional liposomes (Strejan et al., J.
Neuroimmunol. 7: 27 (1984)), in particular cationic liposomes),
micelles, nanoparticles in which the RNA is enclosed or
encapsulated, or a diluent. Pharmaceutically acceptable diluents
include saline and aqueous buffered solutions.
[0389] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, vegetable oils, such as olive
oil, and injectable organic esters, such as ethyl oleate. The
proper fluidity can be maintained, for example, by the use of a
coating material such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the pharmaceutical
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0390] Generally, dispersions are prepared by incorporating the
active agent into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying (lyophilization)
that yield a powder of the active agent plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0391] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate
pharmaceutical compositions in unit dosage form for ease of
administration and uniformity of dosage. Unit dosage form as used
herein refers to physically discrete units suited as unitary
dosages for the individuals to be treated; each unit contains a
predetermined quantity of active agent calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the unit dosage forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active agent and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active agent for the treatment of
sensitivity in individuals. The amount of active agent (in
particular, the amount of RNA) which can be combined with a carrier
material to produce a pharmaceutical composition (such as a single
dosage form) will vary depending upon the individual being treated,
and the particular mode of administration. The amount of active
agent which can be combined with a carrier material to produce a
single dosage form will generally be that amount of the composition
which produces a therapeutic effect.
[0392] Generally, out of 100% (for the pharmaceutical
formulations/compositions), the amount of active agent (in
particular, the amount of the RNA of the present invention,
optionally together with one or more additional/supplementary
active compounds, if present in the pharmaceutical
formulations/compositions) will range from about 0.01% to about
99%, preferably from about 0.1% to about 70%, most preferably from
about 1% to about 30%, wherein the reminder is preferably composed
of the one or more pharmaceutically acceptable excipients.
[0393] The amount of active agent, e.g., an RNA of the invention,
in a unit dosage form and/or when administered to an individual or
used in therapy, may range from about 0.001 mg to about 1000 mg
(for example, from about 0.01 mg to about 500 mg, from about 0.1 mg
to about 100 mg such as from about 1 mg to about 50 mg) per unit,
administration or therapy. In certain embodiments, a suitable
amount of such active agent may be calculated using the mass or
body surface area of the individual, including amounts of between
about 0.1 mg/kg and 10 mg/kg (such as between about 0.2 mg/kg and 5
mg/kg), or between about 0.1 mg/m.sup.2 and about 400 mg/m.sup.2
(such as between about 0.3 mg/m.sup.2 and about 350 mg/m.sup.2 or
between about 1 mg/m.sup.2 and about 200 mg/m.sup.2).
[0394] Regardless of the route of administration selected, the
active agents (i.e., the RNA and optionally one or more
additional/supplementary active compounds), which may be used in a
suitable hydrated form, and/or the pharmaceutical compositions of
the present invention, are formulated into pharmaceutically
acceptable dosage forms by conventional methods known to those of
skill in the art (cf., e.g., Remington, "The Science and Practice
of Pharmacy" edited by Allen, Loyd V., Jr., 22.sup.nd edition,
Pharmaceutical Sciences, September 2012; Ansel et al.,
"Pharmaceutical Dosage Forms and Drug Delivery Systems", 7.sup.th
edition, Lippincott Williams & Wilkins Publishers, 1999.).
[0395] Actual dosage levels of the active agents in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active agent which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. The selected dosage level will depend upon a variety of
pharmacokinetic factors including the activity of the particular
compositions of the present invention employed, the route of
administration, the time of administration, the rate of excretion
of the particular active agent being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0396] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start with doses of the active agents employed
in the pharmaceutical composition at levels lower than that
required in order to achieve the desired therapeutic effect and
gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a pharmaceutical composition
of the invention will be that amount of the active agent which is
the lowest dose effective to produce a therapeutic effect. Such an
effective dose will generally depend upon the factors described
above. It is preferred that administration be parenteral, such as
intravenous, intramuscular, intraperitoneal, or subcutaneous,
preferably administered proximal to the site of the target. The
administration can also be intra-tumoral. If desired, the effective
daily dose of a pharmaceutical composition may be administered as
two, three, four, five, six or more sub-doses administered
separately at appropriate intervals throughout the day, optionally,
in unit dosage forms. While it is possible for an active agent (in
particular RNA) of the present invention to be administered alone,
it is preferable to administer the active agent as a pharmaceutical
formulation/composition.
[0397] In one embodiment, the RNA or pharmaceutical compositions of
the invention may be administered by infusion, preferably slow
continuous infusion over a long period, such as more than 24 hours,
in order to reduce toxic side effects. The administration may also
be performed by continuous infusion over a period of from 2 to 24
hours, such as of from 2 to 12 hours. Such regimen may be repeated
one or more times as necessary, for example, after 6 months or 12
months.
[0398] The pharmaceutical composition of the invention can be
formulated for parenteral administration by injection, for example,
by bolus injection or continuous infusion. Formulations for
injection can be presented in units dosage form (e.g., in phial, in
multi-dose container), and with an added preservative. The
pharmaceutical composition of the invention can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing,
or dispersing agents. Alternatively, the agent can be in powder
form for constitution with a suitable vehicle (e.g., sterile
pyrogen-free water) before use. Typically, pharmaceutical
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the
pharmaceutical composition can also include a solubilizing agent
and a local anesthetic such as lignocaine to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilised powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the pharmaceutical composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients can be mixed prior to administration.
[0399] Pharmaceutical compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
pharmaceutical composition of the invention can be administered
with a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or U.S. Pat. No. 4,596,556.
Examples of well-known implants and modules useful in the present
invention include those described in: U.S. Pat. No. 4,487,603,
which discloses an implantable micro-infusion pump for dispensing
medication at a controlled rate; U.S. Pat. No. 4,486,194, which
discloses a therapeutic device for administering medicaments
through the skin; U.S. Pat. No. 4,447,233, which discloses a
medication infusion pump for delivering medication at a precise
infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable
flow implantable infusion apparatus for continuous drug delivery;
U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery
system having multi-chamber compartments; and U.S. Pat. No.
4,475,196, which discloses an osmotic drug delivery system.
[0400] Many other such implants, delivery systems, and modules are
known to those skilled in the art. In certain embodiments, RNA or
pharmaceutical compositions of the invention can be formulated to
ensure proper distribution in vivo. For example, the blood-brain
barrier (BBB) excludes many highly hydrophilic compounds. To ensure
that the RNA or pharmaceutical compositions of the invention cross
the BBB (if desired), they can be formulated, for example, in
liposomes. For methods of manufacturing liposomes, see, e.g., U.S.
Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may
comprise one or more moieties which are selectively transported
into specific cells or organs, and thus enhance targeted drug
delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:
685). Exemplary targeting moieties include folate or biotin (see,
e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa
et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357: 140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); and
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233: 134).
[0401] In one embodiment of the invention, the RNA of the invention
is formulated in liposomes. In a more preferred embodiment, the
liposomes include a targeting moiety. In a most preferred
embodiment, the RNA in the liposomes is delivered by bolus
injection to a site proximal to the desired area. Such
liposome-based composition should be fluid to the extent that easy
syringability exists, should be stable under the conditions of
manufacture and storage and should be preserved against the
contaminating action of microorganisms such as bacteria and
fungi.
[0402] A "therapeutically effective amount" for treatment can be
measured by objective responses which can either be complete or
partial. A complete response (CR) is defined as no clinical,
radiological or other evidence of a condition, disorder or disease.
