U.S. patent application number 12/676015 was filed with the patent office on 2010-08-12 for complexes of rna and cationic peptides for transfection and for immunostimulation.
This patent application is currently assigned to CureVac GmbH. Invention is credited to Patrick Baumhof, Mariola Fotin-Mleczek.
Application Number | 20100203076 12/676015 |
Document ID | / |
Family ID | 39386081 |
Filed Date | 2010-08-12 |
United States Patent
Application |
20100203076 |
Kind Code |
A1 |
Fotin-Mleczek; Mariola ; et
al. |
August 12, 2010 |
COMPLEXES OF RNA AND CATIONIC PEPTIDES FOR TRANSFECTION AND FOR
IMMUNOSTIMULATION
Abstract
The present invention relates to a complexed RNA, comprising at
least one RNA complexed with one or more oligopeptides, wherein the
oligopeptide, which has the function of cell-penetrating peptide
(CPP), has a length of 8 to 15 amino acids and has the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x with the majority of residues being selected from Arg,
Lys, His, Orn. The invention further relates to a method for
transfecting a cell or an organism, thereby applying the inventive
complexed RNA. Additionally, pharmaceutical compositions and kits
comprising the inventive complexed RNA, as well as the use of the
inventive complexed RNA for transfecting a cell, tissue or an
organism and/or for modulating, preferably inducing or enhancing,
an immune response are disclosed herein.
Inventors: |
Fotin-Mleczek; Mariola;
(Sindelfingen, DE) ; Baumhof; Patrick;
(Dusslingen, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
CureVac GmbH
Tubingen
DE
|
Family ID: |
39386081 |
Appl. No.: |
12/676015 |
Filed: |
September 4, 2008 |
PCT Filed: |
September 4, 2008 |
PCT NO: |
PCT/EP2008/007244 |
371 Date: |
April 30, 2010 |
Current U.S.
Class: |
424/193.1 ;
435/375; 514/1.1; 514/2.4; 530/322 |
Current CPC
Class: |
A61K 47/6455 20170801;
C12N 9/0069 20130101; A61K 48/0075 20130101; A61P 37/02 20180101;
C12Y 304/21022 20130101; C12Y 113/12007 20130101; A61K 2039/53
20130101; A61P 31/12 20180101; A61K 9/0019 20130101; A61K 39/00
20130101; A61K 47/61 20170801; A61K 48/0041 20130101; A61P 35/00
20180101; A61K 38/4846 20130101; C12N 15/87 20130101; A61P 37/08
20180101; A61K 48/0033 20130101; A61P 31/00 20180101; C07K 19/00
20130101; C12Y 204/01007 20130101; A61K 38/45 20130101; A61K
48/0066 20130101; A61K 48/005 20130101; A61P 9/00 20180101 |
Class at
Publication: |
424/193.1 ;
530/322; 514/8; 435/375 |
International
Class: |
A61K 39/385 20060101
A61K039/385; C07K 9/00 20060101 C07K009/00; A61K 38/10 20060101
A61K038/10; C12N 5/02 20060101 C12N005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2007 |
EP |
PCT/EP2007/007702 |
Claims
1. Complexed RNA, comprising at least one RNA (molecule) complexed
with one or more oligopeptides, wherein the oligopeptide has a
length of 8 to 15 amino acids and has the following empirical
formula:
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x
(formula I) wherein l+m+n+o+x=8-15, and l, m, n or o independently
of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall
content of Arg, Lys, His and Orn represents at least 50% of all
amino acids of the oligopeptide; and Xaa may be any amino acid
selected from native or non-native amino acids except of Arg, Lys,
His or Orn; and x may be any number selected from 0, 1, 2, 3, 4, 5,
6, 7, or 8, provided, that the overall content of Xaa does not
exceed 50% of all amino acids of the oligopeptide.
2. Complexed RNA according to claim 1, wherein the at least one RNA
(molecule) is an mRNA.
3. Complexed RNA according to claim 1, wherein the oligopeptide has
a length of 8 to 14, 8 to 13, 8 to 12, or 9 to 12 or 9 to 11 amino
acids.
4. Complexed RNA according to claim 1, wherein the overall content
of Arg, Lys, His and Orn represents at least 60%, 70%, 80%, 90%, or
95% of all amino acids of the oligopeptide of the complexed
RNA.
5. Complexed RNA according to claim 1, wherein Xaa in empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x is selected from amino acids having a neutral side
chain including amino acids having a neutral hydrophobic side chain
and amino acids having a neutral polar side chain.
6. Complexed RNA according to claim 1, wherein the oligopeptide
according to empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x does not comprise amino acids
at one or both terminal ends, which comprise an acidic side
chain.
7. Complexed RNA according to claim 1, wherein the oligopeptide
according to empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x comprises neutral or basic
amino acids at one or both terminal ends, or basic amino acids at
one or both terminal ends or at least one, preferably at least two,
more preferably at least three, basic amino acids at both terminal
ends.
8. Complexed RNA according to claim 1, wherein the oligopeptide
according to empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x comprises a stretch of at
least 3 contiguous basic amino acids within its sequence.
9. Complexed RNA according to claim 1, wherein the oligopeptide
according to empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x comprises at least 1, 2, or 3
non-cationic amino acid(s) at one or both terminal ends, the
non-cationic amino acid preferably being selected from
histidine.
10. Complexed RNA according to claim 1, wherein the at least one
RNA (molecule) of the complexed RNA is an mRNA, wherein the mRNA
codes for a therapeutically active protein or peptide, an
immunostimulating protein or peptide, a tumor antigen or an
antibody.
11. Complexed RNA according to claim 1, wherein the RNA is a
modified RNA, in particular a stabilized RNA, as compared to the wt
RNA.
12. Complexed RNA according to claim 11, wherein the G/C content of
the coding region of the modified RNA is increased compared with
the G/C content of the coding region of the wild-type RNA, and
wherein the coded amino acid sequence of the modified RNA is
preferably not modified compared with the coded amino acid sequence
of the wild-type RNA.
13. Complexed RNA according to claim 1, wherein the mass ratio of
the at least one RNA (molecule) of the complexed RNA to the one or
more oligopeptides is in a range of about 1:100 to about 1:0.5, or
has a value of about 1:50 to about 1:1, about 1:100, about 1:90,
about 1:80, about 1:70, about 1:60, about 1:50, about 1:45, about
1:40, about 1:35, about 1:30, about 1:25, about 1:20, about 1:15,
about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1
or about 1:0.5.
14. Complexed RNA according to claim 1, wherein the molar ratio of
the at least one RNA (molecule) of the complexed RNA to the one or
more oligopeptides is in a range of about 1:20000 to about 1:500 or
1:250, or in a range of about 1:10000 to about 1:1000, or has a
value of about 1:9500, about 1:9000, about 1:8500, about 1:8000,
about 1:7500, about 1:7000, about 1:6500, about 1:6000, about
1:5500, about 1:5000, about 1:4500, about 1:4000, about 1:3500,
about 1:3000, about 1:2500, about 1:2000, about 1:1500, about
1:1000, about 1:500, about 1:450, about 1:400, about 1:350, about
1:300, or about 1:250.
15. Complexed RNA according to claim 1, wherein the
nitrogen/phosphate ratio (N/P-ratio) of the at least one RNA
(molecule) of the complexed RNA to the one or more oligopeptides is
in a range of about 0, 2-50, in a range of about 0.5-50 or in a
range of about 0.75-25.
16. Complexed RNA according to claim 1, wherein the oligopeptide is
selected from the group consisting of: Arg.sub.9His.sub.3,
His.sub.3Arg.sub.9His.sub.3, TyrSerSerArg.sub.9SerSerTyr,
His.sub.3Arg.sub.9SerSerTyr, (ArgLysHis).sub.4,
Tyr(ArgLysHis).sub.2Arg; or from Arg.sub.8, Arg.sub.9, Arg.sub.10,
Arg.sub.11, Arg.sub.12, Arg.sub.13, Arg.sub.14, Arg.sub.15, (SEQ ID
NOs: 1-8); Lys.sub.8, Lys.sub.9, Lys.sub.10, Lys.sub.11,
Lys.sub.12, Lys.sub.13, Lys.sub.14, Lys.sub.15, (SEQ ID NOs: 9-16);
His.sub.8, His.sub.9, His.sub.10, His.sub.11, His.sub.12,
His.sub.13, His.sub.14, His.sub.15, (SEQ ID NOs: 17-24); and
Orn.sub.8, Orn.sub.9, Orn.sub.10, Orn.sub.11, Orn.sub.12,
Orn.sub.13, Orn.sub.14, Orn.sub.15, (SEQ ID NOs: 25-32).
17. Pharmaceutical composition comprising the complexed RNA
according to claim 1 and optionally a pharmaceutically suitable
carrier.
18. A method for transfection of a cell or an organism, comprising
the following steps: a. optionally preparing and/or providing a
complexed RNA according to claim 1, comprising at least one RNA
complexed with one or more oligopeptides having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x; b. transfecting a cell, a tissue or an organism using
the complexed RNA prepared and/or provided according to step
a).
19. Method according to claim 18, wherein the organism is a mammal,
selected from the group comprising humans, goat, cattle, swine,
dog, cat, donkey, monkey, ape or rodents, including mouse, hamster
and rabbit.
20. Use of a complexed RNA according to any of claim 1 for
transfecting a cell or an organism.
21. Use of a complexed RNA according to any of claim 1 for
preparing an agent for treating a disease selected from a tumour or
cancer disease, a cardiovascular disease, an infectious disease, an
(infectious) virus disease, an autoimmune disease, a (mono-)
genetic disease and/or an allergy.
22. Use according to claim 21, wherein transfection of the
complexed RNA elicits an immune response in the organism.
23. Kit according to claim 22 comprising a cytokine or a
composition containing a cytokine and/or at least one additional
adjuvant or a composition containing at least one additional
adjuvant.
Description
[0001] The present invention relates to a complexed RNA, comprising
at least one RNA (molecule) complexed with one or more
oligopeptides, wherein the oligopeptide has a length of 8 to 15
amino acids and has the formula
(Arg).sub.l(Lys).sub.m(His).sub.n(Orn).sub.o(Xaa).sub.x. The
invention further relates to a method for transfecting a cell or an
organism, thereby applying the inventive complexed RNA.
Additionally, pharmaceutical compositions and kits comprising the
inventive complexed RNA, as well as the use of the inventive
complexed RNA for transfecting a cell, tissue or an organism and/or
for modulating, preferably inducing or enhancing, an immune
response are disclosed herein.
[0002] Transfection of nucleic acids into cells or tissues of
patients by methods of gene transfer is a central method of
molecular medicine and plays a critical role in therapy and
prevention of numerous diseases. Methods for transfection of
nucleic acids may lead to immune stimulation of the tissue or
organism. Alternatively or additionally, transfection of nucleic
acids may be followed by processing of the information coded by the
nucleic acids introduced, i.e. translation of desired polypeptides
or proteins. DNA or RNA as nucleic acids form alternative
approaches to gene therapy. Transfection of nucleic acids may also
lead to modulation, e.g. suppression or enhancement of gene
expression, dependent on the type of nucleic acid transfected.
Transfection of these nucleic acids is typically carried out by
using methods of gene transfer.
[0003] Methods of gene transfer into cells or tissues have been
intensively studied in the last decades, however in part with
limited success. Well known methods include physical or
physico-chemical methods such as (direct) injection of (naked)
nucleic acids or biolistic gene transfer. Biolistic gene transfer
(also known as biolistic particle bombardment) is a method
developed at Cornell University, that allows introducing genetic
material into tissues or culture cells. Biolistic gene transfer is
typically accomplished by surface coating metal particles, such as
gold or silver particles, and shooting these metal particles,
comprising the adsorbed DNA, into cells by using a gene gun.
However, biolistic gene transfer methods have not yet been shown to
work with RNA, probably due to its fast degradation. Furthermore,
these methods are not suitable for in vivo applications, a matter
which represents a severe practical limitation.
[0004] An alternative physical or physico-chemical method includes
the method of in vitro electroporation. In vitro electroporation is
based on the use of high-voltage current to make cell membranes
permeable to allow the introduction of new DNA or RNA into the
cell. Therefore, cell walls are typically weakened prior to
transfection either by using chemicals or by a careful process of
freezing to make them "electrocompetent". If electrocompetent
bacteria or cells (e.g. eukaryotic cells) and DNA (or RNA) are
mixed together, the plasmid can be transferred into the cell by
using an electric discharge to carry the DNA (or RNA) into cells in
the path of the spark crossing the reaction chamber.
[0005] Another alternative physical or physico-chemical method
includes use of nanoplexes (nanoparticular systems), lipoplexes
(liposomal systems), or the use of polyplexes or cationic polymers.
Such nanoplexes (nanoparticular systems) involve use of
polyacrylates, polyamides, polystyrene, cyanoacrylates, polylactat
(PLA), poly(lactic-co-glycolic acid) (PLGA), polyethyl, etc., as
carrier systems for the transport of nucleic acids into cells or
tissues. Lipoplexes or liposomal systems typically involve use of
cationic lipids, which are capable to mimic a cell membrane.
Thereby, the positively charged moiety of the lipids interacts with
the negatively charged moiety of the nucleic acids and thus enables
fusion with the cell membrane. Lipoplexes or liposomal systems
include e.g. DOTMA, DOPE, DOSPA, DOTAP, DC-Chol, EDMPC, etc.
Polyplexes (cationic polymers) typically form a complex with
negatively charged nucleic acids leading to a condensation of
nucleic acids and protecting these nucleic acids against
degradation. Transport into cells using polyplexes (cationic
polymers) typically occurs via receptor mediated endocytosis.
Thereby, the DNA is coupled to a distinct molecule, such as
Transferrin, via e.g. the polyplex poly-L-lysine (PLL), which binds
to a surface receptor and triggers endocytosis. Polyplexes
(cationic polymers) include e.g. poly-L-lysine (PLL), chitosan,
polyethylenimine (PEI), polydimethylaminoethylmethacrylate
(PD-MAEMA), polyamidoamine (PAMAM).
[0006] Other well known physical or physico-chemical methods of
gene transfer into cells or organisms include methods such as virus
based transfection methods. As a particular example, DNA viruses
may be used as DNA vehicles. Because of their infection properties,
such viruses have a very high transfection rate. The viruses
typically used are genetically modified in a way, that no
functional infectious particles are formed in the transfected cell.
In spite of this safety precaution, however, a risk of uncontrolled
propagation of the therapeutically active genes introduced and the
viral genes cannot be ruled out e.g. because of possible
recombination events.
[0007] More advantageous in this context is the use of so called
translocatory proteins or of protein transduction domains (PTDs)
for the transport of macromolecules into cells or tissues.
Translocatory proteins are considered as a group of peptides
capable of effecting transport of macromolecules between cells
(translocatory proteins), such as HIV tat (HIV), antennapedia
(Drosophila antennapedia), HSV VP22 (Herpes simplex), FGF or
lactoferrin, etc. In contrast, protein transduction domains (PTDs)
are considered as a group of peptides capable of directing proteins
and peptides covalently bound to these sequences into a cell via
the cell membrane (Leifert and Whitton: Translocatory proteins and
protein transduction domains: a critical analysis of their
biological effects and the underlying mechanisms. Molecular Therapy
Vol. 8 No. 1 2003). Common to translocatory proteins as well as to
PTDs is a basic region, which is regarded as mainly responsible for
transport of the fusion peptides since it is capable of binding
polyanions such as nucleic acids. Without being bound thereto, PTDs
may act similar to cationic transfection reagents using receptor
dependent non-saturatable adsorptive endocytosis. PTDs are
typically coupled to proteins or peptides in order to effect or
enhance a CTL response when administering a peptide based vaccine
(see review: Melikov and Chemomordik, Arginine-rich cell
penetrating peptides: from endosomal uptake to nuclear delivery,
Cell. Mol. Life. Sci. 2005).
[0008] Protein transduction domains (PTDs) are sometimes also
termed "cell penetrating peptides" (CPPs) due to their capability
of penetrating the cell membrane and thus to effect the transport
of (macro-) molecules into cells. CPPs are small peptides and
typically comprise a high content of basic amino acids and exhibit
a length of 7 to 30 amino acids. Macromolecules, which have been
shown to be transported into cells via CPPs, include peptides as
well as DNA, siRNA or PNAs (peptide nucleic acids), wherein the
CPPs are typically bonded to these macromolecules via a covalent
bond and transfected into the cells. Although cell penetrating
peptides (CPPs) have been successfully used to mediate
intracellular delivery of a wide variety of molecules of
pharmacological interest both in vitro and in vivo, the mechanisms
by which cellular uptake occurs still remains unclear. The group of
CPPs is highly diverse and consists of amphipathic, helical
peptides such as transportan, penetratin, hydrophobic peptides such
as MTS, VP22, MAP, KALA, PpTG20, prolin-rich peptides,
MPG-peptides, Pep-1, L-oligomers, calcitonin-peptides, or cationic,
hydrophilic arginine-rich peptides, including arginine-rich CPPs,
which mediate cellular uptake of (covalently) conjugated molecules
via binding to proteoglycanes of the cell, such as the transduction
domain of the HIV-1 Tat protein (Review: Deshayes et al.
Cell-penetrating peptides: tools for intracellular delivery of
therapeutics. Cell. Mol. Life. Sci. 2005). Particularly,
arginine-rich CPPs are described as vehicles for proteins or DNA,
e.g. plasmid DNA, etc. into cells. Poly-arginines may also be used
for the transport of (macro-) molecules into cells, which typically
comprises a length of at least 60 to 80 amino acids (in particular
arginines), more typically from 1000 to 15000 amino acids, and thus
represents a high molecular mass compound. Even though the cellular
uptake mechanism for CPPs in general remains unclear, endocytosis
is suggested as an uptake mechanism for poly-arginine. Endocytosis
is a cellular process by which macromolecules may enter a cell
without passing through the cell membrane, wherein three different
endocytotic mechanisms have been suggested (chlathrin-dependent
endocytosis, caveolin-dependent endocytosis and/or F
actin-dependent endocytosis, see e.g. review: Melikov and
Chemomordik, Arginine-rich cell penetrating peptides: from
endosomal uptake to nuclear delivery, Cell. Mol. Life. Sci. 2005).
Without being bound to any theory, during endocytosis the
CPP-complexed macromolecule first binds to the negatively charged
cell surface glycosaminoglycans (GAGs), including heparans (HS).
Then, the CPP-bound macromolecule enters cell by
chlathrin-dependent endocytosis, caveolin-dependent endocytosis
and/or F actin-dependent endocytosis, e.g. by folding of the
membrane around the CPP-bound macromolecule outside the cell. This
results in the formation of a saclike vesicle into which the
CPP-bound macromolecule is incorporated. Trafficking of the
CPP-bound macromolecule through late endosomes and/or Golgi and/or
endoplasmic reticulum (ER) delivers the CPP-bound macromolecule
into the cytoplasm, wherein this stage may involve CPP-induced
opening of the transient pores in the lipid bilayer. Alternatively,
the CPP-complexed macromolecule may be transported to other
locations in the cell, e.g. into the endosom, dependent on the mode
of action required for the specific purpose. As an example, TLR-7
and TLR-8 receptors are located in the endosome. Thus, transfection
of cells with immunostimulatory RNA, which may e.g. be ligands of
Toll-like receptors (TLRs) selected from ligands of TLR1-TLR13
(Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,
TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13) may lead to transport to
the endosomes and (depeding on the specific interaction and the
interaction partners) to e.g. immunostimulation by the RNA
ligand.
[0009] Cell penetrating peptides (CPPs) as defined above are well
known in the art and widely discussed. However, the use of these
CPPs (as carriers) is established for the transport of peptides,
proteins and DNA as cargo, wherein the CPPs are typically linked to
cargo molecules in a covalent manner. In contrast, cellular
transport of RNAs using CPPs was only shown for a very limited
number of cases, particularly for short RNA sequences, e.g. double
stranded siRNA sequences.
[0010] By way of example, Futaki et al. (The Journal of Biological
Chemistry, Vol 276, No. 8, pp. 5836-5840, 2001) disclose the use of
carrier oligopeptides (Arg).sub.n having a length of 4-16 amino
acids for in vitro transfer of cargo peptides, wherein the carrier
peptides are covalently linked to the cargo peptides. A
translocation optimum was demonstrated for (Arg).sub.n having a
length of 6 or 8 arginines, respectively.
[0011] Trans-membrane transport peptides or peptidomimetics by CPPs
was also shown by Deshayes et al. (2005, supra). Deshayes et al.
(2005, supra) disclose the use of oligopeptides Arg.sub.7 and
Arg.sub.9 for in vitro transfer of cargo peptides and in vivo
transfer of cargo proteins such as cyclosporin or catalase.
[0012] For transfection of cells with macromolecules, such as DNA,
peptides or proteins, high molecular weight polypeptides such as
poly-L-arginines (e.g. typically having a MW of about 5000 Da to 15
kDa) or poly-L-lysines (e.g. typically having a MW of about 54 kDa)
as well as high molecular weight PEI (polyethyleneimin) (e.g.
typically having a MW of about 25 kDa) were used according to the
art (see also Bettinger et al., Nucleic Acids Research, Vol. 29 No.
18 (2001)). However, high molecular weight poly-L-lysine and PEI
appeared to be ineffective as carrier molecules. Further, when
using high molecular weight poly-L-arginines at high
concentrations, toxic effects were observed which lead to
activation of the complement system. Thus, efforts were undertaken
to develop low molecular weight transfection agents, such as, e.g.,
low molecular weight poly-arginines. However, such low molecular
weight poly-arginines typically exhibit a low stability of the
carrier-cargo-complex, i.e. the complex formed of, e.g., a
poly-arginine as carrier and a DNA molecule as a cargo. Thus,
McKenzie et al. (McKenzie et al. A potent new class of reductively
activated peptide gene delivery agents; The Journal of Biological
Chemistry Vol. 274 No. 14, 2000) tried to increase stability of
peptide-DNA-complexes by crosslinking these peptides via
glutaraldehyde to the DNA, thereby forming a Schiff's base.
However, such crosslinking results in extremely slow dissociation
of the complex in the cell and, consequently, expression of the
encoded protein is extremely low over time. In order to circumvent
this problem, McKenzie et al. (2000, supra) introduced cysteine
residues into the CPP carrier, which stabilize the complex by
forming disulfide bonds between CPP and DNA. Upon transfection,
these disulfide bonds are cleaved in the cell due to the reducing
conditions inside the cell, resulting in increased expression of
the encoded peptides. However, such crosslinking is elaborative and
may cause further undesired modifications of the DNA.
[0013] Furthermore, low-molecular weight PEI (e.g. typically having
a MW of about 2000 Da) and low-molecular weight poly-L-lysines
(e.g. typically having a MW of about 3400 Da) may be used for
transfection of such macromolecules as mentioned above. However,
even though an improved transfection was observed for low-molecular
weight PEI or poly-L-lysines in these experiments, expression was
not detectable due to formation of extremely stable complexes of
these carrier molecules with the DNA. As a result, these carrier
molecules do not appear to exhibit dissociation of their complexed
DNA, a necessary step for translation and expression of the encoded
protein (see Bettinger et al., (2001), supra).
[0014] Transport of DNA by CPPs was further shown by Niidome et al.
(The Journal of Biological Chemistry, Vol 272., No. 24, pp.
15307-15312, 1997). Niidome et al. (1997, supra) disclose the use
of CPPs, particularly of cationic alpha-helical peptides with a
defined arginine content of 25% and a length of 12 or 24 amino
acids, respectively, for the transport of plasmid DNA as cargo
moiety. As a result, it was found that long and/or hydrophobic
peptides can strongly bind to the DNA and effect transport of DNA
into cells. Moreover, Niidome et al. (Bioconjugate Chem. 1999, 10,
773-780) showed that peptides having a length of 16 to 17 amino
acids were most efficient for the transport of plasmid DNA.
However, when using small peptides (e.g. of about 12 amino acids)
as CPPs, transfection efficiency of DNA into cells turned out to
decrease significantly.
[0015] In order to enhance cellular transfection efficiency of
short arginine molecules, Futaki et al. (Bioconjugate Chem. 2001,
12, 1005-1011) used stearylated oligopeptides (Arg).sub.n having a
length of 4-16 amino acids. These oligopeptides were used in
transfection experiments in comparison to non-stearylated
oligopeptides (Arg).sub.n having a length of 4-16 amino acids and
poly-arginine (MW 5000-15000) for in vitro transfer of plasmid DNA
coding for luciferase. Accordingly, carrier peptides used for
transfection were mixed with plasmid DNA and formed a carrier/cargo
complex. A translocation optimum was demonstrated for stearylated
(Arg).sub.n having a length of 8 arginines, whereas arginines
having a length of 6-7 and 9-15 arginines showed a significantly
reduced cellular transport activity. Furthermore, transport
activity of non-stearylated arginines and poly-arginine exhibited
poor results, indicating loss of transport activity when using
these carrier peptides. The observed difference of transfection
efficiency shown by Futaki et al. (2001, supra) for stearylated and
non-stearylated carrier peptides is thus due to the presence of
lipid moieties, which significantly change the chemical properties
of the CPPs used in these experiments.
[0016] According to Kim et al. (Kim et al., Basic peptide system
for efficient delivery of foreign genes, Biochimica et Biophysica
Acta 1640 (2003) 129-136), short arginine carrier peptides such as
(Arg).sub.9 to (Arg).sub.15 may be used for complexation and
cellular transfection of DNA, encoding green fluorescent protein
PEGFP-N3. When using arginines (Arg).sub.9 to (Arg).sub.15, optimum
results were obtained with (Arg).sub.15 showing increasing cellular
transfection efficiency from (Arg).sub.9 to (Arg).sub.15. These
results indicate that optimum transport properties for transfecting
cells with DNA may be achieved with an (Arg).sub.n carrier peptide,
wherein n is far beyond 15. However, applicability of short
arginine peptides for transfection purposes was exclusively
documented for DNA molecules as cargo moiety by Kim et al. (2003,
supra).
[0017] Cells may also be transfected by using CPPs in combination
with RNA. However, only a small number of working examples were
carried out for the cellular transport of RNA, probably due to its
fast degradation and low stability in complexes. Thus, transfection
of RNA using CPPs appears to be restricted to more stable double
stranded RNAs, such as siRNA. By way of example, Tonges et al.,
(RNA (2006), 12:1431-1438) used stearylated octa-arginine
(Arg).sub.8 for the in vitro transfer of double stranded short
siRNA into neuronal hippocampus cells, wherein the stearylated
octa-arginine (Arg).sub.8 forms a complex with siRNA. Based on the
results of Tonges et al. (2006, supra) the stearyl component of the
carrier peptides seems to be indispensable for the transport of
siRNA or the transport of other RNA molecules.
[0018] Veldhoen et al. (2006) also published the use of specific
CPPs in a non-covalent complex for cellular transfection of double
stranded short siRNA sequences (Veldhoen et al., Cellular delivery
of small interfering RNA by a non-covalently attached cell
penetrating peptide: quantitative analysis of uptake and biological
effect. Nucleic Acids Research 2006). Peptides used by Veldhoen et
al. (2006) were MPGalpha (Ac-GALFLAFLAAALSLMGLWSQPKKKRKV-Cya) and
MPGalpha-mNLS (Ac-GALFLAFLAAALSLMGLWSQPKSKRKV-Cya). These specific
peptides were additionally modified with an acetyl moiety (Ac) at
the N-terminus and a cysteamide moiety at the C-terminus. Veldhoen
et al. (2006) were able to show transfer of double-stranded siRNA,
having a length of about 18 to 40 nucleotides, into cells by using
the afore-mentioned carrier peptides.
[0019] Summarizing the above, use of CPPs or other carrier peptides
for the cellular transport of macromolecules was basically shown
for peptides and for DNA molecules. Few very specific publications
disclose cell penetrating properties of double stranded siRNA.
[0020] RNA transfer represents an important tool in modern
molecular medicine and exhibits superior properties over DNA cell
transfection, since DNA molecules may lead to serious problems.
E.g. application of DNA molecules bears the risk that the DNA
integrates into the host genome. Integration of foreign DNA into
the host genome can have an influence on expression of the host
genes and possibly triggers expression of an oncogene or
destruction of a tumor suppressor gene. A gene--and therefore the
gene product--which is essential to the host may also be
inactivated by integration of the foreign DNA into the coding
region of this gene. There is a particular danger if integration of
the DNA takes place into a gene which is involved in regulation of
cell growth. In this case, the host cell may enter into a
degenerated state and lead to cancer or tumor formation. Such
undesired integration into the DNA may be even more problematic, if
the DNA transfected into the cell comprises a potent promoter, such
as the viral CMV promoter. Integration of such promoters into the
genome of the treated cell can lead to undesirable changes in the
regulation of gene expression in the cell. A further disadvantage
is that the DNA molecules remain in the cell nucleus for a long
time, either as an episome or, as mentioned, integrated into the
host genome. This phenomenon leads both to production of transgenic
protein which is not limited or cannot be limited in time and to
danger of associated tolerance towards this transgenic protein. The
development of anti-DNA antibodies (Gilkeson et al., J Clin Invest
95, 1398-1402 (1995)) and the induction of autoimmune diseases can
furthermore be triggered by injection of DNA. All these risks
listed are associated with application of DNA. In contrast, they do
not occur if RNA, particularly mRNA, is used instead of DNA. For
example, mRNA does not integrate into the host genome, no viral
sequences, such as promoters etc., are required for effective
transcription etc. A disadvantage resulting from the use of RNA may
be due to its instability as compared to DNA (RNA-degrading
enzymes, so-called RNases (ribonucleases), in particular, but also
numerous other processes which destabilize RNA are responsible for
the instability of RNA). However, methods for stabilizing RNA have
meanwhile been disclosed in the art, such as, for example, in WO
03/051401, WO 02/098443, WO 99/14346, EP-A-1083232, U.S. Pat. No.
5,580,859 and U.S. Pat. No. 6,214,804. Methods have also been
developed for protecting RNA against degradation by ribonucleases,
either using liposomes (Martinon et al., Eur J Immunol 23,
1719-1722 (1993)) or an intra-cytosolic in vivo administration of
the nucleic acid with a ballistic device (gene gun) (Vassilev et
al., Vaccine 19, 2012-2019 (2001)).
[0021] Since RNA molecules as such provide advantageous properties
over DNA as discussed above, it is the object of the present
invention to provide a suitable and efficient carrier for the
transport of RNA into cells. Accordingly, the present invention
provides a solution which allows RNA to transfect cells in an
efficient manner.
[0022] This object of the present invention is achieved by the
embodiments of the present invention as characterized by the
claims. Particularly, the above object is solved by a complexed RNA
(molecule), comprising at least one RNA (molecule), preferably an
mRNA, complexed with one or more oligopeptides, wherein the at
least one oligopeptide has a length of 8 to 15 amino acids, and
wherein the at least one oligopeptide contains I Arg residues, m
Lys residues, n His residues, o Orn residues and x Xaa residues
positioned in any order within the at least one oligopeptide having
the following empirical formula:
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x
(formula I) [0023] wherein [0024] l+m+n+o+x=8-15, and [0025] l, m,
n or o independently of each other may be any number selected from
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided
that the overall content of Arg, Lys, His and Orn represents at
least 50%, e.g. at least 60% or 70%, of all amino acids of the
oligopeptide; and [0026] Xaa may be any amino acid selected from
native (=naturally occurring) or non-native amino acids except of
Arg, Lys, His or Orn; and [0027] x may be any number selected from
0, 1, 2, 3, 4, 5, 6, 7 or 8, provided, that the overall content of
Xaa does not exceed 50%, e.g. not more than 40% or 30%, of all
amino acids of the oligopeptide.
[0028] In the context of the present invention, a complexed RNA is
to be understood as an RNA (molecule) as defined herein, preferably
an mRNA, which is complexed to the one or more oligopeptides
according to empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x by forming a non-covalent
complex between RNA and oligopeptide(s). Herein, "non-covalent"
means that a reversible association of RNA and oligopeptide is
formed by non-covalent interactions of these molecules, wherein the
molecules are associated together by any type of interaction of
electrons, other than a covalent bond, e.g. by van der Waals-bonds,
i.e. a weak electrostatic attraction arising from a nonspecific
attractive force of the complexed molecules. Association of an RNA
and at least one oligopeptide is in equilibrium with dissociation
of that complex. Intracellularly, without being bound to theory,
the equilibrium appears to be shifted towards dissociated RNA and
oligopeptide(s).
[0029] The at least one oligopeptide of the complexed RNA according
to the present invention has a length of 8 to 15 amino acids,
preferably a length of 8 to 14, 8 to 13, 8 to 12, or 9 to 12 or 9
to 11 amino acids, and more preferably a length of 8 to 10, 9 to
11, 10 to 12, 11 to 13, 12 to 14 or 13 to 15 amino acids, or even
more preferably may be selected from a peptide of the above formula
having a length of 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
[0030] The oligopeptide of the complexed RNA according to the
present invention has the empirical formula (Arg).sub.l;
(Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x, as defined
above wherein l+m+n+o+x=8-15, and l, m, n or o independently of
each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14 or 15, or any range formed by two of these
values, provided that the overall content of (the basic amino
acids) Arg, Lys, His and/or Orn represents at least 50% (e.g. at
least 51, 52, 53, 54, 55, 56, 57, 58, or 59%) at least 60% (e.g. at
least 61, 62, 63, 64, 65, 66, 67, 68, or 69%), at least 70% (e.g.
at least 71, 72, 73, 74, 75, 76, 77, 78, or 79%), at least 80%
(e.g. at least 81, 82, 83, 84, 85, 86, 87, 88, or 89%) at least 90%
(e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or even
100% of all amino acids of the oligopeptide of the complexed RNA
according to the present invention. The amino acids Arg, Lys, His
and Orn (three letter code) are to be understood as the amino acids
arginine, lysine, histidine and ornithine, respectively. In this
context, ornithine is an amino acid whose structure is
NH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CHNH.sub.2--COOH. Ornithine
was artificially incorporated as the 21'' amino acid and does not
belong to the "natively occurring" 20 amino acids in the sense that
ornithine is not an amino acid coded for by DNA, and, accordingly,
is not involved in primary protein synthesis. However, ornithine is
provided by enzymatic reaction starting from L-arginine. It is
believed not to be a part of the genetic code because polypeptides
containing unprotected ornithines undergo spontaneous
lactamization. Ornithine is to be regarded as a basic amino acid
since it is one of the products of the reaction of the enzyme
Arginase on L-arginine, creating urea.
[0031] According to a further preferred embodiment the (single)
amino acids of the oligopeptide of the complexed RNA of the present
invention, having the empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x (formula I) as shown above,
may occur in any frequency as defined above for the empirical
formula, i.e. each basic amino acid (as well as Xaa) may occur in
the above defined empirical formula within the above defined values
or ranges, wherein any range may be formed from by two of the
values as defined above. However, it is particularly preferred, if
the content of the basic amino acid Arg in the above empricial
formula is at least 10%, more preferably at least 20%, even more
preferably at least 30%, 40% or even 50%, even more preferably at
least 60%, 70%, 80% 90% or even 100% with respect to the entire
empirical formula. According to another particularly preferred
embodiment the content of the basic amino acid Lys in the above
empricial formula is at least 10%, more preferably at least 20%,
even more preferably at least 30%, 40% or even 50%, even more
preferably at least 60%, 70%, 80% 90% or even 100% with respect to
the entire empirical formula. According to a further particularly
preferred embodiment the content of the basic amino acid His in the
above empricial formula is at least 10%, more preferably at least
20%, even more preferably at least 30%, 40% or even 50%, even more
preferably at least 60%, 70%, 80% 90% or even 100% with respect to
the entire empirical formula. According to one other particularly
preferred embodiment the content of the basic amino acid Orn in the
above empricial formula is at least 10%, more preferably at least
20%, even more preferably at least 30%, 40% or even 50%, even more
preferably at least 60%, 70%, 80% 90% or even 100% with respect to
the entire empirical formula. Any of the above defined contents,
values or ranges of basic amino acids Arg, Lys, His and/or Orn as
defined above may also be combined with each other, preferably
leading to an overall content of all basic amino acids of the
oligopeptide of the complexed RNA of the present invention of at
least 50% (at least 51, 52, 53, 54, 55, 56, 57, 58, or 59%) at
least 60% (at least 61, 62, 63, 64, 65, 66, 67, 68, or 69%), at
least 70% (at least 71, 72, 73, 74, 75, 76, 77, 78, or 79%), at
least 80% (at least 81, 82, 83, 84, 85, 86, 87, 88, or 89%) at
least 90% (at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or
even 100%, as defined initially.
[0032] The amino acids in the above formula (Arg).sub.l;
(Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x, i.e. Arg, Lys,
His and/or Orn may be furthermore be selected from the native
(=naturally occurring) amino acids Arg, Lys, His and Orn or from
non-native (=not naturally occurring) amino acids derived from
these amino acids. As a non-native (=not naturally occurring) amino
acid derived from the amino acids Arg, Lys, His and Orn, any known
derivative of these amino acids may be used, which has been
chemically modified, provided these derivatives are not toxic for
cells or organisms, when provided with the above oligopeptide.
(Such derivatives of amino acids are distributed by different
companies; see e.g. Sigma Aldrich (see
http://www.sigmaaldrich.com).
[0033] Furthermore, the oligopeptide of the complexed RNA according
to the present invention may contain an amino acid Xaa in the above
empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x, which may be any amino acid selected from
native (=naturally occurring) or non-native (=not naturally
occurring) amino acids except of Arg, Lys, His or Orn. Preferably,
Xaa may be selected, without being limited thereto, from naturally
occurring neutral (and hydrophobic) amino acids, i.e. amino acids,
which have neutral (and hydrophobic) side chains, such as alanine
(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline
(Pro), tryptophane (Trp), phenylalanine (Phe), or methionine (Met),
and/or from naturally occurring neutral (and polar) amino acids,
i.e. amino acids, which have neutral (and polar) side chains, such
as glycine (Gly), serine (ser), threonine (Thr), tyrosine (Tyr),
cysteine (Cys), asparagine (Asn), or glutamine (Glu), and/or from
naturally occurring acidic amino acids, i.e. amino acids, which
have acidic side chains, such as aspartic acid (Asp) or glutamic
acid (Glu). Preferably the oligopeptide of the complexed RNA
according to the present invention may contain an amino acid Xaa in
the above empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x, which is selected from amino acids having
no acidic side chain. Even more preferably, Xaa in empirical
formula (Arg).sub.1; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x is selected from amino acids having a neutral side
chain, i.e. from amino acids, which have a neutral (and
hydrophobic) side chain and/or from amino acids, which have a
neutral (and polar) side chain, as defined above. Additionally, any
known derivative of amino acids may be used for Xaa in the above
empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x, i.e. amino acids, which have been
chemically modified, provided these derivatives are not toxic for
cells or organisms, when provided with the above oligopeptide.
(Such derivatives of amino acids are distributed by different
companies, see e.g. Sigma Aldrich (see
http://www.sigmaaldrich.com). Xaa is typically present in the above
formula in a content of 0-30%, 0-40% or 0-50% of all amino acids of
the entire oligopeptide sequence, i.e. the overall content of Xaa
may not exceed 30%, 40% or 50% of all amino acids of the entire
oligopeptide sequence, preferably it may not exceed 20%, even more
preferably not 10%, and most preferably not 5% of all amino acids
of the entire oligopeptide sequence. Thus, x in the empirical
formula (Arg).sub.1; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x as shown above may be any number selected from 0, 1, 2,
3, 4, 5, 6, 7 or 8, provided, that the content of Xaa does not
exceed the above indicated value of 30% (or less), 40% or 50% of
all entire amino acids of the oligopeptide of the complexed
RNA.
[0034] Typically, the amino acids Arg, Lys, His, Orn and Xaa of the
oligopeptide of the complexed RNA according to the present
invention, having the empirical formula (Arg).sub.l; (Lys).sub.m;
(His).sub.n; (Orn).sub.o; (Xaa).sub.x as indicated above, may be
positioned at any position of the oligopeptide sequence.
Accordingly, empirical formula (I) does not determine any specific
order of amino acids, but is rather intended to reflect the type of
amino acids and their frequency of occurrence in the peptide,
indicating that the peptide chain contains a number of l Arg
residues, m Lys residues, n His residues, o Orn residues and x Xaa
residues, without specifying any order of these residues within the
peptide chain.
[0035] However, it is preferred, that the above oligopeptide
comprises amino acids at one or, preferably, both terminal ends,
which do not comprise an acidic side chain. More preferably, the
above oligopeptide sequence comprises neutral or basic amino acids
at one or, preferably, both terminal ends, even more preferably
basic amino acids at one or both terminal ends. In a further
preferred embodiment, the oligopeptide according to the general
formula given above contains at least two, more preferably at least
three, at least four or even at least five terminal basic residues,
in particular Arg, Orn or Lys, at either terminus. According to
just another preferred embodiment, the oligopeptide according to
the general formula given above preferably comprises no cationic
amino acids (i.e. no Arg, Orn or Lys) at one or, preferably, at
both terminal ends, even more preferably no cationic amino acids
(i.e. no Arg, Orn or Lys) at both terminal ends. In other words,
one, or more preferably both, terminal ends of the oligopeptide
according to the general formula given above may comprise any
non-cationic amino acid as defined herein, provided that such
non-cationic amino acid is selected from an amino acid except Arg,
Orn or Lys or any variant or derivative of these cationic amino
acids. The terminal ends may comprise e.g. one, at least two, at
least three, at least four, at least five or even more basic
non-cationic residues as defined above starting from the N- and/or
C-terminal end of the particular sequence.
[0036] According to a further preferred embodiment, one or both
terminal ends of the oligopeptide of the complexed RNA according to
the present invention may comprise at least one histidine residues
at one or both of its terminal ends, e.g. the oligopeptide of the
complexed RNA according to the present invention may comprise one,
two, three or more histidine residues in consecutive order at one
or both terminal ends, provided that the overall length of the
oligopeptide is limited to 8 to 15 amino acids as defined
above.
[0037] Additionally, Xaa residues of the oligopeptide of the
complexed RNA according to the present invention are typically
separated from each other by at least one Arg, Lys, His or Orn.
Such a separation of Xaa residues preferably avoids clusters of
non-basic amino acids in the oligopeptide, as such non-basic
clusters may reduce the advantageous properties of the oligopeptide
as a carrier peptide for the complexed RNA according to the present
invention.
[0038] However, basic amino acid residues of the oligopeptide of
the complexed RNA according to the formula given above are selected
from Arg, Lys, His or Orn as defined above and typically occur in a
cluster of at least 2, preferably at least 3, 4, 5, or even 6 or
more basic amino acids as defined herein. According to a
particularly preferred embodiment, such clusters may also comprise
6, 7, 8, 9, 10, 11, 12, 13, 14 or even 15 amino acids. Such a
cluster of basic amino acids, preferably a cluster of at least 3,
4, 5, or even 6 or more basic amino acids preferably creates a
basic surface or binding region within the oligopeptide, which
provides advantageous properties to the oligopeptide as a carrier
peptide for the complexed RNA according to the present
invention.
