U.S. patent application number 17/393253 was filed with the patent office on 2021-12-23 for nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen.
This patent application is currently assigned to CureVac AG. The applicant listed for this patent is CureVac AG. Invention is credited to Jochen PROBST, Thomas SCHLAKE, Andreas THESS.
Application Number | 20210393755 17/393253 |
Document ID | / |
Family ID | 1000005813213 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210393755 |
Kind Code |
A1 |
THESS; Andreas ; et
al. |
December 23, 2021 |
NUCLEIC ACID COMPRISING OR CODING FOR A HISTONE STEM-LOOP AND A
POLY(A) SEQUENCE OR A POLYADENYLATION SIGNAL FOR INCREASING THE
EXPRESSION OF AN ENCODED TUMOUR ANTIGEN
Abstract
The present invention relates to a nucleic acid sequence,
comprising or coding for a coding region, encoding at least one
peptide or protein comprising a tumour antigen or a fragment,
variant or derivative thereof, at least one histone stem-loop and a
poly(A) sequence or a polyadenylation signal. Furthermore the
present invention provides the use of the nucleic acid for
increasing the expression of said encoded peptide or protein. It
also discloses its use for the preparation of a pharmaceutical
composition, especially a vaccine, e.g. for use in the treatment of
cancer or tumour diseases. The present invention further describes
a method for increasing the expression of a peptide or protein
comprising a tumour antigen or a fragment, variant or derivative
thereof, using the nucleic acid comprising or coding for a histone
stem-loop and a poly(A) sequence or a polyadenylation signal.
Inventors: |
THESS; Andreas;
(Kusterdingen, DE) ; SCHLAKE; Thomas;
(Gundelfingen, DE) ; PROBST; Jochen;
(Wolfschlugen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CureVac AG |
Tubingen |
|
DE |
|
|
Assignee: |
CureVac AG
Tubingen
DE
|
Family ID: |
1000005813213 |
Appl. No.: |
17/393253 |
Filed: |
August 3, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16002695 |
Jun 7, 2018 |
11110156 |
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17393253 |
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14378572 |
Aug 13, 2014 |
10010592 |
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PCT/EP2013/000459 |
Feb 15, 2013 |
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16002695 |
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PCT/EP2012/000674 |
Feb 15, 2012 |
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14378572 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001186 20180801;
A61K 39/001153 20180801; A61K 38/00 20130101; A61K 2039/64
20130101; A61K 39/001193 20180801; A61K 39/001156 20180801; A61K
39/001113 20180801; A61K 39/001122 20180801; A61K 39/001106
20180801; A61K 39/001124 20180801; C12N 15/63 20130101; A61K
39/001134 20180801; A61K 39/0011 20130101; A61K 39/001176 20180801;
A61K 39/001162 20180801; A61K 39/001112 20180801; C07K 14/47
20130101; A61K 39/001191 20180801; A61K 39/001132 20180801; A61K
39/001182 20180801; A61K 39/001189 20180801; A61K 39/001129
20180801; C07K 14/4748 20130101; A61K 39/001195 20180801; A61K
39/001168 20180801; A61K 39/001158 20180801; A61K 39/001197
20180801; A61K 39/001119 20180801; A61K 39/001109 20180801; A61K
39/001151 20180801; A61K 39/001139 20180801; A61K 39/001104
20180801; A61K 39/001188 20180801; A61K 39/001157 20180801; A61K
39/00115 20180801; C12N 15/67 20130101; A61K 39/001184 20180801;
A61K 39/001194 20180801; A61K 2039/53 20130101; A61K 39/001149
20180801; C12N 2830/50 20130101; C07K 14/4703 20130101; A61K
39/001192 20180801; A61K 39/001166 20180801; A61K 39/00117
20180801; A61K 39/001164 20180801 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/47 20060101 C07K014/47; C12N 15/67 20060101
C12N015/67; C12N 15/63 20060101 C12N015/63 |
Claims
1. Nucleic acid sequence comprising or coding for a) a coding
region, encoding at least one peptide or protein; b) at least one
histone stem-loop, and c) a poly(A) sequence or a polyadenylation
signal; wherein said peptide or protein comprises a tumour antigen
a fragment, variant or derivative of said tumour antigen.
2. The nucleic acid sequence according to claim 1, wherein the
tumour antigen is a melanocyte-specific antigen, a cancer-testis
antigen or a tumour-specific antigen, preferably a CT-X antigen, a
non-X CT-antigen, a binding partner for a CT-X antigen or a binding
partner for a non-X CT-antigen or a tumour-specific antigen, more
preferably a CT-X antigen, a binding partner for a non-X CT-antigen
or a tumour-specific antigen or a fragment, variant or derivative
of said tumour antigen.
3. The nucleic acid sequence according to claim 1 or claim 2,
wherein the tumour antigen is selected from the list of: 5T4,
707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin,
alpha-5-beta-6-integrin, alpha-actinin-4/m,
alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,
BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA
15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL,
CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30,
CD33, CD4, CD52, CD55, CD56, CD80, CDCl.sub.27/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-R17I, 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-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, PAP, PART-1, PATE, PDEF,
Pim-1-Kinase, Pin-1, Pml/PARalpha, 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-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2,
TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m,
TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA,
VEGFR1, VEGFR-2/FLK-1, WT1 and a immunoglobulin idiotype of a
lymphoid blood cell or a T cell receptor idiotype of a lymphoid
blood cell, or a fragment, variant or derivative of said tumour
antigen; preferably survivin or a homologue thereof, an antigen
from the MAGE-family or a binding partner thereof or a fragment,
variant or derivative of said tumour antigen.
4. The nucleic acid according to claims 1 to 3, wherein the at
least one histone stem loop is heterologous to the coding region
encoding the at least one peptide or protein, preferably, wherein
the coding region does not encode a histone protein or fragment,
derivate of variant thereof having histone or histone-like
function.
5. The nucleic acid according to claim 1 or 4, wherein the peptide
or protein encoded by the coding region comprises a tumour
antigenic protein or a fragment, variant or derivative thereof, the
fragment, variant or derivative of the tumour antigenic protein
retaining at least 50% of the biological activity of the tumour
antigenic protein.
6. The nucleic acid of any of claims 1 to 5, wherein its coding
region does not encode a reporter protein or a marker or selection
protein.
7. Nucleic acid sequence according to any of claims 1 to 6, wherein
the nucleic acid is an RNA, preferably an mRNA.
8. Nucleic acid sequence according to any of claims 1 to 7, wherein
the at least one histone stem-loop is selected from following
formulae (I) or (II): formula (I) (stem-loop sequence without stem
bordering elements): ##STR00019## formula (II) (stem-loop sequence
with stem bordering elements): ##STR00020## wherein: stem1 or stem2
bordering elements N.sub.1-6 is a consecutive sequence of 1 to 6,
preferably of 2 to 6, more preferably of 2 to 5, even more
preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein
each N is independently from another selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof;
stem1N.sub.0-2GN.sub.3-5 is reverse complementary or partially
reverse complementary with element stem2, and is a consecutive
sequence between of 5 to 7 nucleotides; wherein N.sub.0-2 is a
consecutive sequence of 0 to 2, preferably of 0 to 1, more
preferably of 1 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof; wherein N.sub.5-5 is a consecutive
sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N,
wherein each N is independently from another selected from a
nucleotide selected from A, U, T, G and C or a nucleotide analogue
thereof, and wherein G is guanosine or an analogue thereof, and may
be optionally replaced by a cytidine or an analogue thereof,
provided that its complementary nucleotide cytidine in stem2 is
replaced by guanosine; loop sequence N.sub.0-4(U/T)N.sub.0-4 is
located between elements stem1 and stem2, and is a consecutive
sequence of 3 to 5 nucleotides, more preferably of 4 nucleotides;
wherein each N.sub.0-4 is independent from another a consecutive
sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2
N, wherein each N is independently from another selected from a
nucleotide selected from A, U, T, G and C or a nucleotide analogue
thereof; and wherein U/T represents uridine, or optionally
thymidine; stem2N.sub.3-5CN.sub.0-2 is reverse complementary or
partially reverse complementary with element stem1, and is a
consecutive sequence between of 5 to 7 nucleotides; wherein
N.sub.5-5 is a consecutive sequence of 3 to 5, preferably of 4 to
5, more preferably of 4 N, wherein each N is independently from
another selected from a nucleotide selected from A, U, T, G and C
or a nucleotide analogue thereof; wherein N.sub.0-2 is a
consecutive sequence of 0 to 2, preferably of 0 to 1, more
preferably of 1 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof; and wherein C is cytidine or an
analogue thereof, and may be optionally replaced by a guanosine or
an analogue thereof provided that its complementary nucleotide
guanosine in stem1 is replaced by cytidine; wherein stem1 and stem2
are capable of base pairing with each other forming a reverse
complementary sequence, wherein base pairing may occur between
stem1 and stem2, or forming a partially reverse complementary
sequence, wherein an incomplete base pairing may occur between
stem1 and stem2.
9. The nucleic acid according to claim 8 wherein the at least one
histone stem-loop is selected from at least one of following
formulae (Ia) or (IIa): ##STR00021## formula (Ia) (stem-loop
sequence without stem bordering elements) ##STR00022## formula
(IIa) (stem-loop sequence with stem bordering elements)
10. The nucleic acid sequence according to any of claims 1 to 9,
wherein the poly(A) sequence comprises a sequence of about 25 to
about 400 adenosine nucleotides, preferably a sequence of about 50
to about 400 adenosine nucleotides, more preferably a sequence of
about 50 to about 300 adenosine nucleotides, even more preferably a
sequence of about 50 to about 250 adenosine nucleotides, most
preferably a sequence of about 60 to about 250 adenosine
nucleotides.
11. Nucleic acid sequence according to any of claims 1 to 10,
wherein the polyadenylation signal comprises the consensus sequence
NN(U/T)ANA, preferably AA(U/T)AAA or A(U/T)(U/T)AAA.
12. Nucleic acid sequence according to any of claims 1 to 11,
wherein the nucleic acid sequence is a modified nucleic acid, in
particular a stabilized nucleic acid.
13. Nucleic acid sequence according to claim 12, wherein the G/C
content of the coding region encoding at least one peptide or
protein of said modified nucleic acid is increased compared with
the G/C content of the coding region of the wild-type nucleic acid,
the coded amino acid sequence of said modified nucleic acid
preferably not being modified compared with the coded amino acid
sequence of the wild-type nucleic acid.
14. A composition comprising at least one type of nucleic acid
sequences according to any of claims 1 to 13.
15. The composition according to claim 14, wherein the composition
comprises at least two types of nucleic acid sequences wherein each
type of nucleic acid sequence encodes for a different peptide or
protein, preferably for a different tumour antigen.
16. The composition according to claim 14 or claim 15, wherein one
type of the contained nucleic acid sequences encodes for PSA, PSMA,
PSCA, STEAP-1, NY-ESO-1, 5T4, Survivin, MAGE-C1, or MAGE-C2.
17. The composition according to any of claims 14 to 16, wherein
the nucleic acid sequence does not encode for NY-ESO1, provided
that the composition contains only one type of nucleic acid
sequence.
18. A kit or kit of parts comprising at least one, preferably a
plurality or more than one of nucleic acid sequences each according
to any of claims 1 to 13.
19. The composition or kit or kit of parts according to any of
claims 14 to 18, comprising at least: a) a nucleic acid sequence of
any of claims 1 to 13 wherein said encoded peptide or protein
comprises the tumour antigen PSA, or a fragment, variant or
derivative thereof; and b) a nucleic acid sequence of any of claims
1 to 13 wherein said encoded peptide or protein comprises the
tumour antigen PSMA, or a fragment, variant or derivative thereof;
and c) a nucleic acid sequence of any of claims 1 to 13 wherein
said encoded peptide or protein comprises the tumour antigen PSCA,
or a fragment, variant or derivative thereof; and d) a nucleic acid
sequence of any of claims 1 to 13 wherein said encoded peptide or
protein comprises the tumour antigen STEAP-1, or a fragment,
variant or derivative thereof.
20. A composition or kit or kit of parts according to any of claims
14 to 18, comprising at least: a) a nucleic acid sequence
comprising or coding for i. a coding region, encoding at least one
peptide or protein which comprises the tumour antigen NY-ESO-1, or
a fragment, variant or derivative thereof, ii. at least one histone
stem-loop, and iii. a poly(A) sequence or a polyadenylation signal;
b) a nucleic acid sequence of any of claims 1 to 13 wherein said
encoded peptide or protein comprises the tumour antigen 5T4, or a
fragment, variant or derivative thereof; and c) a nucleic acid
sequence of any of claims 1 to 13 wherein said encoded peptide or
protein comprises the tumour antigen Survivin, or a fragment,
variant or derivative thereof.
21. The composition or kit or kit of parts according to any of
claims 11 to 17, further comprising at least: a) a nucleic acid
sequence of any of claims 1 to 13 wherein said encoded peptide or
protein comprises the tumour antigen MAGE-C1, or a fragment,
variant or derivative thereof; and b) a nucleic acid sequence of
any of claims 1 to 13 wherein said encoded peptide or protein
comprises the tumour antigen MAGE-C2, or a fragment, variant or
derivative thereof.
22. Nucleic acid sequence as defined according to any of claims 1
to 13 or composition or kit or kit of parts as defined according to
any of claims 14 to 21 for use as a medicament.
23. Nucleic acid sequence as defined according to any of claims 1
to 13 or composition or kit or kit of parts as defined according to
any of claims 14 to 21 for use in the treatment of cancer or tumour
diseases.
24. Pharmaceutical composition comprising a nucleic acid sequence
as defined according to any of claims 1 to 13 or a composition as
defined according to any of claims 14 to 21 and optionally a
pharmaceutically acceptable carrier.
25. Use of a nucleic acid sequence as defined according to any of
claims 1 to 13 or a composition or kit or kit of parts as defined
according to any of claims 14 to 21 for increasing the expression
of said encoded peptide or protein.
26. Use of a nucleic acid sequence as defined according to any of
claims 1 to 13 or composition or kit or kit of parts as defined
according to any of claims 14 to 21 for increasing the expression
of said encoded peptide or protein in the treatment of cancer or
tumour diseases.
27. A method for increasing the expression of an encoded peptide or
protein comprising the steps: a) providing the nucleic acid
sequence as defined according to any of claims 1 to 13 or the
composition as defined according to any of claims 14 to 21, b)
applying or administering the nucleic acid sequence or the
composition to a cell-free expression system, a cell, a tissue or
an organism.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 16/002,695, filed Jun. 7, 2018, which is a continuation of U.S.
application Ser. No. 14/378,572, filed Aug. 13, 2014, now U.S. Pat.
No. 10,010,592, which is a national phase application under 35
U.S.C. .sctn. 371 of International Application No.
PCT/EP2013/000459, filed Feb. 15, 2013, which is a continuation of
International Application No. PCT/EP2012/000674, filed Feb. 15,
2012. The entire text of each of the above referenced disclosures
is specifically incorporated herein by reference.
[0002] The present invention relates to a nucleic acid sequence,
comprising or coding for a coding region, encoding at least one
peptide or protein comprising a tumour antigen or a fragment,
variant or derivative thereof, at least one histone stem-loop and a
poly(A) sequence or a polyadenylation signal. Furthermore the
present invention provides the use of the nucleic acid for
increasing the expression of said encoded peptide or protein. It
also discloses its use for the preparation of a pharmaceutical
composition, especially a vaccine, e.g. for use in the treatment of
cancer or tumour diseases. The present invention further describes
a method for increasing the expression of a peptide or protein
comprising a tumour antigen or a fragment, variant or derivative
thereof, using the nucleic acid comprising or coding for a histone
stem-loop and a poly(A) sequence or a polyadenylation signal.
[0003] Apart from cardiovascular diseases and infectious diseases,
the occurrence of tumours and cancer diseases is one of the most
frequent causes of death in modern society and is in most cases
associated with considerable costs in terms of therapy and
subsequent rehabilitation measures. The treatment of tumours and
cancer diseases is greatly dependent, for example, on the type of
tumour that occurs, on the age, the distribution of cancer cells in
the patient to be treated, etc. Cancer therapy is nowadays
conventionally carried out by the use of radiation therapy or
chemotherapy in addition to invasive operations. However, such
conventional therapies typically place extraordinary stress on the
immune system and can be used in some cases to only a limited
extent. In addition, most of these conventional therapies require
long intervals between the individual treatments to allow for
regeneration of the immune system.
[0004] Therefore, supplementary strategies have been investigated
in recent years in addition to such "conventional treatments" to
avoid or at least reduce the impact on the immune system by such
treatments. One such supplementary treatment in particular includes
gene therapeutic approaches or genetic vaccination, which already
have been found to be highly promising for treatment or for
supporting such conventional therapies.
[0005] Gene therapy and genetic vaccination are methods of
molecular medicine which already have been proven in the therapy
and prevention of diseases and generally exhibit a considerable
effect on daily medical practice, in particular on the treatment of
diseases as mentioned above. Both methods, gene therapy and genetic
vaccination, are based on the introduction of nucleic acids into
the patient's cells or tissue and subsequent processing of the
information coded for by the nucleic acid that has been introduced
into the cells or tissue, that is to say the (protein) expression
of the desired polypeptides.