A partial response (PR) results from a reduction in disease of
greater than 50%. Median time to progression is a measure that
characterizes the durability of the objective response.
[0403] A "therapeutically effective amount" for treatment can also
be measured by its ability to stabilize the progression of a
condition, disorder or disease, e.g., by using appropriate animal
model systems and/or in vitro assays known to the skilled person. A
therapeutically effective amount of an active agent (in particular
RNA of the invention) refers to the amount which achieves a desired
reaction or a desired effect alone or together with further doses.
In the case of treatment of a particular disease or of a particular
condition, the desired reaction preferably relates to inhibition of
the course of the disease. This comprises slowing down the progress
of the disease and, in particular, interrupting or reversing the
progress of the disease. The desired reaction in a treatment of a
disease or of a condition may also be delay of the onset or a
prevention of the onset of said disease or said condition. Thus, a
therapeutically effective amount of an active agent can cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the condition, disorder or disease or the symptoms of the
condition, disorder or disease or the predisposition toward the
condition, disorder or disease in an individual. One of ordinary
skill in the art would be able to determine such amounts based on
such factors as the disease, disorder or condition to be treated,
the severity of the disease, disorder or condition, the parameters
of the individual to be treated (including age, physiological
condition, size and weight), the duration of treatment, the type of
an accompanying therapy (if present), the specific route of
administration and similar factors. Accordingly, the doses
administered of the active agents described herein may depend on
various of such parameters. In the case that a reaction in an
individual/patient is insufficient with an initial dose, higher
doses (or effectively higher doses achieved by a different, more
localized route of administration) may be used.
[0404] The pharmaceutical composition of the present invention may
take the form of a vaccine preparation comprising the RNA of the
invention encoding at least one antigen such as an antigen as
discussed above or an fragment thereof (in particular an
immunogenic fragment thereof).
[0405] The pharmaceutical composition of the invention can also, if
desired, be presented in a pack, kit or dispenser device which can
contain one or more unit dosage forms containing the active agent
(i.e., the RNA and optionally one or more additional/supplementary
active compounds). The pack can for example comprise metal or
plastic foil, such as blister pack. The pack, kit or dispenser
device can be accompanied with instruction for administration.
[0406] The one or more additional/supplementary active compounds
may comprise an immunomodulating agent such as anti-CTL-A4 or
anti-PD1 or anti-PDL1 or anti-regulatory T-cell reagents such as an
anti-CD25 antibody or cyclophosphamide.
[0407] The pharmaceutical compositions of the invention may be
administered together with supplementing immunity-enhancing
substances such as one or more adjuvants and may comprise one or
more immunity-enhancing substances to further increase its
effectiveness, preferably to achieve a synergistic effect of
immunostimulation.
[0408] The term "adjuvant" relates to compounds which when
administered in combination with an antigen, an antigen peptide, or
a nucleic acid (such as RNA, preferably mRNA) encoding said antigen
or antigen peptide to an individual prolongs or enhances or
accelerates the immune response. In the context of the present
invention, RNA (preferably mRNA) may be administered with any
adjuvants. It is assumed that adjuvants exert their biological
activity by one or more mechanisms, including an increase of the
surface of the antigen, a prolongation of the retention of the
antigen in the body, a retardation of the antigen release,
targeting of the antigen to macrophages, increase of the uptake of
the antigen, enhancement of antigen processing, stimulation of
cytokine release, stimulation and activation of immune cells such
as B-cells, macrophages, dendritic cells, T-cells and unspecific
activation of immune cells. For example, compounds which allow the
maturation of the DCs, e.g. lipopolysaccharides or CD40 ligand,
form a class of suitable adjuvants. Generally, any agent which
influences the immune system of the type of a "danger signal" (LPS,
GP96, dsRNA etc.) or cytokines, such as GM-CSF, can be used as an
adjuvant which enables an immune response to be intensified and/or
influenced in a controlled manner. CpG oligodeoxynucleotides (Krieg
et al., 1995, Nature 374: 546-549) can optionally also be used in
this context. Further types of adjuvants include oil emulsions
(e.g., Freund's adjuvants), mineral compounds (such as alum),
bacterial products (such as Bordetella pertussis toxin), liposomes,
immune-stimulating complexes, cytokines (e.g., monokines,
lymphokines, interleukins or chemokines, such as IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-.alpha.,
IFN-.gamma., GM-CSF, LT-.alpha., or growth factors, e.g. hGH),
lipopeptides (e.g., Pam3Cys). In case the RNA (preferably mRNA) of
the invention in one embodiment may encode an immunostimulating
agent and said immunostimulating agent encoded by said RNA is to
act as the primary immunostimulant, however, only a relatively
small amount of CpG DNA is necessary (compared with
immunostimulation with only CpG DNA). Examples for adjuvants are
monophosphoryl-lipid-A (MPL SmithKline Beecham). Saponins such as
QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739),
QS7, QS17, QS18, and QS-L1 (So et al., 1997, Mol. Cells 7:
178-186), incomplete Freund's adjuvants, complete Freund's
adjuvants, vitamin E, montanid, alum, CpG oligonucleotides, and
various water-in-oil emulsions which are prepared from biologically
degradable oils such as squalene and/or tocopherol. Particularly
preferred adjuvants are cytokines, such as monokines, lymphokines,
interleukins or chemokines, e.g. IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-.alpha., IFN-.gamma.,
GM-CSF, LT-.alpha., growth factors, e.g. hGH or lipopeptides, such
as Pam3Cys, all of which are suitable for use as adjuvants in the
pharmaceutical compositions of the present invention or when RNA
(in particular mRNA) of the present invention is used in
therapy.
[0409] Treatment may be provided at home, the doctor's office, a
clinic, a hospital's outpatient department, or a hospital.
Treatment generally begins under medical supervision so that
medical personnel can observe the treatment's effects closely and
make any adjustments that are needed. The duration of the treatment
depends on the age and condition of the patient, as well as how the
patient responds to the treatment.
[0410] A person having a greater risk of developing a condition,
disorder or disease may receive prophylactic treatment to inhibit
or delay symptoms of the condition, disorder or disease.
[0411] The term "treatment" is known to the person of ordinary
skill, and includes the application or administration of an active
agent (e.g., a pharmaceutical composition containing said active
agent) as described herein (e.g., RNA such as mRNA or a
pharmaceutical composition comprising RNA such as mRNA) or
procedure to an individual/patient or application or administration
of an active agent (e.g., a pharmaceutical composition containing
said active agent) as described herein (e.g., RNA such as mRNA or a
pharmaceutical composition comprising RNA such as mRNA) or
procedure to a cell, cell culture, cell line, sample, tissue or
organ isolated from an individual, who has a condition, disorder or
disease, a symptom of the condition, disorder or disease or a
predisposition toward a condition, disorder or disease, with the
purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve, affect or prevent the condition, disorder or
disease, the symptoms of the condition, disorder or disease or the
predisposition toward the condition, disorder or disease (e.g., to
prevent or eliminate a disease, including reducing the size of a
tumor or the number of tumors in an individual; arrest or slow a
disease in an individual; inhibit or slow the development of a new
disease in an individual; decrease the frequency or severity of
symptoms and/or recurrences in an individual who currently has or
who previously has had a disease; and/or prolong, i.e. increase the
lifespan of the individual). In particular, the term "treatment of
a disease" includes curing, shortening the duration, ameliorating,
preventing, slowing down or inhibiting progression or worsening, or
preventing or delaying the onset of a disease or the symptoms
thereof. Hence, the term "treatment" can include prophylactic
treatment of a condition, disorder or disease, or the symptom of a
condition, disorder or disease. An active agent, when used in
treatment, includes the RNA of the invention as well as the one or
more additional/supplementary active compounds described herein and
includes, but is not limited to, other therapeutically active
compounds that may be small molecules, peptides, peptidomimetics,
polypeptides/proteins, antibodies, other polynucleotides such as
DNA or dsRNA, cells, viruses, ribozymes, and antisense
oligonucleotides.