[0039] According to a further preferred embodiment the oligopeptide
of the complexed RNA of the present invention, having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x (formula I) as shown above, may be, without being
restricted thereto, selected from the following subgroup of
formulae:
TABLE-US-00001 (SEQ ID NOs: 1-8) Arg.sub.8, Arg.sub.9, Arg.sub.10,
Arg.sub.11, Arg.sub.12, Arg.sub.13, Arg.sub.14, Arg.sub.15,; (SEQ
ID NOs: 9-16) Lys.sub.8, Lys.sub.9, Lys.sub.10, Lys.sub.11,
Lys.sub.12, Lys.sub.13, Lys.sub.14, Lys.sub.15,; (SEQ ID NOs:
17-24) His.sub.8, His.sub.9, His.sub.10, His.sub.11, His.sub.12,
His.sub.13, His.sub.14, His.sub.15,; (SEQ ID NOs: 25-32) Orn.sub.8,
Orn.sub.9, Orn.sub.10, Orn.sub.11, 0rn.sub.12, 0rn.sub.13,
Orn.sub.14, Orn.sub.15,;
[0040] According to a further preferred embodiment the oligopeptide
of the complexed RNA of the present invention, having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x (formula I) as shown above, may be, without being
restricted thereto, selected from following subgroup. This subgroup
exemplarily defines specific inventive oligopeptides, which fall
under empirical formula I as defined above, wherein the following
formulae (as with empirical formula (I)) do not specify any amino
acid order, but are intended to reflect empirical formulae by
exclusively specifying the (number of) amino acids as components of
the respective peptide. Accordingly, empirical formula
Arg.sub.(7-14)Lys.sub.1 is intended to mean that peptides falling
under this formula contain 7 to 14 Arg residues and 1 Lys residue
of whatsoever order. If the peptides contain 7 Arg residues and 1
Lys residue, all variants having 7 Arg residues and 1 Lys residue
are encompassed. The Lys residue may therefore be positioned
anywhere in the e.g. 8 amino acid long sequence composed of 7 Arg
and 1 Lys residues. The subgroup preferably comprises:
TABLE-US-00002 Arg.sub.(7-14)Lys.sub.1, Arg.sub.(7-14)His.sub.1,
Arg.sub.(7-14)Orn.sub.1, Lys.sub.(7-14)His.sub.1,
Lys.sub.(7-14)Orn.sub.1, His.sub.(7-14)Orn.sub.1,;
Arg.sub.(6-13)Lys.sub.2, Arg.sub.(6-13)His.sub.2,
Arg.sub.(6-13)Orn.sub.2, Lys.sub.(6-13)His.sub.2,
Lys.sub.(6-13)Orn.sub.2, His.sub.(6-13)Orn.sub.2,;
Arg.sub.(5-12)Lys.sub.3, Arg.sub.(5-12)His.sub.3,
Arg.sub.(5-12)Orn.sub.3, Lys.sub.(5-12)His.sub.3,
Lys.sub.(5-12)Orn.sub.3, His.sub.(5-12)Orn.sub.3,;
Arg.sub.(4-11)Lys.sub.4, Arg.sub.(4-11)His.sub.4,
Arg.sub.(4-11)Orn.sub.4, Lys.sub.(4-11)His.sub.4,
Lys.sub.(4-11)Orn.sub.4, His.sub.(4-11)Orn.sub.4,;
Arg.sub.(3-10)Lys.sub.5, Arg.sub.(3-10)His.sub.5,
Arg.sub.(3-10)Orn.sub.5, Lys.sub.(3-10)His.sub.5,
Lys.sub.(3-10)Orn.sub.5, His.sub.(3-10)Orn.sub.5,;
Arg.sub.(2-9)Lys.sub.6, Arg.sub.(2-9)His.sub.6,
Arg.sub.(2-9)Orn.sub.6, Lys.sub.(2-9)His.sub.6,
Lys.sub.(2-9)Orn.sub.6, His.sub.(2-9)Orn.sub.6,;
Arg.sub.(1-8)Lys.sub.7, Arg.sub.(1-8)His.sub.7,
Arg.sub.(1-8)Orn.sub.7, Lys.sub.(1-8)His.sub.7,
Lys.sub.(1-8)Orn.sub.7, His.sub.(1-8)Orn.sub.7,;
Arg.sub.(6-13)Lys.sub.1His.sub.1, Arg.sub.(6-13)Lys.sub.1Orn.sub.1,
Arg.sub.(6-13)His.sub.1Orn.sub.1, Arg.sub.1Lys.sub.(6-13)His.sub.1,
Arg.sub.1Lys.sub.(6-13)Orn.sub.1, Lys.sub.(6-13)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(6-13), Arg.sub.1His.sub.(6-13)Orn.sub.1,
Lys.sub.1His.sub.(6-13)Orn.sub.1; Arg.sub.(5-12)Lys.sub.2His.sub.1,
Arg.sub.(5-12)Lys.sub.1His.sub.2, Arg.sub.(5-12)Lys.sub.2Orn.sub.1,
Arg.sub.(5-12)Lys.sub.1Orn.sub.2, Arg.sub.(5-12)His.sub.2Orn.sub.1,
Arg.sub.(5-12)His.sub.1Orn.sub.2, Arg.sub.2Lys.sub.(5-12)His.sub.1,
Arg.sub.1Lys.sub.(5-12)His.sub.2, Arg.sub.2Lys.sub.(5-12)Orn.sub.1,
Arg.sub.1Lys.sub.(5-12)Orn.sub.2, Lys.sub.(5-12)His.sub.2Orn.sub.1,
Lys.sub.(5-12)His.sub.1Orn.sub.2, Arg.sub.2Lys.sub.1His.sub.(5-12),
Arg.sub.1Lys.sub.2His.sub.(5-12), Arg.sub.2His.sub.(5-12)Orn.sub.1,
Arg.sub.1His.sub.(5-12)Orn.sub.2, Lys.sub.2His.sub.(5-12)Orn.sub.1,
Lys.sub.1His.sub.(5-12)Orn.sub.2; Arg.sub.(4-11)Lys.sub.3His.sub.1,
Arg.sub.(4-11)Lys.sub.2His.sub.2, Arg.sub.(4-11)Lys.sub.1His.sub.3,
Arg.sub.(4-11)Lys.sub.3Orn.sub.1, Arg.sub.(4-11)Lys.sub.2Orn.sub.2,
Arg.sub.(4-11)Lys.sub.1Orn.sub.3, Arg.sub.(4-11)His.sub.3Orn.sub.1,
Arg.sub.(4-11)His.sub.2Orn.sub.2, Arg.sub.(4-11)His.sub.1Orn.sub.3,
Arg.sub.3Lys.sub.(4-11)His.sub.1, Arg.sub.2Lys.sub.(4-11)His.sub.2,
Arg.sub.1Lys.sub.(4-11)His.sub.3, Arg.sub.3Lys.sub.(4-11)Orn.sub.1,
Arg.sub.2Lys.sub.(4-11)Orn.sub.2, Arg.sub.1Lys.sub.(4-11)Orn.sub.3,
Lys.sub.(4-11)His.sub.3Orn.sub.1, Lys.sub.(4-11)His.sub.2Orn.sub.2,
Lys.sub.(4-11)His.sub.1Orn.sub.3, Arg.sub.3Lys.sub.1His.sub.(4-11),
Arg.sub.2Lys.sub.2His.sub.(4-11), Arg.sub.1Lys.sub.3His.sub.(4-11),
Arg.sub.3His.sub.(4-11)Orn.sub.1, Arg.sub.2His.sub.(4-11)Orn.sub.2,
Arg.sub.1His.sub.(4-11)Orn.sub.3, Lys.sub.3His.sub.(4-11)Orn.sub.1,
Lys.sub.2His.sub.(4-11)Orn.sub.2, Lys.sub.1His.sub.(4-11)Orn.sub.3;
Arg.sub.(3-10)Lys.sub.4His.sub.1, Arg.sub.(3-10)Lys.sub.3His.sub.2,
Arg.sub.(3-10)Lys.sub.2His.sub.3, Arg.sub.(3-10)Lys.sub.1His.sub.4,
Arg.sub.(3-10)Lys.sub.4Orn.sub.1, Arg.sub.(3-10)Lys.sub.3Orn.sub.2,
Arg.sub.(3-10)Lys.sub.2Orn.sub.3, Arg.sub.(3-10)Lys.sub.1Orn.sub.4,
Arg.sub.(3-10)His.sub.4Orn.sub.1, Arg.sub.(3-10)His.sub.3Orn.sub.2,
Arg.sub.(3-10)His.sub.2Orn.sub.3, Arg.sub.(3-10)His.sub.1Orn.sub.4,
Arg.sub.4Lys.sub.(3-10)His.sub.1, Arg.sub.3Lys.sub.(3-10)His.sub.2,
Arg.sub.2Lys.sub.(3-10)His.sub.3, Arg.sub.1Lys.sub.(3-10)His.sub.4,
Arg.sub.4Lys.sub.(3-10)Orn.sub.1, Arg.sub.3Lys.sub.(3-10)Orn.sub.2,
Arg.sub.2Lys.sub.(3-10)Orn.sub.3, Arg.sub.1Lys.sub.(3-10)Orn.sub.4,
Lys.sub.(3-10)His.sub.4Orn.sub.1, Lys.sub.(3-10)His.sub.3Orn.sub.2,
Lys.sub.(3-10)His.sub.2Orn.sub.3, Lys.sub.(3-10)His.sub.1Orn.sub.4,
Arg.sub.4Lys.sub.1His.sub.(3-10), Arg.sub.3Lys.sub.2His.sub.(3-10),
Arg.sub.2Lys.sub.3His.sub.(3-10), Arg.sub.1Lys.sub.4His.sub.(3-10),
Arg.sub.4His.sub.(3-10)Orn.sub.1, Arg.sub.3His.sub.(3-10)Orn.sub.2,
Arg.sub.2His.sub.(3-10)Orn.sub.3, Arg.sub.1His.sub.(3-10)Orn.sub.4,
Lys.sub.4His.sub.(3-10)Orn.sub.1, Lys.sub.3His.sub.(3-10)Orn.sub.2,
Lys.sub.2His.sub.(3-10)Orn.sub.3, Lys.sub.1His.sub.(3-10)Orn.sub.4;
Arg.sub.(2-9)Lys.sub.5His.sub.1, Arg.sub.(2-9)Lys.sub.4His.sub.2,
Arg.sub.(2-9)Lys.sub.3His.sub.3, Arg.sub.(2-9)Lys.sub.2His.sub.4,
Arg.sub.(2-9)Lys.sub.1His.sub.5, Arg.sub.(2-9)Lys.sub.5Orn.sub.1,
Arg.sub.(2-9)Lys.sub.4Orn.sub.2, Arg.sub.(2-9)Lys.sub.3Orn.sub.3,
Arg.sub.(2-9)Lys.sub.2Orn.sub.4, Arg.sub.(2-9)Lys.sub.1Orn.sub.5,
Arg.sub.(2-9)His.sub.5Orn.sub.1, Arg.sub.(2-9)His.sub.4Orn.sub.2,
Arg.sub.(2-9)His.sub.3Orn.sub.3, Arg.sub.(2-9)His.sub.2Orn.sub.4,
Arg.sub.(2-9)His.sub.1Orn.sub.5, Arg.sub.5Lys.sub.(2-9)His.sub.1,
Arg.sub.4Lys.sub.(2-9)His.sub.2, Arg.sub.3Lys.sub.(2-9)His.sub.3,
Arg.sub.2Lys.sub.(2-9)His.sub.4, Arg.sub.1Lys.sub.(2-9)His.sub.5,
Arg.sub.5Lys.sub.(2-9)Orn.sub.1, Arg.sub.4Lys.sub.(2-9)Orn.sub.2,
Arg.sub.3Lys.sub.(2-9)Orn.sub.3, Arg.sub.2Lys.sub.(2-9)Orn.sub.4,
Arg.sub.1Lys.sub.(2-9)Orn.sub.5, Lys.sub.(2-9)His.sub.5Orn.sub.1,
Lys.sub.(2-9)His.sub.4Orn.sub.2, Lys.sub.(2-9)His.sub.3Orn.sub.3,
Lys.sub.(2-9)His.sub.2Orn.sub.4, Lys.sub.(2-9)His.sub.1Orn.sub.5,
Arg.sub.5Lys.sub.1His.sub.(2-9), Arg.sub.4Lys.sub.2His.sub.(2-9),
Arg.sub.3Lys.sub.3His.sub.(2-9), Arg.sub.2Lys.sub.4His.sub.(2-9),
Arg.sub.1Lys.sub.5His.sub.(2-9), Arg.sub.5His.sub.(2-9)Orn.sub.1,
Arg.sub.4His.sub.(2-9)Orn.sub.2, Arg.sub.3His.sub.(2-9)Orn.sub.3,
Arg.sub.2His.sub.(2-9)Orn.sub.4, Arg.sub.1His.sub.(2-9)Orn.sub.5,
Lys.sub.5His.sub.(2-9)Orn.sub.1, Lys.sub.4His.sub.(2-9)Orn.sub.2,
Lys.sub.3His.sub.(2-9)Orn.sub.3, Lys.sub.2His.sub.(2-9)Orn.sub.4,
Lys.sub.1His.sub.(2-9)Orn.sub.5; Arg.sub.(1-8)Lys.sub.6His.sub.1,
Arg.sub.(1-8)Lys.sub.5His.sub.2, Arg.sub.(1-8)Lys.sub.4His.sub.3,
Arg.sub.(1-8)Lys.sub.3His.sub.4, Arg.sub.(1-8)Lys.sub.2His.sub.5,
Arg.sub.(1-8)Lys.sub.1His.sub.6, Arg.sub.(1-8)Lys.sub.6Orn.sub.1,
Arg.sub.(1-8)Lys.sub.5Orn.sub.2, Arg.sub.(1-8)Lys.sub.4Orn.sub.3,
Arg.sub.(1-8)Lys.sub.3Orn.sub.4, Arg.sub.(1-8)Lys.sub.2Orn.sub.5,
Arg.sub.(1-8)Lys.sub.1Orn.sub.6, Arg.sub.(1-8)His.sub.6Orn.sub.1,
Arg.sub.(1-8)His.sub.5Orn.sub.2, Arg.sub.(1-8)His.sub.4Orn.sub.3,
Arg.sub.(1-8)His.sub.3Orn.sub.4, Arg.sub.(1-8)His.sub.2Orn.sub.5,
Arg.sub.(1-8)His.sub.1Orn.sub.6, Arg.sub.6Lys.sub.(1-8)His.sub.1,
Arg.sub.5Lys.sub.(1-8)His.sub.2, Arg.sub.4Lys.sub.(1-8)His.sub.3,
Arg.sub.3Lys.sub.(1-8)His.sub.4, Arg.sub.2Lys.sub.(1-8)His.sub.5,
Arg.sub.1Lys.sub.(1-8)His.sub.6, Arg.sub.6Lys.sub.(1-8)Orn.sub.1,
Arg.sub.5Lys.sub.(1-8)Orn.sub.2, Arg.sub.4Lys.sub.(1-8)Orn.sub.3,
Arg.sub.3Lys.sub.(1-8)Orn.sub.4, Arg.sub.2Lys.sub.(1-8)Orn.sub.5,
Arg.sub.1Lys.sub.(1-8)Orn.sub.6, Lys.sub.(1-8)His.sub.6Orn.sub.1,
Lys.sub.(1-8)His.sub.5Orn.sub.2, Lys.sub.(1-8)His.sub.4Orn.sub.3,
Lys.sub.(1-8)His.sub.3Orn.sub.4, Lys.sub.(1-8)His.sub.2Orn.sub.5,
Lys.sub.(1-8)His.sub.1Orn.sub.6, Arg.sub.6Lys.sub.1His.sub.(1-8),
Arg.sub.5Lys.sub.2His.sub.(1-8), Arg.sub.4Lys.sub.3His.sub.(1-8),
Arg.sub.3Lys.sub.4His.sub.(1-8), Arg.sub.2Lys.sub.5His.sub.(1-8),
Arg.sub.1Lys.sub.6His.sub.(1-8), Arg.sub.6His.sub.(1-8)Orn.sub.1,
Arg.sub.5His.sub.(1-8)Orn.sub.2, Arg.sub.4His.sub.(1-8)Orn.sub.3,
Arg.sub.3His.sub.(1-8)Orn.sub.4, Arg.sub.2His.sub.(1-8)Orn.sub.5,
Arg.sub.1His.sub.(1-8)Orn.sub.6, Lys.sub.6His.sub.(1-8)Orn.sub.1,
Lys.sub.5His.sub.(1-8)Orn.sub.2, Lys.sub.4His.sub.(1-8)Orn.sub.3,
Lys.sub.3His.sub.(1-8)Orn.sub.4, Lys.sub.2His.sub.(1-8)Orn.sub.5,
Lys.sub.1His.sub.(1-8)Orn.sub.6;
Arg.sub.(5-12)Lys.sub.1His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.(5-12)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(5-12)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.1Orn.sub.(5-12);
Arg.sub.(4-11)Lys.sub.2His.sub.1Orn.sub.1,
Arg.sub.(4-11)Lys.sub.1His.sub.2Orn.sub.1,
Arg.sub.(4-11)Lys.sub.1His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.(4-11)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.(4-11)His.sub.2Orn.sub.1,
Arg.sub.1Lys.sub.(4-11)His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.1His.sub.(4-11)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(4-11)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(4-11)Orn.sub.2,
Arg.sub.2Lys.sub.1His.sub.1Orn.sub.(4-11),
Arg.sub.1Lys.sub.2His.sub.1Orn.sub.(4-11),
Arg.sub.1Lys.sub.1His.sub.2Orn.sub.(4-11);
Arg.sub.(3-10)Lys.sub.3His.sub.1Orn.sub.1,
Arg.sub.(3-10)Lys.sub.2His.sub.2Orn.sub.1,
Arg.sub.(3-10)Lys.sub.2His.sub.1ORn.sub.2,
Arg.sub.(3-10)Lys.sub.1His.sub.2Orn.sub.2,
Arg.sub.(3-10)Lys.sub.1His.sub.1Orn.sub.3,
Arg.sub.3Lys.sub.(3-10)His.sub.1Orn.sub.1,
Arg.sub.2Lys.sub.(3-10)His.sub.2Orn.sub.1,
Arg.sub.2Lys.sub.(3-10)His.sub.1Orn.sub.2,
Arg.sub.1Lys.sub.(3-10)His.sub.2Orn.sub.2,
Arg.sub.1Lys.sub.(3-10)His.sub.1Orn.sub.3,
Arg.sub.3Lys.sub.1His.sub.(3-10)Orn.sub.1,
Arg.sub.2Lys.sub.2His.sub.(3-10)Orn.sub.1,
Arg.sub.2Lys.sub.1His.sub.(3-10)Orn.sub.2,
Arg.sub.1Lys.sub.2His.sub.(3-10)Orn.sub.2,
Arg.sub.1Lys.sub.1His.sub.(3-10)Orn.sub.3,
Arg.sub.3Lys.sub.1His.sub.1Orn.sub.(3-10),
Arg.sub.2Lys.sub.2His.sub.1Orn.sub.(3-10),
Arg.sub.2Lys.sub.1His.sub.2Orn.sub.(3-10),
Arg.sub.1Lys.sub.2His.sub.2Orn.sub.(3-10),
Arg.sub.1Lys.sub.1His.sub.3Orn.sub.(3-10);
Arg.sub.(2-9)Lys.sub.4His.sub.1Orn.sub.1,
Arg.sub.(2-9)Lys.sub.1His.sub.4Orn.sub.1,
Arg.sub.(2-9)Lys.sub.1His.sub.1Orn.sub.4,
Arg.sub.(2-9)Lys.sub.3His.sub.2Orn.sub.1,
Arg.sub.(2-9)Lys.sub.3His.sub.1Orn.sub.2,
Arg.sub.(2-9)Lys.sub.2His.sub.3Orn.sub.1,
Arg.sub.(2-9)Lys.sub.2His.sub.1Orn.sub.3,
Arg.sub.(2-9)Lys.sub.1His.sub.2Orn.sub.3,
Arg.sub.(2-9)Lys.sub.1His.sub.3Orn.sub.2,
Arg.sub.(2-9)Lys.sub.2His.sub.2Orn.sub.2,
Arg.sub.4Lys.sub.(2-9)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.(2-9)His.sub.4Orn.sub.1,
Arg.sub.1Lys.sub.(2-9)His.sub.1Orn.sub.4,
Arg.sub.3Lys.sub.(2-9)His.sub.2Orn.sub.1,
Arg.sub.3Lys.sub.(2-9)His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.(2-9)His.sub.3Orn.sub.1,
Arg.sub.2Lys.sub.(2-9)His.sub.1Orn.sub.3,
Arg.sub.1Lys.sub.(2-9)His.sub.2Orn.sub.3,
Arg.sub.1Lys.sub.(2-9)His.sub.3Orn.sub.2,
Arg.sub.2Lys.sub.(2-9)His.sub.2Orn.sub.2,
Arg.sub.4Lys.sub.1His.sub.(2-9)Orn.sub.1,
Arg.sub.1Lys.sub.4His.sub.(2-9)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(2-9)Orn.sub.4,
Arg.sub.3Lys.sub.2His.sub.(2-9)Orn.sub.1,
Arg.sub.3Lys.sub.1His.sub.(2-9)Orn.sub.2,
Arg.sub.2Lys.sub.3His.sub.(2-9)Orn.sub.1,
Arg.sub.2Lys.sub.1His.sub.(2-9)Orn.sub.3,
Arg.sub.1Lys.sub.2His.sub.(2-9)Orn.sub.3,
Arg.sub.1Lys.sub.3His.sub.(2-9)Orn.sub.2,
Arg.sub.2Lys.sub.2His.sub.(2-9)Orn.sub.2,
Arg.sub.4Lys.sub.1His.sub.1Orn.sub.(2-9),
Arg.sub.1Lys.sub.4His.sub.1Orn.sub.(2-9),
Arg.sub.1Lys.sub.1His.sub.4Orn.sub.(2-9),
Arg.sub.3Lys.sub.2His.sub.1Orn.sub.(2-9),
Arg.sub.3Lys.sub.1His.sub.2Orn.sub.(2-9),
Arg.sub.2Lys.sub.3His.sub.1Orn.sub.(2-9),
Arg.sub.2Lys.sub.1His.sub.3Orn.sub.(2-9),
Arg.sub.1Lys.sub.2His.sub.3Orn.sub.(2-9),
Arg.sub.1Lys.sub.3His.sub.2Orn.sub.(2-9),
Arg.sub.2Lys.sub.2His.sub.2Orn.sub.(2-9);
Arg.sub.(1-8)Lys.sub.5His.sub.1Orn.sub.1,
Arg.sub.(1-8)Lys.sub.1His.sub.5Orn.sub.1,
Arg.sub.(1-8)Lys.sub.1His.sub.1Orn.sub.5,
Arg.sub.(1-8)Lys.sub.4His.sub.2Orn.sub.1,
Arg.sub.(1-8)Lys.sub.2His.sub.4Orn.sub.1,
Arg.sub.(1-8)Lys.sub.2His.sub.1Orn.sub.4,
Arg.sub.(1-8)Lys.sub.1His.sub.2Orn.sub.4,
Arg.sub.(1-8)Lys.sub.1His.sub.4Orn.sub.2,
Arg.sub.(1-8)Lys.sub.4His.sub.1Orn.sub.2,
Arg.sub.(1-8)Lys.sub.3His.sub.3Orn.sub.1,
Arg.sub.(1-8)Lys.sub.3His.sub.1Orn.sub.3,
Arg.sub.(1-8)Lys.sub.1His.sub.3Orn.sub.3,
Arg.sub.5Lys.sub.(1-8)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.(1-8)His.sub.5Orn.sub.1,
Arg.sub.1Lys.sub.(1-8)His.sub.1Orn.sub.5,
Arg.sub.4Lys.sub.(1-8)His.sub.2Orn.sub.1,
Arg.sub.2Lys.sub.(1-8)His.sub.4Orn.sub.1,
Arg.sub.2Lys.sub.(1-8)His.sub.1Orn.sub.4,
Arg.sub.1Lys.sub.(1-8)His.sub.2Orn.sub.4,
Arg.sub.1Lys.sub.(1-8)His.sub.4Orn.sub.2,
Arg.sub.4Lys.sub.(1-8)His.sub.1Orn.sub.2,
Arg.sub.3Lys.sub.(1-8)His.sub.3Orn.sub.1,
Arg.sub.3Lys.sub.(1-8)His.sub.1Orn.sub.3,
Arg.sub.1Lys.sub.(1-8)His.sub.3Orn.sub.3,
Arg.sub.5Lys.sub.1His.sub.(1-8)Orn.sub.1,
Arg.sub.1Lys.sub.5His.sub.(1-8)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(1-8)Orn.sub.5,
Arg.sub.4Lys.sub.2His.sub.(1-8)Orn.sub.1,
Arg.sub.2Lys.sub.4His.sub.(1-8)Orn.sub.1,
Arg.sub.2Lys.sub.1His.sub.(1-8)Orn.sub.4,
Arg.sub.1Lys.sub.2His.sub.(1-8)Orn.sub.4,
Arg.sub.1Lys.sub.4His.sub.(1-8)Orn.sub.2,
Arg.sub.4Lys.sub.1His.sub.(1-8)Orn.sub.2,
Arg.sub.3Lys.sub.3His.sub.(1-8)Orn.sub.1,
Arg.sub.3Lys.sub.1His.sub.(1-8)Orn.sub.3,
Arg.sub.1Lys.sub.3His.sub.(1-8)Orn.sub.3,
Arg.sub.5Lys.sub.1His.sub.1Orn.sub.(1-8),
Arg.sub.1Lys.sub.5His.sub.1Orn.sub.(1-8),
Arg.sub.1Lys.sub.1His.sub.5Orn.sub.(1-8),
Arg.sub.4Lys.sub.2His.sub.1Orn.sub.(1-8),
Arg.sub.2Lys.sub.4His.sub.1Orn.sub.(1-8),
Arg.sub.2Lys.sub.1His.sub.4Orn.sub.(1-8),
Arg.sub.1Lys.sub.2His.sub.4Orn.sub.(1-8),
Arg.sub.1Lys.sub.4His.sub.2Orn.sub.(1-8),
Arg.sub.4Lys.sub.1His.sub.2Orn.sub.(1-8),
Arg.sub.3Lys.sub.3His.sub.1Orn.sub.(1-8),
Arg.sub.3Lys.sub.1His.sub.3Orn.sub.(1-8),
Arg.sub.1Lys.sub.3His.sub.3Orn.sub.(1-8);
[0041] According to one preferred embodiment, the oligopeptide of
the complexed RNA of the present invention, having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x, as shown above, is selected from the subgroup
consisting of: Arg.sub.8, Arg.sub.9, Arg.sub.10, Arg.sub.11,
Arg.sub.12, Arg.sub.13, Arg.sub.14, Arg.sub.15, (SEQ ID NOs: 1-8);
Lys.sub.8, Lys.sub.9, Lys.sub.10, Lys.sub.11, Lys.sub.12,
Lys.sub.13, Lys.sub.14, Lys.sub.15, (SEQ ID NOs: 9-16); His.sub.8,
His.sub.9, His.sub.10, His.sub.11, His.sub.12, His.sub.13,
His.sub.14, His.sub.15, (SEQ ID NOs: 17-24); or Orn.sub.8,
Orn.sub.9, Orn.sub.10, Orn.sub.11, Orn.sub.12, Orn.sub.13,
Orn.sub.14, Orn.sub.15, (SEQ ID NOs: 25-32).
[0042] According to another preferred embodiment, the oligopeptide
of the complexed RNA of the present invention, having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x as shown above, is selected from the subgroup
consisting of general formulas Arg.sub.9 (also termed R9),
Arg.sub.9His.sub.3 (also termed R9H3), His.sub.3Arg.sub.9His.sub.3
(also termed H3R9H3), TyrSerSerArg.sub.9SerSerTyr (also termed
YSSR9SSY), His.sub.3Arg.sub.9SerSerTyr (also termed H3R9SSY),
(ArgLysHis), (also termed (RKH).sub.4), Tyr(ArgLysHis).sub.2Arg
(also termed Y(RKH).sub.2R). Even more preferably, these general
formulas are defined as follows:
TABLE-US-00003 (SEQ ID NO: 2) Arg.sub.9:
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 39)
Arg.sub.9His.sub.3: Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-His
(SEQ ID NO: 40) His.sub.3Arg.sub.9His.sub.3:
His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- His-His-His (SEQ
ID NO: 41) TyrSerSerArg9SerSerTyr:
Tyr-Ser-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr (SEQ
ID NO: 42) His.sub.3Arg.sub.9SerSerTyr:
His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr (SEQ
ID NO: 43) (ArgLysHis).sub.4:
Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His (SEQ ID NO: 44)
Tyr(ArgLysHis).sub.2Arg: Tyr-Arg-Lys-His-Arg-Lys-His-Arg
[0043] The at least one oligopeptide of the complexed RNA
(molecule) of the present invention, having the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x as
shown above, may be additionally modified. Modifications in the
context of the present invention typically comprise any
modification suitable for peptides, provided that these
modifications do not interfere with the transfection capabilities
of the resulting complexed RNA.
[0044] Typical modifications may thus include e.g. the use of
modified amino acids as defined above. Furthermore, the terminal
amino acid residues of the oligopeptide, having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x
as shown above, with their carboxy (C-terminus) and their amino
(N-terminus) groups (as well as carboxy or amide amino acid side
chain groups, see above) may be present in their protected (e.g.
the C terminus protected by an amide group) and/or unprotected
form, using appropriate amino or carboxyl protecting groups. Also,
acid-addition salts of the oligopeptide, having the empirical
formula (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x as shown above, may be used. Common acid addition salts
are hydrohalic acid salts, i.e., HBr, HI, or more preferably,
HCl.
[0045] PEGylation of terminal or side chain carboxyl groups or the
epsilon-amino group of lysine occurring in the oligopeptide, having
the empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x as shown above, confers resistance to
agglomeration and serum degradation and is also within the scope of
the present invention.
[0046] The at least one oligopeptide of the complexed RNA
(molecule) of the present invention, having the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x as
shown above, may furthermore be modified to bind to or be coupled
to at least one specific ligand, wherein the at least one specific
ligand may be bound to or coupled to one or both terminal ends of
the at least one oligopeptide. The at least one specific ligand
bound to or coupled to one or both terminal ends of the
oligopeptide may be identical or different and may be selected from
any compound capable to bind to or interact with a receptor or a
protein or a protein/receptor complex, e.g. at the cell surface, e,
e.g., without being limited thereto, RGD-peptide, transferrin or
mannose, etc.
[0047] Other preferred modifications resulting in derivatives of
the oligopeptide, having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x as shown above, are
based on carbohydrates and/or lipids which may be covalently
coupled to the oligopeptide. It is preferred to couple
carbohydrates and/or lipids to serine, threonine, asparagine,
glutamine or tyrosine or glutamate or aspartate via their reactive
side chain moieties. Alternatively, carbohydrates and/or lipids may
also be linked to the terminal moieties of the oligopeptide as
defined herein. Furthermore, the oligopeptide may be coupled to a
functionally different peptide or protein moiety, which may also
stabilize the oligopeptide and/or may serve to improve the
transport properties of oligopeptide in body fluids, in particular
blood. Suitable peptides or proteins may e.g. be selected from
albumin, transferrin etc., which may be directly coupled to the
oligopeptide, having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x as shown above, or via
a peptide or organic linker sequence. Preferably, these peptides or
proteins are linked to one of the termini of the oligopeptide.
[0048] In this context, it is to be noted that a modification of
the oligopeptide with lipids, having the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x as
shown above, does typically not include the use of (saturated or
non-saturated) fatty acids, particularly not the use of long chain
(saturated or non-saturated) fatty acids (in particular with a
chain length of >C.sub.12, >C.sub.14 or >C.sub.16). Thus,
in the context of the present invention, modification of the
oligopeptide with fatty acids, having the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x as
shown above, does not form an integral part of the present
invention. However, if fatty acids are used at all to modify the
carrier peptide, thex may be selected, without being limited
thereto, from the group comprising e.g. butanoic fatty acid
(butyric fatty acid), pentanoic fatty acid (valeric fatty acid),
hexanoic fatty acid (caproic fatty acid), octanoic fatty acid
(caprylic fatty acid), nonanoic fatty acid (pelargonic fatty acid),
decanoic fatty acid (capric fatty acid), dodecanoic fatty acid
(lauric fatty acid), tetradecanoic fatty acid (myristic fatty
acid), hexadecanoic fatty acid (palmitic fatty acid), heptadecanoic
fatty acid (margaric (daturic) fatty acid), octadecanoic fatty acid
(stearic fatty acid), eicosanoic fatty acid (arachidic fatty acid),
docosanoic fatty acid (behenic fatty acid), tetracosanoic fatty
acid (lignoceric fatty acid), hexacosanoic fatty acid (cerotic
fatty acid), heptacosanoic fatty acid (carboceric fatty acid),
octacosanoic fatty acid (montanic fatty acid), triacontanoic fatty
acid (melissic fatty acid), dotriacontanoic fatty acid (lacceroic
fatty acid), tritriacontanoic fatty acid (ceromelissic (psyllic)
fatty acid), tetratriacontanoic fatty acid (geddic fatty acid),
pentatriacontanoic fatty acid (ceroplastic fatty acid), etc., or
their non-saturated analogs. As a particular example, the present
invention does typically not include the use of octadecanoic fatty
acid (stearic fatty acid) or its non-saturated analogs for
modification of the carrier peptides of formula I, i.e. typically
no stearylated oligopeptides of formula I may be used herein for
complexation of the RNA component of the inventive complex.
[0049] In order to circumvent the problem of degradation of the
oligopeptide, having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x as shown above,
according to another embodiment of the present invention a
retro-inverso isomer of the above oligopeptide composed of D amino
acids or at least partially composed of D amino acids may be used.
The term "retro-inverso isomer" refers to an isomer of a linear
peptide in which the direction of the sequence is reversed and the
chirality of each amino acid residue is inverted (see, e.g.,
Jameson et aZ, Nature, 368, 744-746 (1994); Brady et al., Nature,
368, 692-693 (1994)). With respect to the parent peptide, the
retro-inverso peptide is assembled in reverse order of amino acids,
typically with F-moc amino acid derivatives. Typically, the crude
peptides may be purified by reversed phase HPLC.
[0050] Other modifications, which may be introduced into the
oligopeptide, having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x as shown above, relate
to modifications of the peptide backbone. Preferably, the modified
oligopeptides are scaffold mimetics. Their backbone is different
from the natural occurring backbone, while their side-chain
structures are identical with the oligopeptides or their fragments,
variants or derivatives. In general, scaffold mimetics exhibit a
modification of one or more of the backbone chain members (NH, CH,
CO), either as substitution (preferably) or as an insertion.
Substituents are e.g. (I) --O--, --S--, or --CH.sub.2-- instead of
--NH--; (II) --N--, C-Alkyl-, or --BH-- instead of --CHR-- and
(III) --CS--, --CH.sub.2--, --SO.sub.n--, --P.dbd.O(OH)--, or
--B(OH)-- instead of --CO--. A peptide mimetic of an oligopeptide,
having the empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x, as defined herein, may be a combination
of each of these modifications. In particular, modifications of
each the groups I, II and III may be combined. In a peptide mimetic
each backbone chain member may be modified or, alternatively, only
a certain number of chain members may be exchanged for a
non-naturally occurring moiety. Preferably, all backbone chain
members of an oligopeptide, having the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x, as
defined herein, of either --NH--, --CHR-- or CO are exchanged for
another non-naturally occurring group. In case the amide bond
(--NH--CO--) of the oligopeptide backbone is substituted (in the
entire molecule or at least in one single position), preferable
substitution moieties are bioisosteric, e.g. retro-inverse amide
bonds (--CO--NH--), hydroxyl ethylene (--CH(OH)--CH.sub.2--),
alkene (CH.sub.2.dbd.CH--), carba (CH.sub.2--CH.sub.2--) and/or
--P.dbd.O(OH)--CH.sub.2--). Alternatively, backbone chain
elongation by insertions may occur in a scaffold mimetic of the
oligopeptide, having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x as defined
herein, e.g. by moieties flanking the C-alpha atom. On either side
of the C-alpha atom e.g. --O--, --S--, --CH--, --NH-- may be
inserted.
[0051] Particularly preferred are oligocarbamate peptide backbone
structure of the oligopeptide, having the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x, as
defined herein. Thereby amide bond may be replaced by a carbamate
moiety. The monomeric N-protected amino alkyl carbonates are
accessible via the corresponding amino acids or amino alcohols.
They are converted into active esters, e.g. p-nitro phenyl ester by
using the F-moc moiety or a photo sensitive nitroatryloxycarbonyl
group by solid phase synthesis.
[0052] The complexed RNA of the present invention further comprises
at least one RNA (molecule) suitable for transfection purposes,
wherein this at least one RNA (molecule) is complexed with one or
more oligopeptides, as disclosed above with empirical formula I
((Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
(Xaa).sub.x).
[0053] The at least one RNA (molecule) of the complexed RNA of the
present invention may have any length (preferably dependent on the
type of RNA to be applied as a complexed RNA according to the
present invention). Without being restricted thereto, the at least
one RNA (molecule) may have a length of 5 to 20000 nucleotides,
more preferably a length of 5 to 10000 or of 300 to 10000
nucleotides, even more preferably a length of 5 to 5000
nucleotides, and most preferably a length of 20 to 5000, of 50 to
5000, of 100 to 5000 or of 300 to 10000 nucleotides depending on
the type of RNA to be transfected (see disclosure below).
[0054] The at least one RNA (molecule) of the complexed RNA of the
present invention may be any RNA, preferably, without being limited
thereto, a short RNA oligonucleotide (preferable length 5 to 80 or,
more preferably 20 to 80 nucleotides), a coding RNA, an
immunostimulatory RNA, a siRNA, an antisense RNA, or riboswitches,
ribozymes or aptamers. Furthermore, the at least one RNA (molecule)
of the complexed RNA of the present invention may be a single- or a
double-stranded RNA (which may also be regarded as an RNA
(molecule) due to non-covalent association of two single-stranded
RNA (molecules)) or a partially double-stranded RNA (which is
typically formed by a longer and a shorter single-stranded RNA
molecule or by two single stranded RNA-molecules, which are about
equal in length, wherein one single-stranded RNA molecule is
partially complementary to the other single-stranded RNA molecule
and both thus form a double-stranded RNA molecule in this region).
Preferably, the at least one RNA (molecule) of the complexed RNA of
the present invention may be a single-stranded RNA. The at least
one RNA (molecule) of the complexed RNA of the present invention
may also be a circular or linear RNA, preferably a linear RNA. More
preferably, the at least one RNA (molecule) of the complexed RNA of
the present invention may be a (linear) single-stranded RNA. The at
least one RNA (molecule) of the complexed RNA of the present
invention may be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a
messenger RNA (mRNA), or a viral RNA (vRNA), preferably a mRNA. The
present invention allows all of these RNAs to be transfected into
the cell. In this context, an mRNA is typically an RNA, which is
composed of several structural elements, e.g. an optional 5'-UTR
region, an upstream positioned ribosomal binding site followed by a
coding region, an optional 3'-UTR region, which may be followed by
a poly-A tail (and/or a poly-C-tail). An mRNA may occur as a mono-,
di-, or even multicistronic RNA, i.e. an RNA which carries the
coding sequences of one, two or more proteins. Such coding
sequences in di-, or even multicistronic mRNA may be separated by
at least one IRES sequence, e.g. as defined herein.
Short RNA Oligonucleotides
[0055] In a first embodiment, the at least one RNA (molecule) of
the complexed RNA of the present invention may be a short RNA
oligonucleotide. Short RNA oligonucleotides in the context of the
present invention may comprise any RNA as defined above.
Preferably, the short RNA oligonucleotide may be a single- or a
double-stranded RNA oligonucleotide, more preferably a
single-stranded RNA oligonucleotide. Even more preferably, the
short RNA oligonucleotide may be a linear single-stranded RNA
oligonucleotide.
[0056] Preferably, the short RNA oligonucleotides as used herein
comprise a length as defined above in general for RNA molecules,
more preferably a length of 5 to 100, of 5 to 50, or of 5 of 30,
or, alternatively, a length of 20 to 100, of 20 to 80, or, even
more preferably, of 20 of 60 nucleotides. Short RNA
oligonucleotides may be used for various purposes, e.g. for
(unspecific) immune stimulation, or reducing/suppressing
transcription/translation of genes.
Coding RNA
[0057] In a second embodiment, the at least one RNA (molecule) of
the complexed RNA of the present invention may be a coding RNA. The
coding RNA of the complexed RNA of the present invention may be any
RNA as defined above. Preferably, the coding RNA may be a single-
or a double-stranded RNA, more preferably a single-stranded RNA,
and/or a circular or linear RNA, more preferably a linear RNA. Even
more preferably, the coding RNA may be a (linear) single-stranded
RNA. Most preferably, the coding RNA may be a ((linear)
single-stranded) messenger RNA (mRNA).
[0058] The coding RNA may further encode a protein or a peptide,
which may be selected, without being restricted thereto, e.g. from
therapeutically active proteins or peptides, tumor antigens,
antibodies, immunostimulating proteins or peptides, etc., or from
any other protein or peptide suitable for a specific (therapeutic)
application, wherein the at least one RNA (molecule) encoding the
protein is to be transported into a cell, a tissue or an organism
and the protein is expressed subsequently in this cell, tissue or
organism.
[0059] In this context, therapeutically active proteins may be
selected from any recombinant or isolated proteins known to a
skilled person from the prior art. Without being restricted thereto
therapeutically active proteins as encoded by the at least one RNA
(molecule) of the complexed RNA as defined herein may be selected
from apoptotic factors or apoptosis related proteins including AIF,
Apaf e.g. Apaf-1, Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L),
Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L, Bcl-x.sub.S, bik, CAD,
Calpain, Caspase e.g. Caspase-1, Caspase-2, Caspase-3, Caspase-4,
Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10,
Caspase-11, ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrom C, CdR1,
DcR1, DD, DED, DISC, DNA-PK.sub.CS, DR3, DR4, DR5, FADD/MORT-1,
FAK, Fas (Fas-ligand CD95/fas (receptor)), FLICE/MACH, FLIP,
fodrin, fos, G-Actin, Gas-2, gelsolin, granzyme A/B, ICAD, ICE,
JNK, lamin A/B, MAP, MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD,
NF-.sub.kappaB, NuMa, p53, PAK-2, PARP, perforin, PITSLRE,
PKCdelta, pRb, presenilin, prICE, RAIDD, Ras, RIP,
sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2,
TRAIL-R1, TRAIL-R2, TRAIL-R3, transglutaminase, etc.