[0006] In gene therapy approaches, typically DNA is used even
though RNA is also known in recent developments. Importantly, in
all these gene therapy approaches mRNA functions as messenger for
the sequence information of the encoded protein, irrespectively if
DNA, viral RNA or mRNA is used.
[0007] In general RNA is considered an unstable molecule: RNases
are ubiquitous and notoriously difficult to inactivate.
Furthermore, RNA is also chemically more labile than DNA. Thus, it
is perhaps surprising that the "default state" of an mRNA in a
eukaryotic cell is characterized by a relative stability and
specific signals are required to accelerate the decay of individual
mRNAs. The main reason for this finding appears to be that mRNA
decay within cells is catalyzed almost exclusively by exonucleases.
However, the ends of eukaryotic mRNAs are protected against these
enzymes by specific terminal structures and their associated
proteins: a m7GpppN CAP at the 5' end and typically a poly(A)
sequence at the 3' end. Removal of these two terminal modifications
is thus considered rate limiting for mRNA decay. Although a
stabilizing element has been characterized in the 3' UTR of the
alpha-globin mRNA, RNA sequences affecting turnover of eukaryotic
mRNAs typically act as a promoter of decay usually by accelerating
deadenylation (reviewed in Meyer, S., C. Temme, et al. (2004), Crit
Rev Biochem Mol Biol 39(4): 197-216).
[0008] As mentioned above, the 5' ends of eukaryotic mRNAs are
typically modified posttranscriptionally to carry a methylated CAP
structure, e.g. m7GpppN. Aside from roles in RNA splicing,
stabilization, and transport, the CAP structure significantly
enhances the recruitment of the 40S ribosomal subunit to the 5' end
of the mRNA during translation initiation. The latter function
requires recognition of the CAP structure by the eukaryotic
initiation factor complex eIF4F. The poly(A) sequence additionally
stimulates translation via increased 40S subunit recruitment to
mRNAs, an effect that requires the intervention of poly(A) binding
protein (PABP). PABP, in turn, was recently demonstrated to
interact physically with eIF4G, which is part of the CAP-bound
eIF4F complex. Thus, a closed loop model of translation initiation
on capped, polyadenylated mRNAs was postulated (Michel, Y. M., D.
Poncet, et al. (2000), J Biol Chem 275(41): 32268-76).
[0009] Nearly all eukaryotic mRNAs end with such a poly(A) sequence
that is added to their 3' end by the ubiquitous
cleavage/polyadenylation machinery. The presence of a poly(A)
sequence at the 3' end is one of the most recognizable features of
eukaryotic mRNAs. After cleavage, most pre-mRNAs, with the
exception of replication-dependent histone transcripts, acquire a
polyadenylated tail. In this context, 3' end processing is a
nuclear co-transcriptional process that promotes transport of mRNAs
from the nucleus to the cytoplasm and affects the stability and the
translation of mRNAs. Formation of this 3' end occurs in a two step
reaction directed by the cleavage/polyadenylation machinery and
depends on the presence of two sequence elements in mRNA precursors
(pre-mRNAs); a highly conserved hexanucleotide AAUAAA
(polyadenylation signal) and a downstream G/U-rich sequence. In a
first step, pre-mRNAs are cleaved between these two elements. In a
second step tightly coupled to the first step the newly formed 3'
end is extended by addition of a poly(A) sequence consisting of
200-250 adenylates which affects subsequently all aspects of mRNA
metabolism, including mRNA export, stability and translation
(Dominski, Z. and W. F. Marzluff (2007), Gene 396(2): 373-90).
[0010] The only known exception to this rule are the
replication-dependent histone mRNAs which end with a histone
stem-loop instead of a poly(A) sequence. Exemplary histone
stem-loop sequences are described in Lopez et al. (Davila Lopez,
M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.
doi:10.1261/rna.782308).
[0011] The stem-loops in histone pre-mRNAs are typically followed
by a purine-rich sequence known as the histone downstream element
(HDE). These pre-mRNAs are processed in the nucleus by a single
endonucleolytic cleavage approximately 5 nucleotides downstream of
the stem-loop, catalyzed by the U7 snRNP through base pairing of
the U7 snRNA with the HDE. The 3'-UTR sequence comprising the
histone stem-loop structure and the histone downstream element
(HDE) (binding site of the U7 snRNP) were usually termed as histone
3'-processing signal (see e.g. Chodchoy, N., N. B. Pandey, et al.
(1991). Mol Cell Biol 11(1): 497-509).
[0012] Due to the requirement to package newly synthesized DNA into
chromatin, histone synthesis is regulated in concert with the cell
cycle. Increased synthesis of histone proteins during S phase is
achieved by transcriptional activation of histone genes as well as
posttranscriptional regulation of histone mRNA levels. It could be
shown that the histone stem-loop is essential for all
posttranscriptional steps of histone expression regulation. It is
necessary for efficient processing, export of the mRNA into the
cytoplasm, loading onto polyribosomes, and regulation of mRNA
stability.
[0013] In the above context, a 32 kDa protein was identified, which
is associated with the histone stem-loop at the 3'-end of the
histone messages in both the nucleus and the cytoplasm. The
expression level of this stem-loop binding protein (SLBP) is
cell-cycle regulated and is highest during S-phase when histone
mRNA levels are increased. SLBP is necessary for efficient 3'-end
processing of histone pre-mRNA by the U7 snRNP. After completion of
processing, SLBP remains associated with the stem-loop at the end
of mature histone mRNAs and stimulates their translation into
histone proteins in the cytoplasm. (Dominski, Z. and W. F. Marzluff
(2007), Gene 396(2): 373-90). Interestingly, the RNA binding domain
of SLBP is conserved throughout metazoa and protozoa (Davila Lopez,
M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.
doi:10.1261/rna.782308) and it could be shown that its binding to
the histone stem-loop sequence is dependent on the stem-loop
structure and that the minimum binding site contains at least 3
nucleotides 5' and 2 nucleotides 3' of the stem-loop (Pandey, N.
B., et al. (1994), Molecular and Cellular Biology, 14(3), 1709-1720
and Williams, A. S., & Marzluff, W. F., (1995), Nucleic Acids
Research, 23(4), 654-662).
[0014] Even though histone genes are generally classified as either
"replication-dependent", giving rise to mRNA ending in a histone
stem-loop, or "replacement-type", giving rise to mRNA bearing a
poly(A)-tail instead, naturally occurring mRNAs containing both a
histone stem-loop and poly(A) or oligo(A) 3' thereof have been
identified in some very rare cases. Sanchez et al. examined the
effect of naturally occurring oligo(A) tails appended 3' of the
histone stem-loop of histone mRNA during Xenopus oogenesis using
Luciferase as a reporter protein and found that the oligo(A) tail
is an active part of the translation repression mechanism that
silences histone mRNA during oogenesis and its removal is part of
the mechanism that activates translation of histone mRNAs (Sanchez,
R. and W. F. Marzluff (2004), Mol Cell Biol 24(6): 2513-25).
[0015] Furthermore, the requirements for regulation of replication
dependent histones at the level of pre-mRNA processing and mRNA
stability have been investigated using artificial constructs coding
for the marker protein alpha Globin, taking advantage of the fact
that the globin gene contains introns as opposed to the intron-less
histone genes. For this purpose constructs were generated in which
the alpha globin coding sequence was followed by a histone
stem-loop signal (histone stem-loop followed by the histone
downstream element) and a polyadenylation signal (Whitelaw, E., et
al. (1986). Nucleic Acids Research, 14(17), 7059-7070; Pandey, N.
B., & Marzluff, W. F. (1987). Molecular and Cellular Biology,
7(12), 4557-4559; Pandey, N. B., et al. (1990). Nucleic Acids
Research, 18(11), 3161-3170).
[0016] In another approach Luscher et al. investigated the
cell-cycle dependent regulation of a recombinant histone H4 gene.
Constructs were generated in which the H4 coding sequence was
followed by a histone stem-loop signal and a polyadenylation
signal, the two processing signals incidentally separated by a
galactokinase coding sequence (Luscher, B. et al., (1985). Proc.
Natl. Acad. Sci. USA, 82(13), 4389-4393).
[0017] Additionally, Stauber et al. identified the minimal sequence
required to confer cell-cycle regulation on histone H4 mRNA levels.
For these investigations constructs were used, comprising a coding
sequence for the selection marker Xanthine:guanine phosphoribosyl
transferase (GPT) preceding a histone stem-loop signal followed by
a polyadenylation signal (Stauber, C. et al., (1986). EMBO J,
5(12), 3297-3303).
[0018] Examining histone pre-mRNA processing Wagner et al.
identified factors required for cleavage of histone pre-mRNAs using
a reporter construct placing EGFP between a histone stem-loop
signal and a polyadenylation signal, such that EGFP was expressed
only in case histone pre-mRNA processing was disrupted (Wagner, E.
J. et al., (2007). Mol Cell 28(4), 692-9).
[0019] To be noted, translation of polyadenylated mRNA usually
requires the 3' poly(A) sequence to be brought into proximity of
the 5' CAP. This is mediated through protein-protein interaction
between the poly(A) binding protein and eukaryotic initiation
factor eIF4G. With respect to replication-dependent histone mRNAs,
an analogous mechanism has been uncovered. In this context, Gallie
et al. show that the histone stem-loop is functionally similar to a
poly(A) sequence in that it enhances translational efficiency and
is co-dependent on a 5'-CAP in order to establish an efficient
level of translation. They showed that the histone stem-loop is
sufficient and necessary to increase the translation of a reporter
mRNA in transfected Chinese hamster ovary cells but must be
positioned at the 3'-terminus in order to function optimally.
Therefore, similar to the poly(A) tail on other mRNAs, the 3' end
of these histone mRNAs appears to be essential for translation in
vivo and is functionally analogous to a poly(A) tail (Gallie, D.
R., Lewis, N. J., & Marzluff, W. F. (1996), Nucleic Acids
Research, 24(10), 1954-1962).
[0020] Additionally, it could be shown that SLBP is bound to the
cytoplasmic histone mRNA and is required for its translation. Even
though SLBP does not interact directly with eIF4G, the domain
required for translation of histone mRNA interacts with the
recently identified protein SLIP1. In a further step, SLIP1
interacts with eIF4G and allows to circularize histone mRNA and to
support efficient translation of histone mRNA by a mechanism
similar to the translation of polyadenylated mRNAs.
[0021] As mentioned above, gene therapy approaches normally use DNA
to transfer the coding information into the cell which is then
transcribed into mRNA, carrying the naturally occurring elements of
an mRNA, particularly the 5'-CAP structure and the 3' poly(A)
sequence to ensure expression of the encoded therapeutic or
antigenic protein.
[0022] However, in many cases expression systems based on the
introduction of such nucleic acids into the patient's cells or
tissue and the subsequent expression of the desired polypeptides
coded for by these nucleic acids do not exhibit the desired, or
even the required, level of expression which may allow for an
efficient therapy, irrespective as to whether DNA or RNA is
used.
[0023] In the prior art, different attempts have hitherto been made
to increase the yield of the expression of an encoded protein, in
particular by use of improved expression systems, both in vitro
and/or in vivo. Methods for increasing expression described
generally in the prior art are conventionally based on the use of
expression vectors or cassettes containing specific promoters and
corresponding regulation elements. As these expression vectors or
cassettes are typically limited to particular cell systems, these
expression systems have to be adapted for use in different cell
systems. Such adapted expression vectors or cassettes are then
usually transfected into the cells and typically treated in
dependence of the specific cell line. Therefore, preference is
given primarily to those nucleic acid molecules which are able to
express the encoded proteins in a target cell by systems inherent
in the cell, independent of promoters and regulation elements which
are specific for particular cell types. In this context, there can
be distinguished between mRNA stabilizing elements and elements
which increase translation efficiency of the mRNA.
[0024] mRNAs which are optimized in their coding sequence and which
are in general suitable for such a purpose are described in
application WO 02/098443 (CureVac GmbH). For example, WO 02/098443
describes mRNAs that are stabilised in general form and optimised
for translation in their coding regions. WO 02/098443 further
discloses a method for determining sequence modifications. WO
02/098443 additionally describes possibilities for substituting
adenine and uracil nucleotides in mRNA sequences in order to
increase the guanine/cytosine (G/C) content of the sequences.
According to WO 02/098443, such substitutions and adaptations for
increasing the G/C content can be used for gene therapeutic
applications but also genetic vaccines in the treatment of cancer
or infectious diseases. In this context, WO 02/098443 generally
mentions sequences as a base sequence for such modifications, in
which the modified mRNA codes for at least one biologically active
peptide or polypeptide, which is translated in the patient to be
treated, for example, either not at all or inadequately or with
faults. Alternatively, WO 02/098443 proposes mRNAs coding for
antigens e.g. tumour antigens or viral antigens as a base sequence
for such modifications.
[0025] In a further approach to increase the expression of an
encoded protein the application WO 2007/036366 describes the
positive effect of long poly(A) sequences (particularly longer than
120 bp) and the combination of at least two 3' untranslated regions
of the beta globin gene on mRNA stability and translational
activity.
[0026] However, even though all these latter prior art documents
already try to provide quite efficient tools for gene therapy
approaches and additionally improved mRNA stability and
translational activity, there still remains the problem of a
generally lower stability of RNA-based applications versus DNA
vaccines and DNA based gene therapeutic approaches. Accordingly,
there still exists a need in the art to provide improved tools for
gene therapy approaches and genetic vaccination or as a
supplementary therapy for conventional treatments as discussed
above, which allow for better provision of encoded proteins in
vivo, e.g. via a further improved mRNA stability and/or
translational activity, preferably for gene therapy and genetic
vaccination.
[0027] Furthermore despite of all progress in the art, efficient
expression of an encoded peptide or protein in cell-free systems,
cells or organisms (recombinant expression) is still a challenging
problem.
[0028] The object underlying the present invention is, therefore,
to provide additional and/or alternative methods to increase
expression of an encoded protein, preferably via further
stabilization of the mRNA and/or an increase of the translational
efficiency of such an mRNA with respect to such nucleic acids known
from the prior art for the use in genetic vaccination in the
therapeutic or prophylactic treatment of cancer or tumour
diseases.
[0029] This object is solved by the subject matter of the attached
claims. Particularly, the object underlying the present invention
is solved according to a first aspect by an inventive nucleic acid
sequence comprising or coding for [0030] a) a coding region,
encoding at least one peptide or protein which comprises a tumour
antigen or a fragment, variant or derivative thereof; [0031] b) at
least one histone stem-loop, and [0032] c) a poly(A) sequence or a
polyadenylation signal, preferably for increasing the expression of
said encoded peptide or protein.
[0033] Alternatively, any appropriate stem loop sequence other than
a histone stem loop sequence (derived from histone genes, in
particular histone genes of the families H1, H2A, H2B, H3 and H4))
may be employed by the present invention in all of its aspects and
embodiments.
[0034] In this context it is particularly preferred that the
inventive nucleic acid according to the first aspect of the present
invention is produced at least partially by DNA or RNA synthesis,
preferably as described herein or is an isolated nucleic acid.
[0035] The present invention is based on the surprising finding of
the present inventors, that the combination of a poly(A) sequence
or polyadenylation signal and at least one histone stem-loop, even
though both representing alternative mechanisms in nature, acts
synergistically as this combination increases the protein
expression manifold above the level observed with either of the
individual elements. The synergistic effect of the combination of
poly(A) and at least one histone stem-loop is seen irrespective of
the order of poly(A) and histone stem-loop and irrespective of the
length of the poly(A) sequence.
[0036] Therefore it is particularly preferred that the inventive
nucleic acid sequence comprises or codes for a) a coding region,
encoding at least one peptide or protein which comprises a tumour
antigen or a fragment, variant or derivative thereof; b) at least
one histone stem-loop, and c) a poly(A) sequence or polyadenylation
sequence; preferably for increasing the expression level of said
encoded peptide or protein. In some embodiments, it may be
preferred if the encoded protein is not a histone protein, in
particular no histone protein of the H4, H3, H2A and/or H2B histone
family or a fragment, derivative or variant thereof retaining
histone(-like) function), namely forming a nucleosome. Also, the
encoded protein typically does not correspond to a histone linker
protein of the H1 histone family. The inventive nucleic acid
molecule does typically not contain any regulatory signals (5'
and/or, particularly, 3' of a mouse histone gene, in particular not
of a mouse histone gene H2A and, further, most preferably not of
the mouse histone gene H2A614. In particular, it does not contain a
histone stem loop and/or a histone stem loop processing signal from
a mouse histone gene, in particular not of mouse histone gene H2A
und, most preferably not of mouse histone gene H2A614.
[0037] Also, the inventive nucleic acid typically does not provide
a reporter protein (e.g. Luciferase, GFP, EGFP,
.beta.-Galactosidase, particularly EGFP) galactokinase (galK)
and/or marker or selection protein (e.g. alpha-Globin,
galactokinase and Xanthine:Guanine phosphoribosyl transferase
(GPT)) or a bacterial reporter protein, e.g. chloramphenicol acetyl
transferase (CAT) or other bacterial antibiotics resistance
proteins, e.g. derived from the bacterial neo gene in its element
(a).