[0412] In a preferred embodiment, the pharmaceutical composition of
the invention is substantially free of dsRNA, preferably
substantially free of dsRNA and DNA.
[0413] The term "substantially free of dsRNA" as used herein in
conjunction with an RNA preparation comprising RNA modified with a
5'-cap compound of the present application, in particular a
pharmaceutical composition, especially a pharmaceutical composition
comprising an RNA modified with a 5'-cap compound of the present
application, means that the amount of dsRNA in the RNA preparation
or pharmaceutical composition is such that said RNA preparation or
pharmaceutical composition when administered to an individual does
not substantially induce an undesired response (such as an
undesired induction of inflammatory cytokines (e.g., IFN-.alpha.)
and/or an undesired activation of effector enzyme leading to an
inhibition of protein synthesis from the RNA of the invention) in
said individual. Preferably, the terms "substantially free of
dsRNA" and "does not substantially induce an undesired response"
mean that, when administered to an individual, said RNA preparation
or pharmaceutical composition results in the translation of the RNA
into the peptide or protein for at least 10 h (e.g., at least 12 h,
at least 14 h, at least 16 h, at least 18 h, at least 20 h, at
least 22 h, at least 24 h, at least 30 h, at least 36 h, at least
42 h, at least 48 h, at least 54 h, at least 60 h, at least 66 h,
at least 72 h, at least 78 h, at least 84 h, at least 90 h, or at
least 96 h) after administration. For example, the content of dsRNA
in the RNA preparation or pharmaceutical composition may be at most
5% by weight (preferably at most 4% by weight, at most 3% by
weight, at most 2% by weight, at most 1% by weight, at most 0.5% by
weight, at most 0.1% by weight, at most 0.05% by weight, at most
0.01% by weight, at most 0.005% by weight, at most 0.001% by
weight), based on the total weight of said RNA preparation or
pharmaceutical composition.
[0414] The term "substantially free of DNA" as used herein in
conjunction with an RNA preparation comprising RNA modified with a
5'-cap compound of the present application, in particular a
pharmaceutical composition, especially a pharmaceutical composition
comprising an RNA modified with a 5'-cap compound of the present
application, means that the amount of DNA in the RNA preparation or
pharmaceutical composition may be at most 5% by weight (preferably
at most 4% by weight, at most 3% by weight, at most 2% by weight,
at most 1% by weight, at most 0.5% by weight, at most 0.1% by
weight, at most 0.05% by weight, at most 0.01% by weight, at most
0.005% by weight, at most 0.001% by weight), based on the total
weight of said RNA preparation or pharmaceutical composition.
[0415] The term "substantially free of dsRNA and DNA" as used
herein in conjunction with an RNA preparation comprising RNA
modified with a 5'-cap compound of the present application, in
particular a pharmaceutical composition, especially a
pharmaceutical composition comprising an RNA modified with a 5'-cap
compound of the present application, means that said RNA
preparation or pharmaceutical composition is substantially free of
dsRNA as specified above (e.g., the translation lasts at least 10 h
after administration and/or the dsRNA content is at most 5% by
weight) and is substantially free of DNA as specified above (e.g.,
the DNA content is at most 5% by weight).
[0416] In one embodiment, the pharmaceutical composition of the
invention is a vaccine composition.
[0417] The vaccine composition of the present invention may be
regarded as a pharmaceutical composition of the present invention
for a particular use (i.e., vaccination). Thus, one or more of the
features and embodiments described above in connection with the
pharmaceutical composition of the present invention (e.g.,
administration route; presence of other components (such as one or
more pharmaceutically acceptable carriers, excipients, and/or
diluents and/or adjuvants and/or one or more
additional/supplementary active compounds); amount of active
agent(s); pharmaceutically acceptable salts; etc.) may also apply
to the vaccine composition of the present invention.
[0418] In a preferred embodiment, said vaccine composition further
comprises one or more pharmaceutically acceptable carriers,
excipients, and/or diluents. Said vaccine composition may further
comprise compounds or substances which are capable of enhancing
and/or supporting an immune reaction in an individual. For example,
the vaccine composition of the present invention may further
comprise an adjuvant as described above or cytokines, for example,
interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating
factor (GM-CSF), or interleukin-18 (IL-18). Furthermore, the
vaccine composition according to the present invention may further
comprise RNA stabilizing substances such as RNase inhibitors,
pharmaceutically acceptable salts or buffers, preservatives (such
as benzalkonium chloride, chlorbutanol, parabene, or Thimerosal),
wetting agents, emulsifying agents, and/or additional drugs or
active agents.
[0419] In a particularly preferred embodiment, the RNA is present
in the vaccine composition according to the present invention in
the form of naked RNA.
[0420] It is particularly preferred that the vaccine composition of
the present invention is formulated for parenteral administration,
for example, for intravenous, intraperitoneal, intramuscular,
subcutaneous, intralymphatic, intradermal or intranodal
administration, more preferably for intradermal or intranodal
administration, such as intranodal injection. The vaccine
composition of the invention is most preferably formulated for
injection into lymph nodes, preferably inguinal lymph nodes, for
injection into lymphatic vessels and/or the spleen.
[0421] Preferably, the vaccine composition is in the form of an
aqueous or non-aqueous solution which is isotonic with the blood of
the recipient, i.e., the individual to be vaccinated. For example,
Ringer solution, isotonic sodium chloride solution, or phosphate
buffered saline (PBS) may be used. In particular, the vaccine
composition is preferably sterile and comprises the above specified
RNA in a therapeutically effective amount.
[0422] In a preferred embodiment, the vaccine composition is
substantially free of dsRNA, preferably substantially free of dsRNA
and DNA.
[0423] Cells
[0424] In a further aspect, the present invention provides a cell
comprising an RNA which is modified with a 5'-cap compound of the
present application, wherein the RNA preferably comprises a
nucleotide sequence encoding a peptide or protein. In this
preferred embodiment of the cell, where the RNA of the invention
comprises a nucleotide sequence encoding a peptide or protein, the
cell can be used for producing said peptide or protein, e.g., in
the corresponding method for producing a peptide or protein
described herein, or for expressing said peptide or protein in an
individual by administering said cell to the individual, e.g., in
the corresponding method for expressing a peptide or protein
described herein.
[0425] In a preferred embodiment, the cell is an antigen presenting
cell, such as an immature antigen presenting cell, and may be
selected from the group consisting of macrophages, monocytes,
B-cells, and dendritic cells.
[0426] In a particularly preferred embodiment, the cell according
to the present invention is formulated in a pharmaceutical
composition as described above, said pharmaceutical composition
preferably being suitable to express a peptide or protein, such as
a pharmaceutically active peptide or protein. In an alternative
embodiment, the cell according to the present invention is
formulated in a pharmaceutical composition as described above, said
pharmaceutical composition preferably being suitable to elicit an
immune response when administered to an individual, wherein the
immune response is preferably directed against the protein or
peptide encoded by the RNA or an antigen and/or immunogen comprised
by the protein or peptide encoded by the RNA present in the
immature antigen presenting cell of the present invention. Thus,
the present invention provides a pharmaceutical composition
comprising an immature antigen presenting cell according to the
third aspect of the present invention.