[0060] Therapeutically active proteins as encoded by the at least
one RNA (molecule) of the complexed RNA as defined herein may also
be selected from recombinant proteins, including proteins selected
from the group consisting of OATL3, OFC3, OPA3, OPD2, 4-1BBL, 5T4,
6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4,
ABCB1, ABCB11, ABCB2, ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1,
ABCD3, ABCG5, ABCC8, ABL1, ABO, ABR ACAA1, ACACA, ACADL, ACADM,
ACADS, ACADVL, ACAT1, ACCPN, ACE, ACHE, ACHM3, ACHM1, ACLS, ACPI,
ACTA1, ACTC, ACTN4, ACVRL1, AD2, ADA, ADAMTS13, ADAMTS2, ADFN,
ADH1B, ADH1C, ADLDH3A2, ADRB2, ADRB3, ADSL, AEZ, AFA, AFD1, AFP,
AGA, AGL, AGMX2, AGPS, AGS1, AGT, AGTR1, AGXT, AH02, AHCY, AHDS,
AHHR, AHSG, AIC, AIED, AIH2, AIH3, AIM-2, AIPL1, AIRE, AK1, ALAD,
ALAS2, ALB, HPG1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH1A1, ALDOA,
ALDOB, ALMS1, ALPL, ALPP, ALS2, ALX4, AMACR, AMBP, AMCD, AMCD1,
AMCN, AMELX, AMELY, AMGL, AMH, AMHR2, AMPD3, AMPD1, AMT, ANC, ANCR,
ANK1, ANOP1, AOM, APOA4, APOC2, APOC3, AP3B1, APC, aPKC, APOA2,
APOA1, APOB, APOC3, APOC2, APOE, APOH, APP, APRT, APS1, AQP2, AR,
ARAF1, ARG1, ARHGEF12, ARMET, ARSA, ARSB, ARSC2, ARSE, ART-4,
ARTC1/m, ARTS, ARVD1, ARX, AS, ASAH, ASAT, ASD1, ASL, ASMD, ASMT,
ASNS, ASPA, ASS, ASSP2, ASSP5, ASSP6, AT3, ATD, ATHS, ATM, ATP2A1,
ATP2A2, ATP2C1, ATP6B1, ATP7A, ATP7B, ATP8B1, ATPSK2, ATRX, ATXN1,
ATXN2, ATXN3, AUTS1, AVMD, AVP, AVPR2, AVSD1, AXIN1, AXIN2, AZF2,
B2M, B4GALT7, B7H4, BAGE, BAGE-1, BAX, BBS2, BBS3, BBS4, BCA225,
BCAA, BCH, BCHE, BCKDHA, BCKDHB, BCL10, BCL2, BCL3, BCL5, BCL6,
BCPM, BCR, BCR/ABL, BDC, BDE, BDMF, BDMR, BEST1, beta-Catenin/m,
BF, BFHD, BFIC, BFLS, BFSP2, BGLAP, BGN, BHD, BHR1, BING-4, BIRC5,
BJS, BLM, BLMH, BLNK, BMPR2, BPGM, BRAF, BRCA1, BRCA1/m, BRCA2,
BRCA2/m, BRCD2, BRCD1, BRDT, BSCL, BSCL2, BTAA, BTD, BTK, BUB1,
BWS, BZX, COL2A1, COL6A1, C1NH, C1QA, C1QB, C1QG, C1S, C2, C3, C4A,
C4B, C5, C6, C7, C7orf2, C8A, C8B, C9, CA125, CA15-3/CA 27-29,
CA195, CA19-9, CA72-4, CA2, CA242, CA50, CABYR, CACD, CACNA2D1,
CACNA1A, CACNA1F, CACNA1S, CACNB2, CACNB4, CAGE, CA1, CALB3, CALCA,
CALCR, CALM, CALR, CAM43, CAMEL, CAP-1, CAPN3, CARD15, CASP-5/m,
CASP-8, CASP-8/m, CASR, CAT, CATM, CAV3, CB1, CBBM, CBS, CCA1,
CCAL2, CCAL1, CCAT, CCL-1, CCL-11, CCL-12, CCL-13, CCL-14, CCL-15,
CCL-16, CCL-17, CCL-18, CCL-19, CCL-2, CCL-20, CCL-21, CCL-22,
CCL-23, CCL-24, CCL-25, CCL-27, CCL-3, CCL-4, CCL-5, CCL-7, CCL-8,
CCM1, CCNB1, CCND1, CCO, CCR2, CCR5, CCT, CCV, CCZS, CD1, CD19,
CD20, CD22, CD25, CD27, CD27L, cD3, CD30, CD30, CD30L, CD33, CD36,
CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD44v, CD44v6, CD52,
CD55, CD56, CD59, CD80, CD86, CDAN1, CDAN2, CDAN3, CDC27, CDC27/m,
CDC2L1, CDH1, CDK4, CDK4/m, CDKN1C, CDKN2A, CDKN2A/m, CDKN1A,
CDKN1C, CDL1, CDPD1, CDR1, CEA, CEACAM1, CEACAM5, CECR, CECR9,
CEPA, CETP, CFNS, CFTR, CGF1, CHAC, CHED2, CHED1, CHEK2, CHM, CHML,
CHR39c, CHRNA4, CHRNA1, CHRNB1, CHRNE, CHS, CHS1, CHST6, CHX10,
CIAS1, CIDX, CKN1, CLA2, CLAS, CLA1, CLCA2, CLCN1, CLCN5, CLCNKB,
CLDN16, CLP, CLN2, CLN3, CLN4, CLN5, CLN6, CLN8, C1QA, C1QB, C1QG,
C1R, CLS, CMCWTD, CMDJ, CMD1A, CMD1B, CMH2, MH3, CMH6, CMKBR2,
CMKBR5, CML28, CML66, CMM, CMT2B, CMT2D, CMT4A, CMT1A, CMTX2,
CMTX3, C-MYC, CNA1, CND, CNGA3, CNGA1, CNGB3, CNSN, CNTF, COA-1/m,
COCH, COD2, COD1, COH1, COL10A, COL2A2, COL11A2, COL17A1, COL1A1,
COL1A2, COL2A1, COL3A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1,
COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A2, COL9A3,
COL11A1, COL1A2, COL23A1, COL1A1, COLQ, COMP, COMT, CORD5, CORD1,
COX10, COX-2, CP, CPB2, CPO, CPP, CPS1, CPT2, CPT1A, CPX, CRAT,
CRB1, CRBM, CREBBP, CRH, CRHBP, CRS, CRV, CRX, CRYAB, CRYBA1,
CRYBB2, CRYGA, CRYGC, CRYGD, CSA, CSE, CSF1R, CSF2RA, CSF2RB,
CSF3R, CSF1R, CST3, CSTB, CT, CT7, CT-9/BRD6, CTAA1, CTACK, CTEN,
CTH, CTHM, CTLA4, CTM, CTNNB1, CTNS, CTPA, CTSB, CTSC, CTSK, CTSL,
CTS1, CUBN, CVD1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13,
CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9,
CYB5, CYBA, CYBB, CYBB5, CYFRA 21-1, CYLD, CYLD1, CYMD, CYP11B1,
CYP11B2, CYP17, CYP17A1, CYP19, CYP19A1, CYP1A2, CYP1B1, CYP21A2,
CYP27A1, CYP27B1, CYP2A6, CYP2C, CYP2C19, CYP2C9, CYP2D, CYP2D6,
CYP2D7P1, CYP3A4, CYP7B1, CYPB1, CYP11B1, CYP1A1, CYP1B1, CYRAA,
D40, DADI, DAM, DAM-10/MAGE-B1, DAM-6/MAGE-B2, DAX1, DAZ, DBA, DBH,
DBI, DBT, DCC, DC-CK1, DCK, DCR, DCX, DDB1, DDB2, DDIT3, DDU,
DECR1, DEK-CAN, DEM, DES, DF, DFN2, DFN4, DFN6, DFNA4, DFNA5,
DFNB5, DGCR, DHCR7, DHFR, DHOF, DHS, DIA1, DIAPH2, DIAPH1, DIH1,
DIO1, DISCI, DKC1, DLAT, DLD, DLL3, DLX3, DMBT1, DMD, DM1, DMPK,
DMWD, DNAI1, DNASE1, DNMT3B, DPEP1, DPYD, DPYS, DRD2, DRD4, DRPLA,
DSCR1, DSG1, DSP, DSPP, DSS, DTDP2, DTR, DURS1, DWS, DYS, DYSF,
DYT2, DYT3, DYT4, DYT2, DYT1, DYX1, EBAF, EBM, EBNA, EBP, EBR3,
EBS1, ECA1, ECB2, ECE1, ECGF1, ECT, ED2, ED4, EDA, EDAR, ECA1,
EDN3, EDNRB, EEC1, EEF1A1L14, EEGV1, EFEMP1, EFTUD2/m, EGFR,
EGFR/Her1, EGI, EGR2, EIF2AK3, eIF4G, EKG, EI IS, ELA2, ELF2,
ELF2M, ELK1, ELN, ELONG, EMD, EML1, EMMPRIN, EMX2, ENA-78, ENAM,
END3, ENG, ENO1, ENPP1, ENUR2, ENUR1, EOS, EP300, EPB41, EPB42,
EPCAM, EPD, EphA1, EphA2, EphA3, EphrinA2, EphrinA3, EPHX1, EPM2A,
EPO, EPOR, EPX, ERBB2, ERCC2 ERCC3, ERCC4, ERCC5, ERCC6, ERVR,
ESR1, ETFA, ETFB, ETFDH, ETM1, ETV6-AML1, ETV1, EVC, EVR2, EVR1,
EWSR1, EXT2, EXT3, EXT1, EYA1, EYCL2, EYCL3, EYCL1, EZH2, F10, F11,
F12, F13A1, F13B, F2, F5, F5F8D, F7, F8, F8C, F9, FABP2, FACL6,
FAH, FANCA, FANCB, FANCC, FANCD2, FANCF, FasL, FBN2, FBN1, FBP1,
FCG3RA, FCGR2A, FCGR2B, FCGR3A, FCHL, FCMD, FCP1, FDPSL5, FECH,
FEO, FEOM1, FES, FGA, FGB, FGD1, FGF2, FGF23, FGF5, FGFR2, FGFR3,
FGFR1, FGG, FGS1, FH, FIC1, FIH, F2, FKBP6, FLNA, FLT4, FMO3, FMO4,
FMR2, FMR1, FN, FN1/m, FOXC1, FOXE1, FOXL2, FOXO1A, FPDMM, FPF,
Fra-1, FRAXF, FRDA, FSHB, FSHMD1A, FSHR, FTH1, FTHL17, FTL, FTZF1,
FUCA1, FUT2, FUT6, FUT1, FY, G250, G250/CAIX, G6PC, G6PD, G6PT1,
G6PT2, GAA, GABRA3, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7b, GAGE-8, GALC, GALE, GALK1, GALNS, GALT, GAMT, GAN, GAST,
GASTRIN17, GATA3, GATA, GBA, GBE, GC, GCDH, GCGR, GCH1, GCK, GCP-2,
GCS1, G-CSF, GCSH, GCSL, GCY, GDEP, GDF5, GDI1, GDNF, GDXY, GFAP,
GFND, GGCX, GGT1, GH2, GH1, GHR, GHRHR, GHS, GIF, GINGF, GIP, GJA3,
GJA8, GJB2, GJB3, GJB6, GJB1, GK, GLA, GLB, GLB1, GLC3B, GLC1B,
GLC1C, GLDC, GLI3, GLP1, GLRA1, GLUD1, GM1 (fuc-GM1), GM2A, GM-CSF,
GMPR, GNAI2, GNAS, GNAT1, GNB3, GNE, GNPTA, GNRH, GNRH1, GNRHR,
GNS, GnT-V, gp100, GP1BA, GP1BB, GP9, GPC3, GPD2, GPDS1, GP1,
GP1BA, GPN1LW, GPNMB/m, GPSC, GPX1, GRHPR, GRK1, GRO, GRO, GRO,
GRPR, GSE, GSM1, GSN, GSR, GSS, GTD, GTS, GUCA1A, GUCY2D, GULOP,
GUSB, GUSM, GUST, GYPA, GYPC, GYS1, GYS2, HOKPP2, HOMG2, HADHA,
HADHB, HAGE, HAGH, HAL, HAST-2, HB 1, HBA2, HBA1, HBB, HBBP1, HBD,
HBE1, HBG2, HBG1, HBHR, HBP1, HBQ1, HBZ, HBZP, HCA, HCC-1, HCC-4,
HCF2, HCG, HCL2, HCL1, HCR, HCVS, HD, HPN, HER2, HER2/NEU, HER3,
HERV-K-MEL, HESX1, HEXA, HEXB, HF1, HFE, HF1, HGD, HHC2, HHC3, HHG,
HK1 HLA-A, HLA-A*0201-R170I, HLA-A11/m, HLA-A2/m, HLA-DPB1 HLA-DRA,
HLCS, HLXB9, HMBS, HMGA2, HMGCL, HMI, HMN2, HMOX1, HMS1 HMW-MAA,
HND, HNE, HNF4A, HOAC, HOMEOBOX NKX 3.1, HOM-TES-14/SCP-1,
HOM-TES-85, HOXA1 HOXD13, HP, HPC1, HPD, HPE2, HPE1, HPFH, HPFH2,
HPRT1, HPS1, HPT, HPV-E6, HPV-E7, HR, HRAS, HRD, HRG, HRPT2, HRPT1,
HRX, HSD11B2, HSD17B3, HSD17B4, HSD3B2, HSD3B3, HSN1, HSP70-2M,
HSPG2, HST-2, HTC2, HTC1, hTERT, HTN3, HTR2c, HVBS6, HVBS1, HVEC,
HV1S, HYAL1, HYR, 1-309, IAB, IBGC1, IBM2, ICAM1, ICAM3, iCE, ICHQ,
ICR5, ICR1, ICS1, IDDM2, IDDM1, IDS, IDUA, IF, IFNa/b, IFNGR1,
IGAD1, IGER, IGF-1R, IGF2R, IGF1, IGH, IGHC, IGHG2, IGHG1, IGHM,
IGHR, IGKC, IHG1, IHH, IKBKG, IL1, IL-1RA, IL10, IL-11, IL12,
IL12RB1, IL13, IL-13R.alpha.2, IL-15, IL-16, IL-17, IL18, IL-1a,
IL-1.alpha., IL-1b, IL-1.beta., IL1RAPL1, IL2, IL24, IL-2R, IL2RA,
IL2RG, IL3, IL3RA, IL4, IL4R, IL4R, IL-5, IL6, IL-7, IL7R, IL-8,
IL-9, Immature laminin receptor, IMMP2L, INDX, INFGR1, INFGR2,
INF.alpha., IFN.beta.INF.gamma., INS, INSR, INVS, IP-10, IP2, IPF1,
IP1, IRF6, IRS1, ISCW, ITGA2, ITGA2B, ITGA6, ITGA7, ITGB2, ITGB3,
ITGB4, ITIH1, ITM2B, IV, IVD, JAG1, JAK3, JBS, JBTS1, JMS, JPD,
KAL1, KAL2, KAL1, KLK2, KLK4, KCNA1, KCNE2, KCNE1, KCNH2, KCNJ1,
KCNJ2, KCNJ1, KCNQ2, KCNQ3, KCNQ4, KCNQ1, KCS, KERA, KFM, KFS,
KFSD, KHK, ki-67, KIAA0020, KIAA0205, KIAA0205/m, KIF1B, KIT,
KK-LC-1, KLK3, KLKB1, KM-HN-1, KMS, KNG, KNO, K-RAS/m, KRAS2,
KREV1, KRT1, KRT10, KRT12, KRT13, KRT14, KRT14L1, KRT14L2, KRT14L3,
KRT16, KRT16L1, KRT16L2, KRT17, KRT18, KRT2A, KRT3, KRT4, KRT5,
KRT6A, KRT6B, KRT9, KRTHB1, KRTHB6, KRT1, KSA, KSS, KWE, KYNU,
LOH19CR1, L1CAM, LAGE, LAGE-1, LALL, LAMA2, LAMA3, LAMB3, LAMB1,
LAMC2, LAMP2, LAP, LCA5, LCAT, LCCS, LCCS1, LCFS2, LCS1, LCT, LDHA,
LDHB, LDHC, LDLR, LDLR/FUT, LEP, LEWISY, LGCR, LGGF-PBP, LGI1,
LGMD2H, LGMD1A, LGMD1B, LHB, LHCGR, LHON, LHRH, LHX3, LIF, LIG1,
LIMM, LIMP2, LIPA, LIPA, LIPB, LIPC, LIVIN, L1CAM, LMAN1, LMNA,
LMX1B, LOLR, LOR, LOX, LPA, LPL, LPP, LQT4, LRP5, LRS1, LSFC,
LT-13, LTBP2, LTC4S, LYL1, XCL1, LYZ, M344, MA50, MAA, MADH4,
MAFD2, MAFD1, MAGE, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3,
MAGE-A4, MAGE-A6, MAGE-A9, MAGEB1, MAGE-B10, MAGE-B16, MAGE-B17,
MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2,
MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,
MAGE-H1, MAGEL2, MGB1, MGB2, MAN2A1, MAN2B1, MANBA, MANBB, MAOA,
MAOB, MAPK8IP1, MAPT, MART-1, MART-2, MART2/m, MAT1A, MBL2, MBP,
MBS1, MC1R, MC2R, MC4R, MCC, MCCC2, MCCC1, MCDR1, MCF2, MCKD, MCL1,
MC1R, MCOLN1, MCOP, MCOR, MCP-1, MCP-2, MCP-3, MCP-4, MCPH2, MCPH1,
MCS, M-CSF, MDB, MDCR, MDM2, MDRV, MDS1, MEI, MEI/m, ME2, ME20,
ME3, MEAX, MEB, MEC CCL-28, MECP2, MEFV, MELANA, MELAS, MEN1 MSLN,
MET, MF4, MG50, MG50/PXDN, MGAT2, MGAT5, MGC1 MGCR, MGCT, MGI, MGP,
MHC2TA, MHS2, MHS4, MIC2, MICS, MIDI, MIF, MIP, MIP-5/HCC-2, MITF,
MJD, MKI67, MKKS, MKS1, MLH1, MLL, MLLT2, MLLT3, MLLT7, MLLT1, MLS,
MLYCD, MMA1a, MMP 11, MMVP1, MN/CA IX-Antigen, MNG1, MN1, MOC31,
MOCS2, MOCS1, MOG, MORC, MOS, MOV18, MPD1, MPE, MPFD, MPI, MPIF-1,
MPL, MPO, MPS3C, MPZ, MRE11A, MROS, MRP1, MRP2, MRP3, MRSD, MRX14,
MRX2, MRX20, MRX3, MRX40, MRXA, MRX1, MS, MS4A2, MSD, MSH2, MSH3,
MSH6, MSS, MSSE, MSX2, MSX1, MTATP6, MTCO3, MTCO1, MTCYB, MTHFR,
MTM1, MTMR2, MTND2, MTND4, MTND5, MTND6, MTND1, MTP, MTR, MTRNR2,
MTRNR1, MTRR, MTTE, MTTG, MTTI, MTTK, MTTL2, MTTL1, MTTN, MTTP,
MTTS1, MUC1, MUC2, MUC4, MUC5AC, MUM-1, MUM-1/m, MUM-2, MUM-2/m,
MUM-3, MUM-3/m, MUT, mutant p21 ras, MUTYH, MVK, MX2, MXI1, MY05A,
MYB, MYBPC3, MYC, MYCL2, MYH6, MYH7, MYL2, MYL3, MYMY, MYO15A,
MY01G, MY05A, MYO7A, MYOC, Myosin/m, MYP2, MYP1, NA88-A,
N-acetylglucosaminyltransferase-V, NAGA, NAGLU, NAMSD, NAPB, NAT2,
NAT, NBIA1, NBS1, NCAM, NCF2, NCF1, NDN, NDP, NDUFS4, NDUFS7,
NDUFS8, NDUFV1, NDUFV2, NEB, NEFH, NEM1, Neo-PAP, neo-PAP/m, NEU1,
NEUROD1, NF2, NF1, NFYCJm, NGEP, NHS, NKS1, NKX2E, NM, NME1, NMP22,
NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH1, NP, NPC2, NPC1, NPHL2,
NPHP1, NPHS2, NPHS1, NPM/ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2,
NRAS, NRAS/m, NRL, NROB1, NRTN, NSE, NSX, NTRK1, NUMA1, NXF2,
NY-CO1, NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35,
NYS1, NYX, OA3, OA1, OAP, OASD, OAT, OCA1, OCA2, OCD1, OCRL, OCRL1,
OCT, ODDD, ODT1, OFC1, OFD1, OGDH, OGT, OGT/m, OPA2, OPA1, OPD1,
OPEM, OPG, OPN, OPN1LW, OPN1MW, OPN1SW, OPPG, OPTB1, TTD, ORM1,
ORP1, OS-9, OS-9/m, OSM LIF, OTC, OTOF, OTSC1, OXCT1, OYTES1, P15,
P190 MINOR BCR-ABL, P2RY12, P3, P16, P40, P4HB, P-501, P53, P53/m,
P97, PABPN1, PAFAH1B1, PAFAH1P1, PAGE-4, PAGE-5, PAH, PAI-1, PAI-2,
PAK3, PAP, PAPPA, PARK2, PART-1, PATE, PAX2, PAX3, PAX6, PAX7,
PAX8, PAX9, PBCA, PBCRA1, PBT, PBX1, PBXP1, PC, PCBD, PCCA, PCCB,
PCK2, PCK1, PCLD, PCOS1, PCSK1, PDB1, PDCN, PDE6A, PDE6B, PDEF,
PDGFB, PDGFR, PDGFRL, PDHA1, PDR, PDX1, PECAM1, PEE1, PEO1, PEPD,
PEX10, PEX12, PEX13, PEX3, PEX5, PEX6, PEX7, PEX1, PF4, PFBI, PFC,
PFKFB1, PFKM, PGAM2, PGD, PGK1, PGK1P1, PGL2, PGR, PGS, PHA2A, PHB,
PHEX, PHGDH, PHKA2, PHKA1, PHKB, PHKG2, PHP, PHYH, P1, PI3, PIGA,
PIM1-KINASE, PIN1, PIP5K1B, PITX2, PITX3, PKD2, PKD3, PKD1, PKDTS,
PKHD1, PKLR, PKP1, PKU1, PLA2G2A, PLA2G7, PLAT, PLEC1, PLG, PLI,
PLOD, PLP1, PMEL17, PML, PML/RAR.alpha., PMM2, PMP22, PMS2, PMS1,
PNKD, PNLIP, POF1, POLA, POLH, POMC, PON2, PON1, PORC, POTE,
POU1F1, POU3F4, POU4F3, POU1F1, PPAC, PPARG, PPCD, PPGB, PPH1,
PPKB, PPMX, PPDX, PPP1R3A, PPP2R2B, PPT1, PRAME, PRB, PRB3, PRCA1,
PRCC, PRD, PRDX5/m, PRF1, PRG4, PRKAR1A, PRKCA, PRKDC, PRKWNK4,
PRNP, PROC, PRODH, PROM1, PROP1, PRO51, PRST, PRP8, PRPF31, PRPF8,
PRPH2, PRPS2, PRPS1, PRS, PRSS7, PRSS1, PRTN3, PRX, PSA, PSAP,
PSCA, PSEN2, PSEN1, PSG1, PSGR, PSM, PSMA, PSORS1, PTC, PTCH,
PTCH1, PTCH2, PTEN, PTGS1, PTH, PTHR1, PTLAH, PTOS1, PTPN12, PTPNI
I, PTPRK, PTPRK/m, PTS, PUJO, PVR, PVRL1, PWCR, PXE, PXMP3, PXR1,
PYGL, PYGM, QDPR, RAB27A, RAD54B, RAD54L, RAG2, RAGE, RAGE-1, RAG1,
RAP1, RARA, RASA1, RBAF600/m, RB1, RBP4, RBP4, RBS, RCA1, RCAS1,
RCCP2, RCD1, RCV1, RDH5, RDPA, RDS, RECQL2, RECQL3, RECQL4, REG1A,
REHOBE, REN, RENBP, RENS1, RET, RFX5, RFXANK, RFXAP, RGR, RHAG,
RHAMM/CD168, RHD, RHO, Rip-1, RLBP1, RLN2, RLN1, RLS, RMD1, RMRP,
ROM1, ROR2, RP, RP1, RP14, RP17, RP2, RP6, RP9, RPD1, RPE65, RPGR,
RPGRIP1, RP1, RP10, RPS19, RPS2, RPS4X, RPS4Y, RPS6KA3, RRAS2, RS1,
RSN, RSS, RU1, RU2, RUNX2, RUNX1, RS, RYR1, S-100, SAA1, SACS, SAG,
SAGE, SALL1, SARDH, SART1, SART2, SART3, SAS, SAX1, SCA2, SCA4,
SCA5, SCA7, SCA8, SCA1, SCC, SCCD, SCF, SCLC1, SCN1A, SCN1B, SCN4A,
SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SCP1, SCZD2, SCZD3, SCZD4,
SCZD6, SCZD1, SDF-1.alpha./.beta. SDHA, SDHD, SDYS, SEDL, SERPENA7,
SERPINA3, SERPINA6, SERPINA1, SERPINC1, SERPIND1, SERPINE1,
SERPINF2, SERPING1, SERPINI1, SFTPA1, SFTPB, SFTPC, SFTPD, SGCA,
SGCB, SGCD, SGCE, SGM1, SGSH, SGY-1, SH2D1A, SHBG, SHFM2, SHFM3,
SHFM1, SHH, SHOX, SI, SIAL, SIALYL LEWISX, SIASD, S11, SIM1,
SIRT2/m, SIX3, SJS1, SKP2, SLC10A2, SLC12A1, SLC12A3, SLC17A5,
SLC19A2, SLC22A1L, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC25A4,
SLC25A5, SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1, SLC2A2,
SLC2A4, SLC3A1, SLC4A1, SLC4A4, SLC5A1, SLC5A5, SLC6A2, SLC6A3,
SLC6A4, SLC7A7, SLC7A9, SLC11A1, SLOS, SMA, SMAD1, SMAL, SMARCB1,
SMAX2, SMCR, SMCY, SM1, SMN2, SMN1, SMPD1, SNCA, SNRPN, SOD2, SOD3,
SOD1, SOS1, SOST, SOX9, SOX10, Sp17, SPANXC, SPG23, SPG3A, SPG4,
SPG5A, SPG5B, SPG6, SPG7, SPINK1, SPINK5, SPPK, SPPM, SPSMA, SPTA1,
SPTB, SPTLC1, SRC, SRD5A2, SRPX, SRS, SRY, .beta.hCG, SSTR2, SSX1,
SSX2 (HOM-MEL-40/SSX2), SSX4, ST8, STAMP-1, STAR, STARP1, STATH,
STEAP, STK2, STK11, STn/KLH, STO, STOM, STS, SUOX, SURF1,
SURVIVIN-2B, SYCP1, SYM1, SYN1, SYNS1, SYP, SYT/SSX, SYT-SSX-1,
SYT-SSX-2, TA-90, TAAL6, TACSTD1, TACSTD2, TAG72, TAF7L, TAF1,
TAGE, TAG-72, TALI, TAM, TAP2, TAP1, TAPVR1, TARC, TARP, TAT, TAZ,
TBP, TBX22, TBX3, TBX5, TBXA2R, TBXAS1, TCAP, TCF2, TCF1, TCIRG1,
TCL2, TCL4, TCL1A, TCN2, TCOF1, TCR, TCRA, TDD, TDFA, TDRD1, TECK,
TECTA, TEK, TEUAML1, TELAB1, TEX15, TF, TFAP2B, TFE3, TFR2, TG,
TGF.alpha., TGF.beta., TGF.beta.I, TGF.beta.1, TGF.beta.R2,
TGF.beta.RE, TGF.gamma., TGF.beta.RII, TGIF, TGM-4, TGM1, TH, THAS,
THBD, THC, THC2, THM, THPO, THRA, THRB, TIMM8A, TIMP2, TIMP3,
TIMP1, TITF1, TKCR, TKT, TLP, TLR1, TLR10, TLR2, TLR3, TLR4, TLR4,
TLR5, TLR6, TLR7, TLR8, TLR9, TLX1, TM4SF1, TM4SF2, TMC1, TMD,
TMIP, TNDM, TNF, TNFRSF11A, TNFRSF1A, TNFRSF6, TNFSF5, TNFSF6,
TNF.alpha., TNF.beta., TNNI3, TNNT2, TOC, TOP2A, TOP1, TP53, TP63,
TPA, TPBG, TPI, TPI/m, TPI1, TPM3, TPM1, TPMT, TPO, TPS, TPTA, TRA,
TRAG3, TRAPPC2, TRC8, TREH, TRG, TRH, TRIM32, TRIM37, TRP1, TRP2,
TRP-2/6b, TRP-2/INT2, Trp-p8, TRPS1, TS, TSC2, TSC3, TSC1, TSG101,
TSHB, TSHR, TSP-180, TST, TTGA2B, TTN, TTPA, TTR, TU M2-PK, TULP1,
TWIST, TYH, TYR, TYROBP, TYROBP, TYRP1, TYS, UBE2A, UBE3A, UBE1,
UCHL1, UFS, UGT1A, ULR, UMPK, UMPS, UOX, UPA, UQCRC1, URO5, UROD,
UPK1B, UROS, USH2A, USH3A, USH1A, USH1C, USP9Y, UV24, VBCH, VCF,
VDI, VDR, VEGF, VEGFR-2, VEGFR-1, VEGFR-2/FLK-1, VHL, VIM, VMD2,
VMD1, VMGLOM, VNEZ, VNF, VP, VRNI, VWF, VWS, WAS, WBS2, WFS2, WFS1,
WHCR, WHN, WISP3, WMS, WRN, WS2A, WS2B, WSN, WSS, WT2, WT3, WT1,
WTS, WWS, XAGE, XDH, XIC, XIST, XK, XM, XPA, XPC, XRCC9, XS, ZAP70,
ZFHX1B, ZFX, ZFY, ZIC2, ZIC3, ZNF145, ZNF261, ZNF35, ZNF41, ZNF6,
ZNF198, and ZWS1.
[0061] Additionally, therapeutically active proteins as encoded by
the at least one RNA (molecule) of the complexed RNA as defined
herein may also be selected from growth hormones or growth factors,
for example for promoting growth in a (transgenic) living being,
such as, for example, TGF.alpha. and the IGFs (insulin-like growth
factors), proteins that influence the metabolism and/or
haematopoiesis, such as, for example, .alpha.-anti-trypsin, LDL
receptor, erythropoietin (EPO), insulin, GATA-1, etc., or proteins
such as, for example, factors VIII and XI of the blood coagulation
system, etc. Such proteins further include enzymes, such as, for
example, .beta.-galactosidase (lacZ), DNA restriction enzymes (e.g.
EcoRI, HindIII, etc.), lysozymes, etc., or proteases, such as, for
example, papain, bromelain, keratinases, trypsin, chymotrypsin,
pepsin, renin (chymosin), suizyme, nortase, etc. These proteins may
be encoded by the at least one RNA (molecule) of the complexed RNA
as defined herein. Accordingly, the invention provides a technology
which allows to substitute proteins which are defective in the
organism to be treated (e.g. either due to mutations, due to
defective or missing expression) and thereby effective and
increased expression of proteins, which are not functional in the
organism to be treated, as e.g. occurring in monogenetic disorders,
preferably without leading to an innate immune response.
[0062] Alternatively, therapeutically active proteins as encoded by
the at least one RNA (molecule) of the complexed RNA as defined
herein may also be selected from proteases etc. which allow to cure
a specific disease due to e.g. (over)expression of a dysfunctional
or exogenous proteins causing disorders or diseases. Accordingly,
the invention may be used to therapeutically introduce the
complexed RNA into the organism, which attacks a pathogenic
organism (virus, bacteria etc). E.g. RNA encoding therapeutic
proteases may be used to cleave viral proteins which are essential
to the viral assembly or other essential steps of virus
production.
[0063] Therapeutically active proteins as encoded by the at least
one RNA (molecule) of the complexed RNA as defined herein may also
be selected from proteins which modulate various intracellular
pathways by e.g. signal transmission modulation (inhibition or
stimulation) which may influence pivotal intracellular processes
like apoptosis, cell growth etc, in particular with respect to the
organism's immune system. Accordingly, immune modulators, e.g.
cytokines, lymphokines, monokines, interferones etc. may be
expressed efficiently by the complexed RNA as defined herein.
Preferably, these proteins therefore also include, for example,
cytokines of class I of the cytokine family that contain 4
position-specific conserved cysteine residues (CCCC) and a
conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS), wherein X
represents an unconserved amino acid. Cytokines of class I of the
cytokine family include the GM-CSF sub-family, for example IL-3,
IL-5, GM-CSF, the IL-6 sub-family, for example IL-6, IL-11, IL-12,
or the IL-2 sub-family, for example IL-2, IL-4, IL-7, IL-9, IL-15,
etc., or the cytokines IL-1.alpha., IL-1.beta., IL-10 etc. By
analogy, such proteins can also include cytokines of class II of
the cytokine family (interferon receptor family), which likewise
contain 4 position-specific conserved cysteine residues (CCCC) but
no conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS). Cytokines of
class II of the cytokine family include, for example, IFN-.alpha.,
IFN-.beta., IFN-.gamma., etc. Proteins coded for by the at least
one modified (m)RNA (of the inventive immunosuppressive
composition) used according to the invention can further include
also cytokines of the tumour necrosis family, for example
TNF-.alpha., TNF-.beta., TNF-RI, TNF-RII, CD40, Fas, etc., or
cytokines of the chemokine family, which contain 7 transmembrane
helices and interact with G-protein, for example IL-8, MIP-1,
RANTES, CCR5, CXR4, etc. Such proteins can also be selected from
apoptosis factors or apoptosis-related or -linked proteins,
including AIF, Apaf, for example Apaf-1, Apaf-2, Apaf-3, or APO-2
(L), APO-3 (L), apopain, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L,
Bcl-x.sub.S, bik, CAD, calpain, caspases, for example caspase-1,
caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7,
caspase-8, caspase-9, caspase-10, caspase-11, ced-3, ced-9, c-Jun,
c-Myc, crm A, cytochrome C, CdR1, DcR1, DD, DED, DISC,
DNA-PK.sub.CS, DR3, DR4, DR5, FADD/MORT-1, FAK, Fas (Fas ligand
CD95/fas (receptor)), FLICE/MACH, FLIP, fodrin, fos, G-actin,
Gas-2, gelsolin, granzymes A/B, ICAD, ICE, JNK, lamin A/B, MAP,
MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF-.sub..kappa.B, NuMa, p53,
PAK-2, PARP, perforin, PITSLRE, PKC.delta., pRb, presenilin, prICE,
RAIDD, Ras, RIP, sphingomyelinase, thymidine kinase from Herpes
simplex, TRADD, TRAF2, TRAIL, TRAIL-R1, TRAIL-R2, TRAIL-R3,
transglutaminase, etc.
[0064] Additionally, therapeutically active proteins as encoded by
the at least one RNA (molecule) of the complexed RNA as defined
herein may also code for antigen specific T cell receptors. The T
cell receptor or TCR is a molecule found on the surface of T
lymphocytes (or T cells) that is generally responsible for
recognizing antigens bound to major histocompatibility complex
(MHC) molecules. It is a heterodimer consisting of an alpha and
beta chain in 95% of T cells, while 5% of T cells have TCRs
consisting of gamma and delta chains. Engagement of the TCR with
antigen and MHC results in activation of its T lymphocyte through a
series of biochemical events mediated by associated enzymes,
co-receptors and specialized accessory molecules. Hence, these
proteins allow to specifically target specific antigen and may
support the functionality of the immune system due to their
targeting properties. Accordingly, transfection of cells in vivo by
administering the at least one RNA (molecule) of the complexed RNA
as defined herein coding for these receptors or, preferably, an ex
vivo cell transfection approach (e.g. by transfecting specifically
certain immune cells), may be pursued. The T cell receptor
molecules introduced recognize specific antigens on MHC molecule
and may thereby support the immune system's awareness of antigens
to be attacked.
[0065] The therapeutically active proteins, which may be encoded by
the at least one RNA (molecule) of the complexed RNA as defined
herein, may furthermore comprise an adjuvant protein. In this
context, an adjuvant protein is preferably to be understood as any
protein, which is capable to elicit an innate immune response as
defined herein. Preferably, such an innate immune response
comprises an activation of a pattern recognition receptor, such as
e.g. a receptor selected from the Toll-like receptor (TLR) familiy,
including e.g. a Toll like receptor selected from human TLR1 to
TLR10 or from murine Toll like receptors TLR1 to TLR13. Preferably,
an innate immune response is elicited in a mammal, more preferably
in a human. Preferably, the adjuvant protein is selected from human
adjuvant proteins or from pathogenic adjuvant proteins, in
particular from bacterial adjuvant proteins. In addition, mRNA
encoding human proteins involved in adjuvant effects may be used as
well.
[0066] Human adjuvant proteins, which may be encoded by the at
least one RNA (molecule) of the complexed RNA as defined herein,
typically comprise any human protein, which is capable of eliciting
an innate immune response (in a mammal), e.g. as a reaction of the
binding of an exogenous TLR ligand to a TLR. More preferably, human
adjuvant proteins which may be encoded by the complexed RNA of the
present invention are selected from the group consisting of,
without being limited thereto, cytokines which induce or enhance an
innate immune response, including IL-2, IL-12, IL-15, IL-18,
IL-21CCL21, GM-CSF and TNF-alpha; cytokines which are released from
macrophages, including IL-1, IL-6, IL-8, IL-12 and TNF-alpha; from
components of the complement system including C1q, MBL, C1r, C1s,
C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7,
C8, C9, CR1, CR2, CR3, CR4, C1 qR, C1 INH, C4 bp, MCP, DAF, H, I, P
and CD59; from proteins which are components of the signalling
networks of the pattern recognition receptors including TLR and
IL-1R1, whereas the components are ligands of the pattern
recognition receptors including IL-1 alpha, IL-1 beta,
Beta-defensin, heat shock proteins, such as HSP10, HSP60, HSP65,
HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII repeat extra
domain A of fibronectin; the receptors, including IL-1R1, TLR1,
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; the
signal transducers including components of the Small-GTPases
signalling (RhoA, Ras, Rac1, Cdc42 etc.), components of the PIP
signalling (PI3K, Src-Kinases, etc.), components of the
MyD88-dependent signalling (MyD88, IRAK1, IRAK2, etc.), components
of the MyD88-independent signalling (TICAM1, TICAM2 etc.);
activated transcription factors including e.g. NF-.kappa.B, c-Fos,
c-Jun, c-Myc; and induced target genes including e.g. IL-1 alpha,
IL-1 beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFN beta;
from costimulatory molecules, including CD28 or CD40-ligand or PD1;
protein domains, including LAMP; cell surface proteins; or human
adjuvant proteins including CD80, CD81, CD86, trif, flt-3 ligand,
thymopentin, Gp96 or fibronectin, etc., or any species homolog of
any of the above human adjuvant proteins.
[0067] Pathogenic adjuvant proteins, which may be encoded by the at
least one RNA (molecule) of the complexed RNA as defined herein,
typically comprise any pathogenic (adjuvant) protein, which is
capable of eliciting an innate immune response (in a mammal), more
preferably selected from pathogenic (adjuvant) proteins derived
from bacteria, protozoa, viruses, or fungi, animals, etc., and even
more preferably from pathogenic adjuvant proteins selected from the
group consisting of, without being limited thereto, bacterial
proteins, protozoan proteins (e.g. profilin--like protein of
Toxoplasma gondii), viral proteins, or fungal proteins, animal
proteins, etc.
[0068] In this context, bacterial (adjuvant) proteins which may be
encoded by the at least one RNA (molecule) of the complexed RNA as
defined herein, may comprise any bacterial protein, which is
capable of eliciting an innate immune response (preferably in a
mammal). More preferably, bacterial (adjuvant) proteins, which may
be encoded by the complexed RNA, may comprise bacterial adjuvant
proteins selected from the group consisting of, without being
limited thereto, bacterial flagellins, including flagellins from
organisms including Agrobacterium, Aquifex, Azospirillum, Bacillus,
Bartonella, Bordetella, Borrelia, Burkholderia, Campylobacter,
Cau/obacte, Clostridium, Escherichia, Helicobacter,
Lachnospiraceae, Legionella, Listeria, Proteus, Pseudomonas,
Rhizobium, Rhodobacter, Roseburia, Salmonella, Serpulina, Serratia,
Shigella, Treponema, Vibrio, Wolinella, Yersinia, more preferably
flagellins from the species, without being limited thereto,
Agrobacterium turnefaciens, Aquifex pyrophilus, Azospirillum
brasilense, Bacillus subtilis, Bacillus thuringiensis, Bartonella
bacilliformis, Bordetella bronchiseptica, Borrelia burgdorferi,
Burkholderia cepacia, Campylobacter jejuni, Caulobacter crescentus,
Clostridium botulinum strain Bennett clone 1, Escherichia coli,
Helicobacter pylori, Lachnospiraceae bacterium, Legionella
pneumophila, Listeria monocytogenes, Proteus mirabilis, Pseudomonas
aeroguinosa, Pseudomonas syringae, Rhizobium meliloti, Rhodobacter
sphaeroides, Roseburia cecicola, Roseburis hominis, Salmonella
typhimurium, Salmonella bongori, Salmonella typhi, Salmonella
enteritidis, Serpulina hyodysenteriae, Serratia marcescens,
Shigella boydii, Treponema phagedenis, Vibrio alginolyticus, Vibrio
cholerae, Vibrio parahaemolyticus, Wolinella succinogenes and
Yersinia enterocolitica.
[0069] Bacterial flagellins, which may be encoded by the at least
one RNA (molecule) of the complexed RNA as defined herein, are
particularly preferred and may be selected from any bacterial
flagellin showing adjuvant character, more preferably from
bacterial flagellins selected from the group consisting of
bacterial heat shock proteins or chaperons, including Hsp60, Hsp70,
Hsp90, Hsp100; OmpA (Outer membrane protein) from gram-negative
bacteria; bacterial porins, including OmpF; bacterial toxins,
including pertussis toxin (PT) from Bordetella pertussis, pertussis
adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis,
PT-9K/129G mutant from pertussis toxin, pertussis adenylate cyclase
toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin,
cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from
cholera toxin, CTE112K mutant from CT, Escherichia coli heat-labile
enterotoxin (LT), B subunit from heat-labile enterotoxin (LTB)
Escherichia coli heat-labile enterotoxin mutants with reduced
toxicity, including LTK63, LTR72; phenol-soluble modulin;
neutrophil-activating protein (HP-NAP) from Helicobacter pylori,
Surfactant protein D; Outer surface protein A lipoprotein from
Borrelia burgdorferi, Ag38 (38 kDa antigen) from Mycobacterium
tuberculosis, proteins from bacterial fimbriae; Enterotoxin CT of
Vibrio cholerae, Pilin from pili from gram negative bacteria, and
Surfactant protein A; etc., or any species homolog of any of the
above bacterial (adjuvant) proteins.
[0070] Bacterial flagellins, which may be encoded by the at least
one RNA (molecule) of the complexed RNA as defined herein, even
more preferably comprise a sequence selected from the group
comprising any of the following sequences as referred to their
accession numbers:
TABLE-US-00004 organism species gene name accession No GI No
Agrobacterium Agrobacterium FlaD (flaD) U95165 GI: 14278870
tumefaciens FlhB (flhB) FliG (fliG) FliN (fliN) FliM (fliM) MotA
(motA) FlgF (flgF) FliI (fliI) FlgB (flgB) FlgC (flgC) FliE (fliE)
FlgG (flgG) FlgA (flgA) FlgI (flgI) FlgH (flgH) FliL (fliL) FliP
(fliP) FlaA (flaA) FlaB (flaB) FlaC (flaC) Aquifex Aquifex U17575
GI: 596244 pyrophilus Azospirillum Azospirillum Laf1 U26679 GI:
1173509 brasilense Bacillus Bacillus subtilis hag AB033501 GI:
14278870 Bacillus Bacillus flab X67138 GI: 46019718 thuringiensis
Bartonella Bartonella L20677 GI: 304184 bacilliformis Bordetella
Bordetella flaA L13034 GI: 289453 bronchiseptica Borrelia Borrelia
X16833 GI: 39356 burgdorferi Burkholderia Burkholderia fliC
AF011370 GI: 2935154 cepacia Campylobacter Campylobacter flaA
J05635 GI: 144197 jejuni flaB Caulobacter Caulobacter J01556 GI:
144239 crescentus Clostridium Clostridium FlaA DQ845000 GI:
114054886 botulinum strain Bennett clone 1 Escherichia Escherichia
coli hag M14358 GI: 146311 AJ 884569 (EMBL-SVA) Helicobacter
Helicobacter flaA X60746 GI: 43631 pylori Lachnospiraceae
Lachnospiraceae DQ789131 GI: 113911615 bacterium Legionella
Legionella flaA X83232 GI: 602877 pneumophila Listeria Listeria
flaA X65624 GI: 44097 monocytogenes Proteus Proteus mirabilis FlaD
(flaD) AF221596 GI: 6959881 FlaA (flaA) FlaB (flaB) FliA (fliA)
FliZ (fliZ) Pseudomonas Pseudomonas flaA M57501 GI: 151225
aeroguinosa Pseudomonas Pseudomonas fliC EF544882 GI: 146335619
syringae Rhizobium Rhizobium flaA M24526 GI: 152220 meliloti flaB
Rhodobacter Rhodobacter fliC AF274346 GI: 10716972 sphaeroides
Roseburia Roseburia M20983 GI: 152535 cecicola Roseburia Roseburis
Fla2 DQ789141 GI: 113911632 hominis Salmonella Salmonella D13689
GI: 217062 typhimurium (NCBI ID) Salmonella Salmonella fliC
AY603412 GI: 51342390 bongori Salmonella Salmonella typhi flag
L21912 GI: 397810 Salmonella Salmonella fliC M84980 GI: 154015
enteritidis Serpulina Serpulina flaB2 X63513 GI: 450669
hyodysenteriae Serratia Serratia hag M27219 GI: 152826 marcescens
Shigella Shigella boydii fliC-SB D26165 GI: 442485 Treponema
Treponema flaB2 M94015 GI: 155060 phagedenis Vibrio Vibrio flaA
EF125175 GI: 119434395 alginolyticus Vibrio s Vibrio AF069392 GI:
7327274 parahaemolyticus Wolinella Wolinella flag M82917 GI: 155337
succinogenes Yersinia Yersinia L33467 GI: 496295 enterocolitica
[0071] Protozoan proteins, which may be encoded by the at least one
RNA (molecule) of the complexed RNA as defined herein, may be
selected from any protozoan protein showing adjuvant character,
more preferably, from the group consisting of, without being
limited thereto, Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma
gondii, Protozoan heat shock proteins, LeIF from Leishmania spp.,
profilin-like protein from Toxoplasma gondhii, etc.
[0072] Viral proteins, which may be encoded by the at least one RNA
(molecule) of the complexed RNA as defined herein, may be selected
from any viral protein showing adjuvant character, more preferably,
from the group consisting of, without being limited thereto,
Respiratory Syncytial Virus fusion glycoprotein (F-protein),
envelope protein from MMT virus, mouse leukemia virus protein,
Hemagglutinin protein of wild-type measles virus, etc.
[0073] Fungal proteins, which may be encoded by the at least one
RNA (molecule) of the complexed RNA as defined herein, may be
selected from any fungal protein showing adjuvant character, more
preferably, from the group consisting of, without being limited
thereto, fungal immunomodulatory protein (FIP; LZ-8), etc.
[0074] Finally, pathogenic adjuvant proteins, which may be encoded
by the at least one RNA (molecule) of the complexed RNA as defined
herein, may finally be selected from any further pathogenic protein
showing adjuvant character, more preferably, from the group
consisting of, without being limited thereto, Keyhole limpet
hemocyanin (KLH), OspA, etc.
[0075] The at least one RNA (molecule) of the complexed RNA of the
present invention may alternatively encode an antigen. According to
the present invention, the term "antigen" refers to a substance
which is recognized by the immune system and is capable of
triggering an antigen-specific immune response, e.g. by formation
of antibodies. Antigens can be classified according to their
origin. Accordingly, there are two major classes of antigens:
exogenous and endogenous antigens. Exogenous antigens are antigens
that enter the cell or the body from outside (the cell or the
body), for example by inhalation, ingestion or injection, etc.
These antigens are internalized by antigen-presenting cells
("APCs", such as dendritic cells or macrophages) and processed into
fragments. APCs then present the fragments to T helper cells (e.g.
CD4.sup.+) by the use of MHC II molecules on their surface.
Recognition of these antigen fragments by T cells leads to
activation of the T cells and secretion of cytokines. Cytokines are
substances that can activate proliferation of immune cells such as
cytotoxic T cells, B cells or macrophages. In contrast, endogenous
antigens are antigens which have been generated within the cell,
e.g. as a result of normal cell metabolism. Fragments of these
antigens are presented on MHC I molecules on the surface of APCs.
These antigens are recognized by activated antigen-specific
cytotoxic CD8.sup.+ T cells. After recognition, those T cells react
in secretion of different toxins that cause lysis or apoptosis of
the antigen-presenting cell. Endogenous antigens comprise antigens,
e.g. proteins or peptides encoded by a foreign nucleic acid inside
the cell as well as proteins or peptides encoded by the genetic
information of the cell itself, or antigens from intracellularly
occurring viruses. One class of endogenous antigens is the class of
tumor antigens. Those antigens are presented by the MHC 1 molecules
on the surface of tumor cells. This class can be divided further in
tumor-specific antigens (TSAs) and tumor-associated-antigens
(TAAs). TSAs can only be presented by tumor cells and never by
normal "healthy" cells. They typically result from a tumor specific
mutation. TAAs, which are more common, are usually presented by
both tumor and healthy cells. These antigens are recognized and the
antigen-presenting cell can be destroyed by cytotoxic T cells.
Additionally, tumor antigens can also occur on the surface of the
tumor in the form of e.g. a mutated receptor. In this case, they
can be recognized by antibodies.
[0076] Antigens, which may be encoded by the at least one RNA
(molecule) of the complexed RNA of the present invention, may
include e.g. proteins, peptides or fragments thereof. Preferably,
antigens are proteins and peptides or fragments thereof, such as
epitopes of those proteins or peptides. Epitopes (also called
"antigen determinants"), typically, are fragments located on the
outer surface of such antigenic protein or peptide structures
having 5 to 15, preferably 9 to 15, amino acids (B-cell epitopes
and T-cell epitopes are typically presented on MHC molecules,
wherein e.g. MHC-I typically presents epitopes with a length of
about 9 aa and MHC-II typically presents epitopes with a length of
about 12-15 aa). Furthermore, antigens encoded by the at least one
RNA (molecule) of the complex according to the invention may also
comprise any other biomolecule, e.g., lipids, carbohydrates, etc.,
which may be covalently or non-covalently attached to the RNA
(molecule).
[0077] In accordance with the invention, antigens, which may be
encoded by the at least one RNA (molecule) of the complexed RNA of
the present invention, may be exogenous or endogenous antigens.
Endogenous antigens comprise antigens generated in the cell,
especially in degenerate cells such as tumor cells. These antigens
are referred to as "tumor antigens". Preferably, without being
restricted thereto, they are located on the surface of the cell.
Furthermore, "tumor antigens" means also antigens expressed in
cells which are (were) not by themselves (or originally not by
themselves) degenerate but are associated with the supposed tumor.