[0038] A reporter, marker or selection protein is typically
understood not to be a tumour antigen according to the invention. A
reporter, marker or selection protein or its underlying gene is
commonly used as a research tool in bacteria, cell culture, animals
or plants. They confer on organisms (preferably heterologously)
expressing them an easily identifiable property, which may be
measured or which allows for selection. Specifically, marker or
selection proteins exhibit a selectable function. Typically, such
selection, marker or reporter proteins do not naturally occur in
humans or other mammals, but are derived from other organisms, in
particular from bacteria or plants. Accordingly, proteins with
selection, marker or reporter function originating from species
other than mammals, in particular other than humans, are preferably
excluded from being understood as a protein having the property to
act as a "tumour antigen" according to the present invention. In
particular, a selection, marker or reporter protein allows to
identify transformed cells by in vitro assays based e.g. on
fluorescence or other spectroscopic techniques and resistance
towards antibiotics. Selection, reporter or marker genes awarding
such properties to transformed cells are therefore typically not
understood to be a protein acting as a tumour antigen according to
the invention in vivo.
[0039] In any case, reporter, marker or selection proteins do
usually not exert any tumour antigenic properties and, therefore,
do not exert an immunological effect which therapeutically allows
to treat tumour diseases. If any single reporter, marker or
selection protein should nevertheless do so (in addition to its
reporter, selection or marker function), such a reporter, marker or
selection protein is preferably not understood to be a "tumour
antigen" within the meaning of the present invention.
[0040] In contrast, a protein or peptide acting as a tumour antigen
(in particular excluding histone genes of the families H1, H2A,
H2B, H3 and H4) according to the present invention does typically
not exhibit a selection, marker or reporter function. If any single
"tumour antigen" nevertheless should do so (in addition to its
tumour antigenic function), such a tumour antigen is preferably not
understood to be a "selection, marker or reporter protein" within
the meaning of the present invention.
[0041] It is most preferably understood that a protein acting as
tumour antigen according to the invention is derived from mammals,
in particular humans, in particular from mammalian tumours, and
does not qualify as selection, marker or reporter protein. In
particular, such tumour antigens are derived from mammalian, in
particular from human tumours. These tumour antigenic proteins are
understood to be antigenic, as they are meant to treat the subject
by triggering the subject's immune response such that the subject's
immune system is enabled to combat the subject's tumor cells by TH1
and/or TH2 immune responses. Accordingly, such antigenic tumour
proteins are typically mammalian, in particular human proteins
characterizing the subject's cancer type.
[0042] Accordingly, it is preferred that the coding region (a)
encoding at least one peptide or protein is heterologous to at
least (b) the at least one histone stem loop, or more broadly, to
any appropriate stem loop. In other words, "heterologous" in the
context of the present invention means that the at least one stem
loop sequence does not naturally occur as a (regulatory) sequence
(e.g. at the 3'UTR) of the specific gene, which encodes the (tumour
antigenic) protein or peptide of element (a) of the inventive
nucleic acid. Accordingly, the (histone) stem loop of the inventive
nucleic acid is derived preferably from the 3' UTR of a gene other
than the one comprising the coding region of element (a) of the
inventive nucleic acid. E.g., the coding region of element (a) will
not encode a histone protein or a fragment, variant or derivative
thereof (retaining the function of a histone protein), if the
inventive nucleic is heterologous, but will encode any other
peptide or sequence (of the same or another species) which exerts a
biological function, preferably tumour antigenic function other
than a histone(-like) function, e.g. will encode a protein
(exerting a tumour antigenic function, e.g. in terms of vaccinating
against mammalian, in particular human tumours thereby triggering a
immunological reaction against the subject's tumour cells, which
preferably express the tumour antigen encoded by the inventive
nucleic acid.
[0043] In this context it is particularly preferred that the
inventive nucleic acid comprises or codes for in 5'- to
3'-direction: [0044] a) a coding region, encoding at least one
peptide or protein which comprises a tumour antigen or a fragment,
variant or derivative thereof; [0045] b) at least one histone
stem-loop, optionally without a histone downstream element (HDE) 3'
to the histone stem-loop [0046] c) a poly(A) sequence or a
polyadenylation signal.
[0047] The term "histone downstream element (HDE) refers to a
purine-rich polynucleotide stretch of about 15 to 20 nucleotides 3'
of naturally occurring histone stem-loops, which represents the
binding site for the U7 snRNA involved in processing of histone
pre-mRNA into mature histone mRNA. For example in sea urchins the
HDE is CAAGAAAGA (Dominski, Z. and W. F. Marzluff (2007), Gene
396(2): 373-90).
[0048] Furthermore it is preferable that the inventive nucleic acid
according to the first aspect of the present invention does not
comprise an intron.
[0049] In another particular preferred embodiment, the inventive
nucleic acid sequence according to the first aspect of the present
invention comprises or codes for from 5' to 3': [0050] a) a coding
region, preferably encoding at least one peptide or protein which
comprises a tumour antigen or a fragment, variant or derivative
thereof; [0051] c) a poly(A) sequence; and [0052] b) at least one
histone stem-loop.
[0053] The inventive nucleic acid sequence according to the first
embodiment of the present invention comprise any suitable nucleic
acid, selected e.g. from any (single-stranded or double-stranded)
DNA, preferably, without being limited thereto, e.g. genomic DNA,
plasmid DNA, single-stranded DNA molecules, double-stranded DNA
molecules, or may be selected e.g. from any PNA (peptide nucleic
acid) or may be selected e.g. from any (single-stranded or
double-stranded) RNA, preferably a messenger RNA (mRNA); etc. The
inventive nucleic acid sequence may also comprise a viral RNA
(vRNA). However, the inventive nucleic acid sequence may not be a
viral RNA or may not contain a viral RNA. More specifically, the
inventive nucleic acid sequence may not contain viral sequence
elements, e.g. viral enhancers or viral promotors (e.g. no
inactivated viral promoter or sequence elements, more specifically
not inactivated by replacement strategies), or other viral sequence
elements, or viral or retroviral nucleic acid sequences. More
specifically, the inventive nucleic acid sequence may not be a
retroviral or viral vector or a modified retroviral or viral
vector.
[0054] In any case, the inventive nucleic acid sequence may or may
not contain an enhancer and/or promoter sequence, which may be
modified or not or which may be activated or not. The enhancer and
or promoter may be plant expressible or not expressible, and/or in
eukaryotes expressible or not expressible and/or in prokaryotes
expressible or not expressible. The inventive nucleic acid sequence
may contain a sequence encoding a (self-splicing) ribozyme or
not.
[0055] In specific embodiments the inventive nucleic acid sequence
may be or may comprise a self-replicating RNA (replicon).
[0056] Preferably, the inventive nucleic acid sequence is a plasmid
DNA, or an RNA, particularly an mRNA.
[0057] In particular embodiments of the first aspect of the present
invention, the inventive nucleic acid is a nucleic acid sequence
comprised in a nucleic acid suitable for in vitro transcription,
particularly in an appropriate in vitro transcription vector (e.g.
a plasmid or a linear nucleic acid sequence comprising specific
promoters for in vitro transcription such as T3, T7 or Sp6
promoters).
[0058] In further particular preferred embodiments of the first
aspect of the present invention, the inventive nucleic acid is
comprised in a nucleic acid suitable for transcription and/or
translation in an expression system (e.g. in an expression vector
or plasmid), particularly a prokaryotic (e.g. bacteria like E.
coli) or eukaryotic (e.g. mammalian cells like CHO cells, yeast
cells or insect cells or whole organisms like plants or animals)
expression system.
[0059] The term "expression system" means a system (cell culture or
whole organisms) which is suitable for production of peptides,
proteins or RNA particularly mRNA (recombinant expression).
[0060] The inventive nucleic acid sequence according to the first
aspect of the present invention comprises or codes for at least one
histone stem-loop. In the context of the present invention, such a
histone stem-loop, in general (irrespective of whether it is a
histone stem loop or not), is typically derived from histone genes
and comprises an intramolecular base pairing of two neighbored
entirely or partially reverse complementary sequences, thereby
forming a stem-loop. A stem-loop can occur in single-stranded DNA
or, more commonly, in RNA. The structure is also known as a hairpin
or hairpin loop and usually consists of a stem and a (terminal)
loop within a consecutive sequence, wherein the stem is formed by
two neighbored entirely or partially reverse complementary
sequences separated by a short sequence as sort of spacer, which
builds the loop of the stem-loop structure. The two neighbored
entirely or partially reverse complementary sequences may be
defined as e.g. stem loop elements stem1 and stem2. The stem loop
is formed when these two neighbored entirely or partially reverse
complementary sequences, e.g. stem loop elements stem1 and stem2,
form base-pairs with each other, leading to a double stranded
nucleic acid sequence stretch comprising an unpaired loop at its
terminal ending formed by the short sequence located between stem
loop elements stem1 and stem2 on the consecutive sequence. The
unpaired loop thereby typically represents a region of the nucleic
acid which is not capable of base pairing with either of these stem
loop elements. The resulting lollipop-shaped structure is a key
building block of many RNA secondary structures. The formation of a
stem-loop structure is thus dependent on the stability of the
resulting stem and loop regions, wherein the first prerequisite is
typically the presence of a sequence that can fold back on itself
to form a paired double strand. The stability of paired stem loop
elements is determined by the length, the number of mismatches or
bulges it contains (a small number of mismatches is typically
tolerable, especially in a long double stranded stretch), and the
base composition of the paired region. In the context of the
present invention, a loop length of 3 to 15 bases is conceivable,
while a more preferred loop length is 3-10 bases, more preferably 3
to 8, 3 to 7, 3 to 6 or even more preferably 4 to 5 bases, and most
preferably 4 bases. The stem sequence forming the double stranded
structure typically has a length of between 5 to 10 bases, more
preferably, between 5 to 8 bases.
[0061] In the context of the present invention, a histone stem-loop
is typically derived from histone genes (e.g. genes from the
histone families H1, H2A, H2B, H3, H4) and comprises an
intramolecular base pairing of two neighbored entirely or partially
reverse complementary sequences, thereby forming a stem-loop.
Typically, a histone 3' UTR stem-loop is an RNA element involved in
nucleocytoplasmic transport of the histone mRNAs, and in the
regulation of stability and of translation efficiency in the
cytoplasm. The mRNAs of metazoan histone genes lack polyadenylation
and a poly-A tail, instead 3' end processing occurs at a site
between this highly conserved stem-loop and a purine rich region
around 20 nucleotides downstream (the histone downstream element,
or HDE). The histone stem-loop is bound by a 31 kDa stem-loop
binding protein (SLBP--also termed the histone hairpin binding
protein, or HBP). Such histone stem-loop structures are preferably
employed by the present invention in combination with other
sequence elements and structures, which do not occur naturally
(which means in untransformed living organisms/cells) in histone
genes, but are combined--according to the invention--to provide an
artificial, heterologous nucleic acid. Accordingly, the present
invention is particularly based on the finding that an artificial
(non-native) combination of a histone stem-loop structure with
other heterologous sequence elements, which do not occur in histone
genes or metazoan histone genes and are isolated from operational
and/or regulatory sequence regions (influencing transcription
and/or translation) of genes coding for proteins other than
histones, provide advantageous effects. Accordingly, one aspect of
the invention provides the combination of a histone stem-loop
structure with a poly(A) sequence or a sequence representing a
polyadenylation signal (3'-terminal of a coding region), which does
not occur in metazoan histone genes. According to another preferred
aspect of the invention, a combination of a histone stem-loop
structure with a coding region coding for a tumour antigenic
protein, which does, preferably not occur in metazoan histone
genes, is provided herewith (coding region and histone stem loop
sequence are heterologous). It is preferred, if such tumour
antigenic proteins occur in mammalian, preferably humans, when
suffering from tumour diseases. In a still further preferred
embodiment, all the elements (a), (b) and (c) of the inventive
nucleic acid are heterologous to each other and are combined
artificially from three different sources, e.g. the (a) tumour
antigenic protein coding region from a human gene, (b) the histone
stem loop from the untranslated region of a metazoan, e.g.
mammalian, non-human or human, histone gene and (c) the poly(A)
sequence or the polyadenylation signal from e.g. an untranslated
region of a gene other than a histone gene and other than the
untranslated region of the tumour antigen coding region according
to element (a) of such an inventive nucleic acid.
[0062] A histone stem loop is, therefore, a stem-loop structure as
described herein, which, if preferably functionally defined,
exhibits/retains the property of binding to its natural binding
partner, the stem-loop binding protein (SLBP--also termed the
histone hairpin binding protein, or HBP).
[0063] According to the present invention the histone stem loop
sequence according to component (b) of claim 1 may not derived from
a mouse histone protein. More specifically, the histone stem loop
sequence may not be derived from mouse histone gene H2A614. Also,
the nucleic acid of the invention may neither contain a mouse
histone stem loop sequence nor contain mouse histone gene H2A614.
Further, the inventive nucleic acid sequence may not contain a
stem-loop processing signal, more specifically, a mouse histone
processing signal and, most specifically, may not contain mouse
stem loop processing signal H2kA614, even if the inventive nucleic
acid sequence may contain at least one mammalian histone gene.
However, the at least one mammalian histone gene may not be Seq. ID
No. 7 of WO 01/12824.
[0064] According to one preferred embodiment of the first inventive
aspect, the inventive nucleic acid sequence comprises or codes for
at least one histone stem-loop sequence, preferably according to at
least one of the following formulae (I) or (II): [0065] formula (I)
(stem-loop sequence without stem bordering elements):
[0065] ##STR00001## [0066] formula (II) (stem-loop sequence with
stem bordering elements):
[0066] ##STR00002## [0067] wherein: [0068] stem1 or stem2 bordering
elements N.sub.1-6 is a consecutive sequence of 1 to 6, preferably
of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to
5, most preferably of 4 to 5 or 5 N, wherein each N is
independently from another selected from a nucleotide selected from
A, U, T, G and C, or a nucleotide analogue thereof; [0069] stems
N.sub.0-2GN.sub.3-5 is reverse complementary or partially reverse
complementary with element stem2, and is a consecutive sequence
between of 5 to 7 nucleotides; [0070] wherein N.sub.0-2 is a
consecutive sequence of 0 to 2, preferably of 0 to 1, more
preferably of 1 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof; [0071] wherein N.sub.5-5 is a
consecutive sequence of 3 to 5, preferably of 4 to 5, more
preferably of 4 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof, and [0072] wherein G is guanosine or
an analogue thereof, and may be optionally replaced by a cytidine
or an analogue thereof, provided that its complementary nucleotide
cytidine in stem2 is replaced by guanosine; [0073] loop sequence
N.sub.0-4(U/T)N.sub.0-4 is located between elements stem1 and
stem2, and is a consecutive sequence of 3 to 5 nucleotides, more
preferably of 4 nucleotides; [0074] wherein each N.sub.0-4 is
independent from another a consecutive sequence of 0 to 4,
preferably of 1 to 3, more preferably of 1 to 2 N, wherein each N
is independently from another selected from a nucleotide selected
from A, U, T, G and C or a nucleotide analogue thereof; and wherein
U/T represents uridine, or optionally thymidine; [0075]
stem2N.sub.3-5CN.sub.0-2 is reverse complementary or partially
reverse complementary with element stem1, and is a consecutive
sequence between of 5 to 7 nucleotides; [0076] wherein N.sub.5-5 is
a consecutive sequence of 3 to 5, preferably of 4 to 5, more
preferably of 4 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof; [0077] wherein N.sub.0-2 is a
consecutive sequence of 0 to 2, preferably of 0 to 1, more
preferably of 1 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G or C or a
nucleotide analogue thereof; and [0078] wherein C is cytidine or an
analogue thereof, and may be optionally replaced by a guanosine or
an analogue thereof provided that its complementary nucleotide
guanosine in stem1 is replaced by cytidine; [0079] wherein [0080]
stem1 and stem2 are capable of base pairing with each other forming
a reverse complementary sequence, wherein base pairing may occur
between stem1 and stem2, e.g. by Watson-Crick base pairing of
nucleotides A and U/T or G and C or by non-Watson-Crick base
pairing e.g. wobble base pairing, reverse Watson-Crick base
pairing, Hoogsteen base pairing, reverse Hoogsteen base pairing or
are capable of base pairing with each other forming a partially
reverse complementary sequence, wherein an incomplete base pairing
may occur between stem1 and stem2, on the basis that one or more
bases in one stem do not have a complementary base in the reverse
complementary sequence of the other stem.
[0081] In the above context, a wobble base pairing is typically a
non-Watson-Crick base pairing between two nucleotides. The four
main wobble base pairs in the present context, which may be used,
are guanosine-uridine, inosine-uridine, inosine-adenosine,
inosine-cytidine (G-U/T, I-U/T, I-A and I-C) and adenosine-cytidine
(A-C).
[0082] Accordingly, in the context of the present invention, a
wobble base is a base, which forms a wobble base pair with a
further base as described above. Therefore non-Watson-Crick base
pairing, e.g. wobble base pairing, may occur in the stem of the
histone stem-loop structure according to the present invention.