[0427] Methods and Uses
[0428] In one aspect, the present invention provides a method for
providing an RNA with a 5'-cap structure, said method comprising
performing a transcription reaction using a template nucleic acid
in the presence of a 5'-cap compound of the first aspect. In one
embodiment, the template nucleic acid is DNA. The transcription
reaction may be performed in vivo or in vitro, but is preferably
performed in vitro. In one embodiment, the transcription reaction
is performed using an RNA polymerase selected from the group
consisting of T3, T7 and SP6 RNA polymerases. The RNA may comprise
a nucleotide sequence encoding a peptide or protein, wherein the
peptide or protein is preferably a pharmaceutically active peptide
or protein as described herein. In one embodiment, the method is
performed in the absence of a 2'-O-ribose methyltransferase. In an
alternative embodiment, the method is performed in the presence of
a 2'-O-ribose methyltransferase.
[0429] In another aspect, the present invention provides a method
of increasing the stability of an RNA in cells and/or for
increasing the expression of an RNA in cells, said method
comprising providing said RNA with the structure according to
formula (I) as defined in the first aspect; and transferring said
RNA modified with the structure according to formula (I) into the
cells. Preferably, said cells are antigen presenting cells, such as
immature antigen presenting cells, preferably selected from the
group consisting of monocytes, macrophages, glia cells, B-cells,
and dendritic cells. In order to assess the stability of an RNA in
an immature antigen presenting cell, the skilled person may detect
the presence of the studied RNA or quantify the amount of RNA
within a cell after certain time points after introduction of said
RNA, for example, by using real time RT-PCR. The expression of an
RNA in cells may be determined using an RNA encoding a marker
protein such as luciferase or green fluorescent protein, preferably
d2EGFP but may be any other marker protein known to the skilled
person, and determining the expression of said marker protein at
certain time points after introduction of the RNA. In one
embodiment, the step of providing said RNA with the structure
according to formula (I) is performed in the absence of a
2'-O-ribose methyltransferase. In an alternative embodiment, the
method is performed in the presence of a 2'-O-ribose
methyltransferase.
[0430] In a further aspect, the present invention provides a method
for producing a peptide or protein of interest comprising the step
of using the RNA, RNA composition or cell of the invention, wherein
in each case the RNA comprises a nucleotide sequence encoding the
peptide or protein. In one embodiment, the peptide or protein is a
pharmaceutically active protein, preferably selected from the group
consisting of cytokines, such as erythropoietin; adhesion
molecules, such as an integrin; immunoglobulins; immunologically
active compounds, e.g., antigens, such as tumor-associated
antigens, pathogen-associated antigens (e.g., one or more (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens of a virus, such as one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens of influenza
virus (A, B, or C), CMV, or RSV), allergens, or autoantigens;
hormones, such as vasopressin, insulin or growth hormone; growth
factors, such as VEGFA; enzymes, such as herpes simplex virus type
1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine
hydroxylase, pseudocholinesterase, pancreatic enzymes, or lactase;
receptors, such as growth factor receptors; protease inhibitors,
such as alpha 1-antitrypsin; apoptosis regulators, such as BAX;
transcription factors, such as FOXP3; tumor suppressor proteins,
such as p53; structural proteins, such as surfactant proteins;
reprogramming factors, such as OCT4, SOX2, c-MYC, KLF4, LIN28, or
NANOG; genomic engineering proteins, such as clustered regularly
spaced short palindromic repeat-CRISPR-associated protein 9
(CRISPR-Cas9); and blood proteins, such as fibrinogen. In the
embodiment of the method using the RNA or RNA composition, the
method may comprise the step of transferring said RNA or RNA
composition into a cell. In this respect, any technique which is
suitable to transfer RNA into cells may be used. Preferably, the
RNA is transfected into cells by standard techniques as described
herein, e.g., calcium phosphate precipitation, DEAE transfection,
electroporation, lipofection, or microinjection. The cell may be
any cell which can be transfected with RNA and is preferably an
antigen presenting cell, such as an immature antigen presenting
cell, more preferably selected from the group consisting of
macrophages, monocytes, B-cells, and dendritic cells. The method
for producing a peptide or protein of interest may be performed in
vivo or in vitro, but is preferably performed in vitro.
[0431] In a further aspect, the present invention provides a method
for expressing a peptide or protein in an individual comprising the
step of administering to said individual the RNA, RNA composition
or cell of the invention, wherein in each case the RNA comprises a
nucleotide sequence encoding a peptide or protein. In one
embodiment, the peptide or protein is a pharmaceutically active
protein, preferably selected from the group consisting of
cytokines, such as erythropoietin; adhesion molecules, such as an
integrin; immunoglobulins; immunologically active compounds, e.g.,
antigens, such as tumor-associated antigens, pathogen-associated
antigens (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10) antigens of a virus, such as one or more (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10) antigens of influenza virus (A, B, or C), CMV,
or RSV), allergens, or autoantigens; hormones, such as vasopressin,
insulin or growth hormone; growth factors, such as VEGFA; enzymes,
such as herpes simplex virus type 1 thymidine kinase (HSV1-TK),
hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase,
pancreatic enzymes, or lactase; receptors, such as growth factor
receptors; protease inhibitors, such as alpha 1-antitrypsin;
apoptosis regulators, such as BAX; transcription factors, such as
FOXP3; tumor suppressor proteins, such as p53; structural proteins,
such as surfactant proteins; reprogramming factors, such as OCT4,
SOX2, c-MYC, KLF4, LIN28, or NANOG; genomic engineering proteins,
such as clustered regularly spaced short palindromic
repeat-CRISPR-associated protein 9 (CRISPR-Cas9); and blood
proteins, such as fibrinogen. The RNA, RNA composition or cell of
the invention may be administered by any route, e.g., those
described above with respect to pharmaceutical compositions of the
invention.