Antigens which are connected with tumor-supplying vessels or
(re)formation thereof, in particular those antigens which are
associated with neovascularization, e.g. growth factors, such as
VEGF, bFGF etc., are also included herein. Antigens connected with
a tumor furthermore include antigens from cells or tissues,
typically embedding the tumor. Further, some substances (usually
proteins or peptides) are expressed in patients suffering
(knowingly or not-knowingly) from a cancer disease and they occur
in increased concentrations in the body fluids of said patients,
e.g. proteins, which are associated with tumor cell invasion and
migration. These substances are also referred to as "tumor
antigens", however they are not antigens in the stringent meaning
of an immune response inducing substance. Use thereof is also
encompassed by the scope of the present invention.
[0078] Antigens, which may be encoded by the at least one RNA
(molecule) of the complexed RNA of the present invention, may be
exemplarily selected, without being restricted thereto, e.g. from
any antigen suitable for the specific purpose, e.g. from antigens,
which are relevant (or causal) for specific infection diseases,
such as defined herein, from cancer antigens such as tumor specific
surface antigens, from antigens expressed in cancer diseases, from
mutant antigens expressed in cancer diseases, or from protein
antigens involved in the etiology of further diseases, e.g.
autoimmune diseases, allergies, etc. E.g. these antigens may be
used to desensitize a patient by administering an antigen causing
the patient's allergic or autoimmune status.
[0079] Preferred exemplary antigenic (poly)peptides encoded by the
at least one RNA (molecule) of the complexed RNA as defined herein
include all known antigenic peptides, for example tumour antigens,
etc. Specific examples of tumour antigens are inter alia
tumour-specific surface antigens (TSSAs), for example 5T4,
alpha5beta1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, Bcr-abl, MN/C
IX antigen, CAl25, CAMEL, CAP-1, CASP-8, beta-catenin/m, CD4, CD19,
CD20, CD22, CD25, CDC27/m, CD 30, CD33, CD52, CD56, CD80, CDK4/m,
CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1,
G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R1701,
HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R,
IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski,
MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, PAP, proteinase-3,
p190 minor bcr-abl, Pml/RARalpha, PRAME, PSA, PSM, PSMA, RAGE, RU1
or RU2, SAGE, SART-1 or SART-3, survivin, TEUAML1, TGFbeta, TPI/m,
TRP-1, TRP-2, TRP-2/INT2, VEGF and WT1, or from sequences such as,
for example, NY-Eso-1 or NY-Eso-B. Any class of tumor antigens is
suitable for the purpose of the present invention, e.g. tumor
antigens known to be involved in neovascularization, influencing
the extracellular matrix structure etc. Fragments and analogues of
the above antigens are also encompassed.
[0080] Examples of tumor antigens which may be encoded by the at
least one RNA (molecule) of the complexed RNA of the present
invention are shown in Tables 1 and 2 below. These tables
illustrate specific (protein) antigens (i.e. "tumor antigens") with
respect to the cancer disease, they are associated with. According
to the invention, the terms "cancer diseases" and "tumor diseases"
are used synonymously herein.
TABLE-US-00005 TABLE 1 Antigens expressed in cancer diseases Tumor
antigen Name Expression site 5T4 colorectal cancer, gastric cancer,
ovarian cancer 707-AP 707 alanine proline melanoma 9D7 renal cell
carcinoma AFP alpha-fetoprotein hepatocellular carcinoma,
gallbladder cancer, testicular cancer, ovarian cancer, bladder
cancer AlbZIP HPG1 prostate cancer alpha5beta1- Integrin
alpha5beta6- colon cancer Integrin alpha-methylacyl- prostate
cancer coenzyme A racemase ART-4 adenocarcinoma antigen lung
cancer, head and neck cancer, recognized by T cells 4 leukemia,
esophageal cancer, gastric cancer, cervical cancer, ovarian cancer,
breast cancer, squamous cell carcinoma B7H4 ovarian cancer BAGE-1 B
antigen bladder cancer, head and neck cancer, lung cancer,
melanoma, squamous cell carcinoma BCL-2 leukemia BING-4 melanoma CA
15-3/CA 27-29 breast cancer, ovary cancer, lung cancer, prostate
cancer CA 19-9 gastric cancer, pancreatic cancer, liver cancer,
breast cancer, gallbladder cancer, colon cancer, ovary cancer, lung
cancer CA 72-4 ovarian cancer CA125 ovarian cancer, colorectal
cancer, gastric cancer, liver cancer, pancreatic cancer, uterus
cancer, cervix carcinoma, colon cancer, breast cancer, lung cancer
calreticulin bladder cancer CAMEL CTL-recognized antigen on
melanoma melanoma CASP-8 caspase-8 head and neck cancer cathepsin B
breast cancer cathepsin L breast cancer CD19 B-cell malignancies
CD20 CD22 CD25 CD30 CD33 CD4 CD52 CD55 CD56 CD80 CEA
carcinoembryonic antigen gut carcinoma, colorectal cancer, colon
cancer, hepatocellular cancer, lung cancer, breast cancer, thyroid
cancer, pancreatic cancer, liver cancer cervix cancer, bladder
cancer, melanoma CLCA2 calcium-activated chloride lung cancer
channel-2 CML28 leukemia Coactosin-like pancreatic cancer protein
Collagen XXIII prostate cancer COX-2 ovarian cancer, breast cancer,
colorectal cancer CT-9/BRD6 bromodomain testis-specific protein
Cten C-terminal tensin-like protein prostate cancer cyclin B1
cyclin D1 ovarian cancer cyp-B cyclophilin B bladder cancer, lung
cancer, T-cell leukemia, squamous cell carcinoma, CYPB1 cytochrom
P450 1B1 leukemia DAM-10/MAGE- differentiation antigen melanoma
melanoma, skin tumors, ovarian cancer, B1 10 lung cancer
DAM-6/MAGE-B2 differentiation antigen melanoma 6 melanoma, skin
tumors, ovarian cancer, lung cancer EGFR/Her1 lung cancer, ovarian
cancer, head and neck cancer, colon cancer, pancreatic cancer,
breast cancer EMMPRIN tumor cell-associated lung cancer, breast
cancer, bladder extracellular matrix cancer, ovarian cancer, brain
cancer, metalloproteinase inducer/ lymphoma EpCam epithelial cell
adhesion molecule ovarian cancer, breast cancer, colon cancer, lung
cancer EphA2 ephrin type-A receptor 2 glioma EphA3 ephrin type-A
receptor 2 melanoma, sarcoma, lung cancer ErbB3 breast cancer EZH2
(enhancer of Zeste homolog 2) endometrium cancer, melanoma,
prostate cancer, breast cancer FGF-5 fibroblast growth factor-5
renal cell carcinoma, breast cancer, prostate cancer FN fibronectin
melanoma Fra-1 Fos-related antigen-1 breast cancer, esophageal
cancer, renal cell carcinoma, thyroid cancer G250/CAIX glycoprotein
250 leukemia, renal cell carcinoma, head and neck cancer, colon
cancer, ovarian cancer, cervical cancer GAGE-1 G antigen 1 bladder
cancer, lung cancer, sarcoma, melanoma, head and neck cancer GAGE-2
G antigen 2 bladder cancer, lung cancer, sarcoma, melanoma, head
and neck cancer GAGE-3 G antigen 3 bladder cancer, lung cancer,
sarcoma, melanoma, head and neck cancer GAGE-4 G antigen 4 bladder
cancer, lung cancer, sarcoma, melanoma, head and neck cancer GAGE-5
G antigen 5 bladder cancer, lung cancer, sarcoma, melanoma, head
and neck cancer GAGE-6 G antigen 6 bladder cancer, lung cancer,
sarcoma, melanoma, head and neck cancer GAGE-7b G antigen 7b
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
cancer GAGE-8 G antigen 8 bladder cancer, lung cancer, sarcoma,
melanoma, head and neck cancer GDEP gene differentially expressed
in prostate cancer prostate GnT-V N-acetylglucosaminyltransferase V
glioma, melanoma gp100 glycoprotein 100 kDa melanoma GPC3 glypican
3 hepatocellular carcinoma, melanoma HAGE helicase antigen bladder
cancer HAST-2 human signet ring tumor-2 hepsin prostate
Her2/neu/ErbB2 human epidermal receptor- breast cancer, bladder
cancer, 2/neurological melanoma, ovarian cancer, pancreas cancer,
gastric cancer HERV-K-MEL melanoma HNE human neutrophil elastase
leukemia homeobox NKX prostate cancer 3.1 HOM-TES- ovarian cancer
14/SCP-1 HOM-TES-85 HPV-E6 cervical cancer HPV-E7 cervical cancer
HST-2 gastric cancer hTERT human telomerase reverse breast cancer,
melanoma, lung cancer, transcriptase ovarian cancer, sarcoma,
Non-Hodgkin- lymphoma, acute leukemia iCE intestinal carboxyl
esterase renal cell carcinoma IGF-1R colorectal cancer IL-13Ra2
interleukin 13 receptor alpha 2 glioblastoma chain IL-2R colorectal
cancer IL-5 immature laminin renal cell carcinoma receptor
kallikrein 2 prostate cancer kallikrein 4 prostate cancer Ki67
prostate cancer, breast cancer, Non- Hodgkin-lymphoma, melanoma
KIAA0205 bladder cancer KK-LC-1 Kita-kyushu lung cancer antigen 1
lung cancer KM-HN-1 tongue cancer, hepatocellular carcinomas,
melanoma, gastric cancer, esophageal, colon cancer, pancreatic
cancer LAGE-1 L antigen bladder cancer, head and neck cancer,
melanoma livin bladder cancer, melanoma MAGE-A1 melanoma antigen-A1
bladder cancer, head and neck cancer, melanoma, colon cancer, lung
cancer, sarcoma, leukemia MAGE-A10 melanoma antigen-A10 bladder
cancer, head and neck cancer, melanoma, colon cancer, lung cancer,
sarcoma, leukemia MAGE-A12 melanoma antigen-A12 bladder cancer,
head and neck cancer, melanoma, colon cancer, lung cancer, sarcoma,
leukemia, prostate cancer, myeloma, brain tumors MAGE-A2 melanoma
antigen-A2 bladder cancer, head and neck cancer, melanoma, colon
cancer, lung cancer, sarcoma, leukemia MAGE-A3 melanoma antigen-A3
bladder cancer, head and neck cancer, melanoma, colon cancer, lung
cancer, sarcoma, leukemia MAGE-A4 melanoma antigen-A4 bladder
cancer, head and neck cancer, melanoma, colon cancer, lung cancer,
sarcoma, leukemia MAGE-A6 melanoma antigen-A6 bladder cancer, head
and neck cancer, melanoma, colon cancer, lung cancer, sarcoma,
leukemia MAGE-A9 melanoma-antigen-A9 bladder cancer, head and neck
cancer, melanoma, colon cancer, lung cancer, sarcoma, leukemia
MAGE-B1 melanoma-antigen-B1 melanoma MAGE-B10 melanoma-antigen-B10
melanoma MAGE-B16 melanoma-antigen-B16 melanoma MAGE-B17
melanoma-antigen-B17 melanoma MAGE-B2 melanoma-antigen-B2 melanoma
MAGE-B3 melanoma-antigen-B3 melanoma MAGE-B4 melanoma-antigen-B4
melanoma MAGE-B5 melanoma-antigen-B5 melanoma MAGE-B6
melanoma-antigen-B6 melanoma MAGE-C1 melanoma-antigen-C1 bladder
cancer, melanoma MAGE-C2 melanoma-antigen-C2 melanoma MAGE-C3
melanoma-antigen-C3 melanoma MAGE-D1 melanoma-antigen-D1 melanoma
MAGE-D2 melanoma-antigen-D2 melanoma MAGE-D4 melanoma-antigen-D4
melanoma MAGE-E1 melanoma-antigen-E1 bladder cancer, melanoma
MAGE-E2 melanoma-antigen-E2 melanoma MAGE-F1 melanoma-antigen-F1
melanoma MAGE-H1 melanoma-antigen-H1 melanoma MAGEL2 MAGE-like 2
melanoma mammaglobin A breast cancer MART-1/Melan-A melanoma
antigen recognized by melanoma T cells-1/melanoma antigen A MART-2
melanoma antigen recognized by melanoma T cells-2 matrix protein 22
bladder cancer MC1R melanocortin 1 receptor melanoma M-CSF
macrophage colony-stimulating ovarian cancer factor gene mesothelin
ovarian cancer MG50/PXDN breast cancer, glioblastoma, melanoma MMP
11 M-phase phosphoprotein 11 leukemia MN/CA IX-antigen renal cell
carcinoma MRP-3 multidrug resistance-associated lung cancer protein
3 MUC1 mucin 1 breast cancer MUC2 mucin 2 breast cancer, ovarian
cancer, pancreatic cancer NA88-A NA cDNA clone of patient M88
melanoma N- acetylglucosaminyltransferase-V Neo-PAP Neo-poly(A)
polymerase NGEP prostate cancer NMP22 bladder cancer NPM/ALK
nucleophosmin/anaplastic lymphoma kinase fusion protein NSE
neuron-specific enolase small cell cancer of lung, neuroblastoma,
Wilm' tumor, melanoma, thyroid cancer, kidney cancer, testicle
cancer, pancreas cancer NY-ESO-1 New York esophageous 1 bladder
cancer, head and neck cancer, melanoma, sarcoma, B-lymphoma,
hepatoma, pancreatic cancer, ovarian cancer, breast cancer NY-ESO-B
OA1 ocular albinism type 1 protein melanoma OFA-iLRP oncofetal
antigen-immature leukemia laminin receptor
OGT O-linked N-acetylglucosamine transferase gene OS-9 osteocalcin
prostate cancer osteopontin prostate cancer, breast cancer, ovarian
cancer p15 protein 15 p15 melanoma p190 minor bcr- abl p53 PAGE-4
prostate GAGE-like protein-4 prostate cancer PAI-1 plasminogen
acitvator inhibitor 1 breast cancer PAI-2 plasminogen acitvator
inhibitor 2 breast cancer PAP prostate acic phosphatase prostate
cancer PART-1 prostate cancer PATE prostate cancer PDEF prostate
cancer Pim-1-Kinase Pin1 Propyl isomerase prostate cancer POTE
prostate cancer PRAME preferentially expressed antigen melanoma,
lung cancer, leukemia, head of melanoma and neck cancer, renal cell
carcinoma, sarcoma prostein prostate cancer proteinase-3 PSA
prostate-specific antigen prostate cancer PSCA prostate cancer PSGR
prostate cancer PSM PSMA prostate-specific membrane prostate cancer
antigen RAGE-1 renal antigen bladder cancer, renal cancer, sarcoma,
colon cancer RHAMM/CD168 receptor for hyaluronic acid leukemia
mediated motility RU1 renal ubiquitous 1 bladder cancer, melanoma,
renal cancer RU2 renal ubiquitous 1 bladder cancer, melanoma,
sarcoma, brain tumor, esophagel cancer, renal cancer, colon cancer,
breast cancer S-100 melanoma SAGE sarcoma antigen SART-1 squamous
antigen rejecting tumor 1 esophageal cancer, head and neck cancer,
lung cancer, uterine cancer SART-2 squamous antigen rejecting tumor
1 head and neck cancer, lung cancer, renal cell carcinoma,
melanoma, brain tumour SART-3 squamous antigen rejecting tumor 1
head and neck cancer, lung cancer, leukemia, melanoma, esophageal
cancer SCC squamous cell carcinoma antigen lung cancer Sp17 sperm
protein 17 multiple myeloma SSX-1 synovial sarcoma X breakpoint 1
hepatocellular cell carcinom, breast cancer SSX-2/HOM-MEL- synovial
sarcoma X breakpoint 2 breast cancer 40 SSX-4 synovial sarcoma X
breakpoint 4 bladder cancer, hepatocellular cell carcinoma, breast
cancer STAMP-1 prostate cancer STEAP six transmembrane epithelial
prostate cancer antigen prostate survivin bladder cancer
survivin-2B intron 2-retaining survivin bladder cancer TA-90
melanoma TAG-72 prostate carcinoma TARP prostate cancer TGFb
TGFbeta TGFbRII TGFbeta receptor II TGM-4 prostate-specific
transglutaminase prostate cancer TRAG-3 taxol resistant associated
protein 3 breast cancer, leukemia, and melanoma TRG testin-related
gene TRP-1 tyrosine related protein 1 melanoma TRP-2/6b TRP-2/novel
exon 6b melanoma, glioblastoma TRP-2/INT2 TRP-2/intron 2 melanoma,
glioblastoma Trp-p8 prostate cancer Tyrosinase melanoma UPA
urokinase-type plasminogen breast cancer activator VEGF vascular
endothelial growth factor VEGFR-2/FLK-1 vascular endothelial growth
factor receptor-2 WT1 Wilm' tumor gene gastric cancer, colon
cancer, lung cancer, breast cancer, ovarian cancer, leukemia
TABLE-US-00006 TABLE 2 Mutant antigens expressed in cancer diseases
Mutant antigen Name Expression site alpha-actinin-4/m lung
carcinoma ARTC1/m melanoma bcr/abl breakpoint cluster region- CML
Abelson fusion protein beta-Catenin/m beta-Catenin melanoma BRCA1/m
breast cancer BRCA2/m breast cancer CASP-5/m colorectal cancer,
gastric cancer, endometrial carcinoma CASP-8/m head and neck
cancer, squamous cell carcinoma CDC27/m cell-division-cycle 27
CDK4/m cyclin-dependent kinase 4 melanoma CDKN2A/m melanoma CML66
CML COA-1/m colorectal cancer DEK-CAN fusion protein AML EFTUD2/m
melanoma ELF2/m Elongation factor 2 lung squamous cell carcinoma
ETV6-AML1 Ets variant gene6/acute myeloid ALL leukemia 1 gene
fusion protein FN1/m fibronectin 1 melanoma GPNMB/m melanoma
HLA-A*0201- arginine to isoleucine exchange renal cell carcinoma
R170I at residue 170 of the alpha-helix of the alpha2-domain in the
HLA-A2 gene HLA-A11/m melanoma HLA-A2/m renal cell carcinoma
HSP70-2M heat shock protein 70-2 mutated renal cell carcinoma,
melanoma, neuroblastoma KIAA0205/m bladder tumor K-Ras/m pancreatic
carcinoma, colorectal carcinoma LDLR-FUT LDR-Fucosyltransferase
fusion melanoma protein MART2/m melanoma ME1/m non-small cell lung
carcinoma MUM-1/m melanoma ubiquitous mutated 1 melanoma MUM-2/m
melanoma ubiquitous mutated 2 melanoma MUM-3/m melanoma ubiquitous
mutated 3 melanoma Myosin class I/m melanoma neo-PAP/m melanoma
NFYC/m lung squamous cell carcinoma N-Ras/m melanoma OGT/m
colorectal carcinoma OS-9/m melanoma p53/m Pml/RARa promyelocytic
leukemia/retinoic APL, PML acid receptor alpha PRDX5/m melanoma
PTPRK/m receptor-type protein-tyrosine melanoma phosphatase kappa
RBAF600/m melanoma SIRT2/m melanoma SYT-SSX-1 synaptotagmin
I/synovial sarcoma sarcoma X fusion protein SYT-SSX-2 synaptotagmin
I/synovial sarcoma sarcoma X fusion protein TEL-AML1 translocation
Ets-family AML leukemia/acute myeloid leukemia 1 fusion protein
TGFbRII TGFbeta receptor II colorectal carcinoma TPI/m
triosephosphate isomerase melanoma
[0081] In a preferred embodiment according to the invention,
examples of tumor antigens which may be encoded by the at least one
RNA (molecule) of the complexed RNA of the present invention are
selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AIbZIP
HPG1, 1-integrin, 5 6-integrin, -actinin-4/m, methylacyl-coenzyme A
racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, catenin/m,
BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CAl25,
calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19,
CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80,
CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m,
coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,
cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,
EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3,
ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3,
GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3,
GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL,
HLA-A*0201-R171, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1,
HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2,
hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin
receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,
KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3,
MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2,
MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17,
MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1,
MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A,
MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m,
mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1,
MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A,
N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,
NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1,
OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15,
p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE,
PDEF, Pim-1-Kinase, Pin-1, Pml/PAR.alpha., POTE, PRAME, PRDX5/m,
prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m,
RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1,
SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4,
STAMP-1, STEAP, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90,
TAG-72, TARP, TEL-AML1, TGF.beta., TGF.beta.RII, TGM-4, TPI/m,
TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA,
VEGF, VEGFR-2/FLK-1, and WT1.
[0082] In a preferred embodiment according to the invention,
examples of tumor antigens which may be encoded by the at least one
RNA (molecule) of the complexed RNA of the present invention are
selected from the group consisting of MAGE-A1 [accession number
M77481], MAGE-A6 [accession number NM.sub.--005363], melan-A
[accession number NM.sub.--005511], GP100 [accession number
M77348], tyrosinase [accession number NM.sub.--000372], survivin
[accession number AF077350], CEA [accession number
NM.sub.--004363], Her-2/neu [accession number M11730], WT1
[accession number NM.sub.--000378], PRAME [accession number
NM.sub.--006115], EGFR1 (epidermal growth factor receptor 1)
[accession number AF288738], mucin-1 [accession number
NM.sub.--002456] and SEC61G [accession number NM.sub.--014302].
[0083] As a further alternative, the at least one RNA (molecule) of
the complexed RNA of the present invention may encode an antibody.
According to the present invention, such an antibody may be
selected from any antibody, e.g. any recombinantly produced or
naturally occurring antibodies, known in the art, in particular
antibodies suitable for therapeutic, diagnostic or scientific
purposes, or antibodies which have been identified in relation to
specific cancer diseases. Herein, the term "antibody" is used in
its broadest sense and specifically covers monoclonal and
polyclonal antibodies (including agonist, antagonist, and blocking
or neutralizing antibodies) and antibody species with polyepitopic
specificity. According to the invention, "antibody" typically
comprises any antibody known in the art (e.g. IgM, IgD, IgG, IgA
and IgE antibodies), such as naturally occurring antibodies,
antibodies generated by immunization in a host organism, antibodies
which were isolated and identified from naturally occurring
antibodies or antibodies generated by immunization in a host
organism and recombinantly produced by biomolecular methods known
in the art, as well as chimeric antibodies, human antibodies,
humanized antibodies, bispecific antibodies, intrabodies, i.e.
antibodies expressed in cells and optionally localized in specific
cell compartments, and fragments and variants of the aforementioned
antibodies. In general, an antibody consists of a light chain and a
heavy chain both having variable and constant domains. The light
chain consists of an N-terminal variable domain, V.sub.L, and a
C-terminal constant domain, C.sub.L. In contrast, the heavy chain
of the IgG antibody, for example, is comprised of an N-terminal
variable domain, V.sub.H, and three constant domains, C.sub.H1,
C.sub.H2 and C.sub.H3. Single chain antibodies may be encoded by
the at least one RNA (molecule) of the complexed RNA as defined
herein as well, preferably by a single-stranded RNA, more
preferably by an mRNA.
[0084] According to a first alternative, the at least one RNA
(molecule) of the complexed RNA of the present invention may encode
a polyclonal antibody. In this context, the term, "polyclonal
antibody" typically means mixtures of antibodies directed to
specific antigens or immunogens or epitopes of a protein which were
generated by immunization of a host organism, such as a mammal,
e.g. including goat, cattle, swine, dog, cat, donkey, monkey, ape,
a rodent such as a mouse, hamster and rabbit. Polyclonal antibodies
are generally not identical, and thus usually recognize different
epitopes or regions from the same antigen. Thus, in such a case,
typically a mixture (a composition) of different RNA molecules
complexed as claimed by the present invention will be applied, each
encoding a specific (monoclonal) antibody being directed to
specific antigens or immunogens or epitopes of a protein.
[0085] According to a further alternative, the at least one RNA
(molecule) of the complexed RNA of the present invention may encode
a monoclonal antibody. The term "monoclonal antibody" herein
typically refers to an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
to a single antigenic site. Furthermore, in contrast to
conventional (polyclonal) antibody preparations which typically
include different antibodies directed to different determinants
(epitopes), each monoclonal antibody is directed to a single
determinant on the antigen. For example, monoclonal antibodies as
defined above may be made by the hybridoma method first described
by Kohler and Milstein, Nature, 256:495 (1975), or may be made by
recombinant DNA methods, e.g. as described in U.S. Pat. No.
4,816,567. "Monoclonal antibodies" may also be isolated from phage
libraries generated using the techniques described in McCafferty et
al., Nature, 348:552-554 (1990), for example. According to Kohler
and Milstein, an immunogen (antigen) of interest is injected into a
host such as a mouse and B-cell lymphocytes produced in response to
the immunogen are harvested after a period of time. The B-cells are
combined with myeloma cells obtained from mouse and introduced into
a medium which permits the B-cells to fuse with the myeloma cells,
producing hybridomas. These fused cells (hybridomas) are then
placed in separate wells in microtiter plates and grown to produce
monoclonal antibodies. The monoclonal antibodies are tested to
determine which of them are suitable for detecting the antigen of
interest. After being selected, the monoclonal antibodies can be
grown in cell cultures or by injecting the hybridomas into mice.
However, for the purposes of the present invention, the peptide
sequences of these monoclonal antibodies have to be sequenced and
RNA sequences encoding these antibodies may be prepared according
to procedures well known in the art.
[0086] For therapeutical purposes in humans, non-human monoclonal
or polyclonal antibodies, such as murine antibodies may also be
encoded by the at least one RNA (molecule) of the complexed RNA of
the present invention. However, such antibodies are typically only
of limited use, since they generally induce an immune response by
production of human antibodies directed to the said non-human
antibodies, in the human body. Therefore, a particular non-human
antibody can only be administered once to the human. To solve this
problem, chimeric, humanized non-human and human antibodies can be
encoded by the at least one RNA (molecule) of the complexed RNA of
the present invention. "Chimeric" antibodies, which may be encoded
by the at least one RNA (molecule) of the complexed RNA of the
present invention, are preferably antibodies in which the constant
domains of an antibody described above are replaced by sequences of
antibodies from other organisms, preferably human sequences.
"Humanized" (non-human) antibodies, which may be also encoded by
the at least one RNA (molecule) of the complexed RNA of the present
invention, are antibodies in which the constant and variable
domains (except for the hypervariable domains) described above of
an antibody are replaced by human sequences. According to another
alternative, the at least one RNA (molecule) of the complexed RNA
of the present invention may encode human antibodies, i.e.
antibodies having only human sequences. Such human antibodies can
be isolated from human tissues or from immunized non-human host
organisms which are transgene for the human IgG gene locus,
sequenced RNA sequences may be prepared according to procedures
well known in the art. Additionally, human antibodies can be
provided by the use of a phage display.
[0087] In addition, the at least one RNA (molecule) of the
complexed RNA of the present invention may encode bispecific
antibodies. "Bispecific" antibodies in context of the invention are
preferably antibodies which act as an adaptor between an effector
and a respective target, e.g. for the purposes of recruiting
effector molecules such as toxins, drugs, cytokines etc., targeting
effector cells such as CTL, NK cells, makrophages, granulocytes,
etc. (see for review: Kontermann R. E., Acta Pharmacol. Sin, 2005,
26(1): 1-9). Bispecific antibodies as described herein are, in
general, configured to recognize, e.g. two different antigens,
immunogens, epitopes, drugs, cells (or receptors on cells), or
other molecules (or structures) as described above. Bispecificity
means herewith that the antigen-binding regions of the antibodies
are specific for two different epitopes. Thus, different antigens,
immunogens or epitopes, etc. can be brought close together, what,
optionally, allows a direct interaction of the two components. For
example, different cells such as effector cells and target cells
can be connected via a bispecific antibody. Encompassed, but not
limited, by the present invention are antibodies or fragments
thereof which bind, on the one hand, a soluble antigen as described
herein, and, on the other hand, an antigen or receptor on the
surface of a tumor cell.
[0088] In summary, according to the invention, the at least one RNA
(molecule) of the complexed RNA of the present invention may also
encode antibodies as defined above. Since these antibodies are
intracellularly expressed antibodies, i.e. antibodies which are
encoded by nucleic acids localized in specific compartments of the
cell and also expressed there, such antibodies may also be termed
intrabodies.
[0089] Antibodies as encoded by the at least one RNA (molecule) of
the complexed RNA of the present invention may preferably comprise
full-length antibodies, i.e. antibodies composed of the full heavy
and full light chains, as described above. However, derivatives of
antibodies such as antibody fragments, variants or adducts may be
encoded by the above at least one RNA of the complexed RNA in
accordance with the present invention.
[0090] The at least one RNA (molecule) of the complexed RNA of the
present invention may also encode antibody fragments selected from
Fab, Fab', F(ab').sub.2, Fc, Facb, pFc', Fd and Fv fragments of the
aforementioned antibodies. In general, antibody fragments are known
in the art. For example, a Fab ("fragment, antigen binding")
fragment is composed of one constant and one variable domain of
each of the heavy and the light chain. The two variable domains
bind the epitope on specific antigens. The two chains are connected
via a disulfide linkage. A scFv ("single chain variable fragment")
fragment, for example, typically consists of the variable domains
of the light and heavy chains. The domains are linked by an
artificial linkage, in general a polypeptide linkage such as a
peptide composed of 15-25 glycine, proline and/or serine
residues.
[0091] According to the invention, the at least one RNA (molecule)
of the complexed RNA of the present invention may encode fragments
and/or variants of the aforementioned therapeutically active
proteins, antigens or antibodies, wherein the fragments and/or
variants may have a sequence identity to one of the aforementioned
therapeutically active proteins, antigens or antibodies of at least
70%, 80% or 85%, preferably at least 90%, more preferably at least
95% and most preferably at least 99% over the whole length of the
coding nucleic acid or amino acid sequences encoding these
therapeutically active proteins, antigens or antibodies.
Preferably, the fragments and/or variants have the same biological
function or specific activity compared to the full-length native
therapeutically active proteins, antigens or antibodies, e.g.
specific binding capacity (e.g. of particular antigens), catalytic
activity (e.g. of therapeutically active proteins), etc. In this
context, "biological function" of antibodies described herein also
comprises neutralization of antigens, complement activation or
opsonization. Thereby, antibodies typically recognize either native
epitopes on the cell surface or free antigens. Antibodies as
defined above can interact with the cell-presenting antigens and
initiate different defense mechanisms. On the one hand, the
antibody can initiate signaling mechanisms in the targeted cell
that leads to the cell's self-destruction (apoptosis). On the other
hand, it can mark the cell in such a way that other components or
effector cells of the body's immune system can recognize and
attack. The attack mechanisms are referred to as antibody-dependent
complement-mediated cytotoxicity (CMC) and antibody-dependent
cellular cytotoxicity (ADCC). ADCC involves a recognition of the
antibody by immune cells that engage the antibody-marked cells and
either through their direct action, or through the recruitment of
other cell types, lead to the tagged-cell's death. CMC is a process
where a cascade of different complement proteins becomes activated,
usually when several antibodies are in close proximity to each
other, either resulting in cell lysis or attracting other immune
cells to this location for effector cell function. In the
neutralization of an antigen, the antibody can bind an antigen and
neutralize the same. Such neutralization reaction, in turn, leads
in general to blocking of the antibody. Thus, the antibody can bind
only one antigen, or, in case of a bispecific antibody, two
antigens. In particular, scFv antibody fragments are useful for
neutralization reactions because they don't contain the
functionalities of the constant domain of an antibody. In the
complement activation, the complex system of complement proteins
can be activated via binding of an antibody which is independent of
the Fc part of an antibody. End products of the complement cascade
result in lysis of the cell and generation of an inflammatory
milieu. In the opsonization, pathogens or other non-cellular
particles are made accessible to phagocytes via binding the
constant domain of an antibody. Alternatively, cells recognized as
foreign can be lysed via antibody-dependent cell-mediated
cytotoxicity (ADCC). In particular, NK-cells can display lysis
functions by activating Fc receptors.
[0092] In order to determine the percentage to which two RNA
sequences (nucleic or amino acid) are identical, the sequences can
be aligned in order to be subsequently compared to one another.
Therefore, e.g. gaps can be inserted into the sequence of the first
sequence and the component at the corresponding position of the
second sequence can be compared. If a position in the first
sequence is occupied by the same component as is the case at a
position in the second sequence, the two sequences are identical at
this position. The percentage to which two sequences are identical
is a function of the number of identical positions divided by the
total number of positions.
[0093] The percentage to which two sequences are identical can be
determined using a mathematical algorithm. A preferred, but not
limiting, example of a mathematical algorithm which can be used is
the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or
Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402. Such an
algorithm is integrated in the BLAST program. Sequences which are
identical to the sequences of the RNA of the complexed RNA of the
present invention to a certain extent can be identified by this
program.
[0094] Those at least one RNA molecules (of the complexed RNA of
the present invention) encoding amino acid sequences which have (a)
conservative substitution(s) compared to the physiological sequence
in particular fall under the term variants. Substitutions in which
encoded amino acids which originate from the same class are
exchanged for one another are called conservative substitutions. In
particular, these are encoded amino acids encoded aliphatic side
chains, positively or negatively charged side chains, aromatic
groups in the side chains or encoded amino acids, the side chains
of which can enter into hydrogen bridges, e.g. side chains which
have a hydroxyl function. This means that e.g. an amino acid having
a polar side chain is replaced by another amino acid having a
likewise polar side chain, or, for example, an amino acid
characterized by a hydrophobic side chain is substituted by another
amino acid having a likewise hydrophobic side chain (e.g. serine
(threonine) by threonine (serine) or leucine (isoleucine) by
isoleucine (leucine)). Insertions and substitutions are possible,
in particular, at those sequence positions which cause no
modification to the three-dimensional structure or do not affect
the binding region. Modifications to a three-dimensional structure
by insertion(s) or deletion(s) can easily be determined e.g. using
CD spectra (circular dichroism spectra) (Urry, 1985, Absorption,
Circular Dichroism and ORD of Polypeptides, in: Modern Physical
Methods in Biochemistry, Neuberger et al. (ed.), Elsevier,
Amsterdam).
Immunostimulatory RNA
[0095] According to a third embodiment, the at least one RNA
(molecule) of the complexed RNA of the present invention may be an
immunostimulatory RNA. Thereby, the immunostimulatory RNA may
exhibit an immunostimulatory effect already prior to complexation
of the RNA with the inventive oligopeptide according to formula (I)
as defined above, or, more preferably, an immunostimulatory effect
of the RNA as used herein can be enhanced or even induced by
complexation of the RNA with the inventive oligopeptide according
to formula (I) as defined above. The immunostimulatory RNA of the
complexed RNA of the present invention may be any RNA, e.g. a
coding RNA, as defined above. Preferably, the immunostimulatory RNA
may be a single-stranded, a double-stranded or a partially
double-stranded RNA, more preferably a single-stranded RNA, and/or
a circular or linear RNA, more preferably a linear RNA. More
preferably, the immunostimulatory RNA may be a (linear)
single-stranded RNA. Even more preferably, the immunostimulatory
RNA may be a ((linear) single-stranded) messenger RNA (mRNA). An
immunostimulatory RNA may also occur as a short RNA oligonucleotide
as defined above.
[0096] An immunostimulatory RNA as used herein may furthermore be
selected from any class of RNA molecules, found in nature or being
prepared synthetically, and which can induce an immune response. In
this context, an immune response may occur in various ways. A
substantial factor for a suitable immune response is the
stimulation of different T-cell sub-populations. T-lymphocytes are
typically divided into two sub-populations, the T-helper 1 (Th1)
cells and the T-helper 2 (Th2) cells, with which the immune system
is capable of destroying intracellular (Th1) and extracellular
(Th2) pathogens (e.g. antigens). The two Th cell populations differ
in the pattern of the effector proteins (cytokines) produced by
them. Thus, Th1 cells assist the cellular immune response by
activation of macrophages and cytotoxic T-cells. Th2 cells, on the
other hand, promote the humoral immune response by stimulation of
the B-cells for conversion into plasma cells and by formation of
antibodies (e.g. against antigens). The Th1/Th2 ratio is therefore
of great importance in the immune response. In connection with the
present invention, the Th1/Th2 ratio of the immune response is
preferably shifted in the direction towards the cellular response
(Th1 response) and a cellular immune response is thereby induced.
According to one example, the immune system may be activated by
ligands of Toll-like receptors (TLRs). TLRs are a family of highly
conserved pattern recognition receptor (PRR) polypeptides that
recognize pathogen-associated molecular patterns (PAMPs) and play a
critical role in innate immunity in mammals. Currently at least
thirteen family members, designated TLR1-TLR13 (Toll-like
receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,
TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore, a
number of specific TLR ligands have been identified. It was e.g.
found that unmethylated bacterial DNA and synthetic analogs
thereof. (CpG DNA) are ligands for TLR9 (Hemmi H et al. (2000)
Nature 408:740-5; Bauer S et al. (2001) Proc Natl Acad Sci USA 98,
9237-42). Furthermore, it has been reported that ligands for
certain TLRs include certain nucleic acid molecules and that
certain types of RNA are immunostimulatory in a
sequence-independent or sequence-dependent manner, wherein these
various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or
TLR8, or intracellular receptors such as RIG-I, MDA-5, etc. E.g.
Lipford et al. determined certain G, U-containing
oligoribonucleotides as immunostimulatory by acting via TLR7 and
TLR8 (see WO 03/086280). The immunostimulatory G, U-containing
oligoribonucleotides described by Lipford et al. were believed to
be derivable from RNA sources including ribosomal RNA, transfer
RNA, messenger RNA, and viral RNA.
[0097] According to the present invention, it was found that any
RNA (molecule) as e.g. defined above (irrespective of its specific
length, strandedness, modification and/or nucleotide sequence)
complexed with a carrier peptide according to empirical formula
(Arg).sub.l; (Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x (formula
I) may have immunostimulatory properties, i.e. enhance the immune
response. RNA as defined above complexed with a carrier peptide
according to empirical formula (Arg).sub.l; (Lys).sub.m; (His);
(Orn).sub.o; (Xaa).sub.x (formula I) may thus be used to enhance
(unspecific) immunostimulation, if suitable and desired for a
specific treatment. Accordingly, it can be an intrinsic property of
the complexed RNA of the invention to provide immunostimulatory
effects by complexation of any RNA with a peptide according to
formula (I).
[0098] The at least one (immunostimulatory) RNA (molecule) of the
complexed RNA of the present invention may thus comprise any RNA
sequence known to be immunostimulatory, including, without being
limited thereto, RNA sequences representing and/or encoding ligands
of TLRs, preferably selected from family members TLR1-TLR13, more
preferably from TLR7 and TLR8, ligands for intracellular receptors
for RNA (such as RIG-I or MAD-5, etc.) (see e.g. Meylan, E.,
Tschopp, J. (2006). Toll-like receptors and RNA helicases: two
parallel ways to trigger antiviral responses. Mol. Cell 22,
561-569), or any other immunostimulatory RNA sequence. Furthermore,
(classes of) RNA molecules, which may be used as immunostimulatory
RNA may include any other RNA capable of eliciting an immune
response. Without being limited thereto, such immunostimulatory RNA
may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger
RNA (mRNA), and viral RNA (vRNA).
[0099] Such further (classes of) RNA molecules, which may be used
as the at least one (immunostimulatory) RNA (molecule) of the
complexed RNA of the present invention, may comprise, without being
limited thereto, e.g. an RNA molecule of formula (IIa):
G.sub.lX.sub.mG.sub.n
wherein: [0100] G is guanosine, uracil or an analogue of guanosine
or uracil; [0101] X is guanosine, uracil, adenosine, thymidine,
cytosine or an analogue of the above-mentioned nucleotides; [0102]
l is an integer from 1 to 40, [0103] wherein when l=1 G is
guanosine or an analogue thereof, [0104] when l>1 at least 50%
of the nucleotides are guanosine or an analogue thereof; [0105] m
is an integer and is at least 3; [0106] wherein when m=3 X is
uracil or an analogue thereof, [0107] when m>3 at least 3
successive uracils or analogues of uracil occur; [0108] n is an
integer from 1 to 40, [0109] wherein when n=1 G is guanosine or an
analogue thereof, [0110] when n>1 at least 50% of the
nucleotides are guanosine or an analogue thereof.
[0111] In addition, such further (classes of) RNA molecules, which
may be used as the at least one (immunostimulatory) RNA (molecule)
of the complexed RNA of the present invention may comprise, without
being limited thereto, e.g. an RNA molecule of formula (IIb):
C.sub.lX.sub.mC.sub.n
wherein: [0112] C is cytosine, uracil or an analogue of cytosine or
uracil; [0113] X is guanosine, uracil, adenosine, thymidine,
cytosine or an analogue of the above-mentioned nucleotides; [0114]
l is an integer from 1 to 40, [0115] wherein when l=1 C is cytosine
or an analogue thereof, [0116] when l>1 at least 50% of the
nucleotides are cytosine or an analogue thereof; [0117] m is an
integer and is at least 3; [0118] wherein when m=3 X is uracil or
an analogue thereof, [0119] when m>3 at least 3 successive
uracils or analogues of uracil occur; [0120] n is an integer from 1
to 40, [0121] wherein when n=1 C is cytosine or an analogue
thereof, [0122] when n>1 at least 50% of the nucleotides are
cytosine or an analogue thereof.
[0123] Preferably, the immunostimulatory RNA molecules as used
herein as the at least one RNA (molecule) of the complexed RNA of
the present invention comprise a length as defined above in general
for RNA molecules of the complexed RNA of the present invention,
more preferably a length of 5 to 5000, of 500 to 5000 or, more
preferably, of 1000 to 5000 or, alternatively, of 5 to 1000, 5 to
500, 5 to 250, of 5 to 100, of 5 to 50 or, more preferably, of 5 to
30 nucleotides.
[0124] The at least one immunostimulatory RNA as used herein as the
at least one RNA (molecule) of the complexed RNA of the present
invention may be furthermore modified, preferably "chemically
modified" in order to enhance the immunostimulatory properties of
said DNA. The term "chemical modification" means that the RNA used
as immuostimulatory RNA according to the invention is modified by
replacement, insertion or removal of individual or several atoms or
atomic groups compared with naturally occurring RNA species.
[0125] Preferably, the chemical modification of the RNA comprises
at least one analogue of naturally occurring nucleotides. In a list
which is in no way conclusive, examples which may be mentioned for
nucleotide analogues which can be used according to the invention
are analogues of guanosine, uracil, adenosine, thymidine, cytosine.
The modifications may refer to modifications of the base, the
ribose moiety and/or the phosphate backbone moiety. In this
context, analogues of guanosine, uracil, adenosine, and cytosine
include, without implying any limitation, any naturally occurring
or non-naturally occurring guanosine, uracil, adenosine, thymidine
or cytosine that has been altered chemically, for example by
acetylation, methylation, hydroxylation, etc., including
1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine,
2,2-dimethyl-guanosine, 2,6-diaminopurine,
2'-Amino-2'-deoxyadenosine, 2'-Amino-2'-deoxycytidine,
2'-Amino-2'-deoxyguanosine, 2'-Amino-2'-deoxyuridine,
2-Amino-6-chloropurineriboside, 2-Aminopurine-riboside,
2'-Araadenosine, 2'-Aracytidine, 2'-Arauridine,
2'-Azido-2'-deoxyadenosine, 2'-Azido-2'-deoxycytidine,
2'-Azido-2'-deoxyguanosine, 2'-Azido-2'-deoxyuridine,
2-Chloroadenosine, 2'-Fluoro-2'-deoxyadenosine,
2'-Fluoro-2'-deoxycytidine, 2'-Fluoro-2'-deoxyguanosine,
2'-Fluoro-2'-deoxyuridine, 2'-Fluorothymidine, 2-methyl-adenosine,
2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine,
2'-O-Methyl-2-aminoadenosine, 2'-O-Methyl-2'-deoxyadenosine,
2'-O-Methyl-2'-deoxycytidine, 2'-O-Methyl-2'-deoxyguanosine,
2'-O-Methyl-2'-deoxyuridine, 2'-O-Methyl-5-methyluridine,
2'-O-Methylinosine, 2'-O-Methyl pseudouridine, 2-Thiocytidine,
2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine,
4-Thiouridine, 5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine,
5-Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine,
5-carboxymethylaminomethyl-2-thio-uracil,
5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine,
5-Fluoro-uridine, 5-Iodouridine, 5-methoxycarbonyl methyl-uridine,
5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine,
6-Azauridine, 6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside,
6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside,
7-Deaza-2'-deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine,
8-Azaadenosine, 8-Bromo-adenosine, 8-Bromo-guanosine,
8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole-riboside,
Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine,
N1-Methyladenosine, N6-([6-Aminohexyl]carbamoylmethyl)-adenosine,
N6-isopentenyl-adenosine, N6-methyl-adenosine,
N7-Methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester,
Puromycin, Queosine, Uracil-5-oxyacetic acid, Uracil-5-oxyacetic
acid methyl ester, Wybutoxosine, Xanthosine, and Xylo-adenosine.
The preparation of such analogues is known to a person skilled in
the art, for example from U.S. Pat. Nos. 4,373,071, U.S. Pat. No.
4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S.
Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No.
4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S.
Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642. In the
case of an analogue as described above, particular preference is
given according to the invention to those analogues that increase
the immunogenicity of the immunostimulatory RNA sequence as used
herein as the at least one RNA (molecule) of the complexed RNA of
the present invention and/or do not interfere with a further
modification that has been introduced into said immunostimulatory
RNA.
siRNA
[0126] In a forth embodiment, the at least one RNA (molecule) of
the complexed RNA of the present invention may be in the form of
siRNA. A siRNA is of interest particularly in connection with the
phenomenon of RNA interference. Attention was drawn to the
phenomenon of RNA interference in the course of immunological
research. In recent years, a RNA-based defence mechanism has been
discovered, which occurs both in the kingdom of the fungi and in
the plant and animal kingdom and acts as an "immune system of the
genome". The system was originally described in various species
independently of one another, first in C. elegans, before it was
possible to identify the underlying mechanisms of the processes as
being identical: RNA-mediated virus resistance in plants, PTGS
(posttranscriptional gene silencing) in plants, and RNA
interference in eukaryotes are accordingly based on a common
procedure. The in vitro technique of RNA interference (RNAi) is
based on double-stranded RNA molecules (dsRNA), which trigger the
sequence-specific suppression of gene expression (Zamore (2001)
Nat. Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490:
Hannon (2002) Nature 41: 244-251). In the transfection of mammalian
cells with long dsRNA, the activation of protein kinase R and
RnaseL brings about unspecific effects, such as, for example, an
interferon response (Stark et al. (1998) Annu. Rev. Biochem. 67:
227-264; He and Katze (2002) Viral Immunol. 15: 95-119). These
unspecific effects are avoided when shorter, for example 21- to
23-mer, so-called siRNA (small interfering RNA), is used, because
unspecific effects are not triggered by siRNA that is shorter than
30 by (Elbashir et al. (2001) Nature 411: 494-498). Recently, dsRNA
molecules have also been used in vivo (McCaffrey et al. (2002),
Nature 418: 38-39; Xia et al. (2002), Nature Biotech. 20:
1006-1010; Brummelkamp et al. (2002), Cancer Cell 2: 243-247).
Thus, a siRNA as used for the complexed RNA according to the
present invention typically comprises a (single- or) double
stranded, preferably a double-stranded, RNA sequence with about 8
to 30 nucleotides, preferably 17 to 25 nucleotides, even more
preferably from 20 to 25 and most preferably from 21 to 23
nucleotides. In principle, all the sections having a length of from
17 to 29, preferably from 19 to 25, most preferably from 21 to 23
base pairs that occur in the coding region of a RNA sequence as
mentioned above, e.g. of an (m)RNA sequence, can serve as target
sequence for a siRNA. Equally, siRNAs can also be directed against
nucleotide sequences of a (therapeutically relevant) protein or
antigen described hereinbefore that do not lie in the coding
region, in particular in the 5' non-coding region of the RNA, for
example, therefore, against non-coding regions of the RNA having a
regulatory function. The target sequence of the siRNA can therefore
lie in the translated and/or untranslated region of the RNA and/or
in the region of the control elements. The target sequence of a
siRNA can also lie in the overlapping region of untranslated and
translated sequence; in particular, the target sequence can
comprise at least one nucleotide upstream of the start triplet of
the coding region of the RNA.
Antisense RNA
[0127] According to a fifth embodiment, the at least one RNA
(molecule) of the complexed RNA of the present invention may be an
antisense RNA. In the context of the present invention, an
antisense RNA is preferably a (single-stranded) RNA molecule
transcribed on the basis of the coding, rather than the template,
strand of DNA, so that it is complementary to the sense (messenger)
RNA. An antisense RNA as used herein as the at least one RNA
(molecule) of the complexed RNA of the present invention typically
forms a duplex between the sense and antisense RNA molecules and is
thus capable to block translation of the mRNA. An antisense RNA as
used herein as the at least one RNA (molecule) of the complexed RNA
of the present invention can be directed against (may be
complementary to) any portion of the mRNA sequence, which may
encode a (therapeutically relevant) protein or antigen (e.g. as
described hereinbefore), if thereby translation of the encoded
protein is reduced/suppressed. Accordingly, the target sequence of
the antisense RNA on the targeted mRNA may be located in the
translated and/or untranslated region of the mRNA, e.g. in the
region of the mRNA control elements, in particular in the 5'
non-coding region of the RNA exerting a regulatory function. The
target sequence of an antisense RNA on the targeted mRNA may also
be constructed such that the antisense RNA binds to the mRNA by
covering with its sequence a region which is partially
complementary to the untranslated and to translated (coding)
sequence of the targeted mRNA; in particular, the antisense RNA may
be complementary to the target mRNA sequence by at least one
nucleotide upstream of the start triplet of the coding region of
the targeted mRNA. Preferably, the antisense RNA as used herein as
the at least one RNA (molecule) of the complexed RNA of the present
invention comprises a length as generally defined above for RNA
molecules (of the complexed RNA of the present invention).
Typically the antisense RNA as used herein as the at least one RNA
(molecule) of the complexed RNA of the present invention will be a
fragment of the targeted mRNA. In more detail, the antisense RNA
may have more preferably a length of 5 to 5000, of 500 to 5000,
and, more preferably, of 1000 to 5000 or, alternatively, of 5 to
1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30
nucleotides, or, alternatively, and even more preferably a length
of 20 to 100, of 20 to 80, or of 20 to 60 nucleotides.
Modifications of the RNA
[0128] According to one embodiment, the RNA as used herein as the
at least one RNA (molecule) of the complexed RNA of the present
invention (irrespective of its e.g. specific therapeutic potential,
length, and/or sequence), particularly the short RNA
oligonucleotide, the coding RNA, the immunostimulatory RNA, the
siRNA, the antisense RNA, the riboswitches, ribozymes or aptamers,
may provided as a modified RNA, wherein any modification, in
particular a modification disclosed in the following) may be
introduced (in any combination or as such) into the RNA (molecules)
as defined above. Certain types of modifications may, however, be
more suitable for specific RNA types (e.g. more suitable for coding
RNA), while other modifications may be applied for any RNA
molecule, e.g. as defined herein without being restricted to
specific RNA types. Accordingly, modifications of the RNA may be
introduced in order to achieve specific or complex effects which
may desired for the use of the subject-matter of the invention.
Accordingly, modifications may be designed to e.g. stabilize the
RNA against degradation, to enhance their transfection efficacy, to
improve its translation efficacy, to increase their immunogenic
potential and/or to enhance their therapeutic potential (e.g.
enhance their silencing or antisense properties). It is
particularly preferred, if the modified RNA as component of the
inventive complexed RNA allows to combine improvement of at least
one, more preferably of at least two functional properties, e.g. to
stabilized the RNA and to improve the therapeutic or immunogenic
potential.
[0129] Generally, it is a primary object to stabilize the RNA as
used herein as the at least one RNA (molecule) of the complexed RNA
of the present invention, which allows to extend their half-life
time in vivo. Preferably, the half-life time of a modified RNA
under in vivo conditions is extended (as compared to the unmodified
RNA) by at least 20, more preferably at least 40, more preferably
at least 50 and even more preferably at least 70, 80, 90, 100, 150
or 200%. The stabilization achieved by the modification may extend
the half-life time of the modified mRNA by at least 5, 10, 15, 30
or more preferably at least 60 min as compared to the unmodified
RNA.
[0130] According to one embodiment the at least one RNA (molecule)
of the complexed RNA of the present invention, preferably a coding
RNA, e.g. mRNA, may be stabilized by modifying the G/C content of
e.g. the coding region of the RNA. In a particularly preferred
embodiment of the present invention, the G/C content of the coding
region of the RNA (of the complexed RNA of the present invention)
is altered, particularly increased, compared to the G/C content of
the coding region of its corresponding wild-type RNA, i.e. the
unmodified RNA. The encoded amino acid sequence of the modified RNA
is preferably not altered as compared to the amino acid sequence
encoded by the corresponding wild-type RNA.
[0131] This modification of the RNA as used herein as the at least
one RNA (molecule) of the complexed RNA of the present invention is
based on the fact that the coding sequence of any RNA to be
translated is important for efficient translation of that RNA. In
particular, sequences having an increased G (guanosine)/C
(cytosine) content are more stable than sequences having an
increased A (adenosine)/U (uracil) content. According to the
invention, the codons of the RNA are therefore altered compared to
the wild-type RNA, while retaining the translated amino acid
sequence, such that they include an increased amount of G/C
nucleotides. In respect to the fact that several codons code for
one and the same amino acid (so-called degeneration of the genetic
code), the most favorable codons for the stability can be
determined (so-called alternative codon usage).
[0132] Depending on the amino acid to be encoded by the RNA as used
herein as the at least one RNA (molecule) of the complexed RNA of
the present invention, there are various possibilities for
modification of the RNA sequence, compared to its wild-type
sequence. In the case of amino acids which are encoded by codons
which contain exclusively G or C nucleotides, no modification of
the codon is necessary. Thus, the codons for Pro (CCC or CCG), Arg
(CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no
modification, since no A or U is present.
[0133] In contrast, codons which contain A and/or U nucleotides can
be modified by substitution of other codons which code for the same
amino acids but contain no A and/or U. Examples of these are:
the codons for Pro can be modified from CCU or CCA to CCC or CCG;
the codons for Arg can be modified from CGU or CGA or AGA or AGG to
CGC or CGG; the codons for Ala can be modified from GCU or GCA to
GCC or GCG; the codons for Gly can be modified from GGU or GGA to
GGC or GGG.
[0134] In other cases, although A or U nucleotides cannot be
eliminated from the codons, it is however possible to decrease the
A and U content by using codons which contain a lower content of A
and/or U nucleotides. Examples of these are:
the codons for Phe can be modified from UUU to UUC; the codons for
Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG; the
codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG
or AGC; the codon for Tyr can be modified from UAU to UAC; the
codon for Cys can be modified from UGU to UGC; the codon for His
can be modified from CAU to CAC; the codon for Gln can be modified
from CAA to CAG; the codons for Ile can be modified from AUU or AUA
to AUC; the codons for Thr can be modified from ACU or ACA to ACC
or ACG; the codon for Asn can be modified from AAU to AAC; the
codon for Lys can be modified from AAA to AAG; the codons for Val
can be modified from GUU or GUA to GUC or GUG; the codon for Asp
can be modified from GAU to GAC; the codon for Glu can be modified
from GAA to GAG; the stop codon UAA can be modified to UAG or
UGA.
[0135] In the case of the codons for Met (AUG) and Trp (UGG), on
the other hand, there is no possibility of sequence
modification.
[0136] The substitutions listed above can be used either
individually or in all possible combinations to increase the G/C
content of the RNA as used herein as the at least one RNA
(molecule) of the complexed RNA of the present invention compared
to its particular wild-type RNA (i.e. the original sequence). Thus,
for example, all codons for Thr occurring in the wild-type sequence
can be modified to ACC (or ACG). Preferably, however, for example,
combinations of the above substitution possibilities are used:
substitution of all codons coding for Thr in the original sequence
(wild-type RNA) to ACC (or ACG) and substitution of all codons
originally coding for Ser to UCC (or UCG or AGC); substitution of
all codons coding for Ile in the original sequence to AUC and
substitution of all codons originally coding for Lys to AAG and
substitution of all codons originally coding for Tyr to UAC;
substitution of all codons coding for Val in the original sequence
to GUC (or GUG) and substitution of all codons originally coding
for Glu to GAG and substitution of all codons originally coding for
Ala to GCC (or GCG) and substitution of all codons originally
coding for Arg to CGC (or CGG); substitution of all codons coding
for Val in the original sequence to GUC (or GUG) and substitution
of all codons originally coding for Glu to GAG and substitution of
all codons originally coding for Ala to GCC (or GCG) and
substitution of all codons originally coding for Gly to GGC (or
GGG) and substitution of all codons originally coding for Asn to
AAC; substitution of all codons coding for Val in the original
sequence to GUC (or GUG) and substitution of all codons originally
coding for Phe to UUC and substitution of all codons originally
coding for Cys to UGC and substitution of all codons originally
coding for Leu to CUG (or CUC) and substitution of all codons
originally coding for Gln to CAG and substitution of all codons
originally coding for Pro to CCC (or CCG); etc.
[0137] Preferably, the G/C content of the coding region of the RNA
as used herein as the at least one RNA (molecule) of the complexed
RNA of the present invention is increased by at least 7%, more
preferably by at least 15%, particularly preferably by at least
20%, compared to the G/C content of the coded region of the
wild-type RNA which codes for a protein. According to a specific
embodiment at least 60%, more preferably at least 70%, even more
preferably at least 80% and most preferably at least 90%, 95% or
even 100% of the substitutable codons in the region coding for a
protein or the whole sequence of the wild type RNA sequence are
substituted, thereby increasing or even maximizing the GC/content
of said sequence.
[0138] In this context, it is particularly preferable to increase
the G/C content of the RNA as used herein as the at least one RNA
(molecule) of the complexed RNA of the present invention to the
maximum (i.e. 100% of the substitutable codons), in particular in
the region coding for a protein, compared to the wild-type
sequence.
[0139] According to the invention, a further preferred modification
of the RNA as used herein as the at least one RNA (molecule) of the
complexed RNA of the present invention is based on the finding that
the translation efficiency is also determined by a different
frequency in the occurrence of tRNAs in cells. Thus, if so-called
"rare codons" are present in an RNA sequence to an increased
extent, the corresponding modified RNA sequence is translated to a
significantly poorer degree than in the case where codons coding
for relatively "frequent" tRNAs are present.
[0140] According to the invention, in the RNA as used herein as the
at least one RNA (molecule) of the complexed RNA of the present
invention, the region which codes for the protein is modified
compared to the corresponding region of the wild-type RNA such that
at least one codon of the wild-type sequence which codes for a tRNA
which is relatively rare in the cell is exchanged for a codon which
codes for a tRNA which is relatively frequent in the cell and
carries the same amino acid as the relatively rare tRNA. By this
modification, the RNA sequences are modified such that codons for
which frequently occurring tRNAs are available are inserted. In
other words, according to the invention, by this modification all
codons of the wild-type sequence which code for a tRNA which is
relatively rare in the cell can in each case be exchanged for a
codon which codes for a tRNA which is relatively frequent in the
cell and which, in each case, carries the same amino acid as the
relatively rare tRNA.
[0141] Which tRNAs occur relatively frequently in the cell and
which, in contrast, occur relatively rarely is known to a person
skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001,
11(6): 660-666. The codons which use for the particular amino acid
the tRNA which occurs the most frequently, e.g. the Gly codon,
which uses the tRNA which occurs the most frequently in the (human)
cell, are particularly preferred.
[0142] According to the invention, it is particularly preferable to
link the sequential G/C content which is increased, in particular
maximized, in the RNA as used herein as the at least one RNA
(molecule) of the complexed RNA of the present invention, with the
"frequent" codons without modifying the amino acid sequence of the
protein encoded by the coding region of the RNA. This preferred
embodiment allows provision of a particularly efficiently
translated and stabilized (modified) RNA of the complexed RNA
according to the present invention.
[0143] The determination of the G/C content of an RNA as used
herein as the at least one RNA (molecule) of the complexed RNA of
the present invention (increased G/C content; exchange of tRNAs)
can be carried out using the computer program explained in WO
02/098443--the disclosure content of which is included in its full
scope in the present invention. Using this computer program, the
nucleotide sequence of any desired RNA can be modified with the aid
of the genetic code or the degenerative nature thereof such that a
maximum G/C content results, in combination with the use of codons
which code for tRNAs occurring as frequently as possible in the
cell, the amino acid sequence coded by the RNA (molecule)
preferably not being modified compared to the non-modified
sequence. Alternatively, it is also possible to modify only the G/C
content or only the codon usage compared to the original sequence.
The source code in Visual Basic 6.0 (development environment used:
Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is also
described in WO 02/098443.
[0144] In a further preferred embodiment of the present invention,
the A/U content in the environment of the ribosome binding site of
the at least one RNA (molecule) of the complexed RNA of the present
invention is increased compared to the A/U content in the
environment of the ribosome binding site of its particular
wild-type RNA. This modification (an increased A/U content around
the ribosome binding site) increases the efficiency of ribosome
binding to the modified RNA. An effective binding of the ribosomes
to the ribosome binding site (Kozak sequence: GCCGCCACCAUGG (SEQ ID
NO: 33), the AUG forms the start codon) in turn has the effect of
an efficient translation of the modified RNA.
[0145] According to a further embodiment of the present invention
the at least one RNA (molecule) of the complexed RNA of the present
invention may be modified with respect to potentially destabilizing
sequence elements. Particularly, the coding region and/or the 5'
and/or 3' untranslated region of this RNA may be modified compared
to the particular wild-type RNA such that is contains no
destabilizing sequence elements, the coded amino acid sequence of
the RNA (molecule) preferably not being modified compared to its
particular wild-type RNA. It is known that, for example, in
sequences of eukaryotic RNAs destabilizing sequence elements (DSE)
occur, to which signal proteins bind and regulate enzymatic
degradation of RNA in vivo. For further stabilization of the RNA
(molecule), optionally in the region which encodes for a protein,
one or more such modifications compared to the corresponding region
of the wild-type RNA can therefore be carried out, so that no or
substantially no destabilizing sequence elements are contained
there. According to the invention, DSE present in the untranslated
regions (3'- and/or 5'-UTR) can also be eliminated from the at
least one RNA (molecule) of the complexed RNA of the present
invention by such modifications.
[0146] Such destabilizing sequences are e.g. AU-rich sequences
(AURES), which occur in 3'-UTR sections of numerous unstable RNAs
(Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674).
The RNA of the complexed RNA according to the present invention is
therefore preferably modified compared to the wild-type RNA such
that the RNA contains no such destabilizing sequences. This also
applies to those sequence motifs which are recognized by possible
endonucleases, e.g. the sequence GAACAAG, which is contained in the
3'-UTR segment of the gene which codes for the transferrin receptor
(Binder et al., EMBO J. 1994, 13: 1969 to 1980). These sequence
motifs are also preferably removed according to the invention in
the at least one RNA (molecule) of the complexed RNA of the present
invention.
[0147] According to the present invention, the at least one RNA
(molecule) of the complexed RNA of the present invention can have a
5' cap structure. Examples of cap structures which can be used
according to the invention are m7G(5')ppp, (5'(A,G(5')ppp(5')A and
G(5')ppp(5')G.
[0148] Another modification enhancing the stability of the at least
one RNA (molecule) of the complexed RNA of the present invention is
based on 5'- or 3' elongations of the RNA, typically homonucleotide
elongations of a length of 10 to 200 nucleotides. These elongations
may contain, particularly if the RNA is provided as mRNA, a poly-A
tail at the 3' terminus of typically about 10 to 200 adenosine
nucleotides, preferably about 10 to 100 adenosine nucleotides, more
preferably about 20 to 70 adenosine nucleotides or even more
preferably about 20 to 60 adenosine nucleotides. Alternatively or
additionally, the at least one RNA (molecule) of the complexed RNA
of the present invention may contain, particularly if the RNA is
provided as mRNA, a poly-C tail at the 3' terminus of typically
about 10 to 200 cytosine nucleotides, preferably about 10 to 100
cytosine nucleotides, more preferably about 20 to 70 cytosine
nucleotides or even more preferably about 20 to 60 or even 10 to 40
cytosine nucleotides.
[0149] Another modification, which may occur in the at least one
RNA (molecule) of the complexed RNA of the present invention,
particularly if the RNA is provided as mRNA, refers preferably to
at least one IRES and/or at least one 5' and/or 3' stabilizing
sequence. According to the invention, one or more so-called IRES
(internal ribosomal entry site) may be inserted into the RNA. An
IRES can thus function as the sole ribosome binding site, but it
can also serve to provide a RNA which codes several proteins which
are to be translated by the ribosomes independently of one another
(multicistronic RNA). Examples of IRES sequences which can be used
according to the invention are those from picornaviruses (e.g.
FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis
viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C
viruses (HCV), classical swine fever viruses (CSFV), mouse leukoma
virus (MLV), simian immunodeficiency viruses (SIV) or cricket
paralysis viruses (CrPV).
[0150] According to the invention, the at least one RNA (molecule)
of the complexed RNA of the present invention may exhibit at least
one 5' and/or 3' stabilizing sequence as known from the art. These
stabilizing sequences in the 5' and/or 3' untranslated regions have
the effect of increasing the half-life of the RNA in the cytosol.
These stabilizing sequences can have 100% sequence homology to
naturally occurring sequences which occur in viruses, bacteria and
eukaryotes, but can also be partly or completely synthetic. The
untranslated sequences (UTR) of the globin gene, e.g. from Homo
sapiens or Xenopus laevis may be mentioned as an example of
stabilizing sequences which can be used in the present invention
for a stabilized RNA. Another example of a stabilizing sequence has
the general formula (C/U)CCAN.sub.xCCC(U/A)Py.sub.xUC(C/U)CC (SEQ
ID NO: 34), which is contained in the 3'UTR of the very stable RNA
which codes for globin, (I)-collagen, 15-lipoxygenase or for
tyrosine hydroxylase (cf. Holcik et al., Proc. Natl. Acad. Sci. USA
1997, 94: 2410 to 2414). Such stabilizing sequences can of course
be used individually or in combination with one another and also in
combination with other stabilizing sequences known to a person
skilled in the art. The at least one RNA (molecule) of the
complexed RNA of the present invention is therefore preferably
present as globin UTR (untranslated regions)-stabilized RNA, in
particular as globin UTR-stabilized RNA.
[0151] If desired, the at least one RNA (molecule) of the complexed
RNA of the present invention may contain backbone modifications. A
backbone modification in connection with the present invention is a
modification in which phosphates of the backbone of the nucleotides
contained in the RNA are chemically modified. Such backbone
modifications typically include, without implying any limitation,
modifications from the group consisting of methylphosphonates,
phosphoramidates and phosphorothioates (e.g.
cytidine-5'-O-(1-thiophosphate)).
[0152] The at least one RNA (molecule) of the complexed RNA of the
present invention may additionally or alternatively also contain
sugar modifications. A sugar modification in connection with the
present invention is a chemical modification of the sugar of the
nucleotides present and typically includes, without implying any
limitation, sugar modifications selected from the group consisting
of 2'-deoxy-2'-fluoro-oligoribonucleotide
(2'-fluoro-2'-deoxycytidine-5'-triphosphate,
2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamine
oligoribonucleotide (2'-amino-2'-deoxycytidine-5'-triphosphate,
2'-amino-2'-deoxyuridine-5'-triphosphate), 2'-O-alkyl
oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide
(2'-O-methylcytidine-5'-triphosphate, 2'-methyl
uridine-5'-triphosphate), 2'-C-alkyl oligoribonucleotide, and
isomers thereof (2'-aracytidine-5'-triphosphate,
2'-arauridine-5'-triphosphate), or azidotriphosphate
(2'-azido-2'-deoxycytidine-5'-triphosphate,
2'-azido-2'-deoxyuridine-5'-tri phosphate).
[0153] The at least one RNA (molecule) of the complexed RNA of the
present invention may additionally or alternatively also contain at
least one base modification, which is preferably suitable for
increasing the expression of the protein coded for by the at least
one RNA (molecule) significantly as compared with the unaltered,
i.e. natural native), RNA sequence. Significant in this case means
an increase in the expression of the protein compared with the
expression of the native RNA sequence by at least 20%, preferably
at least 30%, 40%, 50% or 60%, more preferably by at least 70%,
80%, 90% or even 100% and most preferably by at least 150%, 200% or
even 300%. In connection with the present invention, a nucleotide
having a base modification is preferably selected from the group of
the base-modified nucleotides consisting of
2-amino-6-chloropurineriboside-5'-triphosphate,
2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate,
2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5-aminoallylcytidine-5'-triphosphate,
5-aminoallyluridine-5'-triphosphate,
5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate,
5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate,
6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate,
6-chloropurineriboside-5'-triphosphate,
7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-tri
phosphate, 8-azaadenosine-5'-triphosphate,
8-azidoadenosine-5'-triphosphate,
benzimidazole-riboside-5'-triphosphate,
N1-methyladenosine-5'-triphosphate,
N1-methylguanosine-5'-triphosphate,
N6-methyladenosine-5'-triphosphate,
O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate,
or puromycin-5'-triphosphate, xanthosine-5'-triphosphate.
Particular preference is given to nucleotides for base
modifications selected from the group of base-modified nucleotides
consisting of 5-methylcytidine-5'-triphosphate,
7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate,
and pseudouridine-5'-triphosphate.
[0154] The at least one RNA (molecule) of the complexed RNA of the
present invention may additionally or alternatively also contain at
least one modification of a nucleoside of a nucleotide as contained
in the at least one RNA (molecule), which acts immunosuppressive,
i.e. is preferably suitable for preventing or decreasing an immune
response, when administered to a patient in need thereof. Such at
least one modification is preferably selected from nucleoside
modifications selected from: [0155] a) a chemical modification at
the 4-, 5- or 6-position of the pyrimidine base of the nucleosides
of cytidine and/or uridine; [0156] b) a chemical modification at
the 2-, 6-, 7- or 8-position of the purine base of the nucleosides
of adenosine, inosine and/or guanosine; and/or [0157] c) a chemical
modification at the 2'-position of the sugar of the nucleosides of
adenosine, inosine, guanosine, cytidine and/or uridine.
[0158] In this context, an (m)RNA is a nucleic acid chain formed by
a number of nucleotides typically selected from
adenosine-5'-monophosphate, guanosine-5'-monophosphate,
inosine-5'-monophosphate, cytidine-5'-monophosphate and/or
uridine-5'-monophosphate. Those nucleotides are linked to each
other via their monophosphate. Nucleotides comprise nucleosides and
a 5'-monophosphate as a structural component, wherein the
nucleosides are typically formed by a nucleobase, i.e. a pyrimidine
(uracil or cytosine) or a purine (adenine or guanine) base, and a
sugar. Accordingly, a modification of a nucleoside of at least one
RNA (molecule) of the complexed RNA of the present invention is
always intended to mean a modification in the nucleoside structure
of the respective nucleotide of said at least one RNA
(molecule).
[0159] According to a first modification a), at least one
nucleoside of the at least one RNA (molecule) of the complexed RNA
of the present invention, may be modified with a chemical
modification at the 5- or 6-position of the pyrimidine base of the
nucleosides cytidine and/or uridine. Without being limited thereto,
such chemical modifications at the 4-, 5- or 6-position of the base
pyrimidine of the nucleosides cytidine and/or uridine may be
selected from the group consisting of: 4-thio, 5-iodo-/(5-I--),
5-bromo-/(5-Br--), 5-aminoallyl-, 5-fluoro-/(5-F--), 5-hydroxy-,
5-hydro-/(5-H--), 5-nitro-,
5-propynyl-/(5-(C.ident.C--CH.sub.3)--), 5-methyl-,
5-methyl-2-thio-, 5-formyl-, 5-hydroxymethyl-, 5-methoxy-,
5-oxyacetic acid methyl ester-, 5-oxyacetic acid-,
5-carboxyhydroxymethyl-, 5-(carboxyhydroxymethyl)pyrimidine methyl
ester-, 5-methoxycarbonyl methyl-, 5-methoxycarbonylmethyl-2-thio,
5-aminomethyl-, 5-aminomethyl-2-thio-, 5-aminomethyl-2-seleno-,
5-methylaminomethyl-, 5-carbamoylmethyl-,
5-carboxymethylaminomethyl-, 5-carboxymethylaminomethyl-2-thio-,
5-carboxymethyl-, 5-methyldihydro-, 5-taurinomethyl-,
5-taurinomethyl-2-thiouridine, 5-isopentenylaminomethyl-,
5-isopentenylaminomethyl-2-thio-,
5-aminopropyl-/(5-(C.sub.3H.sub.6NH.sub.3)--),
5-methoxy-ethoxy-methyl-/(5-(CH.sub.2--O--C.sub.2H.sub.4--O--CH.sub.3)--)-
, or 6-aza-.
[0160] According to second modification b), at least one nucleoside
of the at least one RNA (molecule) of the complexed RNA of the
present invention, suitable for suppressing and/or avoiding an
(innate) immunostimulatory response in a mammal typically exhibited
when administering the corresponding unmodified at least one RNA
(molecule), may be alternatively modified with a chemical
modification at the 2-, 6-, 7- or 8-position of the purine base of
the nucleosides adenosine, inosine and/or guanosine. Without being
limited thereto, such chemical modifications at the 2-, 6-, 7- or
8-position of the purine base of the nucleosides adenosine, inosine
and/or guanosine may be selected from the group consisting of
2-Amino-, 7-Deaza-, 8-Aza-, or 8-Azido-.
[0161] According to a third modification c), at least one
nucleoside of the at least one RNA (molecule) of the complexed RNA
of the present invention, suitable for suppressing and/or avoiding
an (innate) immunostimulatory response in a mammal typically
exhibited when administering the corresponding unmodified at least
one RNA (molecule), may be modified with at least one chemical
modification at the 2'-position of the sugar of the nucleosides
adenosine, inosine, guanosine, cytidine and/or uridine, when
incorporated in the RNA sequence. Without being limited thereto,
such chemical modifications at the 2'-position of the sugar of the
nucleosides adenosine, inosine, guanosine, cytidine and/or uridine
may be selected from the group consisting of: 2'-deoxy-,
2'-amino-2'-deoxy-, 2'-amino-, 2'-fluoro-2'-deoxy-, 2'-fluoro-,
2'-O-methyl-2'-deoxy- or 2'-O-methyl-.
[0162] According to a particularly preferred embodiment, at least
one nucleoside of the at least one RNA (molecule) of the complexed
RNA of the present invention has been modified at the 4, -5- or
6-position of the base pyrimidine of the nucleosides cytidine
and/or uridine and at the 2'-position of the ribose sugar according
to modifications a) and c) as defined above.
[0163] According to another particularly preferred embodiment, at
least one nucleoside of the at least one RNA (molecule) of the
complexed RNA of the present invention has been modified at the 2-,
6-, 7- or 8-position of the purine base of the nucleosides
adenosine, inosine and/or guanosine and at the 2'-position of the
ribose sugar according to modifications b) and c) as defined above,
more preferably as defined above.
[0164] According to an even more particularly preferred embodiment,
at least one nucleoside of the at least one RNA (molecule) of the
complexed RNA of the present invention has been modified leading to
chemically modified nucleotides (of the (m)RNA) selected from the
following group: 4-thio-uridine-5'-(mono)phosphate,
2-Aminopurine-riboside-5'-(mono)phosphate,
5-Aminoallylcytidine-5'-(mono)phosphate,
5-Aminoallyluridine-5'-(mono)phosphate,
5-Bromocytidine-5'-(mono)phosphate,
5-Bromo-2'-deoxycytidine-5'-(mono)phosphate,
5-Bromouridine-5'-(mono)phosphate,
5-Bromo-2'-deoxyuridine-5'-(mono)phosphate,
5-Iodocytidine-5'-(mono)phosphate,
5-Iodo-2'-deoxycytidine-5'-(mono)phosphate,
5-Iodouridine-5'-(mono)phosphate,
5-Iodo-2'-deoxyuridine-5'-(mono)phosphate,
5-Propynyl-2'-deoxycytidine-5'-(mono)phosphate,
5-Propynyl-2'-deoxyuridine-5'-(mono)phosphate,
5-formylcytidine-5'-(mono)phosphate,
5,2'-O-dimethylcytidine-5'-(mono)phosphate,
5-hydroxymethylcytidine-5'-(mono)phosphate,
5-formyl-2'-O-methylcytidine-5'-(mono)phosphate,
5,2'-O-dimethyluridine-5'-(mono)phosphate,
5-methyl-2-thiouridine-5'-(mono)phosphate,
5-hydroxyuridine-5'-(mono)phosphate,
5-methoxyuridine-5'-(mono)phosphate, uridine 5-oxyacetic
acid-5'-(mono)phosphate, uridine 5-oxyacetic acid methyl
ester-5'-(mono)phosphate,
5-(carboxyhydroxymethyl)uridine-5'-(mono)phosphate,
5-(carboxyhydroxymethyl)uridine methyl ester-5'-(mono)phosphate,
5-methoxycarbonylmethyluridine-5'-(mono)phosphate,
5-methoxycarbonylmethyl-2'-O-methyl uridine-5'-(mono)phosphate,
5-methoxycarbonylmethyl-2-thiouridine-5'-(mono)phosphate,
5-aminomethyl-2-thiouridine-5'-(mono)phosphate,
5-methylaminomethyluridine-5'-(mono)phosphate,
5-methylaminomethyl-2-thiouridine-5'-(mono)phosphate,
5-methylaminomethyl-2-selenouridine-5'-(mono)phosphate,
5-carbamoylmethyluridine-5'-(mono)phosphate,
5-carbamoylmethyl-2'-O-methyluridine-5'-(mono)phosphate,
5-carboxymethylaminomethyl uridine-5'-(mono)phosphate,
5-carboxymethylaminomethyl-2'-O-methyluridine-5'-(mono)phosphate,
5-carboxymethylaminomethyl-2-thiouridine-5'-(mono)phosphate,
5-carboxymethyluridine-5'-(mono)phosphate,
5-methyldihydrouridine-5'-(mono)phosphate, 5-taurinomethyl
uridine-5'-(mono)phosphate,
5-taurinomethyl-2-thiouridine-5'-(mono)phosphate,
5-(isopentenylaminomethyl)uridine-5'-(mono)phosphate,
5-(isopentenylaminomethyl)-2-thiouridine-5'-(mono)phosphate,
5-(isopentenylaminomethyl)-2'-O-methyluridine-5'-(mono)phosphate,
6-Azacytidine-5'-(mono)phosphate,
7-Deazaadenosine-5'-(mono)phosphate,
7-Deazaguanosine-5'-(mono)phosphate,
8-Azaadenosine-5'-(mono)phosphate,
8-Azidoadenosine-5'-(mono)phosphate,
Pseudouridine-5'-(mono)phosphate,
2'-Amino-2'-deoxycytidine-(mono)phosphate,
2'-Fluorothymidine-5'-(mono)phosphate, inosine-5'-(mono)phosphate,
2'-O-Methyl-inosine-5'-(mono)phosphate.
[0165] If desired, the at least one RNA (molecule) of the complexed
RNA of the present invention may contain substitutions, additions
or deletions of nucleotides, which are preferably introduced to
achieve functional effects. These various types of nucleotide
modifications may be introduced, if the RNA, e.g the mRNA, is
derived from a WT sequence. Hereby, a DNA matrix is used for
preparation of the RNA of the complexed RNA according to the
present invention by techniques of the well known site directed
mutagenesis (see e.g. Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed.,
Cold Spring Harbor, N.Y., 2001). In such a process, for preparation
of the RNA, a corresponding DNA molecule may be transcribed in
vitro. This DNA matrix has a suitable promoter, e.g. a T7 or SP6
promoter, for in vitro transcription, which is followed by the
desired nucleotide sequence for the RNA to be prepared and a
termination signal for in vitro transcription. According to the
invention, the DNA molecule which forms the matrix of a RNA of
interest may be prepared by fermentative proliferation and
subsequent isolation as part of a plasmid which can be replicated
in bacteria. Plasmids which may be mentioned as suitable for the
present invention are e.g. the plasmids pT7Ts (GenBank accession
number U26404; Lai et al., Development 1995, 121: 2349 to 2360),
pGEM.RTM. series, e.g. pGEM.RTM.-1 (GenBank accession number
X65300; from Promega) and pSP64 (GenBank accession number X65327);
cf. also Mezei and Storts, Purification of PCR Products, in:
Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC
Press, Boca Raton, Fla., 2001.
[0166] The mass or molar ratio of the components of the RNA complex
according to the present invention, which means the mass or molar
ratio of the RNA (be it single- or double-stranded) to the one or
more oligopeptides typically is by no way restricted and is chosen
as suitable for the particular application. However, the mass or
molar ratio of the one or more oligopeptides and the RNA may be
less than 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,
1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, or less than 1:20.
Alternatively, the mass or molar ratio of the one or more
oligopeptides and the RNA may higher than 1:1, 2:1, 3:1, 4:1, 5:1,
6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,
18:1, 19:1, 20:1. Preferably, the mass or molar ratio of the one or
more oligopeptides and the RNA may not be less than 1:5 with
respect to the content of the one or more oligopeptides. More
preferably, the (molar or) mass ratio of the one or more
oligopeptides and the RNA is from 1:5 to 20:1, more preferably from
1:3 to 15:1.
[0167] According to a particular preferred embodiment, the mass
ratio of the components of the RNA complex according to the present
invention, particularly the mass ratio of the at least one RNA
(molecule) of the complexed RNA to the one or more oligopeptides is
preferably in a range of about 1:100 to about 1:0.5, more
preferably has a value of about 1:50 to about 1:1, or even more
preferably about 1:100, about 1:90, about 1:80, about 1:70, about
1:60, about 1:50, about 1:45, about 1:40, about 1:35, about 1:30,
about 1:25, about 1:20, about 1:15, about 1:10, about 1:5, about
1:4, about 1:3, about 1:2, about 1:1 or even about 1:0.5 regarding
the ratio of RNA:peptide in the complex, wherein any range may be
formed by combining two of the above specifically indicated values.
Most preferably, the mass ratio of the at least one RNA (molecule)
of the complexed RNA to the one or more oligopeptides may be in a
range of about 1:50 to about 1:1.
[0168] Likewise, the molar ratio of the components of the RNA
complex according to the present invention, particularly the molar
ratio of the at least one RNA (molecule) of the complexed RNA to
the one or more oligopeptides is preferably, according to a
particular preferred embodiment, in a range of about 1:20000 to
about 1:500 or even 1:250, more preferably in a range of about
1:10000 to about 1:1000, or even more preferably has a value of
about 1:9500, about 1:9000, about 1:8500, about 1:8000, about
1:7500, about 1:7000, about 1:6500, about 1:6000, about 1:5500,
about 1:5000, about 1:4500, about 1:4000, about 1:3500, about
1:3000, about 1:2500, about 1:2000, about 1:1500, about 1:1000,
about 1:500, about 1:450, about 1:400, about 1:350, about 1:300, or
about 1:250 regarding the ratio of RNA:peptide in the complex,
wherein any range may be formed by combining two of the above
specifically indicated values. Most preferably, the molar ratio of
the at least one RNA (molecule) of the complexed RNA to the one or
more oligopeptides may be in a range of about 1:10000 to about
1:1000. For immunostimulation purposes, the molar ratio of the
components of the RNA complex according to the present invention
may be in a range of about 1:10000 to about 1:100 or even in a
range of about 1:10000 to about 1:500.
[0169] In the context of the present invention, the molar ratio and
the mass ratio are typically dependent on each other, wherein each
of these ratios may be influenced by factors such as RNA length or
peptide length. However, for purposes of determination, the mass
ratio and the molar ratio may be calculated for an average complex
size, wherein a mass ratio of about 1:50-1:1 approximately
corresponds to a molar ratio of about 1:10000-1:1000. An exemplary
schedule of molar and mass ratios is given in the Examples, which
may be used for calculation additionally.
[0170] Furthermore, the ratio of the RNA complex components
according to the present invention, particularly the ratio of the
at least one RNA (molecule) of the complexed RNA to the one or more
oligopeptides, may also be calculated on the basis of the
nitrogen/phosphate ratio (N/P-ratio) of the entire RNA complex. For
example, 1 .mu.g RNA typically contains about 3 nmol phosphate
residues, provided the RNA exhibits a statistical distribution of
bases. Additionally, 1 .mu.g peptide typically contains about x
nmol nitrogen residues, dependent on the molecular weight and the
number of basic amino acids. When exemplarily calculated for
(Arg).sub.9 (molecular weight 1424 g/mol, 9 nitrogen atoms), 1
.mu.g (Arg).sub.9 contains about 700 pmol (Arg).sub.9 and thus
700.times.9=6300 pmol basic amino acids=6.3 nmol nitrogen atoms.
For a mass ratio of about 1:1 RNA/(Arg).sub.9 an N/P ratio of about
2 can be calculated. When exemplarily calculated for protamine
(molecular weight about 4250 g/mol, 21 nitrogen atoms, when
protamine from salmon is used) with a mass ratio of about 2:1 with
2 .mu.g RNA, 6 nmol phosphate are to be calculated for the RNA; 1
.mu.g protamine contains about 235 pmol protamine molecules and
thus 235.times.21=4935 pmol basic nitrogen atoms=4.9 nmol nitrogen
atoms. For a mass ratio of about 2:1 RNA/protamine an N/P ratio of
about 0.81 can be calculated. For a mass ratio of about 8:1
RNA/protamine an N/P ratio of about 0.2 can be calculated. In the
context of the present invention, an N/P-ratio is preferably in the
range of about 0, 2-50, preferably in a range of about 0.5-50 and
most preferably in a range of about 0.75-25 or 1-25 regarding the
ratio of RNA:peptide in the complex, even more preferably in the
range of about 10-50 and most preferably in the range of about
25-50).
[0171] Another embodiment of the present invention relates to a
composition, preferably a pharmaceutical composition, comprising a
complexed RNA according to the present invention and optionally a
(pharmaceutically) suitable carrier and/or further auxiliary
substances and additives. The (pharmaceutical) composition employed
according to the present invention typically comprises a safe and
effective amount of a complexed RNA according to the present
invention. As used herein, a "safe and effective amount" means an
amount of a complexed RNA according to the present invention such
as to provide an effect in cells or tissues in vitro or in vivo,
e.g. to induce significantly an expression (in vitro or in vivo) of
an encoded protein as described above, such as a therapeutically
active protein, an antibody or an antigene, or any other protein or
peptide as described above, to induce a positive change of a state
to be treated (in vivo) in a cell, a tissue or an organism, e.g. a
tumour disease or cancer disease, a cardiovascular disease, an
infectious disease, an autoimmune disease, (mono-)genetic diseases,
etc. as described herein, and/or to induce or enhance an immune
response. At the same time, however, a "safe and effective amount"
is low enough to avoid serious side effects, particularly in the
therapy of diseases as mentioned herein, that is to say to render
possible a reasonable ratio of advantage and risk.
[0172] Determination of these limits typically lies within the
range of reasonable medical judgement. The concentration of the
complexed RNA according to the invention in such (pharmaceutical)
compositions can therefore vary, for example, without being limited
thereto, within a wide range of from e.g. 0.1 ng to 1,000 mg/ml or
even more. Such a "safe and effective amount" of a complexed RNA
according to the invention can vary in connection with the
particular state to be treated and the age and the physical state
of the patient to be treated, the severity of the state, the
duration of the treatment, the nature of the concomitant therapy,
of the particular (pharmaceutically) suitable carrier used and
similar factors within the knowledge and experience of the treating
doctor. The (pharmaceutical) composition described here can be
employed for human and also for veterinary medicine purposes.
[0173] The (pharmaceutical) composition according to the invention
described here can optionally comprise a suitable carrier,
preferably a pharmaceutically suitable carrier. The term "suitable
carrier" used here preferably includes one or more compatible solid
or liquid fillers, or diluents or encapsulating compounds which are
suitable for administration to a person. The term "compatible" as
used here means that the constituents of the composition are
capable of being mixed together with the complexed RNA according to
the invention and the auxiliary substance optionally contained in
the composition, as such and with one another in a manner such that
no interaction which would substantially reduce the
(pharmaceutical) effectiveness of the composition under usual
condition of use occurs, such as e.g. would reduce the
(pharmaceutical) activity of the encoded proteins or even suppress
or impair expression of the coded proteins or e.g. would inhibit
the immunogenic potential of the complexed RNA. Suitable carrier
must of course have a sufficiently high purity and a sufficiently
low toxicity to render them suitable for administration to a person
to be treated.
[0174] Carriers are chosen dependent on the way of administration,
be it in solid or liquid form. Accordingly, the choice of a
(pharmaceutically) suitable carrier as described above is
determined in particular by the mode in which the (pharmaceutical)
composition according to the invention is administered. The
(pharmaceutical) composition according to the invention can be
administered, for example, systemically. Administration routes
include e.g. intra- or transdermal, oral, parenteral, including
subcutaneous, intramuscular, i.a. or intravenous injections,
topical and/or intranasal routes. The suitable amount of the
(pharmaceutical) composition according to the invention which is to
be used can be determined by routine experiments using animal
models. Such models include, but without being limited thereto,
models of the rabbit, sheep, mouse, rat, dog and non-human primate
models.
[0175] If administered in liquid form, e.g. by injection, the
carrier may be selected from pyrogen-free water; isotonic saline
solution and buffered solutions, e.g. phosphate buffered solutions.
Preferred unit dose forms for injection include sterile solutions
of water, physiological saline solution or mixtures thereof, e.g.
Ringer-Lactat solution. The pH of such solutions should be adjusted
to about 7.0 to about 7.6, preferably about 7.4.
[0176] Preferably, the (pharmaceutical) composition contains the
inventive complexed RNA in water. Alternatively, the
(pharmaceutical) composition according to the invention may contain
an injection buffer as carrier for liquid preparation, which
preferably improves transfection and, if the RNA of the complexed
RNA of the present invention codes for a protein, also the
translation of the encoded protein, in cells, tissues or an
organism. The (pharmaceutical) composition according to the
invention can comprise, for example, an aqueous injection buffer or
water which contains, with respect to the total (pharmaceutical)
composition, if this is in liquid form, a sodium salt, preferably
at least 50 mM sodium salt, a calcium salt, preferably at least
0.01 mM calcium and/or magnesium salt, and optionally a potassium
salt, preferably at least 3 mM potassium salt. According to a
preferred embodiment, the sodium salts, calcium and/or magnesium
salts and optionally potassium salts contained in such an injection
buffer are in the form of halides, e.g. chlorides, iodides or
bromides, or in the form of their hydroxides, carbonates,
bicarbonates or sulfates. Examples which are to be mentioned here
are, for the sodium salt NaCl, NaI, NaBr, Na.sub.2CO.sub.3,
NaHCO.sub.3, and/or Na.sub.2SO.sub.4, for the potassium salt
optionally present KCl, KI, KBr, K.sub.2CO.sub.3, KHCO.sub.3,
and/or K.sub.2SO.sub.4, and for the calcium and/or magnesium salt
CaCl.sub.2, Cal.sub.2, CaBr.sub.2, CaCO.sub.3, CaSO.sub.4,
Ca(OH).sub.2, MgCl.sub.2, MgI.sub.2, MgBr.sub.2, MgCO.sub.3,
MgSO.sub.4, and/or Mg(OH).sub.2. The injection buffer can also
contain organic anions of the abovementioned cations. In a
particularly preferred embodiment, such an injection buffer
contains as salts sodium chloride (NaCl), calcium chloride
(CaCl.sub.2) and optionally potassium chloride (KCl), it also being
possible for other anions to be present in addition to the
chlorides.