[0083] In the above context a partially reverse complementary
sequence comprises maximally 2, preferably only one mismatch in the
stem-structure of the stem-loop sequence formed by base pairing of
stem1 and stem2. In other words, stem1 and stem2 are preferably
capable of (full) base pairing with each other throughout the
entire sequence of stem1 and stem2 (100% of possible correct
Watson-Crick or non-Watson-Crick base pairings), thereby forming a
reverse complementary sequence, wherein each base has its correct
Watson-Crick or non-Watson-Crick base pendant as a complementary
binding partner. Alternatively, stem1 and stem2 are preferably
capable of partial base pairing with each other throughout the
entire sequence of stem1 and stem2, wherein at least about 70%,
75%, 80%, 85%, 90%, or 95% of the 100% possible correct
Watson-Crick or non-Watson-Crick base pairings are occupied with
the correct Watson-Crick or non-Watson-Crick base pairings and at
most about 30%, 25%, 20%, 15%, 10%, or 5% of the remaining bases
are unpaired.
[0084] According to a preferred embodiment of the first inventive
aspect, the at least one histone stem-loop sequence (with stem
bordering elements) of the inventive nucleic acid sequence as
defined herein comprises a length of about 15 to about 45
nucleotides, preferably a length of about 15 to about 40
nucleotides, preferably a length of about 15 to about 35
nucleotides, preferably a length of about 15 to about 30
nucleotides and even more preferably a length of about 20 to about
30 and most preferably a length of about 24 to about 28
nucleotides.
[0085] According to a further preferred embodiment of the first
inventive aspect, the at least one histone stem-loop sequence
(without stem bordering elements) of the inventive nucleic acid
sequence as defined herein comprises a length of about 10 to about
30 nucleotides, preferably a length of about 10 to about 20
nucleotides, preferably a length of about 12 to about 20
nucleotides, preferably a length of about 14 to about 20
nucleotides and even more preferably a length of about 16 to about
17 and most preferably a length of about 16 nucleotides.
[0086] According to a further preferred embodiment of the first
inventive aspect, the inventive nucleic acid sequence according to
the first aspect of the present invention may comprise or code for
at least one histone stem-loop sequence according to at least one
of the following specific formulae (Ia) or (IIa): [0087] formula
(Ia) (stem-loop sequence without stem bordering elements):
[0087] ##STR00003## [0088] formula (IIa) (stem-loop sequence with
stem bordering elements):
[0088] ##STR00004## [0089] wherein: [0090] N, C, G, T and U are as
defined above.
[0091] According to a further more particularly preferred
embodiment of the first aspect, the inventive nucleic acid sequence
may comprise or code for at least one histone stem-loop sequence
according to at least one of the following specific formulae (Ib)
or (IIb): [0092] formula (Ib) (stem-loop sequence without stem
bordering elements):
[0092] ##STR00005## [0093] formula (IIb) (stem-loop sequence with
stem bordering elements):
[0093] ##STR00006## [0094] wherein: [0095] N, C, G, T and U are as
defined above.
[0096] According to an even more preferred embodiment of the first
inventive aspect, the inventive nucleic acid sequence according to
the first aspect of the present invention may comprise or code for
at least one histone stem-loop sequence according to at least one
of the following specific formulae (Ic) to (Ih) or (IIc) to (IIh),
shown alternatively in its stem-loop structure and as a linear
sequence representing histone stem-loop sequences as generated
according to Example 1: [0097] formula (Ic): (metazoan and
protozoan histone stem-loop consensus sequence without stem
bordering elements):
[0097] ##STR00007## [0098] formula (IIc): (metazoan and protozoan
histone stem-loop consensus sequence with stem bordering
elements):
[0098] ##STR00008## [0099] formula (Id): (without stem bordering
elements)
[0099] ##STR00009## [0100] formula (IId): (with stem bordering
elements)
[0100] ##STR00010## [0101] formula (Ie): (protozoan histone
stem-loop consensus sequence without stem bordering elements)
[0101] ##STR00011## [0102] formula (IIe): (protozoan histone
stem-loop consensus sequence with stem bordering elements)
[0102] ##STR00012## [0103] formula (If): (metazoan histone
stem-loop consensus sequence without stem bordering elements)
[0103] ##STR00013## [0104] formula (IIe: (metazoan histone
stem-loop consensus sequence with stem bordering elements)
[0104] ##STR00014## [0105] formula (Ig): (vertebrate histone
stem-loop consensus sequence without stem bordering elements)
[0105] ##STR00015## [0106] formula (IIg): (vertebrate histone
stem-loop consensus sequence with stem bordering elements)
[0106] ##STR00016## [0107] formula (Ih): (human histone stem-loop
consensus sequence (Homo sapiens) without stem bordering
elements)
[0107] ##STR00017## [0108] formula (IIh): (human histone stem-loop
consensus sequence (Homo sapiens) with stem bordering elements)
[0108] ##STR00018## [0109] wherein in each of above formulae (Ic)
to (Ih) or (IIc) to (IIh): [0110] N, C, G, A, T and U are as
defined above; [0111] each U may be replaced by T; [0112] each
(highly) conserved G or C in the stem elements 1 and 2 may be
replaced by its complementary nucleotide base C or G, provided that
its complementary nucleotide in the corresponding stem is replaced
by its complementary nucleotide in parallel; and/or [0113] G, A, T,
U, C, R, Y, M, K, S, W, H, B, V, D, and N are nucleotide bases as
defined in the following Table:
TABLE-US-00001 [0113] abbreviation Nucleotide bases remark G G
Guanine A A Adenine T T Thymine U U Uracile C C Cytosine R G or A
Purine Y T/U or C Pyrimidine M A or C Amino K G or T/U Keto S G or
C Strong (3H bonds) W A or T/U Weak (2H bonds) H A or C or T/U Not
G B G or T/U or C Not A V G or C or A Not T/U D G or A or T/U Not C
N G or C or T/U or A Any base * Present or not Base may be present
or not
[0114] In this context it is particularly preferred that the
histone stem-loop sequence according to at least one of the
formulae (I) or (Ia) to (Ih) or (II) or (IIa) to (IIh) of the
present invention is selected from a naturally occurring histone
stem loop sequence, more particularly preferred from protozoan or
metazoan histone stem-loop sequences, and even more particularly
preferred from vertebrate and mostly preferred from mammalian
histone stem-loop sequences especially from human histone stem-loop
sequences.
[0115] According to a particularly preferred embodiment of the
first aspect, the histone stem-loop sequence according to at least
one of the specific formulae (I) or (Ia) to (Ih) or (II) or (IIa)
to (IIh) of the present invention is a histone stem-loop sequence
comprising at each nucleotide position the most frequently
occurring nucleotide, or either the most frequently or the
second-most frequently occurring nucleotide of naturally occurring
histone stem-loop sequences in metazoa and protozoa (FIG. 1),
protozoa (FIG. 2), metazoa (FIG. 3), vertebrates (FIG. 4) and
humans (FIG. 5) as shown in FIG. 1-5. In this context it is
particularly preferred that at least 80%, preferably at least 85%,
or most preferably at least 90% of all nucleotides correspond to
the most frequently occurring nucleotide of naturally occurring
histone stem-loop sequences.
[0116] In a further particular embodiment of the first aspect, the
histone stem-loop sequence according to at least one of the
specific formulae (I) or (Ia) to (Ih) of the present invention is
selected from following histone stem-loop sequences (without
stem-bordering elements) representing histone stem-loop sequences
as generated according to Example 1:
TABLE-US-00002 VGYYYYHHTHRVVRCB (SEQ ID NO: 13 according to formula
(Ic)) SGYYYTTYTMARRRCS (SEQ ID NO: 14 according to formula (Ic))
SGYYCTTTTMAGRRCS (SEQ ID NO: 15 according to formula (Ic))
DGNNNBNNTHVNNNCH (SEQ ID NO: 16 according to formula (Ie))
RGNNNYHBTHRDNNCY (SEQ ID NO: 17 according to formula (Ie))
RGNDBYHYTHRDHNCY (SEQ ID NO: 18 according to formula (Ie))
VGYYYTYHTHRVRRCB (SEQ ID NO: 19 according to formula (If))
SGYYCTTYTMAGRRCS (SEQ ID NO: 20 according to formula (If))
SGYYCTTTTMAGRRCS (SEQ ID NO: 21 according to formula (If))
GGYYCTTYTHAGRRCC (SEQ ID NO: 22 according to formula (Ig))
GGCYCTTYTMAGRGCC (SEQ ID NO: 23 according to formula (Ig))
GGCTCTTTTMAGRGCC (SEQ ID NO: 24 according to formula (Ig))
DGHYCTDYTHASRRCC (SEQ ID NO: 25 according to formula (Ih))
GGCYCTTTTHAGRGCC (SEQ ID NO: 26 according to formula (Ih))
GGCYCTTTTMAGRGCC (SEQ ID NO: 27 according to formula (Ih))
[0117] Furthermore in this context following histone stem-loop
sequences (with stem bordering elements) as generated according to
Example 1 according to one of specific formulae (II) or (IIa) to
(IIh) are particularly preferred:
TABLE-US-00003 H*H*HHVVGYYYYHHTHRVVRCBVHH*N*N* (SEQ ID NO: 28
according to formula (IIc)) M*H*MHMSGYYYTTYTMARRRCSMCH*H*H* (SEQ ID
NO: 29 according to formula (IIc)) M*M*MMMSGYYCTTTTMAGRRCSACH*M*H*
(SEQ ID NO: 30 according to formula (IIc))
N*N*NNNDGNNNBNNTHVNNNCHNHN*N*N* (SEQ ID NO: 31 according to formula
(He)) N*N*HHNRGNNNYHBTHRDNNCYDHH*N*N* (SEQ ID NO: 32 according to
formula (He)) N*H*HHVRGNDBYHYTHRDHNCYRHH*H*H* (SEQ ID NO: 33
according to formula (He)) H*H*MHMVGYYYTYHTHRVRRCBVMH*H*N* (SEQ ID
NO: 34 according to formula (If)) M*M*MMMSGYYCTTYTMAGRRCSMCH*H*H*
(SEQ ID NO: 35 according to formula (If))
M*M*MMMSGYYCTTTTMAGRRCSACH*M*H* (SEQ ID NO: 36 according to formula
(IH)) H*H*MAMGGYYCTTYTHAGRRCCVHN*N*M* (SEQ ID NO: 37 according to
formula (hg)) H*H*AAMGGCYCTTYTMAGRGCCVCH*H*M* (SEQ ID NO: 38
according to formula (hg)) M*M*AAMGGCTCTTTTMAGRGCCMCY*M*M* (SEQ ID
NO: 39 according to formula (hg)) N*H*AAHDGHYCTDYTHASRRCCVHB*N*H*
(SEQ ID NO: 40 according to formula (IIh))
H*H*AAMGGCYCTTTTHAGRGCCVMY*N*M* (SEQ ID NO: 41 according to formula
(IIh)) H*M*AAAGGCYCTTTTMAGRGCCRMY*H*M* (SEQ ID NO: 42 according to
formula (IIh))
[0118] According to a further preferred embodiment of the first
inventive aspect, the inventive nucleic acid sequence comprises or
codes for at least one histone stem-loop sequence showing at least
about 80%, preferably at least about 85%, more preferably at least
about 90%, or even more preferably at least about 95%, sequence
identity with the not to 100% conserved nucleotides in the histone
stem-loop sequences according to at least one of specific formulae
(I) or (Ia) to (Ih) or (II) or (IIa) to (IIh) or with a naturally
occurring histone stem-loop sequence.
[0119] In a preferred embodiment, the histone stem loop sequence
does not contain the loop sequence 5'-UUUC-3'. More specifically,
the histone stem loop does not contain the stems sequence
5'-GGCUCU-3' and/or the stem2 sequence 5'-AGAGCC-3', respectively.
In another preferred embodiment, the stem loop sequence does not
contain the loop sequence 5'-CCUGCCC-3' or the loop sequence
5'-UGAAU-3'. More specifically, the stem loop does not contain the
stem1 sequence 5'-CCUGAGC-3' or does not contain the stem1 sequence
5'-ACCUUUCUCCA-3' and/or the stem2 sequence 5'-GCUCAGG-3' or
5'-UGGAGAAAGGU-3', respectively. Also, as far as the invention is
not limited to histone stem loop sequences specifically, stem loop
sequences are preferably not derived from a mammalian insulin
receptor 3'-untranslated region. Also, preferably, the inventive
nucleic acid may not contain histone stem loop processing signals,
in particular not those derived from mouse histone gene H2A614 gene
(H2kA614).
[0120] The inventive nucleic acid sequence according to the first
aspect of the present invention may optionally comprise or code for
a poly(A) sequence. When present, such a poly(A) sequence comprises
a sequence of about 25 to about 400 adenosine nucleotides,
preferably a sequence of about 30 or, more preferably, of about 50
to about 400 adenosine nucleotides, more preferably a sequence of
about 50 to about 300 adenosine nucleotides, even more preferably a
sequence of about 50 to about 250 adenosine nucleotides, most
preferably a sequence of about 60 to about 250 adenosine
nucleotides. In this context the term "about" refers to a deviation
of .+-.10% of the value(s) it is attached to. Accordingly, the
poly(A) sequence contains at least 25 or more than 25, more
preferably, at least 30, more preferably at least 50 adenosine
nucleotides. Therefore, such a poly (A) sequence does typically not
contain less than 20 adenosine nucleotides. More particularly, it
does not contain 10 and/or less than 10 adenosine nucleotides.
[0121] Preferably, the nucleic acid according of the present
invention does not contain one or two or at least one or all but
one or all of the components of the group consisting of: a sequence
encoding a ribozyme (preferably a self-splicing ribozyme), a viral
nucleic acid sequence, a histone stem-loop processing signal, in
particular a histone-stem loop processing sequence derived from
mouse histone H2A614 gene, a Neo gene, an inactivated promoter
sequence and an inactivated enhancer sequence. Even more
preferably, the nucleic acid according to the invention does not
contain a ribozyme, preferably a self-splicing ribozyme, and one of
the group consisting of: a Neo gene, an inactivated promoter
sequence, an inactivated enhancer sequence, a histone stem-loop
processing signal, in particular a histone-stem loop processing
sequence derived from mouse histone H2A614 gene. Accordingly, the
nucleic acid may in a preferred mode neither contain a ribozyme,
preferably a self-splicing ribozyme, nor a Neo gene or,
alternatively, neither a ribozyme, preferably a self-splicing
ribozyme, nor any resistance gene (e.g. usually applied for
selection). In another preferred mode, the nucleic acid of the
invention may neither contain a ribozyme, preferably a
self-splicing ribozyme nor a histone stem-loop processing signal,
in particular a histone-stem loop processing sequence derived from
mouse histone H2A614 gene
[0122] Alternatively, according to the first aspect of the present
invention, the inventive nucleic sequence optionally comprises a
polyadenylation signal which is defined herein as a signal which
conveys polyadenylation to a (transcribed) mRNA by specific protein
factors (e.g. cleavage and polyadenylation specificity factor
(CPSF), cleavage stimulation factor (CstF), cleavage factors I and
II (CF I and CF II), poly(A) polymerase (PAP)). In this context a
consensus polyadenylation signal is preferred comprising the
NN(U/T)ANA consensus sequence. In a particular preferred aspect the
polyadenylation signal comprises one of the following sequences:
AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usually present in
RNA and thymidine is usually present in DNA). In some embodiments,
the polyadenylation signal used in the inventive nucleic acid does
not correspond to the U3 snRNA, U5, the polyadenylation processing
signal from human gene G-CSF, or the SV40 polyadenylation signal
sequences. In particular, the above polyadenylation signals are not
combined with any antibiotics resistance gene (or any other
reporter, marker or selection gene), in particular not with the
resistance neo gene (neomycin phosphotransferase) (as the gene of
the coding region according to element (a) of the inventive nucleic
acid in such an inventive nucleic acid. And any of the above
polyadenylation signals are preferably not combined with the
histone stem loop or the histone stem loop processing signal from
mouse histone gene H2A614 in an inventive nucleic acid.
[0123] The inventive nucleic acid sequence according to the first
aspect of the present invention may furthermore encode a protein or
a peptide, which comprises a tumour antigen or a fragment, variant
or derivative thereof.
[0124] Tumour antigens are preferably located on the surface of the
(tumour) cell characterizing a mammalian, in particular human
tumour (in e.g. systemic or solid tumour diseases). Tumour antigens
may also be selected from proteins, which are overexpressed in
tumour cells compared to a normal cell. Furthermore, tumour
antigens also includes antigens expressed in cells which are (were)
not themselves (or originally not themselves) degenerated but are
associated with the supposed tumour. Antigens which are connected
with tumour-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 tumour
furthermore include antigens from cells or tissues, typically
embedding the tumour. 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. These
substances are also referred to as "tumour antigens", however they
are not antigens in the stringent meaning of an immune response
inducing substance. The class of tumour antigens can be divided
further into tumour-specific antigens (TSAs) and
tumour-associated-antigens (TAAs). TSAs can only be presented by
tumour cells and never by normal "healthy" cells. They typically
result from a tumour specific mutation. TAAs, which are more
common, are usually presented by both tumour and healthy cells.
These antigens are recognized and the antigen-presenting cell can
be destroyed by cytotoxic T cells. Additionally, tumour antigens
can also occur on the surface of the tumour in the form of, e.g., a
mutated receptor. In this case, they can be recognized by
antibodies.