[0432] In further aspects, the present invention provides (i) the
RNA, RNA composition, or cell of the invention for use in therapy,
in particular for use in a method of treating a disease or disorder
in a subject, (ii) a method of treating a disease or disorder in a
subject comprising the step of administering to said subject the
RNA, RNA composition, or cell of the invention; and (iii) the use
of the RNA, RNA composition, or cell of the invention for the
preparation of a medicament for treating a disease or disorder in a
subject, wherein in each of (i) to (iii) the RNA comprises a
nucleotide sequence encoding a peptide or protein which preferably
is a disease-associated peptide or protein. In one embodiment of
these aspects (i) to (iii), the treatment of a disease or disorder
is selected from the group consisting of protein replacement
therapy, genome engineering, genetic reprogramming, and
immunotherapy, as described herein. Preferably, the peptide or
protein is a pharmaceutically active protein, more preferably
selected from the group consisting of cytokines, such as
erythropoietin; adhesion molecules, such as an integrin;
immunoglobulins; immunologically active compounds, e.g., antigens,
such as tumor-associated antigens, pathogen-associated antigens
(e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)
antigens of a virus, such as one or more (e.g., 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10) antigens of influenza virus (A, B, or C), CMV, or
RSV), allergens, or autoantigens; hormones, such as vasopressin,
insulin or growth hormone; growth factors, such as VEGFA; enzymes,
such as herpes simplex virus type 1 thymidine kinase (HSV1-TK),
hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase,
pancreatic enzymes, or lactase; receptors, such as growth factor
receptors; protease inhibitors, such as alpha 1-antitrypsin;
apoptosis regulators, such as BAX; transcription factors, such as
FOXP3; tumor suppressor proteins, such as p53; structural proteins,
such as surfactant proteins; reprogramming factors, such as OCT4,
SOX2, c-MYC, KLF4, LIN28, or NANOG; genomic engineering proteins,
such as clustered regularly spaced short palindromic
repeat-CRISPR-associated protein 9 (CRISPR-Cas9); and blood
proteins, such as fibrinogen. In one embodiment, the disease or
disorder is selected from the diseases and disorders disclosed
herein, e.g., the illustrative diseases and disorders described
herein with respect to protein replacement therapy, genome
engineering therapy, genetic reprogramming therapy, and/or
immunotherapy. The RNA, RNA composition or cell of the invention
may be administered by any route and/or in any regimen or amount,
e.g., by those routes and/or in those regimens and/or amounts
described above with respect to pharmaceutical compositions of the
invention. In one embodiment, the RNA, RNA composition or cell of
the invention is administered to the subject (e.g., by injection,
such as intraperitoneal, intramuscular, or intradermal injection)
at most once per day, preferably at most once per two days,
preferably at most once per three days or at most once per four
days. In one embodiment, the RNA, RNA composition or cell of the
invention is administered to a chronic patient or long-term patient
(e.g., by injection, such as intraperitoneal, intramuscular, or
intradermal injection) for an extended period of time, in
particular at least 1 week, such as at least 2 weeks, at least 3
weeks, at least 4 weeks, at least 1 month, at least 2 months, at
least 3 months, at least 4 months, at least 5 months, at least 6
months, at least 12 months, at least 2 years, at least 3 years, at
least 4 years, at least 5 years, or at least 10 years, e.g., up to
2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2
months, up to 3 months, up to 4 months, up to 5 months, up to 6
months, up to 12 months, up to 2 years, up to 3 years, or up to 4
years, up to 5 years, up to 10 years, or the entire life of the
patient. In one embodiment, the RNA of the invention (such as the
RNA composition or pharmaceutical composition of the invention) is
administered to a chronic patient or long-term patient (e.g., by
injection, such as intraperitoneal, intramuscular, or intradermal
injection) at most once per day (i.e., the time period between two
administrations is at least 24 h, such as at least 30 h, at least
36 h, or at least 42), preferably at most once per two days (i.e.,
the time period between two administrations is at least 48 h, such
as at least 54 h, at least 60, or at least 66 h), preferably at
most once per three days (i.e., the time period between two
administrations is at least 72 h, such as at least 78 h, at least
84 h, or at least 90 h) or at most once per four days (i.e., the
time period between two administrations is at least 96 h, such as
at least 102 h, at least 108 h, or at least 114 h) for an extended
time period, i.e., at least 1 week, such as at least 2 weeks, at
least 3 weeks, at least 4 weeks, at least 1 month, at least 2
months, at least 3 months, at least 4 months, at least 5 months, at
least 6 months, at least 12 months, at least 2 years, at least 3
years, at least 4 years, at least 5 years, or at least 10 years,
e.g., up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month,
up to 2 months, up to 3 months, up to 4 months, up to 5 months, up
to 6 months, up to 12 months, up to 2 years, up to 3 years, or up
to 4 years, up to 5 years, up to 10 years, or the entire life of
the patient.
[0433] In further aspects, the present invention provides the
following:
[0434] (I) Provided is a method for eliciting an immune response in
an individual comprising the step of administering to said
individual the vaccine composition of the second aspect or the
immature antigen presenting cell of the third aspect. Preferably,
said immune response is specifically directed against the protein
or peptide encoded by the RNA comprised by the vaccine composition
or the immature antigen presenting cell of the present invention or
is specifically directed against an antigen comprised by said
protein or peptide. Said immune response may be therapeutic and/or
protective. It is particularly preferred that said vaccine
composition and said immature antigen presenting cells, preferably
immature dendritic cells, are administered parenterally as
specified above for the second aspect of the present invention,
preferably by intranodal injection, preferably by injection into
inguinal lymph nodes. In one embodiment the method is for eliciting
an immune response against a virus, such as against influenza virus
(A, B, or C), CMV, or RSV.
[0435] (II) Provided is a method of increasing a portion of MHC
molecules which present an antigen of interest (e.g., an antigen of
a virus, such as influenza virus (A, B, or C), CMV, or RSV) on the
surface of an antigen presenting cell, said method comprising
providing an RNA comprising a nucleotide sequence encoding a
peptide or protein comprising said antigen of interest (e.g., an
antigen of a virus, such as influenza virus (A, B, or C), CMV, or
RSV) or an antigen peptide thereof, said RNA being modified with
the structure according to formula (I) as defined in the first
aspect; and transferring said RNA modified with the structure
according to formula (I) into an immature antigen presenting cell.
In one embodiment, the step of providing an RNA comprising a
nucleotide sequence encoding a peptide or protein comprising an
antigen of interest (e.g., an antigen of a virus, such as influenza
virus (A, B, or C), CMV, or RSV) or an antigen peptide thereof,
said RNA being modified with the structure according to formula
(I), is performed in the absence of a 2'-O-ribose
methyltransferase. In an alternative embodiment, said step is
performed in the presence of a 2'-O-ribose methyltransferase.
Without being bound to any theory, it is assumed that modifying an
RNA with a 5'-cap compound of the present invention increases the
stability and/or expression of said RNA, in particular within
immature antigen presenting cells, for example, immature dendritic
cells. This increased stability and/or expression leads to an
accumulation of the protein or peptide encoded by said RNA. Said
protein or peptide may comprise an antigen or antigen peptide.
Thus, after processing of said protein antigens or antigen peptides
may be loaded on MHC molecules on the surface of the antigen
presenting cell. Alternatively, said protein or peptide may be
itself an antigen or antigen peptide and may be loaded on MHC
molecules without processing. It is assumed, that an RNA encoding a
particular protein or peptide comprising an antigen or antigen
peptide which has been modified with a 5'-cap compound of the
present invention increases the portion/fraction of MHC molecules
on the cell surface of an antigen presenting cell which present a
peptide derived from the protein or peptide encoded by said RNA
when compared to the same RNA having a conventional 5'-cap
structure, preferably when compared to a reference RNA, such as the
same RNA having an ARCA 5'-cap structure. Since the density of MHC
molecules presenting a particular antigen on the surface of an
antigen presenting cell is decisive for the intensity of the
induced immune response specific for said particular antigen, it is
assumed that increasing the stability of an antigen encoding RNA
which has been introduced into antigen presenting cells leads to an
increased immune response against said particular antigen.
[0436] (III) Provided is a method for stimulating and/or activating
immune effector cells, said method comprising providing an RNA
comprising a nucleotide sequence encoding a peptide or protein
comprising an antigen of interest (e.g., an antigen of a virus,
such as influenza virus (A, B, or C), CMV, or RSV) or an antigen
peptide thereof, said RNA being modified with the structure
according to formula (I) as defined in the first aspect;
transferring said RNA modified with the structure according to
formula (I) into immature antigen presenting cells; and contacting
the antigen presenting cells with the immune effector cells.