[0177] These salts are typically present in the injection buffer
optionally used in the (pharmaceutical) composition according to
the invention, with respect to the total (pharmaceutical)
composition (if this is in liquid form), in a concentration of at
least 50 mM sodium chloride (NaCl), at least 3 mM potassium
chloride (KCl) and at least 0.01 mM calcium and/or magnesium
chloride (CaCl.sub.2). The injection buffer can be in the form of
both hypertonic and isotonic or hypotonic injection buffers. In
connection with the present invention, in this context the
injection buffer is hypertonic, isotonic or hypotonic in each case
with respect to the particular reference medium, i.e. the injection
buffer has either a higher, the same or a lower salt content
compared with the particular reference medium, such concentrations
of the abovementioned salts which do not lead to damage to the
cells caused by osmosis or other concentration effects preferably
being employed. Reference media here are, for example, liquids
which occur in "in vivo" methods, such as, for example, blood,
lymph fluid, cytosol fluids or other fluids which occur in the
body, or liquids or buffers conventionally employed in "in vitro"
methods. Such liquids and buffers are known to a person skilled in
the art.
[0178] The injection buffer optionally contained in the
(pharmaceutical) composition according to the invention can also
contain further components, for example sugars (mono-, di-, tri- or
polysaccharides), in particular glucose or mannitol. In a preferred
embodiment, however, no sugars are present in the injection buffer
used. It is also preferable for the injection buffer precisely to
contain no non-charged components, such as, for example, sugars.
The injection buffer typically contains exclusively metal cations,
in particular from the group consisting of the alkali or alkaline
earth metals, and anions, in particular the anions described above.
The pH of the injection buffer used, with respect to the total
(pharmaceutical) composition, if this is in liquid form, is
preferably between 1 and 8.5, preferably between 3 and 5, more
preferably between 5.5 and 7.5, in particular between 5.5 and 6.5.
If appropriate, the injection buffer can also contain a buffer
system which fixes the injection buffer at a buffered pH. This can
be, for example, a phosphate buffer system, HEPES or
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4. However, the injection buffer
used very particularly preferably contains none of the
abovementioned buffer systems or contains no buffer system at
all.
[0179] The injection buffer optionally contained in the
(pharmaceutical) composition according to the invention can
contain, in addition to or as an alternative to the monovalent and
divalent cations described, divalent cations, in particular from
the group consisting of alkaline earth metals, such as, for
example, magnesium (Mg.sup.2+), or also iron (Fe.sup.2+), and
monovalent cations, in particular from the groups consisting of
alkali metals, such as, for example, lithium (M. These monovalent
cations are preferably in the form of their salts, e.g. in the form
of halides, e.g. chlorides, iodides or bromides, or in the form of
their hydroxides, carbonates, bicarbonates or sulfates. Examples
which are to be mentioned here are, for the lithium salt LiCl, LiI,
LiBr; Li.sub.2CO.sub.3, LiHCO.sub.3, Li.sub.2SO.sub.4, for the
magnesium salt MgCl.sub.2, MgI.sub.2, MgBr.sub.2, MgCO.sub.3,
MgSO.sub.4, and Mg(OH).sub.2, and for the iron salt FeCl.sub.2,
FeBr.sub.2, FeI.sub.2, FeF.sub.2, Fe.sub.2O.sub.3, FeCO.sub.3,
FeSO.sub.4, Fe(OH).sub.2. All the combinations of di- and/or
monovalent cations, as described above, are likewise included. Such
injection buffers which contain only divalent, only monovalent or
di- and monovalent cations can thus be used in the (pharmaceutical)
composition according to the invention. Such injection buffers
which contain only one type of di- or monovalent cations,
particularly preferably e.g. only Ca.sup.2+ cations, or a salt
thereof, e.g. CaCl.sub.2, can likewise be used. The molarities
given above for Ca.sup.2+ (as a divalent cation) and Na.sup.1+ (as
a monovalent cation) (that is to say typically concentrations of at
least 50 mM Na.sup.+, at least 0.01 mM Ca.sup.2+ and optionally at
least 3 mM K.sup.+) in the injection buffer can also be taken into
consideration if another di- or monovalent cation, in particular
other cations from the group consisting of the alkaline earth
metals and alkali metals, are employed instead of some or all the
Ca.sup.2+ or, respectively, Na.sup.1+ in the injection buffer used
according to the invention for the preparation of the injection
solution. All the Ca.sup.2+ or Na.sup.1+, as mentioned above, can
indeed be replaced by in each case other di- or, respectively,
monovalent cations in the injection buffer used, for example also
by a combination of other divalent cations (instead of Ca.sup.2+)
and/or a combination of other monovalent cations (instead of
Na.sup.1+) (in particular a combination of other divalent cations
from the group consisting of the alkaline earth metals or,
respectively, of other monovalent cations from the group consisting
of the alkali metals), but it is preferable to replace at most some
of the Ca.sup.2+ or Na.sup.1+, i.e. for at least 20%, preferably at
least 40%, even more preferably at least 60% and still more
preferably at least 80% of the particular total molarities of the
mono- and divalent cations in the injection to be occupied by
Ca.sup.2+ and, respectively, Na.sup.1+. However, it is very
particularly preferable if the injection buffer optionally
contained in the pharmaceutical composition according to the
invention contains exclusively Ca.sup.2+ as a divalent cation and
Na.sup.1+ as a monovalent cation, that is to say, with respect to
the total pharmaceutical composition, Ca.sup.2+ represents 100% of
the total molarity of divalent cations, just as Na.sup.1+
represents 100% of the total molarity of monovalent cations. The
aqueous solution of the injection buffer can contain, with respect
to the total pharmaceutical composition, up to 30 mol % of the
salts contained in the solution, preferably up to 25 mol %,
preferably up to 20 mol %, furthermore preferably up to 15 mol %,
more preferably up to 10 mol %, even more preferably up to 5 mol %,
likewise more preferably up to 2 mol % of insoluble or sparingly
soluble salts. Salts which are sparingly soluble in the context of
the present invention are those of which the solubility product is
<10.sup.-4. Salts which are readily soluble are those of which
the solubility product is >10.sup.-4. Preferably, the injection
buffer optionally contained in the pharmaceutical composition
according to the invention is from 50 mM to 800 mM, preferably from
60 mM to 500 mM, more preferably from 70 mM to 250 mM, particularly
preferably 60 mM to 110 mM in sodium chloride (NaCl), from 0.01 mM
to 100 mM, preferably from 0.5 mM to 80 mM, more preferably from
1.5 mM to 40 mM in calcium chloride (CaCl.sub.2) and optionally
from 3 mM to 500 mM, preferably from 4 mM to 300 mM, more
preferably from 5 mM to 200 mM in potassium chloride (KCl). Organic
anions can also occur as further anions in addition to the
abovementioned inorganic anions, for example halides, sulfates or
carbonates. Among these there may be mentioned succinate,
lactobionate, lactate, malate, maleate etc., which can also be
present in combination.
[0180] An injection buffer optionally contained in the
(pharmaceutical) composition according to the invention preferably
contains lactate. If it contains an organic anion, such an
injection buffer particularly preferably contains exclusively
lactate as the organic anion. Lactate in the context of the
invention can be any desired lactate, for example L-lactate and
D-lactate. Lactate salts which occur in connection with the present
invention are typically sodium lactate and/or calcium lactate,
especially if the injection buffer contains only Na.sup.+ as a
monovalent cation and Ca.sup.2+ as a divalent cation. An injection
buffer optionally used in the (pharmaceutical) composition
according to the invention and as described above preferably
contains, with respect to the total pharmaceutical composition,
from 15 mM to 500 mM, more preferably from 15 mM to 200 mM, and
even more most preferably from 15 mM to 100 mM lactate.
[0181] If formulated in non-liquid form (e.g. in solid or
semi-solid form), the pharmaceutical composition of the invention
may be contain compounds which can serve as suitable carriers or
constituents thereof, e.g. sugars, such as, for example, lactose,
glucose and sucrose; starches, such as, for example, corn starch or
potato starch; cellulose and its derivatives, such as, for example,
sodium carboxymethylcellulose, ethylcellulose, cellulose acetate;
pulverized tragacanth; malt; gelatine; tallow; solid lubricants,
such as, for example, stearic acid, magnesium stearate; calcium
sulfate; plant oils, such as, for example, groundnut oil,
cottonseed oil, sesame oil, olive oil, corn oil and oil from
Theobroma; polyols, such as, for example, polypropylene glycol,
glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
However, the above compounds may also be used for the provision of
liquid compositions.
[0182] Other components which may be included in a pharmaceutical
composition of the invention are e.g. emulsifiers, such as, for
example, Tween.RTM.; wetting agents, such as, for example, sodium
lauryl sulfate; colouring agents; flavouring agents; medicament
carriers; tablet-forming agents; stabilizers; antioxidants;
preservatives.
[0183] Other suitable carriers for injection include hydrogels,
devices for controlled or delayed release, polylactic acid and
collagen matrices. Suitable carriers which can be used here include
those which are suitable for use in lotions, creams, gels and the
like. If the compound is to be administered perorally, tablets,
capsules and the like are the preferred unit dose form. The
suitable carriers for the preparation of unit dose forms which can
be used for oral administration are well-known in the prior art.
Their choice will depend on secondary considerations, such as
flavour, cost and storage stability, which are not critical for the
purposes of the present invention and can be implemented without
difficulties by a person skilled in the art.
[0184] The present invention also provides an (in vitro or in vivo)
transfection method for transfecting cells or a tissue with the
complexed RNA of the present invention as described above. The
inventive (in vitro or in vivo) transfection method preferably
comprises the following steps: [0185] a) Optionally preparing
and/or providing a complexed RNA according to the present
invention, comprising at least one RNA complexed with one or more
oligopeptides having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x; [0186] b)
Transfecting a cell, a (living) tissue or an organism (in vitro or
in vivo) using the complexed RNA prepared and/or provided according
to step a).
[0187] Preparing and/or providing a complexed RNA as defined above
according to step a) of the inventive in vitro or in vivo
transfection method for transfecting cells or a tissue with the
complexed RNA of the present invention, may be carried out by any
method known in the art. A complexed RNA as used herein comprises
at least one RNA complexed with one or more oligopeptides having
the empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x. Preparing and/or providing a complexed
RNA as defined above according to step a) may thus comprise the
preparation and/or provision of the least one RNA and the one or
more oligopeptides having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x.
[0188] Methods for preparation of short peptide sequences such as
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x,
are widely known in the art and may employ e.g. solid phase
synthesis such as Fmoc solid phase synthesis or other suitable
methods (see e.g. R. Martin, Ed., Protein Synthesis: Methods and
Protocols. Methods in Molecular Biology, Vol. 77Humana Press
(1998)).
[0189] Preparing and/or providing the at least one RNA (molecule)
as component of the inventive complex as defined above may comprise
according to step a) a first sub-step a1), namely provision and/or
preparation of a nucleic acid template, which typically comprises a
sequence corresponding to the desired RNA. The sequence of the
nucleic acid template may be any nucleic acid, e.g. a single- or
double-stranded DNA, cDNA, genomic DNA or fragments thereof, etc.,
which may code for a therapeutically active protein, an antibody or
an antigene, or any other protein or peptide as described above.
Typically, DNA sequences, e.g. DNA plasmids, preferably in
linearized form, may be employed for this purpose. Preferably, the
sequence of the nucleic acid template may be an (expression)
vector, more preferably an (expression)vector having an RNA
polymerase binding site. Any (expression) vectors known in the
prior art, e.g. commercially available (expression) vectors (see
above), can be used for this. Preferred (expression) vectors are,
for example, those which have an SP6 or a T7 or T3 binding site
upstream and/or downstream of the cloning site. The vector may
comprise a nucleic acid sequence encoding a therapeutically active
protein, an antibody or an antigen, or any other protein or peptide
as described above, which is typically cloned into the (expression)
vector, e.g. via a multiple cloning site of the vector used.
[0190] Prior to transcription the (expression) vector is typically
cleaved with restriction enzymes at the site at which the future 3'
end of the RNA is to be found, using a suitable restriction enzyme,
and the fragment is purified. This prevents the transcribed RNA
from containing vector sequences, and an RNA transcript of defined
length may be obtained. In this context, preferably no restriction
enzymes which generate overhanging ends (such as, e.g., AatII,
ApaI, BanII, BgA, Bsp1286, BstXI, CfoI, HaeII, HgiAI, HhaI, KpnI,
PsA, PvuI, SacI, SacII, SfiI, SphI, etc.) are used. Should such
restriction enzymes nevertheless be used, the overhanging 3' end
preferably may be filled up, e.g. with Klenow or T4 DNA
polymerase.
[0191] As an alternative to the above, the nucleic acid template
used for preparing and/or providing the at least one RNA (molecule)
of the complexed RNA of the invention, may be prepared by employing
a polymerase chain reaction (PCR). The nucleic acid template
preferably and one of the primers used therefore, typically
contains the sequence of an RNA polymerase binding site.
Furthermore, the 5' end of the primer used preferably contains an
extension of about 10-50 further nucleotides, more preferably of
from 15 to 30 further nucleotides and most preferably of about 20
nucleotides.
[0192] Prior to in vitro transcription, the nucleic acid, e.g. the
DNA or cDNA template, used as transcription template, is typically
purified and free from RNase in order to ensure a high yield. In
this context, purification of such template can be carried out with
the aid of any method known in the prior art, for example using a
caesium chloride gradient, ion exchange methods or by purification
via agarose gel electrophoresis.
[0193] Subsequent to preparing and/or providing the nucleic acid
template, an in vitro transcription reaction according to a second
sub-step a2) may be carried out for preparing the desired the at
least one RNA (molecule) of the complexed RNA of the invention
using the nucleic acid template prepared according to first
sub-step a1) as defined above.
[0194] The in vitro transcription reaction according to a second
sub-step a2) is typically carried out in an in vitro transcription
reaction. A suitable in vitro transcription medium initially
comprises the nucleic acid template as described above, for example
about 0.1-10 .mu.g, preferably about 1-5 .mu.g, more preferably 2.5
.mu.g and most preferably about 1 .mu.g of such a nucleic acid. A
suitable in vitro transcription medium furthermore optionally
comprises a reducing agent, e.g. DTT, more preferably about 1-20
.mu.l 50 mM DTT, even more preferably about 5 .mu.l 50 mM DTT. The
in vitro transcription medium typically comprises nucleotides, e.g.
a nucleotide mix, in the case of the present invention comprising a
mixture of nucleotides of A, G, C or U, typically about 0.1-10 mM
per nucleotide, preferably 0.1 to 1 mM per nucleotide, preferably
about 4 mM in total. A suitable in vitro transcription medium
likewise comprises an RNA polymerase, e.g. T7 RNA polymerase (for
example T7-Opti mRNA Kit, CureVac, Tubingen, Germany), T3 RNA
polymerase or SP6, typically about 10 to 500 U, preferably about 25
to 250 U, more preferably about 50 to 150 U, and most preferably
about 100 U of RNA polymerase. The in vitro transcription medium is
furthermore preferably kept free from RNase in order to avoid
degradation of the transcribed the at least one RNA (molecule) of
the complexed RNA of the invention. A suitable in vitro
transcription medium therefore optionally additionally comprises an
RNase inhibitor.
[0195] The nucleic acid template may be then incubated in the in
vitro transcription medium and is transcribed to the at least one
RNA (molecule) of the complexed RNA of the invention, which may
encode for a therapeutically active protein, an antibody or an
antigene, or any other protein or peptide as described above. The
incubation times are typically about 30 to 240 minutes, preferably
about 40 to 120 minutes and most preferably about 90 minutes. The
incubation temperatures are typically about 30-45.degree. C.,
preferably 37-42.degree. C. The incubation temperature depends on
the RNA polymerase used, e.g. for T7 RNA polymerase it is about
37.degree. C. The at least one RNA (molecule) of the complexed RNA
of the invention obtained by the transcription is preferably an
mRNA. The yields obtained in the in vitro transcription are, for
the stated starting amounts employed above, typically in the region
of about 30 .mu.g of RNA per .mu.g of template DNA used. In the
context of the present invention, the yields obtained in the in
vitro transcription can be increased by linear up scaling. For
this, the stated starting amounts employed above are preferably
increased according to the yields required, e.g. by a
multiplication factor of 5, 10, 50, 100, 500, 1,000, 5,000, 10,000,
50,000, 100,000 etc.
[0196] After incubation, a purification of the transcribed at least
one RNA (molecule) of the complexed RNA of the invention can
optionally take place. Any suitable method known in the prior art,
e.g. chromatographic purification methods, e.g. affinity
chromatography, gel filtration etc., can be used for this. By the
purification, non-incorporated, i.e. excess nucleotides and
template DNA can be removed from the in vitro transcription medium
and a clean RNA can be obtained. For example, after the
transcription the reaction mixture with the transcribed RNA can
typically be digested with DNase in order to remove the DNA
template still contained in the reaction mixture. The transcribed
at least one RNA (molecule) of the complexed RNA of the invention
can be subsequently or alternatively precipitated with LiCl.
Purification of the transcribed RNA can then take place via IP
RP-HPLC. This renders in particular effective separation of longer
and shorter fragments from one another possible.
[0197] Preferably, in this context purification of the RNA may take
place via a method for purification of RNA on a preparative scale,
which is distinguished in that the RNA is purified by means of HPLC
using a porous reverse phase as the stationary phase (PURE
Messenger). For example, for the purification a reverse phase can
be employed as the stationary phase for the HPLC purification. For
the chromatography with reverse phases, a non-polar compound
typically serves as stationary phases, and a polar solvent, such as
mixtures of water, which is usually employed in the form of
buffers, with acetonitrile and/or methanol, serves as the mobile
phase for the elution. Preferably, the porous reverse phase has a
particle size of 8.0.+-.2 .mu.m, preferably .+-.1 .mu.m, more
preferably +/-0.5 .mu.m. The reverse phase material can be in the
form of beads. The purification can be carried out in a
particularly favourable manner with a porous reverse phase having
this particle size, optionally in the form of beads, particularly
good separation results being obtained. The reverse phase employed
is preferably porous since with stationary reverse phases which are
not porous, such as are described e.g. by Azarani A. and Hecker K.
H., pressures which are too high are built up, so that preparative
purification of the RNA is possible, if at all, only with great
difficulty. The reverse phase preferably has a pore size of from
200 to 5,000, in particular a pore size of from 300 to 4,000.
Particularly preferred pore sizes for the reverse phases are
200-400, 800-1,200 and 3,500-4,500. With a reverse phase having
these pore sizes, particularly good results are achieved in respect
of the purification of the transcribed RNA. The material for the
reverse phase is preferably a polystyrene-divinylbenzene, and
non-alkylated polystyrene-divinylbenzenes can be employed in
particular. Stationary phases with polystyrene-divinylbenzene are
known per se. For the purification, the polystyrene-divinylbenzenes
which are known per se and already employed for HPLC methods and
are commercially obtainable can be used. A non-alkylated porous
polystyrene-divinylbenzene which in particular has a particle size
of 8.0.+-.0.5 .mu.m and a pore size of 250-300, 900-1,100 or
3,500-4,500 is very particularly preferably used for the
purification. The advantages described above can be achieved in a
particularly favourable manner with this material for the reverse
phases.
[0198] The HPLC purification can be carried out by the ion pair
method, an ion having a positive charge being added to the mobile
phase as a counter-ion to the negatively charged RNA. An ion pair
having a lipophilic character, which is slowed down by the
non-polar stationary phase of the reverse phase system, is formed
in this manner. In practice, the precise conditions for the ion
pair method must be worked out empirically for each specific
separation problem. The size of the counter-ion, its concentration
and the pH of the solution contribute greatly towards the result of
the separation. In a favourable manner, alkylammonium salts, such
as triethylammonium acetate and/or tetraalkylammonium compounds,
such as tetrabutylammonium, are added to the mobile phase.
Preferably, 0.1 M triethylammonium acetate is added and the pH is
adjusted to about 7. The choice of mobile phase depends on the
nature of the desired separation. This means that the mobile phase
found for a specific separation, such as can be known, for example,
from the prior art, cannot be transferred readily to another
separation problem with adequate prospect of success. The ideal
elution conditions, in particular the mobile phase used, must be
determined for each separation problem by empirical experiments. A
mixture of an aqueous solvent and an organic solvent can be
employed as the mobile phase for elution of the RNA by the HPLC
method. In this context, it is favourable if a buffer which has, in
particular, a pH of about 7, for example 6.5-7.5, e.g. 7.0, is used
as aqueous solvent; preferably, the buffer triethylammonium acetate
is used, particularly preferably a 0.1 M triethylammonium acetate
buffer which, as described above, also acts as a counter-ion to the
RNA in the ion pair method. The organic solvent employed in the
mobile phase can be acetonitrile, methanol or a mixture of these
two, very particularly preferably acetonitrile. The purification of
the RNA using an HPLC method as described is carried out in a
particularly favourable manner with these organic solvents. The
mobile phase is particularly preferably a mixture of 0.1 M
triethylammonium acetate, pH 7, and acetonitrile. It has emerged to
be likewise particularly favourable if the mobile phase contains
5.0 vol. % to 20.0 vol. % of organic solvent, based on the mobile
phase, and the remainder to make up 100 vol. % is the aqueous
solvent. It is very particularly favourable for the method
according to the invention if the mobile phase contains 9.5 vol. %
to 14.5 vol. % of organic solvent, based on the mobile phase, and
the remainder to make up 100 vol. % is the aqueous solvent. Elution
of the RNA can subsequently be carried out isocratically or by
means of a gradient separation. In the case of an isocratic
separation, elution of the RNA is carried out with a single eluting
agent or a mixture of several eluting agents which remains
constant, it being possible for the solvents described above in
detail to be employed as the eluting agent.
[0199] Alternatively, the at least one RNA (molecule) according to
step a) of the inventive method of transfection may well be
prepared by chemical synthesis. Hereby, various methods known in
the art may be used. The phosphoroamidite method is used most
widely as a method of chemically synthesizing oligonucleotides,
e.g. RNA fragments (Nucleic Acid Research, 17:7059-7071, 1989). In
general, this phosphoroamidite method makes use of a condensation
reaction between a nucleoside phosphoroamidite and a nucleoside as
a key reaction using tetrazole as an accelerator. Because this
reaction usually occurs competitively on both the hydroxyl group in
a sugar moiety and the amino group in a nucleoside base moiety, the
selective reaction on only the hydroxyl group in a sugar moiety is
required to synthesize a desired nucleotide. Accordingly, the side
reaction on the amino group is usually prevented by protecting the
amino group. The protective group is removed when synthesis is
finished. More specific information about how to synthesize RNA
molecules may be retrieved from Arnold et al., "Chloridite and
Amidite Automated Synthesis of Oligodeoxyribonucleotides Using
Amidine Protected Nucleosides," reported in "7th Symposium Chem.
Nucleic Acid Components," Nucleic Acids Symposium Series, 18,
181-184 (Aug. 30, 1987); Chemical Abstracts, 108(19), p. 692,
Abstr. No. 167875z (May 9, 1988); Hayakawa et al., "Benzimidazolium
Triflate as an Efficient Promoter for Nucleotide Synthesis via the
Phosphoramidite Method," J. Organic Chemistry, 61(23), 7996-7997
(Nov. 15, 1996); Pirrung et al., "Proofing of Photolithographic DNA
Synthesis with 3',5'-Dimethoxybenzoinyloxycarbonyl-Protected
Deoxynucleoside Phosphoramidites," J. Organic Chemistry, 63(2),
241-246 (Jan. 23, 1998); Effenberger et al.,
Trifluoromethanesulfonic Imidazolide--A Convenient Reagent for
Introducing the Triflate Group, Tetrahedron Letters, 1980 (45),
3947-3948 (September 1980), all of them incorporated herein by
reference.
[0200] Preparation of the Complexed RNA According to Step a) of the
Present Invention Typically occurs according to sub-step a3) by
adding a specific amount of the at least one RNA (molecule) to a
specific amount to the one or more oligopeptides having the
empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x. Thereby, molar or mass ratios as
indicated above of the at least one RNA (molecule) and the one or
more oligopeptides having the herein defined empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x,
are typically envisaged. Complex formation typically occurs upon
mixing both components. Thereby, the peptidic component is
typically added to the RNA component, in some cases, however, vice
versa.
[0201] Such a preparation step according to method step a),
however, is optional and may not take place if the complexed RNA
according to the present invention is already available.
Accordingly, sub-steps a1), a2) and a3) as defined above are also
optional and need not to be carried out, if the RNA used for the
complexed RNA is already available. Similarly, the one or more
oligopeptides having the empirical formula (Arg).sub.l;
(Lys).sub.m; (His); (Orn).sub.o; (Xaa).sub.x may be used directly
and need not to be prepared, if already available, e.g. from a
supplier.
[0202] According to step b) of the inventive method for
transfecting cells or tissues in vitro or in vivo a cell or a
tissue may be transfected using the complexed RNA provided and/or
prepared according to step a). Transfection of the cells or tissues
in vitro or in vivo is in general carried out by adding the
complexed RNA provided and/or prepared according to step a) to the
cells or tissue. Preferably, the complexed RNA then enters into the
cells by using cellular mechanisms, e.g. endocytosis. Addition of
the complexed RNA as such to the cells or tissues may occur
according to the invention without addition of any further
components due to the transfectional potential of the complexed RNA
(molecule) of the invention. Alternatively, addition of the
complexed RNA provided and/or prepared according to step a) to the
cells or tissue may occur in the form of a composition, e.g. as
component of an aqueous solution, preferably a pharmaceutical
composition as defined above, which may optionally contain
additional components for further enhancement of the transfection
activity.
[0203] Cells (or host cells) in this context for transfection of
the complexed RNA (provided and/or prepared according to step a))
in vitro includes any cell, and preferably, with out being
restricted thereto, cells, which shall be transfected by any RNA
molecule (as defined above) by using the inventive complexed RNA.
In particular, RNA transfection may allow for expression of a
protein encoded by the RNA of the complexed RNA according to the
invention in the cell or may allow RNA (e.g. siRNA, anti-sense RNA)
of the inventive complex to attenuate or suppress the expression of
a cellular gene. Cells in this context preferably include cultured
eukaryotic cells (e.g. yeast cells, plant cells, animal cells and
human cells) or prokaryotic cells (e.g. bacteria cells etc.) or
induce an immune response. Cells of multicellular organisms are
preferably chosen if posttranslational modifications, e.g.
glycosylation of the encoded protein, are necessary (N- and/or
O-coupled). In contrast to prokaryotic cells, such (higher)
eukaryotic cells render posttranslational modifications of the
protein synthesized possible. The person skilled in the art knows a
large number of such higher eukaryotic cells or cell lines, e.g.
293T (embryonal kidney cell line), HeLa (human cervix carcinoma
cells), CHO (cells from the ovaries of the Chinese hamster) and
further cell lines, including such cells and cell lines developed
for laboratory purposes, such as, for example, hTERT-MSC, HEK293,
Sf9 or COS cells. Suitable eukaryotic cells furthermore include
cells or cell lines which are impaired by diseases or infections,
e.g. cancer cells, in particular cancer cells of any of the types
of cancer mentioned here in the description, cells impaired by HIV,
and/or cells of the immune system or of the central nervous system
(CNS). Suitable cells can likewise be derived from eukaryotic
microorganisms, such as yeast, e.g. Saccharomyces cerevisiae
(Stinchcomb et aZ, Nature, 282:39, (1997)), Schizosaccharomyces
pombe, Candida, Pichia, and filamentous fungi of the genera
Aspergillus, Penicillium, etc. Suitable cells likewise include
prokaryotic cells, such as e.g. bacteria cells, e.g. from
Escherichia coli or from bacteria of the general Bacillus,
Lactococcus, Lactobacillus, Pseudomonas, Streptomyces,
Streptococcus, Staphylococcus, preferably E. coli, etc. Human cells
or animal cells, e.g. of animals as mentioned herein, are
particularly preferred as eukaryotic cells. Furthermore, antigen
presenting cells (APCs) may be used for ex vivo transfection of the
complexed RNA according to the present invention. Particularly
preferred are dendritic cells, which may be used for ex vivo
transfection of the complexed RNA according to the present
invention.
[0204] According to a particularly preferred embodiment, blood
cells and/or haemopoietic cells, or partial populations thereof,
i.e. any type of cells, which may be isolated from (whole) blood
and/or which may be derived from cultivated cell lines derived from
those cells, may be transfected with a complexed RNA as defined
herein using the above method of transfection, e.g. red blood cells
(erythrocytes), granulocytes, mononuclear cells (peripheral blood
mononuclear cells, PBMCs) and/or blood platelets (thrombocytes),
APSs, DCs, etc. Preferably, blood cells are used, especially
partial populations thereof, which are characterized in particular
in that they contain a small proportion of well-differentiated
professional APCs, such as DCs. The transfected cells may contain
preferably less than 5%, particularly preferably no more than 2%,
of DCs when used for transfection. In the context of the present
invention "blood cells" are preferably understood as a mixture or
an enriched to substantially pure population of red blood cells,
granulocytes, mononuclear cells (PBMCs) and/or blood platelets from
whole blood, blood serum or another source, e.g. from the spleen or
lymph nodes, only a small proportion of professional APCs being
present. The blood cells as used according to the present invention
are preferably fresh blood cells, i.e. the period between
collection of the blood cells (especially blood withdrawal) and
transfection being only short, e.g. less than 12 h, preferably less
than 6 h, particularly preferably less than 2 h and very
particularly preferably less than 1 h. Furthermore, the blood cells
to be transfected using the above method for transfecting the
complexed RNA according to the present invention preferably
originate from the actual patient who will be treated with the
pharmaceutical composition of the present invention. The use of
blood cells, haematopoietic cells or partial populations thereof as
defined above is based on the surprising discovery that for
vaccination of a patient to be treated against certain antigens
encoded by an mRNA as defined herein, it is not necessary to
differentiate blood cells, e.g. PBMCs, obtained e.g. from the blood
of an individual, especially the actual patient to be treated, by
means of laborious, lengthy and expensive cell culture techniques,
into a population of cells with a high proportion of professional
antigen presenting cells (APCs), especially dendritic cells (DCs),
but that it is sufficient, for a successful immune stimulation, to
transfect blood cells directly with the mRNA coding for one or more
antigens in order to obtain a pharmaceutical composition which
effects a suitable immune stimulation e.g. in the actual patient
from whom the blood cells, especially the abovementioned partial
populations thereof, have been obtained, said immune stimulation
preferably being directed against one or more antigens from a
tumour or one or more antigens from a pathogenic germ or agent.
Transfection of a complexed RNA as defined herein into blood cells
or cells derived therefrom (either isolated therefrom or from
respective cultivated cell lines) is not limited to antigens and,
of course, relates to any RNA as defined herein used for a
complexed RNA, e.g. any further immunostimulating RNA as defined
herein, any coding RNA, etc.
[0205] While there is the need to transfect cultivated cells in
vitro (e.g. human or animal cells) or to transfect explanted cells
(e.g. human or animal cells) in vitro (before retransplantation
into the host organism), direct administration of the complexed RNA
of the invention to patients for in vivo transfection is envisaged
as well. Accordingly, transfection of the complexed RNA (provided
and/or prepared according to step a)) may also occur in vivo
according to step b), i.e. may be administered to living tissues
and/or organisms. Therefore, the complexed RNA provided according
to step a) of the inventive transfection method may be administered
to a living tissue or an organism either as such or e.g. as
component of a (liquid) composition, in particular an aqueous
composition, e.g. a pharmaceutical composition as defined above. In
this context, an organism (or a being) typically means mammals,
selected from, without being restricted thereto, the group
comprising humans, and animals, including e.g. pig, goat, cattle,
swine, dog, cat, donkey, monkey, ape or rodents, including mouse,
hamster and rabbit. Furthermore, living tissues as mentioned above,
are preferably derived from these organisms. Administration of the
complexed RNA to those living tissues and/or organisms may occur
via any suitable administration route, e.g. systemically, and
include e.g. intra- or transdermal, oral, parenteral, including
subcutaneous, intramuscular or intravenous injections, topical
and/or intranasal routes as defined above.
[0206] Moreover, the method for transfection, which may be used in
vitro or ex vivo, may also be well suited for use in vivo, e.g. as
method of treatment of various diseases as mentioned herein. In a
preferred form of a method of treatment according to the invention
a further step may be included, which may contain administration of
another pharmaceutically effective substance, e.g. an antibody, an
antigen (in particular a pathogenic or a tumor antigen as disclosed
herein) or the administration of at least one cytokine. Both may be
administered separately from the complexed RNA as DNA or RNA coding
for e.g. the cytokine or the antigen or the cytokine or antigen may
be administered as such. The method of treatment may also comprise
the administration of an additional adjuvant (as disclosed herein),
which may further activate the immune system.
[0207] According to a further embodiment of the present invention,
the complexed RNA as defined above, comprising at least one RNA
complexed with one or more oligopeptides, wherein the oligopeptide
shows a length of 8 to 15 amino acids and has the empirical formula
(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x,
may be used for treatment and/or prophylaxis of specific diseases
as mentioned herein. Treatment and/or prophylaxis of specific
diseases is typically dependent on selection of a suitable protein
encoded by the RNA of the complexed RNA of the present invention.
Treatment in this context may occur either by administering the
complexed RNA according to the present invention (encoding this
protein) as such or by administering the (pharmaceutical)
composition according to the present invention as defined
above.
[0208] Without being limited thereto, diseases or states include in
this context, for example, cancer or tumour diseases chosen from
melanomas, malignant melanomas, colon carcinomas, lymphomas,
sarcomas, blastomas, kidney carcinomas, gastrointestinal tumours,
gliomas, prostate tumours, bladder cancer, rectal tumours, stomach
cancer, oesophageal cancer, pancreatic cancer, liver cancer,
mammary carcinomas (=breast cancer), uterine cancer, cervical
cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia
(ALL), chronic myeloid leukaemia (CML), chronic lymphocytic
leukaemia (CLL), hepatomas, diverse virus-induced tumours, such as
e.g. papilloma virus-induced carcinomas (e.g. cervix
carcinoma=cervical cancer), adenocarcinomas, herpes virus-induced
tumours (e.g. Burkitt's lymphoma, EBV-induced B cell lymphoma),
hepatitis B-induced tumours (hepatocell carcinomas), HTLV-1- and
HTLV-2-induced lymphomas, acusticus neurinoma, lung carcinomas
(=lung cancer=bronchial carcinoma), small cell lung carcinomas,
throat cancer, anal carcinoma, glioblastoma, rectum carcinoma,
astrocytoma, brain tumours, retinoblastoma, basalioma, brain
metastases, medulloblastomas, vaginal cancer, testicular cancer,
thyroid carcinoma, Hodgkin's syndrome, meningeomas, Schneeberger's
disease, pituitary tumour, mycosis fungoides, carcinoids,
neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer, kidney
cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's
lymphomas, urethral cancer, CUP syndrome, head/neck tumours,
oligodendroglioma, vulval cancer, intestinal cancer, colon
carcinoma, oesophageal carcinoma (=Oesophageal cancer), wart
conditions, small intestine tumours, craniopharyngeomas, ovarian
carcinoma, soft tissue tumours, ovarian cancer (=Ovarian
carcinoma), pancreatic carcinoma (=pancreatic cancer), endometrium
carcinoma, liver metastases, penis cancer, tongue cancer,
gallbladder cancer, leukaemia, plasmocytoma, lid tumour, prostate
cancer (=prostate tumours) etc.
[0209] Diseases or states may also include in this context
infectious diseases chosen from, e.g., viral infectious diseases
chosen from, without being limited thereto, SARS, yellow fever,
Lyme, anthrax, AIDS, condyloma acuminata, molluscum contagiosum,
dengue fever, three-day fever, Ebola virus, colds, early summer
meningoencephalitis (ESME), influenza, shingles, hepatitis, herpes
simplex type I, herpes simplex type II, herpes zoster, influenza,
Japanese encephalitis, Lassa fever, Marburg virus, measles, foot
and mouth disease, mononucleosis, mumps, Norwalk virus infection,
Pfeiffer's glandular fever, smallpox, polio (poliomyelitis),
pseuodcroup, infectious erythema, rabies, warts, West Nile fever,
chicken-pox, cytomegalovirus (CMV), bacterial infectious diseases,
such as abortion (prostate inflammation), anthrax, appendicitis
(inflammation of the caecum), borreliosis, botulism, Campylobacter,
Chlamydia trachomatis (inflammation of the urethra, conjunctiva),
cholera, diphtheria, donavonosis, epiglottitis, louse-borne typhus,
typhoid fever, gas gangrene, gonorrhoea, hare plague, Helicobacter
pylori, whooping-cough, climatic bubo, osteomyelitis, legionnaires'
disease, leprosy, listeriosis, pneumonia, meningitis, bacterial
meningitis, anthrax, inflammation of the middle ear, Mycoplasma
hominis, neonatal sepsis (chorioamnionitis), noma, paratyphoid
fever, plague, Reiter's syndrome, Rocky Mountain spotted fever,
Salmonella paratyphoid fever, Salmonella typhoid fever, scarlet
fever, syphilis, tetanus, gonorrhoea, tsutsugamushi fever,
tuberculosis, typhus, vaginitis (colpitis), soft chancre and
infectious diseases caused by parasites, protozoa or fungi, such as
amoebic dysentery, bilharziosis, Chagas' disease, Echinococcus,
fish tapeworm, ichthyotoxism (ciguatera), fox tapeworm, mycosis
pedis, dog tapeworm, candiosis, ptyriasis, the itch (scabies),
leishmaniasis, cutaneous leishmaniasis, lamblian dysentery
(giadiasis), lice, malaria, microscopy, onchocercosis (river
blindness), fungal diseases, beef tapeworm, schistosomiasis,
sleeping sickness, pork tapeworm, toxoplasmosis, trichomoniasis,
trypanosomiasis (sleeping sickness), visceral leishmaniasis, nappy
dermatitis or dwarf tapeworm.
[0210] Diseases in the context of the present invention likewise
include, without being limited thereto, (infectious) virus diseases
caused by viruses chosen from, without being limited thereto, HIV,
orthopox variola virus, orthopox alastrim virus, parapox ovis
virus, molluscum contagiosum virus, herpes simplex virus 1, herpes
simplex virus 2, herpes B virus, varicella zoster virus,
pseudorabies virus, human cytomegaly virus, human herpes virus 6,
human herpes virus 7, Epstein-Barr virus, human herpes virus 8,
hepatitis B virus, chikungunya virus, O'nyong'nyong virus,
rubivirus, hepatitis C virus, GB virus C, West Nile virus, dengue
virus, yellow fever virus, louping ill virus, St. Louis
encephalitis virus, Japan B encephalitis virus, Powassan virus,
FSME virus, SARS-associated corona virus, human corona virus 229E,
human corona virus 0c43, Torovirus, human T cell lymphotropic virus
type I, human T cell lymphotropic virus type II, human
immunodeficiency virus type 1, human immunodeficiency virus type 2,
Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus,
Junin virus, Machupo virus, Borna disease virus, Bunyamwera virus,
California encephalitis virus, Rift Valley fever virus, sand fly
fever virus, Toscana virus, Crimean-Congo haemorrhagic fever virus,
Hazara virus, Khasan virus, Hantaan virus, Seoul virus, Prospect
Hill virus, Puumala virus, Dobrava Belgrade virus, Tula virus, sin
nombre virus, Lake Victoria Marburg virus, Zaire Ebola virus, Sudan
Ebola virus, Ivory Coast Ebola virus, influenza virus A, influenza
virus B, influenza viruses C, parainfluenza virus, measles virus,
mumps virus, respiratory syncytial virus, human metapneumovirus,
vesicular stomatitis Indiana virus, rabies virus, Mokola virus,
Duvenhage virus, European bat lyssavirus 1+2, Australian bat
lyssavirus, adenoviruses A-F, human papilloma viruses, condyloma
virus 6, condyloma virus 11, polyoma viruses, adeno-associated
virus 2, rotaviruses, or orbiviruses etc. These diseases may e.g.
be treated by a vaccine according to the invention.
[0211] Additionally, diseases or states may include cardiovascular
diseases chosen from, without being limited thereto, coronary heart
disease, arteriosclerosis, apoplexy and hypertension, and neuronal
diseases chosen from Alzheimer's disease, amyotrophic lateral
sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's
disease etc.
[0212] Diseases or states may also include in this context an
allergic disorder or disease. Allergy is a condition that typically
involves an abnormal, acquired immunological hypersensitivity to
certain foreign antigens or allergens. Allergies normally result in
a local or systemic inflammatory response to these antigens or
allergens and leading to an immunity in the body against these
allergens. Allergens in this context include e.g. grasses, pollens,
molds, drugs, or numerous environmental triggers, etc. Without
being bound to any theory, several different disease mechanisms are
supposed to be involved in the development of allergies. According
to a classification scheme by P. Gell and R. Coombs the word
"allergy" was restricted to type I hypersensitivities, which are
caused by the classical IgE mechanism. Type I hypersensitivity is
characterised by excessive activation of mast cells and basophils
by IgE, resulting in a systemic inflammatory response that can
result in symptoms as benign as a runny nose, to life-threatening
anaphylactic shock and death. Well known types of allergies
include, without being limited thereto, allergic asthma (leading to
swelling of the nasal mucosa), allergic conjunctivitis (leading to
redness and itching of the conjunctiva), allergic rhinitis ("hay
fever"), anaphylaxis, angiodema, atopic dermatitis (eczema),
urticaria (hives), eosinophilia, respiratory, allergies to insect
stings, skin allergies (leading to and including various rashes,
such as eczema, hives (urticaria) and (contact) dermatitis), food
allergies, allergies to medicine, etc. With regard to the present
invention, e.g. a pharmaceutical composition is provided, which
contains e.g. an RNA coding for an allergen (e.g. from a cat
allergen, a dust allergen, a mite antigen, a plant antigen (e.g. a
birch antigen) etc.) as a complex of the invention. Hereby, the
encoded allergen may desensitize the patient' immune response.
Alternatively, the pharmaceutical compositions of the present
invention may shift the (exceeding) immune response to a stronger
TH1 response, thereby suppressing or attenuating the undesired IgE
response from the patient suffers.
[0213] Furthermore, diseases or states as defined herin may include
autoimmune diseases. Autoimmune diseases can be broadly divided
into systemic and organ-specific or localised autoimmune disorders,
depending on the principal clinico-pathologic features of each
disease. Autoimmune disease may be divided into the categories of
systemic syndromes, including SLE, Sjorgen's syndrome, Scleroderma,
rheumatoid arthritis and polyomyositis or local syndromes which may
be endocrinologic (DM Type 1, Hashimoto's thyroiditis, Addison's
disease, etc.), dermatologic (pemphigus vulgaris), haematologic
(autoimmune haemolytic anaemia), neural (multiple sclerosis) or can
involve virtually any circumscribed mass of body tissue. The
autoimmune diseases to be treated may be selected from the group
consisting of type I autoimmune diseases or type II autoimmune
diseases or type III autoimmune diseases or type IV autoimmune
diseases, such as, for example, multiple sclerosis (MS), rheumatoid
arthritis, diabetes, type I diabetes (Diabetes mellitus), systemic
lupus erythematosus (SLE), chronic polyarthritis, Basedow's
disease, autoimmune forms of chronic hepatitis, colitis ulcerosa,
allergy type I diseases, allergy type II diseases, allergy type III
diseases, allergy type IV diseases, fibromyalgia, hair loss,
Bechterew's disease, Crohn's disease, Myasthenia gravis,
neurodermitis, Polymyalgia rheumatica, progressive systemic
sclerosis (PSS), psoriasis, Reiter's syndrome, rheumatic arthritis,
psoriasis, vasculitis, etc, or type II diabetes.
[0214] While the exact mode as to why the immune system induces a
immune reaction against autoantigens has not been elucidated so
far, there are several findings with regard to the etiology.
Accordingly, the autoreaction may be due to a T-Cell Bypass. A
normal immune system requires the activation of B-cells by T-cells
before the former can produce antibodies in large quantities. This
requirement of a T-cell can be by-passed in rare instances, such as
infection by organisms producing super-antigens, which are capable
of initiating polyclonal activation of B-cells, or even of T-cells,
by directly binding to the -subunit of T-cell receptors in a
non-specific fashion. Another explanation deduces autoimmune
diseases from a molecular mimicry. An exogenous antigen may share
structural similarities with certain host antigens; thus, any
antibody produced against this antigen (which mimics the
self-antigens) can also, in theory, bind to the host antigens and
amplify the immune response. The most striking form of molecular
mimicry is observed in Group A beta-haemolytic streptococci, which
shares antigens with human myocardium, and is responsible for the
cardiac manifestations of rheumatic fever. The present invention
allows therefore to provide an RNA coding for an autoantigen as
component of the complexed RNA of the invention (or a (liquid)
composition containing such a complexed RNA of the invention) or to
provide a pharmaceutical composition containing an autoantigen (as
protein, mRNA or DNA encoding for a autoantigen protein) and a
complexed RNA of the invention all of which typically allow the
immune system to be desensitized.
Finally, diseases to be treated in the context of the present
invention likewise include monogenetic diseases, i.e. (hereditary)
diseases, or genetic diseases in general. Such genetic diseases are
typically caused by genetic defects, e.g. due to gene mutations
resulting in loss of protein activity or regulatory mutations which
do not allow transcription or translation of the protein.