[0125] Further, tumour associated antigens may be classified as
tissue-specific antigens, also called melanocyte-specific antigens,
cancer-testis antigens and tumour-specific antigens. Cancer-testis
antigens are typically understood to be peptides or proteins of
germ-line associated genes which may be activated in a wide variety
of tumours. Human cancer-testis antigens may be further subdivided
into antigens which are encoded on the X chromosome, so-called CT-X
antigens, and those antigens which are not encoded on the X
chromosome, the so-called (non-X CT antigens). Cancer-testis
antigens which are encoded on the X-chromosome comprises, for
example, the family of melanoma antigen genes, the so-called
MAGE-family. The genes of the MAGE-family may be characterised by a
shared MAGE homology domain (MHD). Each of these antigens, i.e.
melanocyte-specific antigens, cancer-testis antigens and
tumour-specific antigens, may elicit autologous cellular and
humoral immune response. Accordingly, the tumour antigen encoded by
the inventive nucleic acid sequence is preferably a
melanocyte-specific antigen, a cancer-testis antigen or a
tumour-specific antigens, preferably it may be a CT-X antigen, a
non-X CT-antigens, a binding partner for a CT-X antigen or a
binding partner for a non-X CT-antigen or a tumour-specific
antigen, more preferably a CT-X antigen, a binding partner for a
non-X CT-antigen or a tumour-specific antigen.
[0126] Particular preferred tumour antigens are selected from the
list consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,
alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,
alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4,
ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4,
BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,
calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19,
CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80,
CDCl.sub.27/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-R17I, 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-B, NY-ESO-1, 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, PAP, PART-1,
PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, 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-1, survivin, survivin-2B,
SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta,
TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2,
TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and WT1. Such
tumour antigens preferably may be selected from the group
consisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1,
MAGE-A3, Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA, PSCA,
PSMA, STEAP-1, VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more
preferably from PAP, MAGE-A3, WT1, and MUC-1. Such tumour antigens
preferably may be selected from the group consisting of MAGE-A1
(e.g. MAGE-A1 according to accession number M77481), MAGE-A2,
MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accession number
NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according to
accession number NM_005511), GP100 (e.g. GP100 according to
accession number M77348), tyrosinase (e.g. tyrosinase according to
accession number NM_000372), surviving (e.g. survivin according to
accession number AF077350), CEA (e.g. CEA according to accession
number NM_004363), Her-2/neu (e.g. Her-2/neu according to accession
number M11730), WT1 (e.g. WT1 according to accession number
NM_000378), PRAME (e.g. PRAME according to accession number
NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g. EGFRI
(epidermal growth factor receptor 1) according to accession number
AF288738), MUC1, mucin-1 (e.g. mucin-1 according to accession
number NM_002456), SEC61G (e.g. SEC61G according to accession
number NM_014302), hTERT (e.g. hTERT accession number NM_198253),
5T4 (e.g. 5T4 according to accession number NM_006670), TRP-2 (e.g.
TRP-2 according to accession number NM_001922), STEAP1, PCA, PSA,
PSMA, etc.
[0127] Furthermore tumour antigens also may encompass idiotypic
antigens associated with a cancer or tumour disease, particularly
lymphoma or a lymphoma associated disease, wherein said idiotypic
antigen is an immunoglobulin idiotype of a lymphoid blood cell or a
T cell receptor idiotype of a lymphoid blood cell.
[0128] Tumour antigenic proteins for the treatment of cancer or
tumour diseases, are typically proteins of mammalian origin,
preferably of human origin. Their selection for treatment of the
subject depends on the tumour type to be treated and the expression
profile of the individual tumour. A human suffering from prostate
cancer, is e.g. preferably treated by a tumour antigen, which is
typically expressed (or overexpressed) in prostate carcinoma or
specifically overexpressed in the subject to be treated, e.g. any
of PSMA, PSCA, and/or PSA.
[0129] The coding region of the inventive nucleic acid according to
the first aspect of the present invention may occur as a mono-,
di-, or even multicistronic nucleic acid, i.e. a nucleic acid which
carries the coding sequences of one, two or more proteins or
peptides. Such coding sequences in di-, or even multicistronic
nucleic acids may be separated by at least one internal ribosome
entry site (IRES) sequence, e.g. as described herein or by signal
peptides which induce the cleavage of the resulting polypeptide
which comprises several proteins or peptides.
[0130] According to the first aspect of the present invention, the
inventive nucleic acid sequence comprises a coding region, encoding
a peptide or protein which comprises a tumour antigen or a
fragment, variant or derivative thereof. Preferably, the encoded
tumour antigen is no histone protein. In the context of the present
invention such a histone protein is typically a strongly alkaline
protein found in eukaryotic cell nuclei, which package and order
the DNA into structural units called nucleosomes. Histone proteins
are the chief protein components of chromatin, act as spools around
which DNA winds, and play a role in gene regulation. Without
histones, the unwound DNA in chromosomes would be very long (a
length to width ratio of more than 10 million to one in human DNA).
For example, each human cell has about 1.8 meters of DNA, but wound
on the histones it has about 90 millimeters of chromatin, which,
when duplicated and condensed during mitosis, result in about 120
micrometers of chromosomes. More preferably, in the context of the
present invention such a histone protein is typically defined as a
highly conserved protein selected from one of the following five
major classes of histones: H1/H5, H2A, H2B, H3, and H4'',
preferably selected from mammalian histone, more preferably from
human histones or histone proteins. Such histones or histone
proteins are typically organised into two super-classes defined as
core histones, comprising histones H2A, H2B, H3 and H4, and linker
histones, comprising histones H1 and H5.
[0131] In this context, linker histones, preferably excluded from
the scope of protection of the pending invention, preferably
mammalian linker histones, more preferably human linker histones,
are typically selected from H1, including H1F, particularly
including H1F0, H1FNT, H1FOO, H1FX, and H1H1, particularly
including HIST1H1A, HIST1H18, HIST1H1C, HIST1H1D, HIST1H1E,
HIST1H1T; and
[0132] Furthermore, core histones, preferably excluded from the
scope of protection of the pending invention, preferably mammalian
core histones, more preferably human core histones, are typically
selected from H2A, including H2AF, particularly including H2AFM,
H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ, and
H2A1, particularly including HIST1H2AA, HIST1H2AB, HIST1H2AC,
HIST1H2AD, HIST1H2AE, HIST1H2AG, HIST1H2AI, HIST1H2AJ, HIST1H2AK,
HIST11-12AL, HIST11-12AM, and H2A2, particularly including
HIST2H2AA3, HIST2H2AC; H2B, including H2BF, particularly including
H2BFM, H2BFO, H2BFS, H2BFWT H2B1, particularly including
HIST11-12BA, HIST11-12BB, HIST1H2BC, HIST11-12BD, HIST1H2BE,
HIST1H2BF, HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK,
HIST1H2BL, HIST1H2BM, HIST11-12BN, HIST11-12B0, and H2B2,
particularly including HIST2H2BE; H3, including H3A1, particularly
including HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E,
HIST1H3F, HIST11-13G, HIST11-13H, HIST11-13I, HIST11-13J, and H3A2,
particularly including HIST2H3C, and H3A3, particularly including
HIST3H3; H4, including H41, particularly including HIST1H4A,
HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G,
HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, and H44,
particularly including HIST4H4, and H5.
[0133] According to the first aspect of the present invention, the
inventive nucleic acid sequence comprises a coding region, encoding
a peptide or protein which comprises a tumour antigen or a
fragment, variant or derivative thereof. Preferably, the encoded
tumour antigen is no reporter protein (e.g. Luciferase, Green
Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein
(EGFP), .beta.-Galactosidase) and no marker or selection protein
(e.g. alpha-Globin, Galactokinase and Xanthine:guanine
phosphoribosyl transferase (GPT)). Preferably, the nucleic acid
sequence of the invention does not contain a (bacterial)
antibiotics resistance gene, in particular not a neo gene sequence
(Neomycin resistance gene) or CAT gene sequence (chloramphenicol
acetyl transferase, chloramphenicol resistance gene).
[0134] The inventive nucleic acid as define above, comprises or
codes for a) a coding region, encoding a peptide or protein which
comprises a tumour antigen or a fragment, variant or derivative
thereof; b) at least one histone stem-loop, and c) a poly(A)
sequence or polyadenylation signal; preferably for increasing the
expression of said encoded peptide or protein, wherein the encoded
peptide or protein is preferably no histone protein, no reporter
protein and/or no marker or selection protein, as defined above.
The elements b) to c) of the inventive nucleic acid may occur in
the inventive nucleic acid in any order, i.e. the elements a), b)
and c) may occur in the order a), b) and c) or a), c) and b) from
5' to 3' direction in the inventive nucleic acid sequence, wherein
further elements as described herein, may also be contained, such
as a 5'-CAP structure, a poly(C) sequence, stabilization sequences,
IRES sequences, etc. Each of the elements a) to c) of the inventive
nucleic acid, particularly a) in di- or multicistronic constructs
and/or each of the elements b) and c), more preferably element b)
may also be repeated at least once, preferably twice or more in the
inventive nucleic acid. As an example, the inventive nucleic acid
may show its sequence elements a), b) and optionally c) in e.g. the
following order: [0135] 5'-coding region-histone stem-loop-poly(A)
sequence-3'; or [0136] 5'-coding region-histone
stem-loop-polyadenylation signal-3'; or [0137] 5'-coding
region-poly(A) sequence-histone stem-loop-3'; or [0138] 5'-coding
region-polyadenylation signal-histone stem-loop-3'; or [0139]
5'-coding region-coding region-histone stem-loop-polyadenylation
signal-3'; or [0140] 5'-coding region-histone stem-loop-histone
stem-loop-poly(A) sequence-3'; or [0141] 5'-coding region-histone
stem-loop-histone stem-loop-polyadenylation signal-3'; etc.
[0142] In this context it is particularly preferred that the
inventive nucleic acid sequence comprises or codes for a) a coding
region, encoding a peptide or protein which comprises a tumour
antigen or fragment, variant or derivative thereof; b) at least one
histone stem-loop, and c) a poly(A) sequence or polyadenylation
sequence; preferably for increasing the expression level of said
encoded peptide or protein, wherein the encoded protein is
preferably no histone protein, no reporter protein (e.g.
Luciferase, GFP, EGFP, .beta.-Galactosidase, particularly EGFP)
and/or no marker or selection protein (e.g. alpha-Globin,
Galactokinase and Xanthine:Guanine phosphoribosyl transferase
(GPT)).
[0143] In a further preferred embodiment of the first aspect the
inventive nucleic acid sequence as defined herein may also occur in
the form of a modified nucleic acid.
[0144] In this context, the inventive nucleic acid sequence as
defined herein may be modified to provide a "stabilized nucleic
acid", preferably a stabilized RNA, more preferably an RNA that is
essentially resistant to in vivo degradation (e.g. by an exo- or
endo-nuclease). A stabilized nucleic acid may e.g. be obtained by
modification of the G/C content of the coding region of the
inventive nucleic acid sequence, by introduction of nucleotide
analogues (e.g. nucleotides with backbone modifications, sugar
modifications or base modifications) or by introduction of
stabilization sequences in the 3'- and/or 5'-untranslated region of
the inventive nucleic acid sequence.
[0145] As mentioned above, the inventive nucleic acid sequence as
defined herein may contain nucleotide analogues/modifications e.g.
backbone modifications, sugar modifications or base modifications.
A backbone modification in connection with the present invention is
a modification in which phosphates of the backbone of the
nucleotides contained in inventive nucleic acid sequence as defined
herein are chemically modified. A sugar modification in connection
with the present invention is a chemical modification of the sugar
of the nucleotides of the inventive nucleic acid sequence as
defined herein. Furthermore, a base modification in connection with
the present invention is a chemical modification of the base moiety
of the nucleotides of the nucleic acid molecule of the inventive
nucleic acid sequence. In this context nucleotide analogues or
modifications are preferably selected from nucleotide analogues
which are applicable for transcription and/or translation.
[0146] In a particular preferred embodiment of the first aspect of
the present invention the herein defined nucleotide
analogues/modifications are selected from base modifications which
additionally increase the expression of the encoded protein and
which are preferably selected from
2-amino-6-chloropurMeriboside-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-chloropurMeriboside-5'-triphosphate,
7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate,
8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate,
benzimidazole-riboside-5'-triphosphate,
N-methyladenosine-5'-triphosphate,
N-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.
[0147] According to a further embodiment, the inventive nucleic
acid sequence as defined herein can contain a lipid modification.
Such a lipid-modified nucleic acid typically comprises a nucleic
acid as defined herein. Such a lipid-modified nucleic acid molecule
of the inventive nucleic acid sequence as defined herein typically
further comprises at least one linker covalently linked with that
nucleic acid molecule, and at least one lipid covalently linked
with the respective linker. Alternatively, the lipid-modified
nucleic acid molecule comprises at least one nucleic acid molecule
as defined herein and at least one (bifunctional) lipid covalently
linked (without a linker) with that nucleic acid molecule.
According to a third alternative, the lipid-modified nucleic acid
molecule comprises a nucleic acid molecule as defined herein, at
least one linker covalently linked with that nucleic acid molecule,
and at least one lipid covalently linked with the respective
linker, and also at least one (bifunctional) lipid covalently
linked (without a linker) with that nucleic acid molecule. In this
context it is particularly preferred that the lipid modification is
present at the terminal ends of a linear inventive nucleic acid
sequence.
[0148] According to another preferred embodiment of the first
aspect of the invention, the inventive nucleic acid sequence as
defined herein, particularly if provided as an (m)RNA, can
therefore be stabilized against degradation by RNases by the
addition of a so-called "5' CAP" structure.
[0149] According to a further preferred embodiment of the first
aspect of the invention, the inventive nucleic acid sequence as
defined herein can be modified by a sequence of at least 10
cytidines, preferably at least 20 cytidines, more preferably at
least 30 cytidines (so-called "poly(C) sequence"). Particularly,
the inventive nucleic acid sequence may contain or code for a
poly(C) sequence of typically about 10 to 200 cytidine nucleotides,
preferably about 10 to 100 cytidine nucleotides, more preferably
about 10 to 70 cytidine nucleotides or even more preferably about
20 to 50 or even 20 to 30 cytidine nucleotides. This poly(C)
sequence is preferably located 3' of the coding region comprised in
the inventive nucleic acid according to the first aspect of the
present invention.
[0150] In a particularly preferred embodiment of the present
invention, the G/C content of the coding region, encoding at least
one peptide or protein which comprises a tumour antigen or a
fragment, variant or derivative thereof of the inventive nucleic
acid sequence as defined herein, is modified, particularly
increased, compared to the G/C content of its particular wild type
coding region, i.e. the unmodified coding region. The encoded amino
acid sequence of the coding region is preferably not modified
compared to the coded amino acid sequence of the particular wild
type coding region.
[0151] The modification of the G/C-content of the coding region of
the inventive nucleic acid sequence as defined herein is based on
the fact that the sequence of any mRNA region to be translated is
important for efficient translation of that mRNA. Thus, the
composition and the sequence of various nucleotides are important.
In particular, mRNA sequences having an increased G (guanosine)/C
e)/C (cytosine) content are more stable than mRNA sequences having
an increased A (adenosine)/U (uracil) content. According to the
invention, the codons of the coding region are therefore varied
compared to its wild type coding region, 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 favourable codons for the stability
can be determined (so-called alternative codon usage).
[0152] Depending on the amino acid to be encoded by the coding
region of the inventive nucleic acid sequence as defined herein,
there are various possibilities for modification of the nucleic
acid sequence, e.g. the coding region, compared to its wild type
coding region. 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.
[0153] 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:
[0154] the codons for Pro can be modified from CCU or CCA to CCC or
CCG; [0155] the codons for Arg can be modified from CGU or CGA or
AGA or AGG to CGC or CGG; [0156] the codons for Ala can be modified
from GCU or GCA to GCC or GCG; [0157] the codons for Gly can be
modified from GGU or GGA to GGC or GGG.
[0158] 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: [0159] the codons for
Phe can be modified from UUU to UUC; [0160] the codons for Leu can
be modified from UUA, UUG, CUU or CUA to CUC or CUG; [0161] the
codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG
or AGC; [0162] the codon for Tyr can be modified from UAU to UAC;
[0163] the codon for Cys can be modified from UGU to UGC; [0164]
the codon for His can be modified from CAU to CAC; [0165] the codon
for Gln can be modified from CAA to CAG; [0166] the codons for Ile
can be modified from AUU or AUA to AUC; [0167] the codons for Thr
can be modified from ACU or ACA to ACC or ACG; [0168] the codon for
Asn can be modified from AAU to AAC; [0169] the codon for Lys can
be modified from AAA to AAG; [0170] the codons for Val can be
modified from GUU or GUA to GUC or GUG; [0171] the codon for Asp
can be modified from GAU to GAC; [0172] the codon for Glu can be
modified from GAA to GAG; [0173] the stop codon UAA can be modified
to UAG or UGA.
[0174] In the case of the codons for Met (AUG) and Trp (UGG), on
the other hand, there is no possibility of sequence
modification.
[0175] The substitutions listed above can be used either
individually or in all possible combinations to increase the G/C
content of the coding region of the inventive nucleic acid sequence
as defined herein, compared to its particular wild type coding
region (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).