Preferably, said immune effector cells are antigen-specifically
activated and/or stimulated. Preferably, by this method, the amount
of antigen-specific effector cells, preferably T-cells, is
increased. Preferably, the immature antigen presenting cells are
immature dendritic cells. In one embodiment, the step of providing
an RNA comprising a nucleotide sequence encoding a peptide or
protein comprising an antigen of interest (e.g., an antigen of a
virus, such as influenza virus (A, B, or C), CMV, or RSV) or an
antigen peptide thereof, said RNA being modified with the structure
according to formula (I), is performed in the absence of a
2'-O-ribose methyltransferase. In an alternative embodiment, said
step is performed in the presence of a 2'-O-ribose
methyltransferase. In a preferred embodiment, the immune effector
cells are T-cells, preferably CD4.sup.+ and/or CD8.sup.+ cells. In
one embodiment, the step of transferring said RNA into immature
antigen presenting cells is performed in vitro by any nucleic acid
transfer method, e.g., a transfection method, known to the skilled
person such as lipofection, electroporation, or microinjection as
described above. In another embodiment, the step of transferring
said RNA into immature antigen presenting cells is performed in
vivo, for example, by administering the RNA to an individual,
preferably by parenteral administration, preferably by
intralymphatic administration, preferably by injection into lymph
node(s), i.e., by intranodal injection, by injection into lymphatic
vessels, or by injection into the spleen. Preferably, said
administration is by intranodal injection of the RNA which is
preferably taken up by immature dendritic cells in the lymph
node(s). The administered RNA is preferably in the form of naked
RNA. In one embodiment, the step of contacting the antigen
presenting cells with the immune effector cells is performed in
vitro, for example, in a tissue culture dish. In another
embodiment, the step of contacting the antigen presenting cells
with the immune effector cells is performed in vivo. In this
embodiment, the step of transferring the RNA into immature antigen
presenting cells may be performed in vitro or in vivo as described
above. For contacting the antigen presenting cells into which the
RNA has been transferred in vitro with immune effector cells in
vivo, the antigen presenting cells are administered to an
individual, preferably by parenteral administration, for example,
by intravenous, intramuscular, subcutaneous, intranodal,
intralymphatic, or intraperitoneal injection, preferably by
injection into the lymphatic system such as by injection into
lymphatic vessel(s), the spleen, and/or lymph node(s), preferably
inguinal lymph node(s). In an embodiment, the method may further
comprise the step of differentiating the immature antigen
presenting cells into mature antigen presenting cells after
transferring the RNA into the immature antigen presenting cells and
before contacting the antigen presenting cells with the immune
effector cells. The differentiation step may be performed in vitro
or in vivo. For example, the RNA may be transferred into the
immature antigen presenting cells, preferably into immature
dendritic cells, the immature antigen presenting cells are
differentiated in vitro, and the differentiated mature antigen
presenting cells, preferably the mature dendritic cells, are
contacted with immune effector cells in vitro or in vivo as
described above, preferably in vivo. The immature antigen
presenting cells into which the RNA is transferred in vitro may be
isolated from an individual, for example a patient to be immunized,
or they may be differentiated from hematopoietic stem cells.
[0437] A stimulation and/or activation of immune effector cells, in
particular of T-cells, is typically associated with expansion,
cytotoxic reactivity, and/or cytokine secretion of the immune
effector cells. Thus, the skilled person may determine whether
immune effector cells are stimulated and/or activated by simple in
vitro tests, typically performed using T cells. Such T cells may be
provided by transformed T cell lines such as T cell hybridomas or T
cells which have been isolated from a mammal such as from a rodent,
e.g., a mouse or a rat. Suitable T cell hybridomas are commercially
available or may be generated by known methods. T cells may be
isolated from a mammal by known methods (cf. Shimonkevitz et al.,
1983, J. Exp. Med. 158: 303-316). A suitable experimental setting
to test for T cell activation and/or stimulation is described below
in steps (1) to (4). T cells express a suitable marker which may be
tested and which indicates T cell activation or modulation of T
cell activity. The murine T cell hybridoma DO11.10 may be used for
this purpose, since said hybridoma expresses interleukin-2 (IL-2)
upon activation. IL-2 concentrations may be determined to verify T
cell activation/stimulation or to determine whether a composition
is capable of modulating the activity of said T cell hybridoma.
This test is performed by the following steps: (1) providing T
cells from a T cell hybridoma or by isolation from a mammal, (2)
cultivating the T cells under conditions which allow for
proliferation, (3) contacting the proliferating T cells with an
antigen presenting cell which has been contacted with an antigen or
a nucleic acid encoding therefore, and (4) testing the T cells for
a marker, for example, the IL-2 production is determined. Cells
which are used for the test are cultured under conditions which
allow for proliferation. For example, the DO11.10 T cell hybridoma
is adequately cultured at 37.degree. C. and 5% CO.sub.2 in complete
medium (RPMI 1640, supplemented with 10% FBS,
penicillin/streptomycin, L-glutamine and 5.times.10.sup.5 M
2-mercaptoethanol). T cell activation signals are provided by
antigen presenting cells which have been loaded with an appropriate
antigenic peptide.
[0438] Alternatively, modulation of T cell activity may be verified
by determining alterations or proliferation of antigen-specific T
cells, which may be measured, for example, by known radiolabeling
methods. For example, a labeled nucleotide may be added to a test
culture medium. The incorporation of such labeled nucleotides into
the DNA may serve as indicator for T cell proliferation. This test
is not applicable for T cells that do not require antigen
presentation for their proliferation such as T cell hybridomas.
This test is useful for determining modulation of T cell activity
in the case of untransformed T-cells which have been isolated from
a mammal.
[0439] (IV) Provided is a method for inducing an immune response in
an individual, said method comprising providing an RNA comprising a
nucleotide sequence encoding a peptide or protein comprising an
antigen of interest (e.g., an antigen of a virus, such as influenza
virus (A, B, or C), CMV, or RSV) or an antigen peptide thereof,
said RNA being modified with the structure according to formula (I)
as defined in the first aspect; and administering said RNA modified
with the structure according to formula (I) to said individual. In
one embodiment, the RNA is administered by intranodal injection or
is administered intradermally. The antigen of interest may be any
antigen (e.g., an antigen of a virus (A, B, or C), such as
influenza virus, CMV, or RSV) and is preferably as defined above.
In a preferred embodiment, said RNA is administered in the form of
naked RNA, preferably by parenteral administration, for example, by
intravenous, intramuscular, subcutaneous, intranodal, intradermal,
intralymphatic, or intraperitoneal injection, preferably by
injection into the lymphatic system such as by injection into
lymphatic vessel(s), the spleen, and/or lymph node(s), preferably
inguinal lymph node(s). Preferably, the administered RNA is taken
up by immature dendritic cells of the individual. Preferably, the
immune response is protective and/or therapeutic, for example, is
useful for treating and/or preventing diseases such as cancerous
diseases or infectious diseases. In one embodiment, the step of
providing an RNA comprising a nucleotide sequence encoding a
peptide or protein comprising an antigen of interest (e.g., an
antigen of a virus, such as influenza virus (A, B, or C), CMV, or
RSV) or an antigen peptide thereof, said RNA being modified with
the structure according to formula (I), is performed in the absence
of a 2'-O-ribose methyltransferase.