Frequently, these diseases lead to metabolic disorders or other
symptoms, e.g. muscle dystrophy. Accordingly, the present invention
allows to treat these diseases by providing the complexed RNA as
defined herein. Insofar, the following diseases may be treated:
3-beta-hydroxysteroid dehydrogenase deficiency (type II);
3-ketothiolase deficiency; 6-mercaptopurine sensitivity;
Aarskog-Scott syndrome; Abetalipoproteinemia; Acatalasemia;
Achondrogenesis; Achondrogenesis-hypochondrogenesis;
Achondroplasia; Achromatopsia; Acromesomelic dysplasia
(Hunter-Thompson type); ACTH deficiency; Acyl-CoA dehydrogenase
deficiency (short-chain, medium chain, long chain); Adenomatous
polyposis coli; Adenosin-deaminase deficiency; Adenylosuccinase
deficiency; Adhalinopathy; Adrenal hyperplasia, congenital (due to
11-beta-hydroxylase deficiency; due to 17-alpha-hydroxylase
deficiency; due to 21-hydroxylase deficiency); Adrenal hypoplasia,
congenital, with hypogonadotropic hypogonadism; Adrenogenital
syndrom; Adrenoleukodystrophy; Adrenomyeloneuropathy;
Afibrinogenemia; Agammaglobulinemia; Alagille syndrome; Albinism
(brown, ocular, oculocutaneous, rufous); Alcohol intolerance,
acute; Aldolase A deficiency; Aldosteronism,
glucocorticoid-remediable; Alexander disease; Alkaptonuria;
Alopecia universalis; Alpha-1-antichymotrypsin deficiency;
Alpha-methylacyl-CoA racemase deficiency; Alpha-thalassemia/mental
retardation syndrome; Alport syndrome; Alzheimer disease-1
(APP-related); Alzheimer disease-3; Alzheimer disease-4;
Amelogenesis imperfecta; Amyloid neuropathy (familial, several
allelic types); Amyloidosis (Dutch type; Finnish type; hereditary
renal; renal; senile systemic); Amytrophic lateral sclerosis;
Analbuminemia; Androgen insensitivity; Anemia (Diamond-Blackfan);
Anemia (hemolytic, due to PK deficiency); Anemia (hemolytic,
Rh-null, suppressor type); Anemia (neonatal hemolytic, fatal and
nearfatal); Anemia (sideroblastic, with ataxia); Anemia
(sideroblastic/hypochromic); Anemia due to G6PD deficiency;
Aneurysm (familial arterial); Angelman syndrome; Angioedema;
Aniridia; Anterior segment anomalies and cataract; Anterior segment
mesenchymal dysgenesis; Anterior segment mesenchymal dysgenesis and
cataract; Antithrombin III deficiency; Anxiety-related personality
traits; Apert syndrome; Apnea (postanesthetic); ApoA-I and apoC-III
deficiency (combined); Apolipoprotein A-II deficiency;
Apolipoprotein B-100 (ligand-defective); Apparent mineralocorticoid
excess (hypertension due to); Argininemia;
Argininosuccinicaciduria; Arthropathy (progressive
pseudorheumatoid, of childhood); Aspartylglucosaminuria; Ataxia
(episodic); Ataxia with isolated vitamin E deficiency;
Ataxia-telangiectasia; Atelosteogenesis II; ATP-dependent DNA
ligase I deficiency; Atrial septal defect with atrioventricular
conduction defects; Atrichia with papular lesions; Autism
(succinylpurinemic); Autoimmune polyglandular disease, type I;
Autonomic nervous system dysfunction; Axenfeld anomaly;
Azoospermia; Bamforth-Lazarus syndrome; Bannayan-Zonana syndrome;
Barthsyndrome; Bartter syndrome (type 2 or type 3); Basal cell
carcinoma; Basal cell nevus syndrome; BCG infection;
Beare-Stevenson cutis gyrata syndrome; Becker muscular dystrophy;
Beckwith-Wiedemann syndrome; Bernard-Soulier syndrome (type B; type
C); Bethlem myopathy; Bile acid malabsorption, primary; Biotimidase
deficiency; Bladder cancer; Bleeding disorder due to defective
thromboxane A2 receptor; Bloom syndrome; Brachydactyly (type B1 or
type C); Branchiootic syndrome; Branchiootorenal syndrome; Breast
cancer (invasive intraductal; lobular; male, with Reifenstein
syndrome; sporadic); Breast cancer-1 (early onset); Breast cancer-2
(early onset); Brody myopathy; Brugada syndrome; Brunner syndrome;
Burkitt lymphoma; Butterfly dystrophy (retinal); C1q deficiency
(type A; type B; type C); C1r/C1s deficiency; C1s deficiency,
isolated; C2 deficiency; C3 deficiency; C3b inactivator deficiency;
C4 deficiency; C8 deficiency, type II; C9 deficiency; Campomelic
dysplasia with autosomal sex reversal;
Camptodactyly-arthropathy-coxa varapericarditis syndrome; Canavan
disease; Carbamoylphosphate synthetase I deficiency;
Carbohydrate-deficient glycoprotein syndrome (type I; type Ib; type
II); Carcinoid tumor of lung; Cardioencephalomyopathy (fatal
infantile, due to cytochrome c oxidase deficiency); Cardiomyopathy
(dilated; X-linked dilated; familial hypertrophic; hypertrophic);
Carnitine deficiency (systemic primary); Carnitine-acylcarnitine
translocase deficiency; Carpal tunnel syndrome (familial); Cataract
(cerulean; congenital; crystalline aculeiform; juvenile-onset;
polymorphic and lamellar; punctate; zonular pulverulent); Cataract,
Coppock-like; CD59 deficiency; Central core disease; Cerebellar
ataxia; Cerebral amyloid angiopathy; Cerebral arteriopathy with
subcortical infarcts and leukoencephalopathy; Cerebral cavernous
malformations-1; Cerebrooculofacioskeletal syndrome; Cerebrotendi
nous xanthomatosis; Cerebrovascular disease; Ceroid lipofuscinosis
(neuronal, variant juvenile type, with granular osmiophilic
deposits); Ceroid lipofuscinosis (neuronal-1, infantile);
Ceroid-lipofuscinosis (neuronal-3, juvenile); Char syndrome;
Charcot-Marie-Tooth disease; Charcot-Marie-Tooth neuropathy;
Charlevoix-Saguenay type; Chediak-Higashi syndrome; Chloride
diarrhea (Finnish type); Cholestasis (benign recurrent
intrahepatic); Cholestasis (familial intrahepatic); Cholestasis
(progressive familial intrahepatic); Cholesteryl ester storage
disease; Chondrodysplasia punctata (brachytelephalangic;
rhizomelic; X-linked dominant; X-linked recessive; Grebe type);
Chondrosarcoma; Choroideremia; Chronic granulomatous disease
(autosomal, due to deficiency of CYBA); Chronic granulomatous
disease (X-linked); Chronic granulomatous disease due to deficiency
of NCF-1; Chronic granulomatous disease due to deficiency of NCF-2;
Chylomicronemia syndrome, familial; Citrullinemia; classical
Cockayne syndrome-1; Cleft lip, cleft jaw, cleft palate; Cleft
lip/palate ectodermal dysplasia syndrome; Cleidocranial dysplasia;
CMO II deficiency; Coats disease; Cockayne syndrome-2, type B;
Coffin-Lowry syndrome; Colchicine resistance; Colon adenocarcinoma;
Colon cancer; Colorblindness (deutan; protan; tritan); Colorectal
cancer; Combined factor V and VIII deficiency; Combined
hyperlipemia (familial); Combined immunodeficiency (X-linked,
moderate); Complex I deficiency; Complex neurologic disorder; Cone
dystrophy-3; Cone-rod dystrophy 3; Cone-rod dystrophy 6; Cone-rod
retinal dystrophy-2; Congenital bilateral absence of vas deferens;
Conjunctivitis, ligneous; Contractural arachnodactyly;
Coproporphyria; Cornea plana congenita; Corneal clouding; Corneal
dystrophy (Avellino type; gelatinous drop-like; Groenouw type I;
lattice type I; Reis-Bucklers type); Cortisol resistance; Coumarin
resistance; Cowden disease; CPT deficiency, hepatic (type I; type
II); Cramps (familial, potassium-aggravated);
Craniofacial-deafness-hand syndrome; Craniosynostosis (type 2);
Cretinism; Creutzfeldt-Jakob disease; Crigler-Najjar syndrome;
Crouzon syndrome; Currarino syndrome; Cutis laxa; Cyclic
hematopoiesis; Cyclic ichthyosis; Cylindromatosis; Cystic fibrosis;
Cystinosis (nephropathic); Cystinuria (type II; type III);
Daltonism; Darier disease; D-bifunctional protein deficiency;
Deafness, autosomal dominant 1; Deafness, autosomal dominant 11;
Deafness, autosomal dominant 12; Deafness, autosomal dominant 15;
Deafness, autosomal dominant 2; Deafness, autosomal dominant 3;
Deafness, autosomal dominant 5; Deafness, autosomal dominant 8;
Deafness, autosomal dominant 9; Deafness, autosomal recessive 1;
Deafness, autosomal recessive 2; Deafness, autosomal recessive 21;
Deafness, autosomal recessive 3; Deafness, autosomal recessive 4;
Deafness, autosomal recessive 9; Deafness, nonsyndromic
sensorineural 13; Deafness, X-linked 1; Deafness, X-linked 3;
Debrisoquine sensitivity; Dejerine-Sottas disease; Dementia
(familial Danish); Dementia (frontotemporal, with parkinsonism);
Dent disease; Dental anomalies; Dentatorubro-pallidoluysian
atrophy; Denys-Drash syndrome; Dermatofibrosarcoma protuberans;
Desmoid disease; Diabetes insipidus (nephrogenic); Diabetes
insipidus (neurohypophyseal); Diabetes mellitus
(insulin-resistant); Diabetes mellitus (rare form); Diabetes
mellitus (type II); Diastrophic dysplasia; Dihydropyrimidinuria;
Dosage-sensitive sex reversal; Doyne honeycomb degeneration of
retina; Dubin-Johnson syndrome; Duchenne muscular dystrophy;
Dyserythropoietic anemia with thrombocytopenia; Dysfibrinogenemia
(alpha type; beta type; gamma type); Dyskeratosis congenita-1;
Dysprothrombinemia; Dystonia (DOPAresponsive); Dystonia
(myoclonic); Dystonia-1 (torsion); Ectodermal dysplasia; Ectopia
lentis; Ectopia pupillae; Ectrodactyly (ectodermal dysplasia, and
cleft lip/palate syndrome 3); Ehlers-Danlos syndrome (progeroid
form); Ehlers-Danlos syndrome (type I; type II; type III; type IV;
type VI; type VII); Elastin Supravalvar aortic stenosis;
Elliptocytosis-1; Elliptocytosis-2; Elliptocytosis-3; Ellis-van
Creveld syndrome; Emery-Dreifuss muscular dystrophy; Emphysema;
Encephalopathy; Endocardial fibroelastosis-2; Endometrial
carcinoma; Endplate acetylcholinesterase deficiency; Enhanced
S-cone syndrome; Enlarged vestibular aqueduct; Epidermolysis
bullosa; Epidermolysis bullosa dystrophica (dominant or recessive);
Epidermolysis bullosa simplex; Epidermolytic hyperkeratosis;
Epidermolytic palmoplantar keratoderma; Epilepsy (generalize;
juvenile; myoclonic; nocturnal frontal lobe; progressive
myoclonic); Epilepsy, benign, neonatal (type1 or type2); Epiphyseal
dysplasia (multiple); Episodic ataxia (type 2); Episodic
ataxia/myokymia syndrome; Erythremias (alpha-; dysplasia);
Erythrocytosis; Erythrokeratoderma; Estrogen resistance; Exertional
myoglobinuria due to deficiency of LDH-A; Exostoses, multiple (type
1; type 2); Exudative vitreoretinopathy, X-linked; Fabry disease;
Factor H deficiency; Factor VII deficiency; Factor X deficiency;
Factor XI deficiency; Factor XII deficiency; Factor XIIIA
deficiency; Factor XIIIB deficiency; Familial Mediterranean fever;
Fanconi anemia; Fanconi-Bickel syndrome; Farber lipogranulomatosis;
Fatty liver (acute); Favism; Fish-eye disease; Foveal hypoplasia;
Fragile X syndrome; Frasier syndrome; Friedreich ataxia;
fructose-bisphosphatase Fructose intolerance; Fucosidosis; Fumarase
deficiency; Fundus albipunctatus; Fundus flavimaculatus; G6PD
deficiency; GABA-transaminase deficiency; Galactokinase deficiency
with cataracts; Galactose epimerase deficiency; Galactosemia;
Galactosialidosis; GAMT deficiency; Gardner syndrome; Gastric
cancer; Gaucher disease; Generalized epilepsy with febrile seizures
plus; Germ cell tumors; Gerstmann-Straussler disease; Giant cell
hepatitis (neonatal); Giant platelet disorder; Giant-cell
fibroblastoma; Gitelman syndrome; Glanzmann thrombasthenia (type A;
type B); Glaucoma 1A; Glaucoma 3A; Glioblastoma multiforme;
Glomerulosclerosis (focal segmental); Glucose transport defect
(blood-brain barrier); Glucose/galactose malabsorption; Glucosidase
I deficiency; Glutaricaciduria (type I; type IIB; type IIC);
Gluthation synthetase deficiency; Glycerol kinase deficiency;
Glycine receptor (alpha-1 polypeptide); Glycogen storage disease I;
Glycogen storage disease II; Glycogen storage disease III; Glycogen
storage disease IV; Glycogen storage disease VI; Glycogen storage
disease VII; Glycogenosis (hepatic, autosomal); Glycogenosis
(X-linked hepatic); GM1-gangliosidosis; GM2-gangliosidosis; Goiter
(adolescent multinodular); Goiter (congenital); Goiter (nonendemic,
simple); Gonadal dysgenesis (XY type); Granulomatosis, septic;
Graves disease; Greig cephalopolysyndactyl)-syndrome; Griscelli
syndrome; Growth hormone deficient dwarfism; Growth retardation
with deafness and mental retardation; Gynecomastia (familial, due
to increased aromatase activity); Gyrate atrophy of choroid and
retina with ornithinemia (B6 responsive or unresponsive);
Hailey-Hailey disease; Haim-Munk syndrome; Hand-foot-uterus
syndrome; Harderoporphyrinuria; HDL deficiency (familial); Heart
block (nonprogressive or progressive); Heinz body anemia; HELLP
syndrome; Hematuria (familial benign); Heme oxygenase-1 deficiency;
Hemiplegic migraine; Hemochromotosis; Hemoglobin H disease;
Hemolytic anemia due to ADA excess; Hemolytic anemia due to
adenylate kinase deficiency; Hemolytic anemia due to band 3 defect;
Hemolytic anemia due to glucosephosphate isomerase deficiency;
Hemolytic anemia due to glutathione synthetase deficiency;
Hemolytic anemia due to hexokinase deficiency; Hemolytic anemia due
to PGK deficiency; Hemolytic-uremic syndrome; Hemophagocytic
lymphohistiocytosis; Hemophilia A; Hemophilia B; Hemorrhagic
diathesis due to factor V deficiency; Hemosiderosis (systemic, due
to aceruloplasminemia); Hepatic lipase deficiency; Hepatoblastoma;
Hepatocellular carcinoma; Hereditary hemorrhagic telangiectasia-1;
Hereditary hemorrhagic telangiectasia-2; Hermansky-Pudlak syndrome;
Heterotaxy (X-linked visceral); Heterotopia (periventricular);
Hippel-Lindau syndrom; Hirschsprung disease; Histidine-rich
glycoprotein Thrombophilia due to HRG deficiency; HMG-CoA lyase
deficiency; Holoprosencephaly-2; Holoprosencephaly-3;
Holoprosencephaly-4; Holoprosencephaly-5; Holt-Oram syndrome;
Homocystinuria; Hoyeraal-Hreidarsson; HPFH (deletion type or
nondeletion type); HPRT-related gout; Huntington disease;
Hydrocephalus due to aqueductal stenosis; Hydrops fetal is;
Hyperbetalipoproteinemia; Hypercholesterolemia, familial;
Hyperferritinemia-cataract syndrome; Hyperglycerolemia;
Hyperglycinemia; Hyperimmunoglobulinemia D and periodic fever
syndrome; Hyperinsulinism; Hyperinsulinism-hyperammonemia syndrome;
Hyperkalemic periodic paralysis; Hyperlipoproteinemia;
Hyperlysinemia; Hypermethioninemia (persistent, autosomal,
dominant, due to methionine, adenosyltransferase I/III deficiency);
Hyperornithinemia-hyperammonemiahomocitrullinemia syndrome;
Hyperoxaluria; Hyperparathyroidism; Hyperphenylalaninemia due to
pterin-4-acarbinolamine dehydratase deficiency;
Hyperproinsulinemia; Hyperprolinemia; Hypertension; Hyperthroidism
(congenital); Hypertriglyceridemia; Hypoalphalipoproteinemia;
Hypobetalipoproteinemia; Hypocalcemia; Hypochondroplasia;
Hypochromic microcytic anemia; Hypodontia; Hypofibrinogenemia;
Hypoglobulinemia and absent B cells; Hypogonadism
(hypergonadotropic); Hypogonadotropic (hypogonadism); Hypokalemic
periodic paralysis; Hypomagnesemia; Hypomyelination (congenital);
Hypoparathyroidism; Hypophosphatasia (adult; childhood; infantile;
hereditary); Hypoprothrombinemia; Hypothyroidism (congenital;
hereditary congenital; nongoitrous); Ichthyosiform erythroderma;
Ichthyosis; Ichthyosis bullosa of Siemens; IgG2 deficiency;
Immotile cilia syndrome-1; Immunodeficiency (T-cell receptor/CD3
complex); Immunodeficiency (X-linked, with hyper-IgM);
Immunodeficiency due to defect in CD3-gamma;
Immunodeficiency-centromeric instabilityfacial anomalies syndrome;
Incontinentia pigmenti; Insensitivity to pain (congenital, with
anhidrosis); Insomnia (fatal familial); Interleukin-2 receptor
deficiency (alpha chain); Intervertebral disc disease;
Iridogoniodysgenesis; Isolated growth hormone deficiency (Illig
type with absent GH and Kowarski type with bioinactive GH);
Isovalericacidemia; Jackson-Weiss sydnrome; Jensen syndrome;
Jervell and Lange-Nielsen syndrome; Joubert syndrom; Juberg-Marsidi
syndrome; Kallmann syndrome; Kanzaki disease; Keratitis;
Keratoderma (palmoplantar); Keratosis palmoplantaris striata I;
Keratosis palmoplantaris striata II; Ketoacidosis due to SCOT
deficiency; Keutel syndrome; Klippel-Trenaurnay syndrom; Kniest
dysplasia; Kostmann neutropenia; Krabbe disease;
Kurzripp-Polydaktylie syndrom; Lacticacidemia due to PDX1
deficiency; Langer mesomelic dysplasia; Laron dwarfism;
Laurence-Moon-Biedl-Bardet syndrom; LCHAD deficiency; Leber
congenital amaurosis; Left-right axis malformation; Leigh syndrome;
Leiomyomatosis (diffuse, with Alport syndrome); Leprechaunism;
Leri-Weill dyschondrosteosis; Lesch-Nyhan syndrome; Leukemia (acute
myeloid; acute promyelocytic; acute T-cell lymphoblastic; chronic
myeloid; juvenile myelomonocytic; Leukemia-1 (T-cell acute
lymphocytic); Leukocyte adhesion deficiency; Leydig cell adenoma;
Lhermitte-Duclos syndrome; Liddle syndrome; Li-Fraumeni syndrome;
Lipoamide dehydrogenase deficiency; Lipodystrophy; Lipoid adrenal
hyperplasia; Lipoprotein lipase deficiency; Lissencephaly
(X-linked); Lissencephaly-1; liver Glycogen storage disease (type
0); Long QT syndrome-1; Long QT syndrome-2; Long QT syndrome-3;
Long QT syndrome-5; Long QT syndrome-6; Lowe syndrome; Lung cancer;
Lung cancer (nonsmall cell); Lung cancer (small cell); Lymphedema;
Lymphoma (B-cell
non-Hodgkin); Lymphoma (diffuse large cell); Lymphoma (follicular);
Lymphoma (MALT); Lymphoma (mantel cell); Lymphoproliferative
syndrome (X-linked); Lysinuric protein intolerance; Machado-Joseph
disease; Macrocytic anemia refractory (of 5q syndrome); Macular
dystrophy; Malignant mesothelioma; Malonyl-CoA decarboxylase
deficiency; Mannosidosis, (alpha- or beta-); Maple syrup urine
disease (type Ia; type Ib; type II); Marfan syndrome;
Maroteaux-Lamy syndrome; Marshall syndrome; MASA syndrome; Mast
cell leukemia; Mastocytosis with associated hematologic disorder;
McArdle disease; McCune-Albright polyostotic fibrous dysplasia;
McKusick-Kaufman syndrome; McLeod phenotype; Medullary thyroid
carcinoma; Medulloblastoma; Meesmann corneal dystrophy;
Megaloblastic anemia-1; Melanoma; Membroproliferative
glomerulonephritis; Meniere disease; Meningioma (NF2-related;
SIS-related); Menkes disease; Mental retardation (X-linked);
Mephenyloin poor metabolizer; Mesothelioma; Metachromatic
leukodystrophy; Metaphyseal chondrodysplasia (Murk Jansen type;
Schmid type); Methemoglobinemia; Methionine adenosyltransferase
deficiency (autosomal recessive); Methylcobalamin deficiency (cbl G
type); Methylmalonicaciduria (mutase deficiency type);
Mevalonicaciduria; MHC class II deficiency; Microphthalmia
(cataracts, and iris abnormalities); Miyoshi myopathy; MODY;
Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency (type A
or type B); Monilethrix; Morbus Fabry; Morbus Gaucher;
Mucopolysaccharidosis; Mucoviscidosis; Muencke syndrome; Muir-Torre
syndrome; Mulibrey nanism; Multiple carboxylase deficiency
(biotinresponsive); Multiple endocrine neoplasia; Muscle
glycogenosis; Muscular dystrophy (congenital merosindeficient);
Muscular dystrophy (Fukuyama congenital); Muscular dystrophy
(limb-girdle); Muscular dystrophy) Duchenne-like); Muscular
dystrophy with epidermolysis bullosa simplex; Myasthenic syndrome
(slow-channel congenital); Mycobacterial infection (atypical,
familial disseminated); Myelodysplastic syndrome; Myelogenous
leukemia; Myeloid malignancy; Myeloperoxidase deficiency;
Myoadenylate deaminase deficiency; Myoglobinuria/hemolysis due to
PGK deficiency; Myoneurogastrointestinal encephalomyopathy
syndrome; Myopathy (actin; congenital; desmin-related;
cardioskeletal; distal; nemaline); Myopathy due to CPT II
deficiency; Myopathy due to phosphoglycerate mutase deficiency;
Myotonia congenita; Myotonia levior; Myotonic dystrophy; Myxoid
liposarcoma; NAGA deficiency; Nailpatella syndrome; Nemaline
myopathy 1 (autosomal dominant); Nemaline myopathy 2 (autosomal
recessive); Neonatal hyperparathyroidism; Nephrolithiasis;
Nephronophthisis (juvenile); Nephropathy (chronic
hypocomplementemic); Nephrosis-1; Nephrotic syndrome; Netherton
syndrome; Neuroblastoma; Neurofibromatosis (type 1 or type 2);
Neurolemmomatosis; neuronal-5 Ceroid-lipofuscinosis; Neuropathy;
Neutropenia (alloimmune neonatal); Niemann-Pick disease (type A;
type B; type C1; type D); Night blindness (congenital stationary);
Nijmegen breakage syndrome; Noncompaction of left ventricular
myocardium; Nonepidermolytic palmoplantar keratoderma; Norrie
disease; Norum disease; Nucleoside phosphorylase deficiency;
Obesity; Occipital hornsyndrome; Ocular albinism (Nettleship-Falls
type); Oculopharyngeal muscular dystorphy; Oguchi disease;
Oligodontia; Omenn syndrome; Opitz G syndrome; Optic nerve coloboma
with renal disease; Ornithine transcarbamylase deficiency;
Oroticaciduria; Orthostatic intolerance; OSMED syndrome;
Ossification of posterior longitudinal ligament of spine;
Osteoarthrosis; Osteogenesis imperfecta; Osteolysis; Osteopetrosis
(recessive or idiopathic); Osteosarcoma; Ovarian carcinoma; Ovarian
dysgenesis; Pachyonychia congenita (Jackson-Lawler type or
Jadassohn-Lewandowsky type); Paget disease of bone; Pallister-Hall
syndrome; Pancreatic agenesis; Pancreatic cancer; Pancreatitis;
Papillon-Lefevre syndrome; Paragangliomas; Paramyotonia congenita;
Parietal foramina; Parkinson disease (familial or juvenile);
Paroxysmal nocturnal hemoglobinuria; Pelizaeus-Merzbacher disease;
Pendred syndrome; Perineal hypospadias; Periodic fever; Peroxisomal
biogenesis disorder; Persistent hyperinsulinemic hypoglycemia of
infancy; Persistent Mullerian duct syndrome (type II); Peters
anomaly; Peutz-Jeghers syndrome; Pfeiffer syndrome;
Phenylketonuria; Phosphoribosyl pyrophosphate synthetaserelated
gout; Phosphorylase kinase deficiency of liver and muscle;
Piebaldism; Pilomatricoma; Pinealoma with bilateral retinoblastoma;
Pituitary ACTH secreting adenoma; Pituitary hormone deficiency;
Pituitary tumor; Placental steroid sulfatase deficiency; Plasmin
inhibitor deficiency; Plasminogen deficiency (types I and II);
Plasminogen Tochigi disease; Platelet disorder; Platelet
glycoprotein IV deficiency; Platelet-activating factor acetyl
hydrolase deficiency; Polycystic kidney disease; Polycystic
lipomembranous osteodysplasia with sclerosing
leukenencephalophathy; Polydactyl), postaxial; Polyposis; Popliteal
pterygium syndrome; Porphyria (acute hepatic or acute intermittent
or congenital erythropoietic); Porphyria cutanea tarda; Porphyria
hepatoerythropoietic; Porphyria variegata; Prader-Willi syndrome;
Precocious puberty; Premature ovarian failure; Progeria Typ I;
Progeria Typ II; Progressive external opthalmoplegia; Progressive
intrahepatic cholestasis-2; Prolactinoma (hyperparathyroidism,
carcinoid syndrome); Prolidase deficiency; Propionicacidemia;
Prostate cancer; Protein S deficiency; Proteinuria; Protoporphyria
(erythropoietic); Pseudoachondroplasia; Pseudohermaphroditism;
Pseudohypoaldosteronism; Pseudohypoparathyroidism; Pseudovaginal
perineoscrotal hypospadias; Pseudovitamin D deficiency rickets;
Pseudoxanthoma elasticum (autosomal dominant; autosomal recessive);
Pulmonary alveolar proteinosis; Pulmonary hypertension; Purpura
fulminans; Pycnodysostosis; Pyropoikilocytosis; Pyruvate
carboxylase deficiency; Pyruvate dehydrogenase deficiency;
Rabson-Mendenhall syndrome; Refsum disease; Renal cell carcinoma;
Renal tubular acidosis; Renal tubular acidosis with deafness; Renal
tubular acidosis-osteopetrosis syndrome; Reticulosis (familial
histiocytic); Retinal degeneration; Retinal dystrophy; Retinitis
pigmentosa; Retinitis punctata albescens; Retinoblastoma; Retinol
binding protein deficiency; Retinoschisis; Rett syndrome; Rh(mod)
syndrome; Rhabdoid predisposition syndrome; Rhabdoid tumors;
Rhabdomyosarcoma; Rhabdomyosarcoma (alveolar); Rhizomelic
chondrodysplasia punctata; Ribbing-Syndrom; Rickets (vitamin
D-resistant); Rieger anomaly; Robinow syndrome; Rothmund-Thomson
syndrome; Rubenstein-Taybi syndrome; Saccharopinuria;
Saethre-Chotzen syndrome; Salla disease; Sandhoff disease
(infantile, juvenile, and adult forms); Sanfilippo syndrome (type A
or type B); Schindler disease; Schizencephaly; Schizophrenia
(chronic); Schwannoma (sporadic); SCID (autosomal recessive,
T-negative/B positive type); Secretory pathway wITMD; SED
congenita; Segawa syndrome; Selective T-cell defect; SEMD
(Pakistani type); SEMD (Strudwick type); Septooptic dysplasia;
Severe combined immunodeficiency (B cell negative); Severe combined
immunodeficiency (T-cell negative, B-cell/natural killer
cell-positive type); Severe combined immunodeficiency (Xlinked);
Severe combined immunodeficiency due to ADA deficiency; Sex
reversal (XY, with adrenal failure); Sezary syndrome;
Shah-Waardenburg syndrome; Short stature; Shprintzen-Goldberg
syndrome; Sialic acid storage disorder; Sialidosis (type I or type
II); Sialuria; Sickle cell anemia; Simpson-Golabi-Behmel syndrome;
Situs ambiguus; Sjogren-Larsson syndrome; Smith-Fineman-Myers
syndrome; Smith-Lemli-Opitz syndrome (type I or type II);
Somatotrophinoma; Sorsby fundus dystrophy; Spastic paraplegia;
Spherocytosis; Spherocytosis-1; Spherocytosis-2; Spinal and bulbar
muscular atrophy of Kennedy; Spinal muscular atrophy;
Spinocerebellar ataxia; Spondylocostal dysostosis;
Spondyloepiphyseal dysplasia tarda; Spondylometaphyseal dysplasia
(Japanese type); Stargardt disease-1; Steatocystoma multiplex;
Stickler syndrome; Sturge-Weber syndrom; Subcortical laminal
heteropia; Subcortical laminar heterotopia; Succinic semialdehyde
dehydrogenase deficiency; Sucrose intolerance; Sutherland-Haan
syndrome; Sweat chloride elevation without CF; Symphalangism;
Synostoses syndrome; Synpolydactyly; Tangier disease; Tay-Sachs
disease; T-cell acute lymphoblastic leukemia; T-cell
immunodeficiency; T-cell prolymphocytic leukemia; Thalassemia
(alpha- or delta-); Thalassemia due to Hb Lepore; Thanatophoric
dysplasia (types I or II); Thiamine-responsive megaloblastic anemia
syndrome; Thrombocythemia; Thrombophilia (dysplasminogenemic);
Thrombophilia due to heparin cofactor II deficiency; Thrombophilia
due to protein C deficiency; Thrombophilia due to thrombomodulin
defect; Thyroid adenoma; Thyroid hormone resistance; Thyroid iodine
peroxidase deficiency; Tietz syndrome; Tolbutamide poor
metabolizer; Townes-Brocks syndrome; Transcobalamin II deficiency;
Treacher Collins mandibulofacial dysostosis; Trichodontoosseous
syndrome; Trichorhinophalangeal syndrome; Trichothiodystrophy;
Trifunctional protein deficiency (type I or type II); Trypsinogen
deficiency; Tuberous sclerosis-1; Tuberous sclerosis-2; Turcot
syndrome; Tyrosine phosphatase; Tyrosinemia; Ulnar-mammary
syndrome; Urolithiasis (2,8-dihydroxyadenine); Usher syndrome (type
1B or type 2A); Venous malformations; Ventricular tachycardia;
Virilization; Vitamin K-dependent coagulation defect; VLCAD
deficiency; Vohwinkel syndrome; von Hippel-Lindau syndrome; von
Willebrand disease; Waardenburg syndrome; Waardenburg
syndrome/ocular albinism; Waardenburg-Shah neurologic variant;
Waardenburg-Shah syndrome; Wagner syndrome; Warfarin sensitivity;
Watson syndrome; Weissenbacher-Zweymuller syndrome; Werner
syndrome; Weyers acrodental dysostosis; White sponge nevus;
Williams-Beuren syndrome; Wilms tumor (type1); Wilson disease;
Wiskott-Aldrich syndrome; Wolcott-Rallison syndrome; Wolfram
syndrome; Wolman disease; Xanthinuria (type I); Xeroderma
pigmentosum; X-SCID; Yemenite deaf-blind hypopigmentation syndrome;
ypocalciuric hypercalcemia (type I); Zellweger syndrome;
Zlotogora-Ogur syndrome.
[0216] Preferred diseases to be treated which have a genetic
inherited background and which are typically caused by a single
gene defect and are inherited according to Mendel's laws are
preferably selected from the group consisting of
autosomal-recessive inherited diseases, such as, for example,
adenosine deaminase deficiency, familial hypercholesterolaemia,
Canavan's syndrome, Gaucher's disease, Fanconi anaemia, neuronal
ceroid lipofuscinoses, mucoviscidosis (cystic fibrosis), sickle
cell anaemia, phenylketonuria, alcaptonuria, albinism,
hypothyreosis, galactosaemia, alpha-1-anti-trypsin deficiency,
Xeroderma pigmentosum, Ribbing's syndrome, mucopolysaccharidoses,
cleft lip, jaw, palate, Laurence Moon Biedl Bardet sydrome, short
rib polydactylia syndrome, cretinism, Joubert's syndrome, type II
progeria, brachydactylia, adrenogenital syndrome, and X-chromosome
inherited diseases, such as, for example, colour blindness, e.g.
red/green blindness, fragile X syndrome, muscular dystrophy
(Duchenne and Becker-Kiener type), haemophilia A and B, G6PD
deficiency, Fabry's disease, mucopolysaccharidosis, Norrie's
syndrome, Retinitis pigmentosa, septic granulomatosis, X-SCID,
ornithine transcarbamylase deficiency, Lesch-Nyhan syndrome, or
from autosomal-dominant inherited diseases, such as, for example,
hereditary angiooedema, Marfan syndrome, neurofibromatosis, type I
progeria, Osteogenesis imperfecta, Klippel-Trenaurnay syndrome,
Sturge-Weber syndrome, Hippel-Lindau syndrome and tuberosis
sclerosis.
[0217] The present invention also allows treatment of diseases,
which have not been inherited, or which may not be summarized under
the above categories. Such dieseases may include e.g. the treatment
of patients, which are in need of a specific protein factor, e.g. a
specific therapeutically active protein as mentioned above. This
may e.g. include dialysis patients, e.g. patients which undergo a
(regular) a kidney or renal dialysis, and which may be in need of
specific therapeutically active proteins as defined above, e.g.
erythropoietin (EPO), etc.
[0218] According to another embodiment, the present invention
comprises the use of the at least one complexed RNA according to
the present invention for transfecting a cell or an organism.
Transfection of the cell or the organism may preferably be carried
out using the above (in vitro or in vivo) transfection method for
transfecting cells or a tissue with the complexed RNA of the
present invention.
[0219] According to one further embodiment, the present invention
comprises the use of at least one complexed RNA according to the
present invention (for the preparation of an agent) for the
treatment of any of the above mentioned diseases, disorders,
conditions or pathological states. An agent in this context may be
e.g. a pharmaceutical composition as defined above or an injection
buffer as defined herein, additionally containing the inventive
complexed RNA, a vaccine, etc. If more than one complexed RNA
molecule type is used, the complexed RNAs may be different by their
RNA (molecules) thereby forming a mixture of at least two distinct
complexed RNA (molecule) types. If more than one complexed RNA is
used (for the preparation of an agent) for the treatment of any of
the above mentioned diseases the same or (at least two) different
RNA (molecule) types may be contained in these complexed RNA
mixtures. In this context, any of the above mentioned RNA
(molecules) may be used for the inventive complexed RNA, e.g. a
short RNA oligonucleotide, a coding RNA, an immunostimulatory RNA,
a siRNA, an antisense RNA, or riboswitches, ribozymes or aptamers,
etc. More preferably a coding RNA (molecule), even more preferably
a linear coding RNA (molecule), and most preferably an mRNA may be
used. Preferably, such a coding RNA (molecule), more preferably a
linear coding RNA (molecule), and more preferably an mRNA is used
for the complexed RNA, the RNA (molecule) typically encodes a
protein or peptide suitable for the therapy of the specific
disease, e.g. an antibody, which is cabable of binding to a
specific cancer antigen, or a tumor antigen, when treating a
(specific) cancer, etc. The combinations of suitable RNA
(molecules) are known to a skilled person from the art and from the
disclosure of the present invention.
[0220] According to another embodiment of the present invention, it
may be preferred to (additionally) elicit, e.g. induce or enhance,
an immune response during therapy. In this context, an immune
response may occur in various ways. A substantial factor for a
suitable immune response is the stimulation of different T-cell
sub-populations. T-lymphocytes are typically divided into two
sub-populations, the T-helper 1 (Th1) cells and the T-helper 2
(Th2) cells, with which the immune system is capable of destroying
intracellular (Th1) and extracellular (Th2) pathogens (e.g.
antigens). The two Th cell populations differ in the pattern of the
effector proteins (cytokines) produced by them. Thus, Th1 cells
assist the cellular immune response by activation of macrophages
and cytotoxic T-cells. Th2 cells, on the other hand, promote the
humoral immune response by stimulation of the B-cells for
conversion into plasma cells and by formation of antibodies (e.g.
against antigens). The Th1/Th2 ratio is therefore of major
importance for the immune response. For various diseases to be
treated by the present invention, the Th1/Th2 ratio of the immune
response is preferably shifted in the direction towards the
cellular response (Th1 response) and a cellular immune response is
thereby induced. Accordingly, the present invention may also be
used to revert this immune response shift. Therefore, the present
invention encompasses also the use of at least one complexed RNA
according to the present invention (for the preparation of an
agent) for the treatment of any of the above mentioned diseases,
wherein the agent (and/or the complexed RNA) may be capable to
elicit, e.g. induce or enhance, an immune response in a tissue or
an organism as defined above. Again, an agent in this context may
be e.g. a pharmaceutical composition as defined above, or an
injection buffer as defined herein, which contains the inventive
complexed RNA, etc. If more than one complexed RNA type is used in
this context (for the preparation of an agent) for the treatment of
any of the above mentioned diseases, the complexed RNA types may be
different with respect to their RNA (molecules) and may form a
mixture of distinct RNA types.
[0221] However, for the present embodiment, it is preferred that at
least one of these complexed RNAs induces or enhances the immune
response during therapy, while other complexed RNA(s) need not to
induce or enhance the immune response or may be used to prevent an
imune response. In this context, any of the above mentioned RNA
(molecules) may be used for the inventive complexed RNA, e.g. a
short RNA oligonucleotide, a coding RNA, an immunostimulatory RNA,
a siRNA, an antisense RNA, or riboswitches, ribozymes or aptamers,
etc. More preferably, a coding RNA (molecule), even more preferably
a linear coding RNA (molecule), and most preferably an mRNA may be
used for the complexed RNA. If the RNA (molecule) is a coding RNA
(molecule), more preferably a linear coding RNA (molecule), and
more preferably an mRNA, it typically encodes a protein or peptide
suitable for the therapy of the specific disease, e.g. an antibody,
which is cabable of binding to a specific cancer antigen, when
treating a (specific) cancer, etc. If more than one complexed RNA
is contained in the agent, different combinations of proteins or
peptides may be selected. Such combinations of suitable RNA
(molecules) (and, if a coding RNA is used, of encoded proteins or
peptides) are known to a skilled person from the art or may be
combined from RNAs encoding therapeutically effective proteins,
etc., as defined in the disclosure of the present invention.
Induction or enhancement of the immune response concurrent to the
treatment of a specific disease using one pharmaceutical
composition or agent as defined above may be particularly
advantageous in cases where an induced or enhanced immune response
supports the treatment of a specific disease as mentioned
above.
[0222] Alternatively, treatment of the disease and induction or
enhancement of the immune response may be carried out by using
different pharmaceutical compositions or agents as defined above in
a time staggered manner. E.g. one may induce or enhance the immune
response by administering a pharmaceutical composition or an agent
as defined herein, containing an inventive complexed
(immunostimulatory) RNA, prior to (or concurrent to) administering
another pharmaceutical composition or an agent as defined herein
which may contain an inventive complexed RNA, e.g. a short RNA
oligonucleotide, a coding RNA, an immunostimulatory RNA, a siRNA,
an antisense RNA, or riboswitches, ribozymes or aptamers, etc.,
which is suitable for the therapy of the specific disease.
[0223] According to one embodiment, the present invention
furthermore comprises the use of at least one complexed RNA
according to the present invention (for the preparation of an
agent) for modulating, preferably to induce or enhance, an immune
response in a tissue or an organism as defined above, more
preferably to support a disease or state as mentioned herein.
Hereby, the inventive complexed RNA may be used to activate the
immune system unspecifically, e.g. to trigger the production of
certain cytokines. The complexed RNA may therefore be used to
support the specific immune response, which is elicited by e.g. an
antigen derived from pathogens or tumors. An agent in this context
may be e.g. a pharmaceutical composition as defined above or an
injection buffer as defined herein, containing the inventive
complexed RNA, a vaccine, etc. The immune response may be modulated
either by the at least one complexed RNA due to the one or more
oligopeptides having a length of 8 to 15 amino acids and showing
the empirical formula (Arg).sub.l; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x, and/or by the immunostimulatory
properties of the protein encoded by the RNA of the complexed
RNA.
[0224] The present invention may therefore, whenever appropriate,
well serve to achieve various objects. A complexed RNA as such or
as a component of an inventive composition may by itself improve
the transfection properties of the RNA as component of the
inventive complex. This underlying property of the inventive
complexed RNA is beneficial to a wide variety of applications.
Whenever it is intended to introduce an RNA into a cell, improved
transfection efficacy is ensured by the present invention. This
property as such may allow the present invention to be used for the
treatment of a huge variety of diseases, e.g. the treatment of
monogenetic or genetic diseases as defined above.
[0225] In addition, the present invention may be used whenever
treatment of immune disorders, e.g. allergies or autoimmune
diseases, is envisaged. Moreover, the present invention may
activate the patients's immune system by enhancing its unspecific
or specific immune response. Accordingly, it may elicit an
unspecific immune response, whenever appropriate, to cure a
disease. And, whenever required, it may elicit a specific immune
response as such (e.g. by encoding an antigen by the RNA as
component of the inventive complex) or by a combination of the
inventive complexed RNA with an antigen, e.g. in the same
composition. Whenever required, the inventive complexed RNA may be
preferably an antigen or an antibody, or any other protein or
peptide as defined above, capable of modulating the immune response
(preferably of inducing or enhancing same or, in case of allergies
or autoimmune diseases by desensitizing the patient's immune system
towards a specific allergen or autoantigen). In order to modulate,
e.g. induce or enhance, an immune response in a tissue or an
organism the complexed RNA may be administered to this tissue or
organism as defined above either as such or as an agent as defined
above. The administration modes, which may be used, may be the same
as described above for pharmaceutical compositions. Administration
of the agent may occur prior, concurrent and/or subsequent to a
therapy of diseases or states as mentioned herein, e.g. by
administration of the agent prior, concurrent and/or subsequent to
a therapy or an administration of a therapeutic suitable for these
diseases or states.
[0226] According to an alternative embodiment, the present
invention also encompasses the use of the peptide (Arg).sub.7 in a
complex with an RNA (molecule) as defined herein or the peptide
(Arg).sub.7 alone (for the preparation of an agent) for modulating,
preferably to elicit, e.g. to induce or enhance, an immune
response, preferably an unspecific immune response by e.g.
triggering the production of cytokines, in a tissue or an organism
as defined above, and preferably to support a disease or state as
mentioned herein. While determining the ranges of the peptide of
the inventive formula (I), the present inventors have surprisingly
found that (Arg).sub.7 is capable to significantly induce or
enhance an immune response in hPBMCs, even if no transfection of
nucleic acids, particularly RNA into hPBMCs, was observed. An RNA
(molecule) may be any RNA (molecule) as defined herein, preferably,
without being limited thereto, a short RNA oligonucleotide, a
coding RNA, an immunostimulatory RNA, a siRNA, an antisense RNA, or
riboswitches, ribozymes or aptamers. Again, an agent in this
context may be e.g. a pharmaceutical composition as defined above,
or an injection buffer as defined herein, which additionally
contains the inventive complexed RNA, etc., wherein in the
inventive complexed RNA in the agent as defined herein has been
replaced by the peptide (Arg).sub.7 in a complex with an RNA
(molecule) as defined herein or the peptide (Arg).sub.7 alone.
[0227] According to another alternative embodiment, the present
invention also encompasses the use of the peptide (Arg).sub.7 in a
complex with an RNA (molecule) as defined herein or the peptide
(Arg).sub.7 alone (for the preparation of an agent) for the
treatment of any of the above mentioned diseases or states.
[0228] According to a final embodiment, the present invention also
provides kits containing a complexed RNA according to the invention
and/or a pharmaceutical composition according to the invention as
well as, optionally, technical instructions with information on the
administration and dosage of the complexed RNA according to the
invention and/or the pharmaceutical composition according to the
invention. The kit may separately further comprise one or more of
the following group of components: at least one antigen or at least
one antibody or a composition containing an antigen or an antibody,
an additional adjuvant or a composition containing at least one
adjuvant and/or at least one cytokine or a composition containing
at least one cytokine. The antigen, antibody and/or the cytokine
may be provided as such (proteins) or may be provided as DNA or RNA
coding for the antigen, antibody or cytokine.
[0229] The present invention also provides kits containing the
peptide (Arg).sub.7 in a complex with an RNA (molecule) as defined
herein or the peptide (Arg).sub.7 alone as well as, optionally,
technical instructions with information on the administration and
dosage of the peptide (Arg).sub.7. Such kits may applied e.g. for
any of the above mentioned applications or uses, preferably for the
use of at least one complexed RNA according to the present
invention (for the preparation of an agent) for the treatment of
any of the above mentioned diseases. The kits may also be applied
for the use of at least one complexed RNA according to the present
invention (for the preparation of an agent) for the treatment of
any of the above mentioned diseases, wherein the agent (and/or the
complexed RNA) may be capable to induce or enhance an immune
response in a tissue or an organism as defined above. Such kits may
further be applied for the use of at least one complexed RNA
according to the present invention (for the preparation of an
agent) for modulating, preferably to elicit, e.g. to induce or
enhance, an immune response in a tissue or an organism as defined
above, and preferably to support a disease or state as mentioned
herein.
FIGURES
[0230] The following Figures are intended to illustrate the
invention further. They are not intended to limit the subject
matter of the invention thereto.