[0176] In the above context, mRNA codons are shown. Therefore
uridine present in an mRNA may also be present as thymidine in the
respective DNA coding for the particular mRNA.
[0177] Preferably, the G/C content of the coding region of the
inventive nucleic acid sequence as defined herein 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 wild
type coding region. According to a specific embodiment at least 5%,
10%, 20%, 30%, 40%, 50%, 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 coding region
encoding at least one peptide or protein which comprises a tumour
antigen or a fragment, variant or derivative thereof are
substituted, thereby increasing the G/C content of said coding
region.
[0178] In this context, it is particularly preferable to increase
the G/C content of the coding region of the inventive nucleic acid
sequence as defined herein, to the maximum (i.e. 100% of the
substitutable codons), compared to the wild type coding region.
[0179] According to the invention, a further preferred modification
of the coding region encoding at least one peptide or protein which
comprises a tumour antigen or a fragment, variant or derivative
thereof of the inventive nucleic acid sequence as defined herein,
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 the coding
region of the inventive nucleic acid sequence as defined herein, to
an increased extent, the corresponding modified nucleic acid
sequence is translated to a significantly poorer degree than in the
case where codons coding for relatively "frequent" tRNAs are
present.
[0180] In this context the coding region of the inventive nucleic
acid sequence is preferably modified compared to the corresponding
wild type coding region 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 coding region
of the inventive nucleic acid sequence as defined herein, is
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 coding region
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.
[0181] 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.
[0182] According to the invention, it is particularly preferable to
link the sequential G/C content which is increased, in particular
maximized, in the coding region of the inventive nucleic acid
sequence as defined herein, with the "frequent" codons without
modifying the amino acid sequence of the peptide or protein encoded
by the coding region of the nucleic acid sequence. This preferred
embodiment allows provision of a particularly efficiently
translated and stabilized (modified) inventive nucleic acid
sequence as defined herein.
[0183] According to another preferred embodiment of the first
aspect of the invention, the inventive nucleic acid sequence as
defined herein, preferably has additionally at least one 5' and/or
3' stabilizing sequence. These stabilizing sequences in the 5'
and/or 3' untranslated regions have the effect of increasing the
half-life of the nucleic acid, particularly of the mRNA in the
cytosol. These stabilizing sequences can have 100% sequence
identity 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 (alpha-)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 nucleic acid. 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: 55), which is
contained in the 3'-UTRs of the very stable RNAs which code for
(alpha-) globin, type(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. In this context it is particularly preferred
that the 3' UTR sequence of the alpha globin gene is located 3' of
the coding region encoding at least one peptide or protein which
comprises a tumour antigen or a fragment, variant or derivative
thereof comprised in the inventive nucleic acid sequence according
to the first aspect of the present invention.
[0184] Substitutions, additions or eliminations of bases are
preferably carried out with the inventive nucleic acid sequence as
defined herein, using a DNA matrix for preparation of the nucleic
acid sequence by techniques of the well-known site directed
mutagenesis or with an oligonucleotide ligation strategy (see e.g.
Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, N.Y.,
2001).
[0185] Any of the above modifications may be applied to the
inventive nucleic acid sequence as defined herein and further to
any nucleic acid as used in the context of the present invention
and may be, if suitable or necessary, be combined with each other
in any combination, provided, these combinations of modifications
do not interfere with each other in the respective nucleic acid. A
person skilled in the art will be able to take his choice
accordingly.
[0186] Nucleic acid sequences used according to the present
invention as defined herein may be prepared using any method known
in the art, including synthetic methods such as e.g. solid phase
synthesis, as well as in vitro methods, such as in vitro
transcription reactions or in vivo reactions, such as in vivo
propagation of DNA plasmids in bacteria.
[0187] In such a process, for preparation of the inventive nucleic
acid sequence as defined herein, especially if the nucleic acid is
in the form of an mRNA, a corresponding DNA molecule may be
transcribed in vitro. This DNA matrix preferably comprises a
suitable promoter, e.g. a T7 or SP6 promoter, for in vitro
transcription, which is followed by the desired nucleotide sequence
for the nucleic acid molecule, e.g. mRNA, to be prepared and a
termination signal for in vitro transcription. The DNA molecule,
which forms the matrix of the at least one 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.RTM. genetic sequence database
accession number U26404; Lai et al., Development 1995, 121: 2349 to
2360), pGEM.RTM. series, e.g. pGEM.RTM.-1 (GENBANK.RTM. genetic
sequence database accession number X65300; from Promega) and pSP64
(GENBANK.RTM. genetic sequence database 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.
[0188] The inventive nucleic acid sequence as defined herein as
well as proteins or peptides as encoded by this nucleic acid
sequence may comprise fragments or variants of those sequences.
Such fragments or variants may typically comprise a sequence having
a sequence identity with one of the above mentioned nucleic acids,
or with one of the proteins or peptides or sequences, if encoded by
the inventive nucleic acid sequence, of at least 5%, 10%, 20%, 30%,
40%, 50%, 60%, preferably at least 70%, more preferably at least
80%, equally more preferably at least 85%, even more preferably at
least 90% and most preferably at least 95% or even 97%, 98% or 99%,
to the entire wild type sequence, either on nucleic acid level or
on amino acid level.
[0189] "Fragments" of proteins or peptides in the context of the
present invention (e.g. as encoded by the inventive nucleic acid
sequence as defined herein) may comprise a sequence of a protein or
peptide as defined herein, which is, with regard to its amino acid
sequence (or its encoded nucleic acid molecule), N-terminally,
ally, C-terminally and/or intrasequentially truncated/shortened
compared to the amino acid sequence of the original (native)
protein (or its encoded nucleic acid molecule). Such truncation may
thus occur either on the amino acid level or correspondingly on the
nucleic acid level. A sequence identity with respect to such a
fragment as defined herein may therefore preferably refer to the
entire protein or peptide as defined herein or to the entire
(coding) nucleic acid molecule of such a protein or peptide.
Likewise, "fragments" of nucleic acids in the context of the
present invention may comprise a sequence of a nucleic acid as
defined herein, which is, with regard to its nucleic acid molecule
5'-, 3'- and/or intrasequentially truncated/shortened compared to
the nucleic acid molecule of the original (native) nucleic acid
molecule. A sequence identity with respect to such a fragment as
defined herein may therefore preferably refer to the entire nucleic
acid as defined herein and the preferred sequence identity level
typically is as indicated herein. Fragments have the same
biological function or specific activity or at least retain an
activity of the natural full length protein of at least 50%, more
preferably at least 70%, even more preferably at least 90%
(measured in an appropriate functional assay, e.g. an assay
assessing the antigenic property by a appropriate assay system
which e.g. measures an immunological reaction, e.g. expression
and/or secretion of an appropriate cytokine (as an indicator of the
immune reaction)) as compared to the full-length native peptide or
protein, e.g. its specific antigenic or therapeutic property.
Accordingly, in a preferred embodiment, the "fragment" is a portion
of the full-length (naturally occurring) tumour antigenic protein,
which exerts tumour antigenic properties on the immune system as
indicated herein.
[0190] Fragments of proteins or peptides in the context of the
present invention (e.g. as encoded by the inventive nucleic acid
sequence as defined herein) may furthermore comprise a sequence of
a protein or peptide as defined herein, which has a length of about
6 to about 20 or even more amino acids, e.g. fragments as processed
and presented by MHC class I molecules, preferably having a length
of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6,
7, 11, or 12 amino acids), or fragments as processed and presented
by MHC class II molecules, preferably having a length of about 13
or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even
more amino acids, wherein these fragments may be selected from any
part of the amino acid sequence. These fragments are typically
recognized by T-cells in form of a complex consisting of the
peptide fragment and an MHC molecule, i.e. the fragments are
typically not recognized in their native form. Fragments of
proteins or peptides as defined herein may comprise at least one
epitope of those proteins or peptides. Furthermore, also domains of
a protein, like the extracellular domain, the intracellular domain
or the transmembrane domain and shortened or truncated versions of
a protein may be understood to comprise a fragment of a
protein.
[0191] Fragments of proteins or peptides as defined herein (e.g. as
encoded by the inventive nucleic acid sequence as defined herein)
may also comprise epitopes of those proteins or peptides. T cell
epitopes or parts of the proteins in the context of the present
invention may comprise fragments preferably having a length of
about 6 to about 20 or even more amino acids, e.g. fragments as
processed and presented by MHC class I molecules, preferably having
a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or
even 11, or 12 amino acids), or fragments as processed and
presented by MHC class II molecules, preferably having a length of
about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20
or even more amino acids, wherein these fragments may be selected
from any part of the amino acid sequence. These fragments are
typically recognized by T cells in form of a complex consisting of
the peptide fragment and an MHC molecule, i.e. the fragments are
typically not recognized in their native form.
[0192] B cell epitopes are typically fragments located on the outer
surface of (native) protein or peptide antigens as defined herein,
preferably having 5 to 15 amino acids, more preferably having 5 to
12 amino acids, even more preferably having 6 to 9 amino acids,
which may be recognized by antibodies, i.e. in their native
form.
[0193] Such epitopes of proteins or peptides may furthermore be
selected from any of the herein mentioned variants of such proteins
or peptides. In this context antigenic determinants can be
conformational or discontinuous epitopes which are composed of
segments of the proteins or peptides as defined herein that are
discontinuous in the amino acid sequence of the proteins or
peptides as defined herein but are brought together in the
three-dimensional structure or continuous or linear epitopes which
are composed of a single polypeptide chain.
[0194] "Variants" of proteins or peptides as defined in the context
of the present invention may be encoded by the inventive nucleic
acid sequence as defined herein. Thereby, a protein or peptide may
be generated, having an amino acid sequence which differs from the
original sequence in one or more (2, 3, 4, 5, 6, 7 or more)
mutation(s), such as one or more substituted, inserted and/or
deleted amino acid(s). The preferred level of sequence identity of
"variants" in view of the full-length natural protein sequence
typically is as indicated herein. Preferably, variants have the
same biological function or specific activity or at least retain an
activity of the natural full length protein of at least 50%, more
preferably at least 70%, even more preferably at least 90%
(measured in an appropriate functional assay, e.g. by an assay
assessing the immunological reaction towards the tumour antigen by
the secretion and/or expression of one or more cytokines) compared
to the full-length native peptide or protein, e.g. its specific
antigenic property. Accordingly, in a preferred embodiment, the
"variant" is a variant of a tumour antigenic protein, which exerts
tumour antigenic properties to the extent as indicated herein.
[0195] "Variants" of proteins or peptides as defined in the context
of the present invention (e.g. as encoded by a nucleic acid as
defined herein) may comprise conservative amino acid
substitution(s) compared to their native, i.e. non-mutated
physiological, sequence. Those amino acid sequences as well as
their encoding nucleotide sequences in particular fall under the
term variants as defined herein. Substitutions in which amino
acids, which originate from the same class, are exchanged for one
another are called conservative substitutions. In particular, these
are amino acids having aliphatic side chains, positively or
negatively charged side chains, aromatic groups in the side chains
or 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).
[0196] Furthermore, variants of proteins or peptides as defined
herein, which may be encoded by the inventive nucleic acid sequence
as defined herein, may also comprise those sequences, wherein
nucleotides of the nucleic acid are exchanged according to the
degeneration of the genetic code, without leading to an alteration
of the respective amino acid sequence of the protein or peptide,
i.e. the amino acid sequence or at least part thereof may not
differ from the original sequence in one or more mutation(s) within
the above meaning.
[0197] In order to determine the percentage to which two sequences
are identical, e.g. nucleic acid sequences or amino acid sequences
as defined herein, preferably the amino acid sequences encoded by
the inventive nucleic acid sequence as defined herein or the amino
acid sequences themselves, the sequences can be aligned in order to
be subsequently compared to one another. Therefore, e.g. a position
of a first sequence may be compared with the corresponding position
of the second sequence. 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.
If this is not the case, the sequences differ at this position. If
insertions occur in the second sequence in comparison to the first
sequence, gaps can be inserted into the first sequence to allow a
further alignment. If deletions occur in the second sequence in
comparison to the first sequence, gaps can be inserted into the
second sequence to allow a further alignment. The percentage to
which two sequences are identical is then a function of the number
of identical positions divided by the total number of positions
including those positions which are only occupied in one sequence.
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 present invention to a certain
extent can be identified by this program.
[0198] The inventive nucleic acid sequence as defined herein may
encode derivatives of a peptide or protein. Such a derivative of a
peptide or protein is a molecule that is derived from another
molecule, such as said peptide or protein. A "derivative" typically
contains the full-length sequence of the natural peptide or protein
and additional sequence features, e.g. at the N- or at the
C-terminus, which may exhibit an additional function to the natural
full-length peptide/protein. Again such derivatives have the same
biological function or specific activity or at least retain an
activity of the natural full length protein of at least 50%, more
preferably at least 70%, even more preferably at least 90%
(measured in an appropriate functional assay, see above, e.g. as
expressed by cytokine expression and/or secretion in an
immunological reaction) as compared to the full-length native
peptide or protein, e.g. its specific therapeutic property.
Thereby, a "derivative" of a peptide or protein also encompasses
(chimeric) fusion peptides/proteins comprising a peptide or protein
used in the present invention or a natural full-length protein (or
variant/fragment thereof) fused to a distinct peptide/protein
awarding e.g. two or more biological functions to the fusion
peptide/protein. For example, the fusion comprises a label, such
as, for example, an epitope, e.g., a FLAG epitope or a V5 epitope
or an HA epitope. For example, the epitope is a FLAG epitope. Such
a tag is useful for, for example, purifying the fusion protein.
[0199] In this context, a "variant" of a protein or peptide may
have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid
identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of
such protein or peptide. Analogously, a "variant" of a nucleic acid
sequence, or particularly, a fragment, may have at least 70%, 75%,
80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch
of 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid
sequence; typically, however, referring to the naturally occurring
full-length sequences. In case of "fragments" typically, sequence
identity is determined for the fragment over length (of the
fragment) on the portion of the full-length protein (reflecting the
same length as the fragment), which exhibits the highest level of
sequence identity.
[0200] In a further preferred embodiment of the first aspect of the
present invention the inventive nucleic acid sequence is associated
with a vehicle, transfection or complexation agent for increasing
the transfection efficiency and/or the immunostimulatory properties
of the inventive nucleic acid sequence. Particularly preferred
agents in this context suitable for increasing the transfection
efficiency are cationic or polycationic compounds, including
protamine, nucleoline, spermine or spermidine, or other cationic
peptides or proteins, such as poly-L-lysine (PLL), poly-arginine,
basic polypeptides, cell penetrating peptides (CPPs), including
HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides,
Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes
simplex), MAP, KALA or protein transduction domains (PTDs), PpT620,
prolin-rich peptides, arginine-rich peptides, lysine-rich peptides,
MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),
Antennapedia-derived peptides (particularly from Drosophila
antennapedia), pAntp, pIs1, FGF, Lactoferrin, Transportan,
Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides,
SAP, or histones. Additionally, preferred cationic or polycationic
proteins or peptides may be selected from the following proteins or
peptides having the following total formula:
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x,
wherein 1+m+n+o+x=8-15, and 1, 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
(=naturally occurring) or non-native amino acids except of Arg,
Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3
or 4, provided, that the overall content of Xaa does not exceed 50%
of all amino acids of the oligopeptide. Particularly preferred
cationic peptides in this context are e.g. Arg.sub.7, Arg.sub.8,
Arg.sub.9, H.sub.5R.sub.9, R.sub.9H.sub.5, H.sub.5R.sub.9H.sub.5,
YSSR.sub.9SSY, (RKH).sub.4, Y(RKH).sub.2R, etc. Further preferred
cationic or polycationic compounds, which can be used as
transfection agent may include cationic polysaccharides, for
example chitosan, polybrene, cationic polymers, e.g.
polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:
[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,
DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC,
DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC,
DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:
Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:
dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:
O,O-ditetradecanoyl-N-(.alpha.-trimethylammonioacetyl)diethanolamine
chloride, CLIP1:
rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
chloride, CLIP6:
rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,
CLIPS:
rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylamm-
onium, oligofectamine, or cationic or polycationic polymers, e.g.
modified polyaminoacids, such as .beta.-aminoacid-polymers or
reversed polyamides, etc., modified polyethylenes, such as PVP
(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified
acrylates, such as pDMAEMA (poly(dimethylaminoethyl
methylacrylate)), etc., modified Amidoamines such as pAMAM
(poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such
as diamine end modified 1,4 butanediol
diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such
as polypropylamine dendrimers or pAMAM based dendrimers, etc.,
polyimine(s), such as PEI: poly(ethyleneimine),
poly(propyleneimine), etc., polyallylamine, sugar backbone based
polymers, such as cyclodextrin based polymers, dextran based
polymers, chitosan, etc., silan backbone based polymers, such as
PMOXA-PDMS copolymers, etc., blockpolymers consisting of a
combination of one or more cationic blocks (e.g. selected from a
cationic polymer as mentioned above) and of one or more hydrophilic
or hydrophobic blocks (e.g polyethyleneglycole); etc.