[0440] In an alternative embodiment, said step is performed in the
presence of a 2'-O-ribose methyltransferase. (V) Provided is a
method for inducing an immune response in an individual, said
method comprising providing an RNA comprising a nucleotide sequence
encoding a peptide or protein comprising an antigen of interest
(e.g., an antigen of a virus, such as influenza virus (A, B, or C),
CMV, or RSV) or an antigen peptide thereof, said RNA being modified
with the structure according to formula (I) as defined in the first
aspect; transferring said RNA modified with the structure according
to formula (I) into immature antigen presenting cells; and
administering the antigen presenting cells to said individual. In
one embodiment, the step of providing an RNA comprising a
nucleotide sequence encoding a peptide or protein comprising an
antigen of interest (e.g., an antigen of a virus, such as influenza
virus (A, B, or C), CMV, or RSV) or an antigen peptide thereof,
said RNA being modified with the structure according to formula
(I), is performed in the absence of a 2'-O-ribose
methyltransferase. In an alternative embodiment, said step is
performed in the presence of a 2'-O-ribose methyltransferase. In
this aspect of the present invention, the RNA is transferred into
immature antigen presenting cells in vitro by any nucleic acid
transfer method, e.g., transfection such as lipofection,
electroporation, or microinjection, known to the skilled person as
described above. Preferably, the immature antigen presenting cells
are immature dendritic cells. The immature antigen presenting cells
into which the RNA is transferred in vitro may be isolated from an
individual, for example, a patient to be immunized, or they may be
differentiated from hematopoietic stem cells, wherein the
hematopoietic stem cells may be isolated from the individual. The
immature antigen presenting cells or the hematopoietic stem cells
may be isolated from the individual by leukapheresis. Preferably,
the immature antigen presenting cells are immature dendritic cells.
Preferably, the immature antigen presenting cells are isolated from
the individual to be immunized, the RNA is transferred into said
isolated cells, and the cells are transferred back to said
individual, preferably by parenteral administration, for example,
by intravenous, intramuscular, subcutaneous, intranodal,
intralymphatic, or intraperitoneal injection, preferably by
injection into the lymphatic system such as by injection into
lymphatic vessel(s), the spleen, and/or lymph node(s), preferably
inguinal lymph node(s).
[0441] The ability to induce an immune reaction, including the
suitability for vaccination against a target disease, may be
readily determined by in vivo tests. For example, a composition,
e.g., a vaccine composition or a pharmaceutical composition, may be
administered to a mammal such as a laboratory animal, e.g., a
mouse, rat, rabbit, etc., and blood samples may be taken from said
animal before administration of the composition and at defined time
points after administration of the composition, for example, 1, 2,
3, 4, 5, 6, 7, and 8 weeks after administration. Serum may be
generated from the blood samples and the development of antibodies
generated upon administration/immunization may be determined. For
example, the concentration of antibodies may be determined.
Furthermore, T cells may be isolated from the blood and/or the
lymphatic system of the mammal, which may be tested for their
reactivity against the antigen used for the immunization. Any
readout system which is known to the skilled person may be used,
for example, proliferation assays, cytokine secretion assays,
assays to test for cytotoxic activity, or tetramer analysis etc.
may be used. Furthermore, the increase of immune reactivity may
also be determined by determining the number of antigen-specific
T-cells, their cytotoxic potential, or their cytokine secretion
pattern as set forth above.
[0442] As demonstrated in the examples of the present application,
the present inventors have surprisingly found that by using the
5'-cap compounds of the present invention it is possible to
incorporate beta-S-ARCA cap1 structures into RNA in one step
thereby combining the positive effect of the thio-substitution with
the cap1-defining 2'-O-methylation.
[0443] The present invention is illustrated by the following
examples which illustrate preferred embodiments of the invention
and should not be interpreted to limit the scope of the present
invention as defined in the claims. Those examples which are not
covered by the appending claims are given for comparative purposes
only.
EXAMPLES
Abbreviations
[0444] h: hour(s) [0445] mM: millimolar (10.sup.-3 mol/l) [0446]
NTP: nucleoside triphosphate
Example 1--Synthesis of Cap Analogs
[0447] To obtain co-transcriptionally capped in vitro transcribed
mRNA, a 5'-cap compound of the invention (Compound 1, a compound of
formula (I)) as shown in FIG. 2 (OR.dbd.OCH.sub.3) was designed.
Compound 1 contains the phosphorothioate substitution at the
beta-position of the 5'-5' triphosphate bridge, a terminal 2'-0
methylation to avoid incorporation in the reverse orientation, an
internal 2'-0 methylation reflecting the cap1 structure plus an
additional guanosine moiety, which allows incorporation by the
phage RNA polymerase. Synthesis and usage of the modified cap
trinucleotide is described in the following.
[0448] 5'-cap compounds of the present invention and other cap
analogs can be synthesized starting from commercially available
oligonucleotides such as (pN).sub.2-4 using standard procedures.
For m.sub.2.sup.7,2-OGppspm.sup.2'-OGpG (Compound 1),
5'-phosphorylated 2'-O-methylated diguanosine 5',3'-dinucleotide
(pm.sup.2'-OGpG) was commercially obtained and used as starting
material without further treatment. The dinucleotide was converted
into the corresponding P-imidazolide derivative (Im-pm.sup.2'-OGpG)
by reacting with 10 equiv. of imidazole in DMF in the presence of 3
equiv. of 2,2'-dithiodipyridine/triphenylphosphine activation
system (cf. FIG. 1; Mukaiyama and Hashimoto 1971). The nucleotide
subunit, 2'-O-methyl guanosine 5'-O-(2-thiodiphosphate)
(m.sub.2.sup.7,2'-OMeGDP.beta.S), was synthesized as described
earlier (Kowalska et al 2008). Then, m.sub.2.sup.7,2'-OMeGDP.beta.S
and the P-imidazolide Im-p.sup.m2'-OGpG were coupled in DMF in
presence of ZnCl.sub.2 excess (8 equiv.) to the cap analog
(m.sub.2.sup.7,2-OGppspm.sup.2'-OGpG, 38% HPLC yield; FIG. 2) as a
mixture of two diastereoisomers (D1 and D2, named according to the
elution order from RP HPLC column). The diastereoisomers were
separated by RP HPLC (Discovery Amide RP C.sub.16 column) into pure
diastereoisomers D1 and D2.
[0449] Spectroscopic Data:
[0450] m.sub.2.sup.7,2'-OGpp.sub.sp.sup.2'-OGpG (Compound 1)
[0451] (D1): .sup.1H NMR (400 MHz, D.sub.2O) .delta. 9.07 (s, 1H),
8.16 (s, 1H), 8.07 (s, 1H), 5.97 (d, J=2.24 Hz, 1H), 5.87 (d,
J=5.98 Hz, 1H), 5.82 (d, J=5.48 Hz, 1H), 4.85-5.00 (m, 1H),
4.74-4.87 (m, 2H, partially overlapped with signal of HDO), 4.61
(t, J=5.35 Hz, 1H), 4.51 (t, J=4.36 Hz, 2H), 4.12-4.48 (m, 9H),
4.07 (s, 3H), 3.58 (s, 3H), 3.43 (s, 3H); .sup.31P NMR (162 MHz,
D.sub.2O) .delta. 30.93 (t, J=26.9 Hz, 1P) -0.59 (br. s., 1P)
-11.94 (d, J=26.9 Hz, 1P) -12.08 (d, J=26.9 Hz, 1P);
[0452] (D2): .sup.1H NMR (400 MHz, D.sub.2O) .delta. 9.08 (s, 1H),
8.16 (s, 1H), 8.10 (s, 1H), 5.89 (d, J=2.49 Hz, 1H), 5.87 (d,
J=5.98 Hz, 1H), 5.82 (d, J=5.98 Hz, 1H), 4.97 (m, 1H), 4.58-4.55
(.about.t, 1H) 4.85-4.73 (m, 2H, overlap with signal of HDO),
4.54-4.47 (m, 2H), 4.13-4.46 (m, 9H), 4.07 (s, 3H), 3.57 (s, 3H),
3.44 (s, 3H); .sup.31P NMR (162 MHz, D.sub.2O) .delta. 30.40 (dt,
J=26.9 Hz, 1P) -0.60 (br. s., 1P) -11.98 (d, J=26.9 Hz, 1 P)
-12.43-12.06 (d, J=26.9 Hz, 1P)
[0453] HRMS calcd. m/z for
C.sub.33H.sub.44N.sub.15O.sub.24P.sub.4S.sup.- [M-H].sup.-
1190.1360, recorded. 1190.13936
Example 2--In Vitro Synthesis of Capped mRNA by In Vitro
Transcription Using Cap Analogs
[0454] For incorporation of different cap analogs during in vitro
transcription the same protocol as for incorporation of regular
beta-S-ARCA dinucleotide caps can be used. In the example given
here, 3 mM cap analog and 7.5 mM NTPs were added to the
transcription reaction. Yield (in mg RNA per ml reaction volume)
and integrity of RNA produced with one of the diastereomers (D1/D2)
of Compound 1 or one of the diastereomers (D1/D2) of beta-S-ARCA
were comparable as given in the following table.