[0231] FIG. 1: depicts the sequence of a stabilized luciferase mRNA
sequence, wherein the native luciferase encoding mRNA is modified
with a poly-A/poly-C-tag (A70-C30). This first construct (construct
CAP-Ppluc(wt)-muag-A70-C30, SEQ ID NO: 35) contained following
sequence elements: [0232] stabilizing sequences from the
alpha-Globin gene, [0233] 70.times.Adenosin at the 3'-terminal end
(poly-A-tail), [0234] 30.times.Cytosin at the 3'-terminal end
(poly-C-tail); [0235] represented by following symbols: [0236]
=coding sequence [0237] =3'-UTR of the alpha globin gene [0238]
=poly-A-tail [0239] =poly-C-tail
[0240] FIG. 2: shows the sequence of a stabilized luciferase mRNA
sequence, wherein the construct according to SEQ ID NO: 35 (see
FIG. 1) is further modified with a GC-optimized sequence for a
better codon usage. The final construct (construct
CAP-Ppluc(GC)-muag-A70-C30, SEQ ID NO: 36) contained following
sequence elements: [0241] GC-optimized sequence for a better codon
usage [0242] stabilizing sequences from the alpha-Globin gene
[0243] 70.times.Adenosin at the 3'-terminal end (poly-A-tail),
[0244] 30.times.Cytosin at the 3'-terminal end (poly-C-tail);
[0245] represented by following symbols: [0246] =coding sequence
[0247] =modified 3'-UTR of the alpha globin gene [0248]
=poly-A-tail [0249] =poly-C-tail
[0250] FIG. 3: shows the coding sequence of the sequence according
to SEQ ID NO: 35 (SEQ ID NO: 37) (see FIG. 1).
[0251] FIG. 4: shows the GC-optimized coding sequence of the
sequence according to SEQ ID NO: 36 (SEQ ID NO: 38) (see FIG. 2).
The GC-optimized codons are underlined.
[0252] FIG. 5: shows the immunostimulatory effect of RNA complexed
with nona-arginine ((Arg).sub.9) in hPBMC cells by measuring IL-6
production. As can be seen, hPBMC cells show a significant IL-6
production, i.e. a significant immunostimulatory effect of RNA
complexed with nona-arginine ((Arg).sub.9).
[0253] FIG. 6: shows the immunostimulatory effect of RNA complexed
with nona-arginine ((Arg).sub.9) in hPBMC cells by measuring
TNF-alpha production. As can be seen, hPBMC cells show a
significant TNF-alpha production, i.e. a significant
immunostimulatory effect of RNA complexed with nona-arginine
((Arg).sub.9).
[0254] FIG. 7: shows in an comparative example the comparison of
immunostimulatory effects of RNA complexed with either
nona-arginine ((Arg).sub.9) or poly-L-arginine, respectively, in
hPBMCs. Advantageously, a significant immunostimulatory effect can
be observed for mass ratios lower than 1:5 (RNA:nona-arginine)
(1:10; 1:8; 1:5; 1:2; 1:1; 2:1). However, when using mass ratios of
RNA:nona-arginine (5:1) no significant TNFalpha production can be
observed. The same applies to stimulation experiments, using
nona-arginine ((Arg).sub.9) or mRNA alone. Additionally, it was
observed, that complexation of mRNA with poly-L-arginine leads to
significantly lower induction of TNF-alpha production in comparison
to nona-arginine ((Arg).sub.9). Apparently, higher concentrations
of poly-L-arginine appear to be toxic for cells transfected
therewith, particularly when using a mass ratio of 1:2
RNA:poly-L-arginine:RNA or higher, since the cells were lysed.
[0255] FIG. 8: shows luciferase expression upon transfection of
complexes of RNA with nona-arginine ((Arg).sub.9) in HeLa cells. As
may be derived from FIG. 8 a mass ratio of less than 2:1
(RNA:nona-arginine) appears to be advantageous. In contrast,
complexation with (high molecular mass) poly-L-arginine does not
lead to a significant luciferase-activity. Thus, (high molecular
mass) poly-L-arginine does not appear to be suitable for
transfection of mRNA.
[0256] FIG. 9: depicts in a comparative example the luciferase
expression upon transfection of complexes of RNA with
hepta-arginine ((Arg).sub.7) in HeLa cells. As may be derived from
FIG. 9, transfection of complexes of RNA with hepta-arginine
((Arg).sub.7) does not lead to a significant luciferase-activity.
Thus, hepta-arginine ((Arg).sub.7) does also not appear to be
suitable for transfection of mRNA.
[0257] FIG. 10: shows the immunostimulatory effect of RNA complexed
with hepta-arginine ((Arg).sub.7) in hPBMC cells by measuring IL-6
production. As can be seen, hPBMC cells show a significant IL-6
production, i.e. a significant immunostimulatory effect of RNA
complexed with hepta-arginine ((Arg).sub.7).
[0258] FIG. 11: shows the immunostimulatory effect of RNA complexed
with hepta-arginine ((Arg).sub.7) in hPBMC cells by measuring
TNF-alpha production. As can be seen, hPBMC cells show a
significant TNF-alpha production, i.e. a significant
immunostimulatory effect of RNA complexed with hepta-arginine
((Arg).sub.7).
[0259] FIG. 12: shows the effect of RNA complexed with R9 peptide
on the expression of luciferase in HeLa cells.
[0260] FIG. 13: shows the effect of RNA complexed with R9H3 peptide
on the expression of luciferase in HeLa cells.
[0261] FIG. 14: shows the effect of RNA complexed with H3R9H3
peptide on the expression of luciferase in HeLa cells.
[0262] FIG. 15: shows the effect of RNA complexed with YYYR9SSY
peptide on the expression of luciferase in HeLa cells.
[0263] FIG. 16: shows the effect of RNA complexed with H3R9SSY
peptide on the expression of luciferase in HeLa cells.
[0264] FIG. 17: shows the effect of RNA complexed with (RKH)4
peptide on the expression of luciferase in HeLa cells.
[0265] FIG. 18: shows the effect of RNA complexed with Y(RKH)2R
peptide on the expression of luciferase in HeLa cells.
[0266] FIG. 19: shows the effect of Histidin in terminal positions
on the transfection efficacy.
[0267] FIG. 20: shows the effect of neutral amino acids in terminal
positions on the transfection efficacy.
[0268] FIG. 21: shows the immunostimulatory effect of RNA complexed
with R9H3 on secretion of TNFalpha in hPBMCs.
[0269] FIG. 22: shows the immunostimulatory effect of RNA complexed
with R9H3 on secretion of IL-6 in hPBMCs.
EXAMPLES
[0270] The following examples are intended to illustrate the
invention further. They are not intended to limit the subject
matter of the invention thereto.
Example 1
Preparation of Luciferase mRNA Constructs
[0271] In the following experiments a stabilized luciferase mRNA
sequence was prepared and used for transfection experiments,
wherein the native luciferase encoding mRNA was modified with a
poly-A/poly-C-tag (A70-C30) and was GC-optimized for a better
codon-usage and further stabilized.
[0272] A first construct (construct CAP-Ppluc(wt)-muag-A70-C30, SEQ
ID NO: 35) contained following sequence elements: [0273]
stabilizing sequences from the alpha-Globin gene [0274] 70
(Adenosin at the 3'-terminal end [0275] 30 (Cytosin at the
3'-terminal end
[0276] The final construct (construct CAP-Ppluc(GC)-muag-A70-C30,
SEQ ID NO: 36), as used herein for the following experiments,
contained following sequence elements: [0277] GC-optimized sequence
for a better codon usage [0278] stabilizing sequences from the
alpha-Globin gene [0279] 70 (Adenosin at the 3'-terminal end [0280]
30 (Cytosin at the 3'-terminal end
[0281] These sequences are also shown in FIGS. 1 and 2 (SEQ ID NOs:
35 and 36). The respective coding sequences are shown in FIGS. 3
and 4 (SEQ ID NOs: 35 and 36)
Example 2
In Vitro Transcription of Stabilized Luciferase mRNA
[0282] The stabilized luciferase mRNA according to SEQ ID NO: 35 or
36 (Luc-RNActive) was transcribed in vitro using T7-Polymerase
(T7-Opti mRNA Kit, CureVac, Tubingen, Deutschland) following the
manufactures instructions.
[0283] All mRNA-transkripts contained a 70 bases poly-A-tail and a
5'-Cap-structure. The 5'-Cap-structure was obtained by adding an
excess of N7-Methyl-Guanosin-5'-Triphosphat-5'-Guanosin.
Example 3
Forming a Complex of RNA with Nona-Arginine ((Arg).sub.9),
poly-L-arginine or Further Peptides Based on (Arg).sub.9,
Respectively
[0284] 15 .mu.g RNA stabilized luciferase mRNA according to SEQ ID
NO: 36 (Luc-RNActive) were mixed in different mass ratios with
nona-arginine (Arg.sub.9) or poly-L-arginine (Sigma-Aldrich; P4663;
5000-15000 g/mol), thereby forming a complex. Following mass ratios
were used as shown exemplarily for ((Arg).sub.9). Poly-L-arginine
was used for comparative examples following the same
instructions.
TABLE-US-00007 (Arg).sub.9 (Arg).sub.9 RNA (Arg).sub.9 H.sub.20
Concentration Ratio RNA (Arg).sub.9 .mu.g .mu.g .mu.l .mu.l .mu.l
(Arg).sub.9 [.mu.M] (Arg).sub.9/RNA 1 Mock 70.0 0 2 (Arg).sub.9
alone 150 3 67.0 151.32 3 RNA alone 15 3.8 66.3 0.00 4 1 10 15
150.0 3.8 3.0 63.3 151.32 10:1 5 1 8 15 120.0 3.8 2.4 63.9 121.06
8:1 6 1 5 15 75.0 3.8 1.5 64.8 75.66 5:1 7 1 2 15 30.0 3.8 0.6 65.7
30.26 2:1 8 1 1 15 15.0 3.8 15.0 51.3 15.13 1:1 9 2 1 15 7.5 3.8
7.5 58.8 7.57 1:2 10 5 1 15 3.0 3.8 3.0 63.3 3.03 1:5 11 8 1 15 1.9
3.8 1.9 64.4 1.89 1:8 12 10 1 15 1.5 3.8 1.5 64.8 1.51 1:10
[0285] Additionally, further complexed RNAs based on (Arg).sub.9
were prepared above using the following peptides for
complexation:
TABLE-US-00008 R9: Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg R9H3:
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-His H3R9H3:
His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- His-His-His
YSSR9SSY: Tyr-Ser-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Ser-Ser-Tyr H3R9SSY:
His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr
(RKH)4: Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His Y(RKH)2R:
Tyr-Arg-Lys-His-Arg-Lys-His-Arg
[0286] For complexation, 4 .mu.g stabilized luciferase mRNA
according to SEQ ID NO: 36 (Luc-RNActive) were mixed in molar
ratios with the respectively peptide (according to formula I),
thereby forming a complex. Afterwards the resulting solution was
adjusted with water to a final volume of 50 .mu.l and incubated for
30 minutes at room temperature. The used ratios are indicated in
the tables given below. HeLa-cells (150.times.10.sup.3/well) were
then seeded 1 day prior to transfection on 24-well microtiter
plates leading to a 70% confluence when transfection was carried
out.
TABLE-US-00009 R9: Formulation -> N/P R9 Molar ratio Mass ratio
RNA R9 RNA .mu.g Peptid .mu.g N/P 1.00 10000.00 1.00 23.72 50.00
1.00 5000.00 1.00 11.86 25.00 1.00 2500.00 1.00 5.93 12.50 1.00
1000.00 1.00 2.37 5.00 1.00 500.00 1.00 1.19 2.50 1.00 100.00 1.00
0.24 0.50 1.00 10.00 1.00 0.02 0.05
TABLE-US-00010 R9H3: Formulation -> N/P R9H3 Molar ratio Mass
ratio RNA R9H3 RNA .mu.g Peptid .mu.g N/P 1.00 10000.00 1.00 30.58
50.00 1.00 5000.00 1.00 15.29 25.00 1.00 2500.00 1.00 7.65 12.50
1.00 1000.00 1.00 3.06 5.00 1.00 500.00 1.00 1.53 2.50 1.00 100.00
1.00 0.31 0.50 1.00 10.00 1.00 0.03 0.05
TABLE-US-00011 H3R9H3: Formulation -> N/P H3R9H3 Molar ratio
Mass ratio RNA H3R9H3 RNA .mu.g Peptid .mu.g N/P 1.00 10000.00 1.00
37.43 50.00 1.00 5000.00 1.00 18.72 25.00 1.00 2500.00 1.00 9.36
12.50 1.00 1000.00 1.00 3.74 5.00 1.00 500.00 1.00 1.87 2.50 1.00
100.00 1.00 0.37 0.50 1.00 10.00 1.00 0.04 0.05
TABLE-US-00012 YSSR9SSY: Formulation -> N/P YSSR9SSY Molar ratio
Mass ratio RNA YSSR9SSY RNA .mu.g Peptid .mu.g N/P 1.00 10000.00
1.00 34.95 50.00 1.00 5000.00 1.00 17.48 25.00 1.00 2500.00 1.00
8.74 12.50 1.00 1000.00 1.00 3.50 5.00 1.00 500.00 1.00 1.75 2.50
1.00 100.00 1.00 0.35 0.50 1.00 10.00 1.00 0.03 0.05
TABLE-US-00013 H3R9SSY: Formulation -> N/P H3R9SSY Molar ratio
Mass ratio RNA H3R9SSY RNA .mu.g Peptid .mu.g N/P 1.00 10000.00
1.00 36.18 50.00 1.00 5000.00 1.00 18.09 25.00 1.00 2500.00 1.00
9.05 12.50 1.00 1000.00 1.00 3.62 5.00 1.00 500.00 1.00 1.81 2.50
1.00 100.00 1.00 0.36 0.50 1.00 10.00 1.00 0.04 0.05
TABLE-US-00014 (RKH)4: Formulation -> N/P (RKH)4 Molar ratio
Mass ratio RNA (RKH)4 RNA .mu.g Peptid .mu.g N/P 1.00 10000.00 1.00
28.38 44.44 1.00 5000.00 1.00 14.19 22.22 1.00 2500.00 1.00 7.10
11.11 1.00 1000.00 1.00 2.84 4.44 1.00 500.00 1.00 1.42 2.22 1.00
100.00 1.00 0.28 0.44 1.00 10.00 1.00 0.03 0.04
TABLE-US-00015 Y(RKH)2R: Formulation -> N/P Y(RKH)2R Molar ratio
Mass ratio RNA Y(RKH)2R RNA .mu.g Peptid .mu.g N/P 1.00 10000.00
1.00 19.67 27.78 1.00 5000.00 1.00 9.83 13.89 1.00 2500.00 1.00
4.92 6.94 1.00 1000.00 1.00 1.97 2.78 1.00 500.00 1.00 0.98 1.39
1.00 100.00 1.00 0.20 0.28 1.00 10.00 1.00 0.02 0.03
Example 4
Nona-arginine((Arg).sub.9)-mediated Transfection and Expression of
Stabilized Luciferase mRNA According to SEQ ID NO: 35 or 36
(Luc-RNActive) in HeLa-cells
[0287] Hela-cells (150.times.10.sup.3/well) were seeded 1 day prior
to transfection on 24-well microtiter plates leading to a 70%
confluence when transfection was carried out. For transfection (40
.mu.l) 50 .mu.l of the RNA/(peptide)-solution as disclosed in
Example 3 were mixed with 250 .mu.l serum free medium and added to
the cells (final RNA concentration: 13 .mu.g/ml). Prior to addition
of the transfection solution the HeLa-cells were washed gently and
carefully 2 times with 1 ml Optimen (Invitrogen) per well. Then,
the transfection solution (300 .mu.l per well) was added to the
cells and the cells were incubated for 4 h at 37.degree. C.
Subsequently 300 .mu.l RPMI-medium (Camprex) containing 10% FCS was
added per well and the cells were incubated for additional 20 h at
37.degree. C. The transfection solution was sucked off 24 h after
transfection and the cells were lysed in 300 .mu.l lysis buffer (25
mM Tris-PO.sub.4, 2 mM EDTA, 10% glycerol, 1% Triton-X 100, 2 mM
DTT). The supernatants were then mixed with luciferin buffer (25 mM
Glycylglycin, 15 mM MgSO.sub.4, 5 mM ATP, 62.5 .mu.M luciferin) and
luminiscence was detected using a luminometer (Lumat LB 9507
(Berthold Technologies, Bad Wildbad, Germany)). The results of
these experiments are shown in FIGS. 8 and 12 to 18.
Example 5
Immune Stimulation Upon Transfection of Complexes of RNA with
Nona-Arginine ((Arg).sub.9) or poly-L-arginine (Comparative
Example)
a) Transfection Experiments
[0288] HPBMC cells from peripheral blood of healthy donors were
isolated using a Ficoll gradient and washed subsequently with
1.times.PBS (phosphate-buffered saline). The cells were then seeded
on 96-well microtiter plates (200.times.10.sup.3/well). The hPBMC
cells were incubated for 24 h, as described under Example 4, supra,
with 10 .mu.l of the RNA/peptide complex (RNA final concentration:
6 .mu.g/ml; the same amounts of RNA were used) in X-VIVO 15 Medium
(BioWhittaker) (final RNA Concentration: 10 .mu.g/ml). The
immunostimulatory effect upon the hPBMC cells was measured by
detecting the cytokine production (Interleukin-6 and Tumor necrose
factor alpha). Therefore, ELISA microtiter plates (Nunc Maxisorb)
were incubated over night (o/n) with binding buffer (0.02% NaN3, 15
mM Na2CO3, 15 mM NaHCO3, pH 9.7), additionally containing a
specific cytokine antibody. Cells were then blocked with
1.times.PBS, containing 1% BSA (bovine serum albumin). The cell
supernatant was added and incubated for 4 h at 37.degree. C.
Subsequently, the microtiter plate was washed with 1.times.PBS,
0.05% Tween-20 and then incubated with a Biotin-labelled secondary
antibody (BD Pharmingen, Heidelberg, Germany). Streptavidin-coupled
horseraddish peroxidase was added to the plate. Then, the plate was
again washed with 1.times.PBS, containing 0.05% Tween-20, and ABTS
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added
as a substrate. The amount of cytokine was determined by measuring
the absorption at 405 nm (OD405) using a standard curve with
recombinant Cytokines (BD Pharmingen, Heidelberg, Germany) with the
Sunrise ELISA-Reader from Tecan (Crailsheim, Germany).
b) Results
[0288] [0289] i) Immunostimulatory effect of RNA complexed with
nona-arginine ((Arg).sub.9) [0290] i1) HPBMC cells were incubated
with RNA complexed with nona-arginine ((Arg).sub.9) for 24 h as
disclosed above, wherein the mass ratio of RNA:(Arg).sub.9 was 1:1.
Then, IL-6 production was measured in the cell supernatants using
ELISA. As a result, HPBMC cells showed a significant IL-6
production, i.e. a significant immunostimulatory effect of RNA
complexed with nona-arginine ((Arg).sub.9) (see FIG. 5). [0291] i2)
HPBMC cells were incubated with RNA complexed with nona-arginine
((Arg).sub.9) for 24 h as disclosed above, wherein the mass ratio
of RNA:(Arg).sub.9 was 1:1. Then, THF-alpha production was measured
in the cell supernatants using ELISA. As a result, HPBMC cells
showed a significant TNF-alpha production, i.e. a significant
immunostimulatory effect of RNA complexed with nona-arginine
((Arg).sub.9) (see FIG. 6). [0292] ii) Comparison of
immunostimulatory effect of RNA complexed with either nona-arginine
((Arg).sub.9) or poly-L-arginine, respectively (Comparative
Example) [0293] hPBMCs were incubated in different mass ratios
(RNA:nona-arginine 1:10; 1:8; 1:5; 1:2; 1:1; 2:1, 5:1; 8:1 and
10:1) with a complex of RNA and nona-arginine ((Arg).sub.9) or
poly-L-arginine, etc., respectively, for 24 h. Subsequently
TNF-alpha production was measured using ELISA. [0294]
Advantageously, a significant immunostimulatory effect can be
observed for mass ratios lower than 5:1 (RNA:nona-arginine) (1:10;
1:8; 1:5; 1:2; 1:1; 2:1) (see FIG. 7). When using mass ratios of
RNA:nona-arginine (5:1) no significant TNFalpha production can be
observed. The same applies to stimulation experiments, using
nona-arginine ((Arg).sub.9) or mRNA alone (see FIG. 7, left).
[0295] Furthermore, complexation of mRNA with poly-L-arginine leads
to significantly lower induction of TNF-alpha production in
comparison to nona-arginine ((Arg).sub.9) (see FIG. 7, right).
Additionally, it was observed that higher concentrations of
poly-L-arginine appear to be toxic for cells transfected therewith,
particularly when using a mass ratio of 1:2 RNA:poly-L-arginine or
lower, since the cells were lysed.
Example 6
Luciferase Expression Upon Transfection of Complexes of RNA with
Nona-Arginine ((Arg).sub.9) or Poly-L-Arginine, Respectively, in
HeLa Cells (Comparative Example)
[0295] [0296] a) Luciferase expression upon transfection of
complexes of RNA with nona-arginine ((Arg).sub.9) in HeLa cells.
HeLa-Cells were transfected with RNActive encoding luciferase,
which has been complexed with different ratios of nona-arginine or
Poly-L-Arginine, respectively. 24 h later luciferase-activity was
measured. Apparently, a mass ration of less than 2:1
(RNA:nona-arginine) appears to be advantageous (see FIG. 8). [0297]
b) In comparison, complexation with (high molecular mass)
poly-L-arginine does not increase luciferase-activity at a
significant level. Thus, (high molecular mass) poly-L-arginine does
not appear to be suitable for transfection of mRNA (see FIG.
8).
Example 7
Luciferase Expression Upon Transfection of Complexes of RNA with
Hepta-Arginine ((Arg).sub.7) in HeLa Cells (Comparative
Example)
[0298] HeLa-Cells were transfected with RNActive encoding
luciferase, which has been complexed with different ratios of
hepta-arginine ((Arg).sub.7). 24 h later luciferase-activity was
measured. Apparently, complexation with hepta-arginine
((Arg).sub.7) does not increase luciferase-activity at a
significant level. Thus, hepta-arginine ((Arg).sub.7) does not
appear to be suitable for transfection of mRNA (see FIG. 9).
Example 8
Immune Stimulation Upon Transfection of Complexes of RNA with
Hepta-Arginine ((Arg).sub.7) (Comparative Example)
a) Transfection Experiments
[0299] Transfection Experiments were carried out for hepta-arginine
((Arg).sub.7) analogously to the experiments in Example 5 as shown
above. b) Results of Immunostimulatory Effect of RNA Complexed with
hepta-arginine ((Arg).sub.7) [0300] i) HPBMC cells were incubated
with RNA complexed with hepta-arginine ((Arg).sub.7) for 24 h as
disclosed above, wherein the mass ratio of RNA:(Arg).sub.7 was 1:1.
Then, IL-6 production was measured in the cell supernatants using
ELISA. As a result, HPBMC cells showed a significant IL-6
production, i.e. a significant immunostimulatory effect of RNA
complexed with hepta-arginine ((Arg).sub.7) (see FIG. 10). [0301]
ii) HPBMC cells were furthermore incubated with RNA complexed with
hepta-arginine ((Arg).sub.7) for 24 h as disclosed above, wherein
the mass ratio of RNA:(Arg).sub.7 was 1:1. Then, THF-alpha
production was measured in the cell supernatants using ELISA. As a
result, HPBMC cells also showed a significant TNF-alpha production,
i.e. a significant immunostimulatory effect of RNA complexed with
hepta-arginine ((Arg).sub.7) (see FIG. 11).
Example 9
Determination of the Effect of Histidin on the Transfection
Efficiency
[0302] To determine the effect of Histidin on the transfection
efficiency a transfection was carried out analogously to the
transfection experiments above using peptides with different
Histidine content. Therefore, 4 .mu.g stabilized luciferase mRNA
according to SEQ ID NO: 36 (Luc-RNActive) were mixed in molar
ratios with the respectively peptide (according to formula I),
particularly R9, R9H3 or H3R9H3, thereby forming a complex.
Afterwards the resulting solution was adjusted with water to a
final volume of 50 .mu.l and incubated for 30 minutes at room
temperature. The used ratios are in each experiment 1:10000, 1:5000
and 1:1000. HeLa-cells (150.times.10.sup.3/well) were then seeded 1
day prior to transfection on 24-well microtiter plates leading to a
70% confluence when transfection was carried out. For transfection
50 .mu.l of the RNA/(peptide)-solution were mixed with 250 .mu.l
serum free medium and added to the cells (final RNA concentration:
13 .mu.g/ml). Prior to addition of the transfection solution the
HeLa-cells were washed gently and carefully 2 times with 1 ml
Optimen (Invitrogen) per well. Then, the transfection solution (300
.mu.l per well) was added to the cells and the cells were incubated
for 4 h at 37.degree. C. Subsequently 300 .mu.l RPMI-medium
(Camprex) containing 10% FCS was added per well and the cells were
incubated for additional 20 h at 37.degree. C. The transfection
solution was sucked off 24 h after transfection and the cells were
lysed in 300 .mu.l lysis buffer (25 mM Tris-PO.sub.4, 2 mM EDTA,
10% glycerol, 1% Triton-X 100, 2 mM DTT). The supernatants were
then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM
MgSO.sub.4, 5 mM ATP, 62.5 .mu.M luciferin) and luminiscence was
detected using a luminometer (Lumat LB 9507 (Berthold Technologies,
Bad Wildbad, Germany)).
[0303] The results are shown in FIG. 19. As can be seen, a stretch
of 3 histidines at one terminal end already increases the
transfection efficacy of the complexed RNA, wherein a stretch of 3
histidines at both terminal ends significantly increases the
transfection efficacy of the complexed RNA.
Example 10
Determination of the Effect of Neutral Amino Acids on the
Transfection Efficiency
[0304] To determine the effect of neutral amino acids on the
transfection efficiency a further transfection experiment was
carried out analogously to the transfection experiments above in
Example 9 using the peptide H3R9CCS. The results of theis
additional experiment are shown in FIG. 20.
Example 11
Immunostimulation Using R9H3 in hPBMCs
[0305] The effect of R9H3 on immunostimulation was tested in
hPBMCs. Therefore, a complex of R9H3 and RNA as shown above in
Example 3 was prepared. Furthermore, HPBMC cells from peripheral
blood of healthy donors were isolated using a Ficoll gradient and
washed subsequently with 1.times.PBS (phophate-buffered saline).
The cells were then seeded on 96-well microtiter plates
(200.times.10.sup.3/well). The hPBMC cells were incubated for 24 h,
as described under Example 4, supra, with 10 .mu.l of the
RNA/peptide complex (RNA final concentration: 6 .mu.g/ml; the same
amounts of RNA were used) in X-VIVO 15 Medium (BioWhittaker). The
immunostimulatory effect upon the hPBMC cells was measured by
detecting the cytokine production (Interleukin-6 and Tumor necrose
factor alpha). Therefore, ELISA microtiter plates (Nunc Maxisorb)
were incubated over night (o/n) with binding buffer (0.02% NaN3, 15
mM Na2CO3, 15 mM NaHCO3, pH 9.7), additionally containing a
specific cytokine antibody. Cells were then blocked with
1.times.PBS, containing 1% BSA (bovine serum albumin). The cell
supernatant was added and incubated for 4 h at 37.degree. C.
Subsequently, the microtiter plate was washed with 1.times.PBS,
0.05% Tween-20 and then incubated with a Biotin-labelled secondary
antibody (BD Pharmingen, Heidelberg, Germany). Streptavidin-coupled
horseraddish peroxidase was added to the plate. Then, the plate was
again washed with 1.times.PBS, containing 0.05% Tween-20, and ABTS
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added
as a substrate. The amount of cytokine was determined by measuring
the absorption at 405 nm (OD405) using a standard curve with
recombinant Cytokines (BD Pharmingen, Heidelberg, Germany) with the
Sunrise
[0306] ELISA-Reader from Tecan (Crailsheim, Germany). The results
are seen in FIGS. 21 and 22. As can be seen, a significant
immunostimulation was exhibited at a ratio of 1:5000 RNA:R9H3.
Sequence CWU 1
1
4418PRTArtificial SequenceExemplary oligopeptide according to
formula I 1Arg Arg Arg Arg Arg Arg Arg Arg1 529PRTArtificial
SequenceExemplary oligopeptide according to formula I 2Arg Arg Arg
Arg Arg Arg Arg Arg Arg1 5310PRTArtificial SequenceExemplary
oligopeptide according to formula I 3Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg1 5 10411PRTArtificial SequenceExemplary oligopeptide
according to formula I 4Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg1 5 10512PRTArtificial SequenceExemplary oligopeptide according
to formula I 5Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg1 5
10613PRTArtificial SequenceExemplary oligopeptide according to
formula I 6Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg1 5
10714PRTArtificial SequenceExemplary oligopeptide according to
formula I 7Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg1
5 10815PRTArtificial SequenceExemplary oligopeptide according to
formula I 8Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg1 5 10 1598PRTArtificial SequenceExemplary oligopeptide
according to formula I 9Lys Lys Lys Lys Lys Lys Lys Lys1
5109PRTArtificial SequenceExemplary oligopeptide according to
formula I 10Lys Lys Lys Lys Lys Lys Lys Lys Lys1 51110PRTArtificial
SequenceExemplary oligopeptide according to formula I 11Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys1 5 101211PRTArtificial
SequenceExemplary oligopeptide according to formula I 12Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys1 5 101312PRTArtificial
SequenceExemplary oligopeptide according to formula I 13Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 101413PRTArtificial
SequenceExemplary oligopeptide according to formula I 14Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 101514PRTArtificial
SequenceExemplary oligopeptide according to formula I 15Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 101615PRTArtificial
SequenceExemplary oligopeptide according to formula I 16Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10
15178PRTArtificial SequenceExemplary oligopeptide according to
formula I 17His His His His His His His His1 5189PRTArtificial
SequenceExemplary oligopeptide according to formula I 18His His His
His His His His His His1 51910PRTArtificial SequenceExemplary
oligopeptide according to formula I 19His His His His His His His
His His His1 5 102011PRTArtificial SequenceExemplary oligopeptide
according to formula I 20His His His His His His His His His His
His1 5 102112PRTArtificial SequenceExemplary oligopeptide according
to formula I 21His His His His His His His His His His His His1 5
102213PRTArtificial SequenceExemplary oligopeptide according to
formula I 22His His His His His His His His His His His His His1 5
102314PRTArtificial SequenceExemplary oligopeptide according to
formula I 23His His His His His His His His His His His His His
His1 5 102415PRTArtificial SequenceExemplary oligopeptide according
to formula I 24His His His His His His His His His His His His His
His His1 5 10 15258PRTArtificial SequenceExemplary oligopeptide
according to formula I 25Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5269PRTArtificial SequenceExemplary oligopeptide according to
formula I 26Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 52710PRTArtificial
SequenceExemplary oligopeptide according to formula I 27Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 102811PRTArtificial
SequenceExemplary oligopeptide according to formula I 28Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 102912PRTArtificial
SequenceExemplary oligopeptide according to formula I 29Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 103013PRTArtificial
SequenceExemplary oligopeptide according to formula I 30Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 103114PRTArtificial
SequenceExemplary oligopeptide according to formula I 31Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 103215PRTArtificial
SequenceExemplary oligopeptide according to formula I 32Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
153313RNAArtificial SequenceDescription of Artificial Sequence
Kozak- Sequence 33gccgccacca ugg 133413RNAArtificial
SequenceDescription of Artificial Sequence Example of a stabilizing
sequence which is contained in the 3 prime UTR of the very stable
RNA which codes for alpha-globin, alpha-(I)- collagen,
15-lipoxygenase or tyrosine hydroxylase 34nccacccnuc ncc
13351882RNAArtificial SequenceDescription of Artificial Sequence
construct CAP-Ppluc(wt)-muag-A70-C30 35gggagaaagc uuggcauucc
gguacuguug guaaagccac cauggaagac gccaaaaaca 60uaaagaaagg cccggcgcca
uucuauccgc uggaagaugg aaccgcugga gagcaacugc 120auaaggcuau
gaagagauac gcccugguuc cuggaacaau ugcuuuuaca gaugcacaua
180ucgaggugga caucacuuac gcugaguacu ucgaaauguc cguucgguug
gcagaagcua 240ugaaacgaua ugggcugaau acaaaucaca gaaucgucgu
augcagugaa aacucucuuc 300aauucuuuau gccgguguug ggcgcguuau
uuaucggagu ugcaguugcg cccgcgaacg 360acauuuauaa ugaacgugaa
uugcucaaca guaugggcau uucgcagccu accguggugu 420ucguuuccaa
aaagggguug caaaaaauuu ugaacgugca aaaaaagcuc ccaaucaucc
480aaaaaauuau uaucauggau ucuaaaacgg auuaccaggg auuucagucg
auguacacgu 540ucgucacauc ucaucuaccu cccgguuuua augaauacga
uuuugugcca gaguccuucg 600auagggacaa gacaauugca cugaucauga
acuccucugg aucuacuggu cugccuaaag 660gugucgcucu gccucauaga
acugccugcg ugagauucuc gcaugccaga gauccuauuu 720uuggcaauca
aaucauuccg gauacugcga uuuuaagugu uguuccauuc caucacgguu
780uuggaauguu uacuacacuc ggauauuuga uauguggauu ucgagucguc
uuaauguaua 840gauuugaaga agagcuguuu cugaggagcc uucaggauua
caagauucaa agugcgcugc 900uggugccaac ccuauucucc uucuucgcca
aaagcacucu gauugacaaa uacgauuuau 960cuaauuuaca cgaaauugcu
ucugguggcg cuccccucuc uaaggaaguc ggggaagcgg 1020uugccaagag
guuccaucug ccagguauca ggcaaggaua ugggcucacu gagacuacau
1080cagcuauucu gauuacaccc gagggggaug auaaaccggg cgcggucggu
aaaguuguuc 1140cauuuuuuga agcgaagguu guggaucugg auaccgggaa
aacgcugggc guuaaucaaa 1200gaggcgaacu gugugugaga gguccuauga
uuauguccgg uuauguaaac aauccggaag 1260cgaccaacgc cuugauugac
aaggauggau ggcuacauuc uggagacaua gcuuacuggg 1320acgaagacga
acacuucuuc aucguugacc gccugaaguc ucugauuaag uacaaaggcu
1380aucagguggc ucccgcugaa uuggaaucca ucuugcucca acaccccaac
aucuucgacg 1440caggugucgc aggucuuccc gacgaugacg ccggugaacu
ucccgccgcc guuguuguuu 1500uggagcacgg aaagacgaug acggaaaaag
agaucgugga uuacgucgcc agucaaguaa 1560caaccgcgaa aaaguugcgc
ggaggaguug uguuugugga cgaaguaccg aaaggucuua 1620ccggaaaacu
cgacgcaaga aaaaucagag agauccucau aaaggccaag aagggcggaa
1680agaucgccgu guaauucuag uuauaagacu gacuagcccg augggccucc
caacgggccc 1740uccuccccuc cuugcaccga gauuaauaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
auauuccccc cccccccccc cccccccccc 1860cccccucuag acaauuggaa uu
1882361857RNAArtificial SequenceDescription of Artificial Sequence
construct CAP-Ppluc(GC)-muag-A70-C30 36gggagaaagc uugaggaugg
aggacgccaa gaacaucaag aagggcccgg cgcccuucua 60cccgcuggag gacgggaccg
ccggcgagca gcuccacaag gccaugaagc gguacgcccu 120ggugccgggc
acgaucgccu ucaccgacgc ccacaucgag gucgacauca ccuacgcgga
180guacuucgag augagcgugc gccuggccga ggccaugaag cgguacggcc
ugaacaccaa 240ccaccggauc guggugugcu cggagaacag ccugcaguuc
uucaugccgg ugcugggcgc 300ccucuucauc ggcguggccg ucgccccggc
gaacgacauc uacaacgagc gggagcugcu 360gaacagcaug gggaucagcc
agccgaccgu gguguucgug agcaagaagg gccugcagaa 420gauccugaac
gugcagaaga agcugcccau cauccagaag aucaucauca uggacagcaa
480gaccgacuac cagggcuucc agucgaugua cacguucgug accagccacc
ucccgccggg 540cuucaacgag uacgacuucg ucccggagag cuucgaccgg
gacaagacca ucgcccugau 600caugaacagc agcggcagca ccggccugcc
gaagggggug gcccugccgc accggaccgc 660cugcgugcgc uucucgcacg
cccgggaccc caucuucggc aaccagauca ucccggacac 720cgccauccug
agcguggugc cguuccacca cggcuucggc auguucacga cccugggcua
780ccucaucugc ggcuuccggg ugguccugau guaccgguuc gaggaggagc
uguuccugcg 840gagccugcag gacuacaaga uccagagcgc gcugcucgug
ccgacccugu ucagcuucuu 900cgccaagagc acccugaucg acaaguacga
ccugucgaac cugcacgaga ucgccagcgg 960gggcgccccg cugagcaagg
aggugggcga ggccguggcc aagcgguucc accucccggg 1020cauccgccag
ggcuacggcc ugaccgagac cacgagcgcg auccugauca cccccgaggg
1080ggacgacaag ccgggcgccg ugggcaaggu ggucccguuc uucgaggcca
agguggugga 1140ccuggacacc ggcaagaccc ugggcgugaa ccagcggggc
gagcugugcg ugcgggggcc 1200gaugaucaug agcggcuacg ugaacaaccc
ggaggccacc aacgcccuca ucgacaagga 1260cggcuggcug cacagcggcg
acaucgccua cugggacgag gacgagcacu ucuucaucgu 1320cgaccggcug
aagucgcuga ucaaguacaa gggcuaccag guggcgccgg ccgagcugga
1380gagcauccug cuccagcacc ccaacaucuu cgacgccggc guggccgggc
ugccggacga 1440cgacgccggc gagcugccgg ccgcgguggu ggugcuggag
cacggcaaga ccaugacgga 1500gaaggagauc gucgacuacg uggccagcca
ggugaccacc gccaagaagc ugcggggcgg 1560cgugguguuc guggacgagg
ucccgaaggg ccugaccggg aagcucgacg cccggaagau 1620ccgcgagauc
cugaucaagg ccaagaaggg cggcaagauc gccguguaag acuaguuaua
1680agacugacua gcccgauggg ccucccaacg ggcccuccuc cccuccuugc
accgagauua 1740auaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1800aaaaaauauu cccccccccc cccccccccc
cccccccccc ucuagacaau uggaauu 1857371653RNAArtificial
SequenceDescription of Artificial Sequence coding sequence of
construct CAP-Ppluc(wt)-muag-A70-C30 (the sequence according to SEQ
ID NO 35) 37auggaagacg ccaaaaacau aaagaaaggc ccggcgccau ucuauccgcu
ggaagaugga 60accgcuggag agcaacugca uaaggcuaug aagagauacg cccugguucc
uggaacaauu 120gcuuuuacag augcacauau cgagguggac aucacuuacg
cugaguacuu cgaaaugucc 180guucgguugg cagaagcuau gaaacgauau
gggcugaaua caaaucacag aaucgucgua 240ugcagugaaa acucucuuca
auucuuuaug ccgguguugg gcgcguuauu uaucggaguu 300gcaguugcgc
ccgcgaacga cauuuauaau gaacgugaau ugcucaacag uaugggcauu
360ucgcagccua ccgugguguu cguuuccaaa aagggguugc aaaaaauuuu
gaacgugcaa 420aaaaagcucc caaucaucca aaaaauuauu aucauggauu
cuaaaacgga uuaccaggga 480uuucagucga uguacacguu cgucacaucu
caucuaccuc ccgguuuuaa ugaauacgau 540uuugugccag aguccuucga
uagggacaag acaauugcac ugaucaugaa cuccucugga 600ucuacugguc
ugccuaaagg ugucgcucug ccucauagaa cugccugcgu gagauucucg
660caugccagag auccuauuuu uggcaaucaa aucauuccgg auacugcgau
uuuaaguguu 720guuccauucc aucacgguuu uggaauguuu acuacacucg
gauauuugau auguggauuu 780cgagucgucu uaauguauag auuugaagaa
gagcuguuuc ugaggagccu ucaggauuac 840aagauucaaa gugcgcugcu
ggugccaacc cuauucuccu ucuucgccaa aagcacucug 900auugacaaau
acgauuuauc uaauuuacac gaaauugcuu cugguggcgc uccccucucu
960aaggaagucg gggaagcggu ugccaagagg uuccaucugc cagguaucag
gcaaggauau 1020gggcucacug agacuacauc agcuauucug auuacacccg
agggggauga uaaaccgggc 1080gcggucggua aaguuguucc auuuuuugaa
gcgaagguug uggaucugga uaccgggaaa 1140acgcugggcg uuaaucaaag
aggcgaacug ugugugagag guccuaugau uauguccggu 1200uauguaaaca
auccggaagc gaccaacgcc uugauugaca aggauggaug gcuacauucu
1260ggagacauag cuuacuggga cgaagacgaa cacuucuuca ucguugaccg
ccugaagucu 1320cugauuaagu acaaaggcua ucagguggcu cccgcugaau
uggaauccau cuugcuccaa 1380caccccaaca ucuucgacgc aggugucgca
ggucuucccg acgaugacgc cggugaacuu 1440cccgccgccg uuguuguuuu
ggagcacgga aagacgauga cggaaaaaga gaucguggau 1500uacgucgcca
gucaaguaac aaccgcgaaa aaguugcgcg gaggaguugu guuuguggac
1560gaaguaccga aaggucuuac cggaaaacuc gacgcaagaa aaaucagaga
gauccucaua 1620aaggccaaga agggcggaaa gaucgccgug uaa
1653381653RNAArtificial SequenceDescription of Artifical Sequence
the GC-optimized coding sequence of construct CAP-Ppluc(GC)-muag-
A70-C30 (the sequence according to SEQ ID NO 36) 38auggaggacg
ccaagaacau caagaagggc ccggcgcccu ucuacccgcu ggaggacggg 60accgccggcg
agcagcucca caaggccaug aagcgguacg cccuggugcc gggcacgauc
120gccuucaccg acgcccacau cgaggucgac aucaccuacg cggaguacuu
cgagaugagc 180gugcgccugg ccgaggccau gaagcgguac ggccugaaca
ccaaccaccg gaucguggug 240ugcucggaga acagccugca guucuucaug
ccggugcugg gcgcccucuu caucggcgug 300gccgucgccc cggcgaacga
caucuacaac gagcgggagc ugcugaacag cauggggauc 360agccagccga
ccgugguguu cgugagcaag aagggccugc agaagauccu gaacgugcag
420aagaagcugc ccaucaucca gaagaucauc aucauggaca gcaagaccga
cuaccagggc 480uuccagucga uguacacguu cgugaccagc caccucccgc
cgggcuucaa cgaguacgac 540uucgucccgg agagcuucga ccgggacaag
accaucgccc ugaucaugaa cagcagcggc 600agcaccggcc ugccgaaggg
gguggcccug ccgcaccgga ccgccugcgu gcgcuucucg 660cacgcccggg
accccaucuu cggcaaccag aucaucccgg acaccgccau ccugagcgug
720gugccguucc accacggcuu cggcauguuc acgacccugg gcuaccucau
cugcggcuuc 780cggguggucc ugauguaccg guucgaggag gagcuguucc
ugcggagccu gcaggacuac 840aagauccaga gcgcgcugcu cgugccgacc
cuguucagcu ucuucgccaa gagcacccug 900aucgacaagu acgaccuguc
gaaccugcac gagaucgcca gcgggggcgc cccgcugagc 960aaggaggugg
gcgaggccgu ggccaagcgg uuccaccucc cgggcauccg ccagggcuac
1020ggccugaccg agaccacgag cgcgauccug aucacccccg agggggacga
caagccgggc 1080gccgugggca aggugguccc guucuucgag gccaaggugg
uggaccugga caccggcaag 1140acccugggcg ugaaccagcg gggcgagcug
ugcgugcggg ggccgaugau caugagcggc 1200uacgugaaca acccggaggc
caccaacgcc cucaucgaca aggacggcug gcugcacagc 1260ggcgacaucg
ccuacuggga cgaggacgag cacuucuuca ucgucgaccg gcugaagucg
1320cugaucaagu acaagggcua ccagguggcg ccggccgagc uggagagcau
ccugcuccag 1380caccccaaca ucuucgacgc cggcguggcc gggcugccgg
acgacgacgc cggcgagcug 1440ccggccgcgg ugguggugcu ggagcacggc
aagaccauga cggagaagga gaucgucgac 1500uacguggcca gccaggugac
caccgccaag aagcugcggg gcggcguggu guucguggac 1560gaggucccga
agggccugac cgggaagcuc gacgcccgga agauccgcga gauccugauc
1620aaggccaaga agggcggcaa gaucgccgug uaa 16533912PRTArtificial
SequenceDescription of Artificial Sequence exemplary oligopeptide
according to generic formula (I) 39Arg Arg Arg Arg Arg Arg Arg Arg
Arg His His His1 5 104015PRTArtificial SequenceDescription of
Artificial Sequence exemplary oligopeptide according to generic
formula (I) 40His His His Arg Arg Arg Arg Arg Arg Arg Arg Arg His
His His1 5 10 154115PRTArtificial SequenceDescription of Artificial
Sequence exemplary oligopeptide according to generic formula (I)
41Tyr Ser Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg Ser Ser Tyr1 5 10
154215PRTArtificial SequenceDescription of Artificial Sequence
exemplary oligopeptide according to generic formula (I) 42His His
His Arg Arg Arg Arg Arg Arg Arg Arg Arg Ser Ser Tyr1 5 10
154312PRTArtificial SequenceDescription of Artificial Sequence
exemplary oligopeptide according to generic formula (I) 43Arg Lys
His Arg Lys His Arg Lys His Arg Lys His1 5 10448PRTArtificial
SequenceDescription of Artificial Sequence exemplary oligopeptide
according to generic formula (I) 44Tyr Arg Lys His Arg Lys His Arg1
5
* * * * *
References