[0201] In this context it is particularly preferred that the
inventive nucleic acid is complexed at least partially with a
cationic or polycationic compound, preferably cationic proteins or
peptides. Partially means that only a part of the inventive nucleic
acid is complexed with a cationic or polycationic compound and that
the rest of the inventive nucleic acid is in uncomplexed form
("free"). Preferably the ratio of complexed nucleic acid to: free
nucleic acid is selected from a range. of about 5:1 (w/w) to about
1:10 (w/w), more preferably from a range of about 4:1 (w/w) to
about 1:8 (w/w), even more preferably from a range of about 3:1
(w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the
ratio of complexed nucleic acid to free nucleic acid is selected
from a ratio of about 1:1 (w/w).
[0202] It is preferred that the nucleic acid sequence of the
invention is provided in either naked form or complexed, e.g. by
polycationic compounds of whatever chemical structure, preferably
polycationic (poly)peptides or synthetic polycationic compounds.
Preferably, the nucleic acid sequence is not provided together with
a packaging cell.
[0203] In a further aspect the invention provides for a composition
or kit or kit of parts comprising a plurality or more than one,
preferably 2 to 10, more preferably 2 to 5, most preferably 2 to 4
of the inventive nucleic acid sequences as defined herein. These
inventive compositions comprise more than one inventive nucleic
acid sequences, preferably encoding different peptides or proteins
which comprise preferably different tumour antigens or fragments,
variants or derivatives thereof.
[0204] In a preferred embodiment the inventive composition or kit
or kit of parts comprising a plurality (which means typically more
than 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10,
preferably 2 to 5 nucleic acids) of inventive nucleic acid
sequences, particularly for use in the treatment of prostate cancer
(PCa) comprises at least: [0205] a) an inventive nucleic acid
encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen PSA, or a fragment,
variant or derivative thereof; and [0206] b) an inventive nucleic
acid encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen PSMA, or a
fragment, variant or derivative thereof; and [0207] c) an inventive
nucleic acid encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen PSCA, or a
fragment, variant or derivative thereof; and [0208] d) an inventive
nucleic acid encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen STEAP-1, or
a fragment, variant or derivative thereof.
[0209] In a further preferred embodiment the inventive composition
or kit or kit of parts comprising a plurality (which means
typically more than 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids,
e.g. 2 to 10, preferably 2 to 5 nucleic acids) of inventive nucleic
acid sequences, particularly for use in the treatment of non-small
lung cancer (NSCLC) comprises at least: [0210] a) a nucleic acid
sequence comprising or coding for [0211] i. a coding region,
encoding at least one peptide or protein which comprises the tumour
antigen NY-ESO-1, or a fragment, variant or derivative thereof,
[0212] ii. at least one histone stem-loop, and [0213] iii. a
poly(A) sequence or a polyadenylation signal; [0214] b) an
inventive nucleic acid encoding at least one peptide or protein,
wherein said encoded peptide or protein comprises the tumour
antigen 5T4, or a fragment, variant or derivative thereof; and
[0215] c) an inventive nucleic acid encoding at least one peptide
or protein, wherein said encoded peptide or protein comprises the
tumour antigen Survivin, or a fragment, variant or derivative
thereof.
[0216] Furthermore in an alternative, the inventive composition or
kit or kit of parts comprising a plurality (which means typically
more than 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to
10, preferably 2 to 5 nucleic acids) of inventive nucleic acid
sequences, particularly for use in the treatment of non-small lung
cancer (NSCLC) comprises at least: [0217] a) a nucleic acid
sequence comprising or coding for [0218] i. a coding region,
encoding at least one peptide or protein which comprises the tumour
antigen NY-ESO-1, or a fragment, variant or derivative thereof,
[0219] ii. at least one histone stem-loop, and [0220] iii. a
poly(A) sequence or a polyadenylation signal; [0221] b) an
inventive nucleic acid encoding at least one peptide or protein,
wherein said encoded peptide or protein comprises the tumour
antigen 5T4, or a fragment, variant or derivative thereof; and
[0222] c) an inventive nucleic acid encoding at least one peptide
or protein, wherein said encoded peptide or protein comprises the
tumour antigen Survivin, or a fragment, variant or derivative
thereof; and [0223] d) an inventive nucleic acid encoding at least
one peptide or protein, wherein said encoded peptide or protein
comprises the tumour antigen MAGE-C1, or a fragment, variant or
derivative thereof; and [0224] e) an inventive nucleic acid
encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen MAGE-C2, or a
fragment, variant or derivative thereof.
[0225] According to a further aspect, the present invention also
provides a method for increasing the expression of an encoded
peptide or protein comprising the steps, e.g. a) providing the
inventive nucleic acid sequence as defined herein or the inventive
composition comprising a plurality (which means typically more than
1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10,
preferably 2 to 5 nucleic acids) of inventive nucleic acid
sequences as defined herein, b) applying or administering the
inventive nucleic acid sequence as defined herein or the inventive
composition comprising a plurality of inventive nucleic acid
sequences as defined herein to an expression system, e.g. to a
cell-free expression system, a cell (e.g. an expression host cell
or a somatic cell), a tissue or an organism. The method may be
applied for laboratory, for research, for diagnostic, for
commercial production of peptides or proteins and/or for
therapeutic purposes. In this context, typically after preparing
the inventive nucleic acid sequence as defined herein or of the
inventive composition comprising a plurality of inventive nucleic
acid sequences as defined herein, it is typically applied or
administered to a cell-free expression system, a cell (e.g. an
expression host cell or a somatic cell), a tissue or an organism,
e.g. in naked or complexed form or as a pharmaceutical composition
or vaccine as described herein, preferably via transfection or by
using any of the administration modes as described herein. The
method may be carried out in vitro, in vivo or ex vivo. The method
may furthermore be carried out in the context of the treatment of a
specific disease, particularly in the treatment of cancer or tumour
diseases, preferably as defined herein.
[0226] In this context in vitro is defined herein as transfection
or transduction of the inventive nucleic acid as defined herein or
of the inventive composition comprising a plurality of inventive
nucleic acid sequences as defined herein into cells in culture
outside of an organism; in vivo is defined herein as transfection
or transduction of the inventive nucleic acid or of the inventive
composition comprising a plurality of inventive nucleic acid
sequences into cells by application of the inventive nucleic acid
or of the inventive composition to the whole organism or individual
and ex vivo is defined herein as transfection or transduction of
the inventive nucleic acid or of the inventive composition
comprising a plurality of inventive nucleic acid sequences (which
means typically more than 1, 2, 3, 4, 5, 6 or more than 10 nucleic
acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) into cells
outside of an organism or individual and subsequent application of
the transfected cells to the organism or individual.
[0227] Likewise, according to another aspect, the present invention
also provides the use of the inventive nucleic acid sequence as
defined herein or of the inventive composition comprising a
plurality of inventive nucleic acid sequences as defined herein,
preferably for diagnostic or therapeutic purposes, for increasing
the expression of an encoded peptide or protein, e.g. by applying
or administering the inventive nucleic acid sequence as defined
herein or of the inventive composition comprising a plurality of
inventive nucleic acid sequences as defined herein, e.g. to a
cell-free expression system, a cell (e.g. an expression host cell
or a somatic cell), a tissue or an organism. The use may be applied
for laboratory, for research, for diagnostic for commercial
production of peptides or proteins and/or for therapeutic purposes.
In this context, typically after preparing the inventive nucleic
acid sequence as defined herein or of the inventive composition
comprising a plurality of inventive nucleic acid sequences as
defined herein, it is typically applied or administered to a
cell-free expression system, a cell (e.g. an expression host cell
or a somatic cell), a tissue or an organism, preferably in naked
form or complexed form, or as a pharmaceutical composition or
vaccine as described herein, preferably via transfection or by
using any of the administration modes as described herein. The use
may be carried out in vitro, in vivo or ex vivo. The use may
furthermore be carried out in the context of the treatment of a
specific disease, particularly in the treatment of cancer or tumour
diseases, preferably as defined herein.
[0228] In yet another aspect the present invention also relates to
an inventive expression system comprising an inventive nucleic acid
sequence or expression vector or plasmid according to the first
aspect of the present invention. In this context the expression
system may be a cell-free expression system (e.g. an in vitro
transcription/translation system), a cellular expression system
(e.g. mammalian cells like CHO cells, insect cells, yeast cells,
bacterial cells like E. coli) or organisms used for expression of
peptides or proteins (e.g. plants or animals like cows).
[0229] Additionally, according to another aspect, the present
invention also relates to the use of the inventive nucleic acid as
defined herein or of the inventive composition comprising a
plurality of inventive nucleic acid sequences as defined herein for
the preparation of a pharmaceutical composition for increasing the
expression of an encoded peptide or protein, e.g. for treating a
cancer or tumour disease, preferably as defined herein, e.g.
applying or administering the inventive nucleic acid as defined
herein or of the inventive composition comprising a plurality of
inventive nucleic acid sequences as defined herein to a cell (e.g.
an expression host cell or a somatic cell), a tissue or an
organism, preferably in naked form or complexed form or as a
pharmaceutical composition or vaccine as described herein, more
preferably using any of the administration modes as described
herein.
[0230] Accordingly, in a particular preferred aspect, the present
invention also provides a pharmaceutical composition, comprising an
inventive nucleic acid as defined herein or an inventive
composition comprising a plurality of inventive nucleic acid
sequences as defined herein and optionally a pharmaceutically
acceptable carrier and/or vehicle.
[0231] As a first ingredient, the inventive pharmaceutical
composition comprises at least one inventive nucleic acid as
defined herein.
[0232] As a second ingredient the inventive pharmaceutical
composition may optional comprise at least one additional
pharmaceutically active component. A pharmaceutically active
component in this connection is a compound that has a therapeutic
effect to heal, ameliorate or prevent a particular indication or
disease as mentioned herein, preferably cancer or tumour diseases.
Such compounds include, without implying any limitation, peptides
or proteins, preferably as defined herein, nucleic acids,
preferably as defined herein, (therapeutically active) low
molecular weight organic or inorganic compounds (molecular weight
less than 5000, preferably less than 1000), sugars, antigens or
antibodies, preferably as defined herein, therapeutic agents
already known in the prior art, antigenic cells, antigenic cellular
fragments, cellular fractions; cell wall components (e.g.
polysaccharides), modified, attenuated or de-activated (e.g.
chemically or by irradiation) pathogens (virus, bacteria etc.),
adjuvants, preferably as defined herein, etc.
[0233] Furthermore, the inventive pharmaceutical composition may
comprise a pharmaceutically acceptable carrier and/or vehicle. In
the context of the present invention, a pharmaceutically acceptable
carrier typically includes the liquid or non-liquid basis of the
inventive pharmaceutical composition. If the inventive
pharmaceutical composition is provided in liquid form, the carrier
will typically be pyrogen-free water; isotonic saline or buffered
(aqueous) solutions, e.g. phosphate, citrate etc. buffered
solutions. The injection buffer may be hypertonic, isotonic or
hypotonic with reference to the specific reference medium, i.e. the
buffer may have a higher, identical or lower salt content with
reference to the specific reference medium, wherein preferably such
concentrations of the afore mentioned salts may be used, which do
not lead to damage of cells due to osmosis or other concentration
effects. Reference media are e.g. liquids occurring in "in vivo"
methods, such as blood, lymph, cytosolic liquids, or other body
liquids, or e.g. liquids, which may be used as reference media in
"in vitro" methods, such as common buffers or liquids. Such common
buffers or liquids are known to a skilled person. Ringer-Lactate
solution is particularly preferred as a liquid basis.
[0234] However, one or more compatible solid or liquid fillers or
diluents or encapsulating compounds may be used as well for the
inventive pharmaceutical composition, which are suitable for
administration to a patient to be treated. The term "compatible" as
used here means that these constituents of the inventive
pharmaceutical composition are capable of being mixed with the
inventive nucleic acid as defined herein in such a manner that no
interaction occurs which would substantially reduce the
pharmaceutical effectiveness of the inventive pharmaceutical
composition under typical use conditions.
[0235] According to a specific embodiment, the inventive
pharmaceutical composition may comprise an adjuvant. In this
context, an adjuvant may be understood as any compound, which is
suitable to initiate or increase an immune response of the innate
immune system, i.e. a non-specific immune response. With other
words, when administered, the inventive pharmaceutical composition
preferably elicits an innate immune response due to the adjuvant,
optionally contained therein. Preferably, such an adjuvant may be
selected from an adjuvant known to a skilled person and suitable
for the present case, i.e. supporting the induction of an innate
immune response in a mammal, e.g. an adjuvant protein as defined
above or an adjuvant as defined in the following.
[0236] Particularly preferred as adjuvants suitable for depot and
delivery are cationic or polycationic compounds as defined above
for the inventive nucleic acid sequence as vehicle, transfection or
complexation agent.
[0237] The inventive pharmaceutical composition can additionally
contain one or more auxiliary substances in order to increase its
immunogenicity or immunostimulatory capacity, if desired. A
synergistic action of the inventive nucleic acid sequence as
defined herein and of an auxiliary substance, which may be
optionally contained in the inventive pharmaceutical composition,
is preferably achieved thereby. Depending on the various types of
auxiliary substances, various mechanisms can come into
consideration in this respect. For example, compounds that permit
the maturation of dendritic cells (DCs), for example
lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class
of suitable auxiliary substances. In general, it is possible to use
as auxiliary substance any agent that influences the immune system
in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines,
such as GM-CFS, which allow an immune response to be enhanced
and/or influenced in a targeted manner. Particularly preferred
auxiliary substances are cytokines, such as monokines, lymphokines,
interleukins or chemokines, that further promote the innate immune
response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,
IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth
factors, such as hGH.
[0238] Further additives which may be included in the inventive
pharmaceutical composition are emulsifiers, such as, for example,
TWEEN.RTM., non-ionic detergent; wetting agents, such as, for
example, sodium lauryl sulfate; colouring agents; taste-imparting
agents, pharmaceutical carriers; tablet-forming agents;
stabilizers; antioxidants; preservatives.
[0239] The inventive pharmaceutical composition can also
additionally contain any further compound, which is known to be
immunostimulating due to its binding affinity (as ligands) to human
Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, or due to its binding affinity (as ligands) to murine
Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, TLR11, TLR12 or TLR13.
[0240] The inventive pharmaceutical composition may be administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term parenteral as used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-synovial, intrasternal,
intrathecal, intrahepatic, intralesional, intracranial,
transdermal, intradermal, intrapulmonal, intraperitoneal,
intracardial, intraarterial, and sublingual injection or infusion
techniques.
[0241] Preferably, the inventive pharmaceutical composition may be
administered by parenteral injection, more preferably by
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional, intracranial, transdermal, intradermal,
intrapulmonal, intraperitoneal, intracardial, intraarterial, and
sublingual injection or via infusion techniques. Particularly
preferred is intradermal and intramuscular injection. Sterile
injectable forms of the inventive pharmaceutical compositions may
be aqueous or oleaginous suspension. These suspensions may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents and suspending agents.
[0242] The inventive pharmaceutical composition as defined herein
may also be administered orally in any orally acceptable dosage
form including, but not limited to, capsules, tablets, aqueous
suspensions or solutions.
[0243] The inventive pharmaceutical composition may also be
administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
e.g. including diseases of the skin or of any other accessible
epithelial tissue. Suitable topical formulations are readily
prepared for each of these areas or organs. For topical
applications, the inventive pharmaceutical composition may be
formulated in a suitable ointment, containing the inventive nucleic
acid as defined herein suspended or dissolved in one or more
carriers.
[0244] The inventive pharmaceutical composition typically comprises
a "safe and effective amount" of the components of the inventive
pharmaceutical composition, particularly of the inventive nucleic
acid sequence(s) as defined herein. As used herein, a "safe and
effective amount" means an amount of the inventive nucleic acid
sequence(s) as defined herein as such that is sufficient to
significantly induce a positive modification of a disease or
disorder as defined herein. At the same time, however, a "safe and
effective amount" is small enough to avoid serious side-effects and
to permit a sensible relationship between advantage and risk. The
determination of these limits typically lies within the scope of
sensible medical judgment.
[0245] The inventive pharmaceutical composition may be used for
human and also for veterinary medical purposes, preferably for
human medical purposes, as a pharmaceutical composition in general
or as a vaccine.
[0246] According to another particularly preferred aspect, the
inventive pharmaceutical composition (or the inventive nucleic acid
sequence as defined herein or the inventive composition comprising
a plurality of inventive nucleic acid sequences as defined herein)
may be provided or used as a vaccine. Typically, such a vaccine is
as defined above for pharmaceutical compositions. Additionally,
such a vaccine typically contains the inventive nucleic acid as
defined herein or the inventive composition comprising a plurality
of inventive nucleic acid sequences as defined herein.
[0247] The inventive vaccine may also comprise a pharmaceutically
acceptable carrier, adjuvant, and/or vehicle as defined herein for
the inventive pharmaceutical composition. In the specific context
of the inventive vaccine, the choice of a pharmaceutically
acceptable carrier is determined in principle by the manner in
which the inventive vaccine is administered. The inventive vaccine
can be administered, for example, systemically or locally. Routes
for systemic administration in general include, for example,
transdermal, oral, parenteral routes, including subcutaneous,
intravenous, intramuscular, intraarterial, intradermal and
intraperitoneal injections and/or intranasal administration routes.