TABLE-US-00001 Yield of reaction RNA integrity (mg RNA/ml reaction
volume) (BIOANALYZER) beta-S-ARCA (D1) 6.54 82% Compound 1 (D1)
6.39 87% beta-S-ARCA (D2) 6.42 82% Compound 1 (D2) 5.81 80%
Example 3--Protein Expression from Differently Capped mRNA in Cell
Culture
[0455] As a functional assay of cap analog incorporation, and as a
test of translatability of the resulting cap structure, differently
capped mRNA encoding a Luciferase reporter was transfected into
human immature dendritic cells (hiDCs). For that, 700 ng capped RNA
per well were formulated with liposomes as described in Kranz et
al., Nature 534, 396-401 (2016), and added to a 96-well containing
5E04 hiDCs. Subsequently, reporter activity was recorded over 72 h.
The results are shown in FIG. 3.
[0456] mRNAs co-transcriptionally capped with the D1 or D2
diastereomer of Compound 1 were functional and translated in hiDCs
at comparable levels as mRNA capped with the corresponding D1 or D2
diastereomer of beta-S-ARCA, indicating that also with the
compounds of formula (I) according to the present invention the
advantages of the beta-S-ARCA modification were still active (FIG.
3).
Example 4--mRNA Modified with a 5'-Cap Compound of Formula (I)
Combines Improved mRNA Stability and Translation Efficiency with
Evasion of an Immune Response Via IFIT1
[0457] Luciferase mRNAs were co-transcriptionally capped with D1 or
D2 diastereomer of Compound 1 or with the corresponding D1 or D2
diastereomer of beta-S-ARCA, as described above. In addition,
triphosphate Luc mRNA (i.e., transcribed in the absence of any cap
analog) was enzymatically capped using the NEB vaccinia capping
enzyme kit (enzymatic Cap0/Cap1 RNA). To obtain enzymatic cap0
structures the vaccinia capping enzyme was used as is, for
enzymatic cap1 structures the vaccinia methyltransferase was also
added. Subsequently, the resulting capped mRNA preparations were
purified in order to decrease or eliminate the amount of double
stranded RNA. Furthermore, the small amount of uncapped RNA present
in the co-transcriptionally capped mRNA preparations, which in
previous experiments has been shown to interfere with the analysis,
was enzymatically converted into cap0 (for mRNAs capped with
dinucleotides) or cap1 structures (for mRNAs capped with
trinucleotides). The resulting mRNA preparations were then
formulated with F12 and given intraveneously in Balb/c mice. Per
group, five mice were tested, with a dosage of 10 .mu.g RNA per
mouse. Strength and kinetics of luciferase expression was monitored
by bioluminescence in vivo imaging 6 h, 24 h and 48 h after
application.
[0458] While in this setting the beta-S-substitution has--if at
all--only a minor effect, as observed by the similar expression
profiles of enzymatically capped cap0 RNA compared to
beta-S-ARCA(D1) and (D2) cap0 RNAs, the main factor driving in vivo
expression is the 2'-O-methylation of the cap1 structure.
Accordingly, the enzymatically capped cap1 mRNA preparations give
the highest protein expression at any time point measured. However,
RNAs capped with Compound 1 demonstrate similar protein levels 20 h
after application, and only slightly lower levels at the other time
points (and always higher levels compared to all cap0 RNAs). Thus,
the 5'-cap compounds of the present invention allow incorporation
of beta-S-ARCA cap1 structures into RNA that combine the positive
effect of the thio-substitution with the cap1-defining
2'-O-methylation.
Example 5--Protein Expression from Differently Capped mRNA In
Vivo
[0459] Murine erythropoietin (mEPO) mRNAs containing
1-methylpseudouridine (m1.PSI.) were co-transcriptionally capped
with ARCA G or with the D1 diastereomer of beta-S-ARCA (designated
as D1), as described above. In addition, triphosphate mEPO mRNA
(i.e., transcribed in the absence of any cap analog) was
enzymatically capped using the NEB vaccinia capping enzyme kit
(enzymatic Cap0/Cap1 RNA). To obtain enzymatic cap0 structures
(designated as Ecap0) the vaccinia capping enzyme with RNA
triphosphatase and guanylyltransferase activities was used as is,
for enzymatic cap1 structures (designated as Ecap1) the vaccinia
methyltransferase with 2'-O-methyltransferase activity was also
added. Subsequently, the resulting differently capped mRNA
preparations were purified in order to decrease or eliminate the
amount of double stranded RNA. Furthermore, the small amount of
uncapped RNA present in the co-transcriptionally capped mRNA
preparations, which in previous experiments has been shown to
interfere with the analysis, was enzymatically converted into cap0
and then into cap1 structures. In the mRNA preparation using D1
structures the resulting product after the treatment with the
enzymes was designated as D1+Ecap1, whereas in the case of ARCA G
the resulting product after the treatment with the enzymes was
designated as ARCA G+Ecap1. The mRNA preparations were then
formulated with TransIT.RTM. and injected intraperitoneally into
Balb/c mice. Per group, five mice were tested, with a dosage of 3
.mu.g RNA per mouse. Translation of the mEPO mRNA was monitored by
ELISA in the plasma collected at 6 h, 24 h, 48 h and 72 h after
application.
[0460] As can be seen from FIG. 5A-5B, the presence of a cap1
structure of the invention in RNA results in much higher expression
levels of mEPO compared to RNA having a cap0 structure, in
particular 24 hours after injection. Moreover, FIG. 5A-5B shows
that by using RNA containing a cap1 structure of the invention it
is possible to maintain high mEPO plasma levels for at least 72
hours. Thus, this example demonstrates that it is not necessary to
administer RNA comprising a nucleotide sequence encoding a peptide
or protein at least twice per day in order to maintain high
expression levels of the peptide or protein. Rather, by using the
present invention it is possible to administer the RNA at most once
per day, preferably at most once per two days, preferably at most
once per three days or at most once per four days while maintaining
high expression levels of the peptide or protein. This has the
advantage for the patient that the number of administrations (e.g.,
injections) can be significantly reduced which is particularly
beneficial with patients who receive their treatment (e.g., a
pharmaceutical composition) over an extended period of time, such
as chronic or long-term patients.
* * * * *