Routes for local administration in general include, for example,
topical administration routes but also intradermal, transdermal,
subcutaneous, or intramuscular injections or intralesional,
intracranial, intrapulmonal, intracardial, and sublingual
injections. More preferably, vaccines may be administered by an
intradermal, subcutaneous, or intramuscular route. Inventive
vaccines are therefore preferably formulated in liquid (or
sometimes in solid) form.
[0248] The inventive vaccine can additionally contain one or more
auxiliary substances in order to increase its immunogenicity or
immunostimulatory capacity, if desired. Particularly preferred are
adjuvants as auxiliary substances or additives as defined for the
pharmaceutical composition.
[0249] The present invention furthermore provides several
applications and uses of the inventive nucleic acid sequence as
defined herein, of the inventive composition comprising a plurality
of inventive nucleic acid sequences as defined herein, of the
inventive pharmaceutical composition, of the inventive vaccine, all
comprising the inventive nucleic acid sequence as defined herein or
of kits comprising same.
[0250] According to one specific aspect, the present invention is
directed to the first medical use of the inventive nucleic acid
sequence as defined herein or of the inventive composition
comprising a plurality of inventive nucleic acid sequences as
defined herein as a medicament, preferably as a vaccine
particularly in the treatment of cancer or tumour diseases.
[0251] According to another aspect, the present invention is
directed to the second medical use of the inventive nucleic acid
sequence as defined herein or of the inventive composition
comprising a plurality of inventive nucleic acid sequences as
defined herein, for the treatment of cancer and tumour diseases as
defined herein, preferably to the use of the inventive nucleic acid
sequence as defined herein, of the inventive composition comprising
a plurality of inventive nucleic acid sequences as defined herein,
of a pharmaceutical composition or vaccine comprising same or of
kits comprising same for the preparation of a medicament for the
prophylaxis, treatment and/or amelioration of cancer or tumour
diseases as defined herein. Preferably, the pharmaceutical
composition or a vaccine is used or to be administered to a patient
in need thereof for this purpose.
[0252] Preferably, diseases as mentioned herein are selected from
cancer or tumour diseases which preferably include e.g. Acute
lymphoblastic leukemia, Acute myeloid leukemia, Adrenocortical
carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal
cancer, Appendix cancer, Astrocytoma, Basal cell carcinoma, Bile
duct cancer, Bladder cancer, Bone cancer, Osteosarcoma/Malignant
fibrous histiocytoma, Brainstem glioma, Brain tumor, cerebellar
astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,
medulloblastoma, supratentorial primitive neuroectodermal tumors,
visual pathway and hypothalamic glioma, Breast cancer, Bronchial
adenomas/carcinoids, Burkitt lymphoma, childhood Carcinoid tumor,
gastrointestinal Carcinoid tumor, Carcinoma of unknown primary,
primary Central nervous system lymphoma, childhood Cerebellar
astrocytoma, childhood Cerebral astrocytoma/Malignant glioma,
Cervical cancer, Childhood cancers, Chronic lymphocytic leukemia,
Chronic myelogenous leukemia, Chronic myeloproliferative disorders,
Colon Cancer, Cutaneous T-cell lymphoma, Desmoplastic small round
cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer,
Ewing's sarcoma in the Ewing family of tumors, Childhood
Extracranial germ cell tumor, Extragonadal Germ cell tumor,
Extrahepatic bile duct cancer, Intraocular melanoma,
Retinoblastoma, Gallbladder cancer, Gastric (Stomach) cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor
(GIST), extracranial, extragonadal, or ovarian Germ cell tumor,
Gestational trophoblastic tumor, Glioma of the brain stem,
Childhood Cerebral Astrocytoma, Childhood Visual Pathway and
Hypothalamic Glioma, Gastric carcinoid, Hairy cell leukemia, Head
and neck cancer, Heart cancer, Hepatocellular (liver) cancer,
Hodgkin lymphoma, Hypopharyngeal cancer, childhood Hypothalamic and
visual pathway glioma, Intraocular Melanoma, Islet Cell Carcinoma
(Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renal cell
cancer), Laryngeal Cancer, Leukemias, acute lymphoblastic Leukemia,
acute myeloid Leukemia, chronic lymphocytic Leukemia, chronic
myelogenous Leukemia, hairy cell Leukemia, Lip and Oral Cavity
Cancer, Liposarcoma, Liver Cancer, Non-Small Cell Lung Cancer,
Small Cell Lung Cancer, Lymphomas, AIDS-related Lymphoma, Burkitt
Lymphoma, cutaneous T-Cell Lymphoma, Hodgkin Lymphoma, Non-Hodgkin
Lymphomas, Primary Central Nervous System Lymphoma, Waldenstrom
Macroglobulinemia, Malignant Fibrous Histiocytoma of
Bone/Osteosarcoma, Childhood Medulloblastoma, Melanoma, Intraocular
(Eye) Melanoma, Merkel Cell Carcinoma, Adult Malignant
Mesothelioma, Childhood Mesothelioma, Metastatic Squamous Neck
Cancer with Occult Primary, Mouth Cancer, Childhood Multiple
Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell
Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,
Myelodysplastic/Myeloproliferative Diseases, Chronic Myelogenous
Leukemia, Adult Acute Myeloid Leukemia, Childhood Acute Myeloid
Leukemia, Multiple Myeloma (Cancer of the Bone-Marrow), Chronic
Myeloproliferative Disorders, Nasal cavity and paranasal sinus
cancer, Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer,
Oropharyngeal cancer, Osteosarcoma/malignant fibrous histiocytoma
of bone, Ovarian cancer, Ovarian epithelial cancer (Surface
epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low
malignant potential tumor, Pancreatic cancer, islet cell Pancreatic
cancer, Paranasal sinus and nasal cavity cancer, Parathyroid
cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal
astrocytoma, Pineal germinoma, childhood Pineoblastoma and
supratentorial primitive neuroectodermal tumors, Pituitary adenoma,
Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma,
Primary central nervous system lymphoma, Prostate cancer, Rectal
cancer, Renal cell carcinoma (kidney cancer), Cancer of the Renal
pelvis and ureter, Retinoblastoma, childhood Rhabdomyosarcoma,
Salivary gland cancer, Sarcoma of the Ewing family of tumors,
Kaposi Sarcoma, soft tissue Sarcoma, uterine Sarcoma, Sezary
syndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Merkel
cell Skin carcinoma, Small intestine cancer, Squamous cell
carcinoma, metastatic Squamous neck cancer with occult primary,
childhood Supratentorial primitive neuroectodermal tumor,
Testicular cancer, Throat cancer, childhood Thymoma, Thymoma and
Thymic carcinoma, Thyroid cancer, childhood Thyroid cancer,
Transitional cell cancer of the renal pelvis and ureter,
gestational Trophoblastic tumor, Urethral cancer, endometrial
Uterine cancer, Uterine sarcoma, Vaginal cancer, childhood Visual
pathway and hypothalamic glioma, Vulvar cancer, Waldenstrom
macroglobulinemia, and childhood Wilms tumor (kidney cancer).
[0253] In a further preferred aspect, the inventive nucleic acid
sequence as defined herein or the inventive composition comprising
a plurality of inventive nucleic acid sequences as defined herein
may be used for the preparation of a pharmaceutical composition or
a vaccine, particularly for purposes as defined herein.
[0254] The inventive pharmaceutical composition or vaccine may
furthermore be used for the treatment of a disease or a disorder,
preferably of cancer or tumour diseases as defined herein.
[0255] According to a final aspect, the present invention also
provides kits, particularly kits of parts. Such kits, particularly
kits of parts, typically comprise as components alone or in
combination with further components as defined herein at least one
inventive nucleic acid sequence as defined herein, the inventive
pharmaceutical composition or vaccine comprising the inventive
nucleic acid sequence. The at least one inventive nucleic acid
sequence as defined herein, is optionally in combination with
further components as defined herein, whereby the at least one
nucleic acid of the invention is provided separately (first part of
the kit) from at least one other part of the kit comprising one or
more other components. The inventive pharmaceutical composition
and/or the inventive vaccine may e.g. occur in one or different
parts of the kit. As an example, e.g. at least one part of the kit
may comprise at least one inventive nucleic acid sequence as
defined herein, and at least one further part of the kit at least
one other component as defined herein, e.g. at least one other part
of the kit may comprise at least one pharmaceutical composition or
vaccine or a part thereof, e.g. at least one part of the kit may
comprise the inventive nucleic acid sequence as defined herein, at
least one further part of the kit at least one other component as
defined herein, at least one further part of the kit at least one
component of the inventive pharmaceutical composition or vaccine or
the inventive pharmaceutical composition or vaccine as a whole, and
at least one further part of the kit e.g. at least one
pharmaceutical carrier or vehicle, etc. In case the kit or kit of
parts comprises a plurality of inventive nucleic acid sequences,
one component of the kit can comprise only one, several or all
inventive nucleic acid sequences comprised in the kit. In an
alternative embodiment each/every inventive nucleic acid sequence
may be comprised in a different/separate component of the kit such
that each component forms a part of the kit. Also, more than one
nucleic acid may be comprised in a first component as part of the
kit, whereas one or more other (second, third etc.) components
(providing one or more other parts of the kit) may either contain
one or more than one inventive nucleic acids, which may be
identical or partially identical or different from the first
component. The kit or kit of parts may furthermore contain
technical instructions with information on the administration and
dosage of the inventive nucleic acid sequence, the inventive
pharmaceutical composition or the inventive vaccine or of any of
its components or parts, e.g. if the kit is prepared as a kit of
parts.
[0256] Taken together, the invention provides a nucleic acid
sequence comprising or coding for [0257] a) a coding region,
encoding at least one peptide or protein; [0258] b) at least one
histone stem-loop, and [0259] c) a poly(A) sequence or a
polyadenylation signal; wherein said peptide or protein comprises a
tumour antigen a fragment, variant or derivative of said tumour
antigen, preferably, wherein the tumour antigen is a
melanocyte-specific antigen, a cancer-testis antigen or a
tumour-specific antigen, preferably a CT-X antigen, a non-X
CT-antigen, a binding partner for a CT-X antigen or a binding
partner for a non-X CT-antigen or a tumour-specific antigen, more
preferably a CT-X antigen, a binding partner for a non-X CT-antigen
or a tumour-specific antigen or a fragment, variant or derivative
of said tumour antigen.
[0260] In a further preferred embodiment, the invention relates to
a composition comprising at least one type of nucleic acid sequence
comprising or coding for [0261] a) a coding region, encoding at
least one peptide or protein; [0262] b) at least one histone
stem-loop, and [0263] c) a poly(A) sequence or a polyadenylation
signal; [0264] wherein said peptide or protein comprises a tumour
antigen a fragment, variant or derivative of said tumour antigen,
preferably, wherein the tumour antigen is a melanocyte-specific
antigen, a cancer-testis antigen or a tumour-specific antigen,
preferably a CT-X antigen, a non-X CT-antigen, a binding partner
for a CT-X antigen or a binding partner for a non-X CT-antigen or a
tumour-specific antigen, more preferably a CT-X antigen, a binding
partner for a non-X CT-antigen or a tumour-specific antigen or a
fragment, variant or derivative of said tumour antigen; and wherein
each of the nucleic acid sequences encodes a different peptide or
protein.
[0265] The composition may comprise further an pharmaceutically
acceptable carrier and/or pharmaceutically acceptable adjuvants as
defined herein. The composition may be used as a vaccine or for
treatment of a disease associated with cancer or tumour.
[0266] In a further preferred embodiment, the invention provides a
composition comprising at least two, preferably two or more, more
preferably a plurality of nucleic acid sequences sequence (which
means typically more than 1, 2, 3, 4, 5, 6 or more than 10 nucleic
acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) comprising or
coding for [0267] a) a coding region, encoding at least one peptide
or protein; [0268] b) at least one histone stem-loop, and [0269] c)
a poly(A) sequence or a polyadenylation signal; [0270] wherein said
peptide or protein comprises a tumour antigen a fragment, variant
or derivative of said tumour antigen, preferably, wherein the
tumour antigen is a melanocyte-specific antigen, a cancer-testis
antigen or a tumour-specific antigen, preferably a CT-X antigen, a
non-X CT-antigen, a binding partner for a CT-X antigen or a binding
partner for a non-X CT-antigen or a tumour-specific antigen, more
preferably a CT-X antigen, a binding partner for a non-X CT-antigen
or a tumour-specific antigen or a fragment, variant or derivative
of said tumour antigen; and wherein each of the nucleic acid
sequences encodes a different peptide or protein.
[0271] The composition may comprise further an pharmaceutically
acceptable carrier and/or pharmaceutically acceptable adjuvants as
defined herein. The composition may be used as a vaccine or for
treatment of a disease associated with cancer or tumour.
[0272] In a further preferred embodiment, the invention provides a
composition comprising at least two, preferably two or more, more
preferably a plurality (which means typically more than 1, 2, 3, 4,
5, 6 or more than 10 nucleic acids, e.g. 2 to 10, preferably 2 to 5
nucleic acids) of nucleic acid sequences sequence comprising or
coding for [0273] a) a coding region, encoding at least one peptide
or protein; [0274] b) at least one histone stem-loop, and [0275] c)
a poly(A) sequence or a polyadenylation signal; [0276] wherein said
peptide or protein comprises a tumour antigen a fragment, variant
or derivative of said tumour antigen, preferably, wherein the
tumour antigen is a melanocyte-specific antigen, a cancer-testis
antigen or a tumour-specific antigen, preferably a CT-X antigen, a
non-X CT-antigen, a binding partner for a CT-X antigen or a binding
partner for a non-X CT-antigen or a tumour-specific antigen, more
preferably a CT-X antigen, a binding partner for a non-X CT-antigen
or a tumour-specific antigen or a fragment, variant or derivative
of said tumour antigen; and wherein each of the nucleic acid
sequences encodes a different peptide or protein; and preferably
wherein each type of nucleic acid sequence encodes for a different
peptide or protein, preferably for a different tumour antigen.
[0277] The composition may comprise further an pharmaceutically
acceptable carrier and/or pharmaceutically acceptable adjuvants as
defined herein. The composition may be used as a vaccine or for
treatment of a disease associated with cancer or tumour.
[0278] In a further preferred embodiment, the invention provides a
composition comprising at least two, preferably two or more, more
preferably a plurality of nucleic acid sequences sequence (which
means typically more than 1, 2, 3, 4, 5, 6 or more than 10 nucleic
acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) comprising or
coding for [0279] a) a coding region, encoding at least one peptide
or protein; [0280] b) at least one histone stem-loop, and [0281] c)
a poly(A) sequence or a polyadenylation signal; [0282] wherein said
peptide or protein comprises a tumour antigen a fragment, variant
or derivative of said tumour antigen, preferably, wherein the
tumour antigen is a melanocyte-specific antigen, a cancer-testis
antigen or a tumour-specific antigen, preferably a CT-X antigen, a
non-X CT-antigen, a binding partner for a CT-X antigen or a binding
partner for a non-X CT-antigen or a tumour-specific antigen, more
preferably a CT-X antigen, a binding partner for a non-X CT-antigen
or a tumour-specific antigen or a fragment, variant or derivative
of said tumour antigen; and wherein each of the nucleic acid
sequences encodes a different peptide or protein; and preferably
wherein each type of nucleic acid sequence encodes for a different
peptide or protein, preferably for a different tumour antigen, more
preferably, wherein one type of the contained nucleic acid
sequences encodes for PSA, PSMA, PSCA, STEAP-1, NY-ESO-1, 5T4,
Survivin, MAGE-C1, or MAGE-C2.
[0283] The composition may comprise further an pharmaceutically
acceptable carrier and/or pharmaceutically acceptable adjuvants as
defined herein. The composition may be used as a vaccine or for
treatment of a disease associated with cancer or tumour.
[0284] In some embodiments, it may be preferred, provided that the
composition contains only one type of nucleic acid sequence, if the
nucleic acid sequence does not encode for NY-ESO1, provided that
the composition contains only one type of nucleic acid
sequence.
[0285] In a further preferred embodiment, the invention provides a
composition comprising at least two nucleic acid sequences sequence
comprising or coding for [0286] a) a coding region, encoding at
least one peptide or protein; [0287] b) at least one histone
stem-loop, and [0288] c) a poly(A) sequence or a polyadenylation
signal; [0289] wherein said peptide or protein comprises a tumour
antigen a fragment, variant or derivative of said tumour antigen,
preferably, wherein the tumour antigen is a melanocyte-specific
antigen, a cancer-testis antigen or a tumour-specific antigen,
preferably a CT-X antigen, a non-X CT-antigen, a binding partner
for a CT-X antigen or a binding partner for a non-X CT-antigen or a
tumour-specific antigen, more preferably a CT-X antigen, a binding
partner for a non-X CT-antigen or a tumour-specific antigen or a
fragment, variant or derivative of said tumour antigen; and wherein
each of the nucleic acid sequences encodes a different peptide or
protein; and wherein at least one of the nucleic acid sequences
encodes for 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,
alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,
alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4,
ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4,
BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,
calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19,
CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80,
CDCl.sub.27/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-R17I, 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-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, PAP, PART-1, PATE, PDEF,
Pim-1-Kinase, Pin-1, Pml/PARalpha, 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-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2,
TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m,
TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA,
VEGFR1, VEGFR-2/FLK-1, WT1 and a immunoglobulin idiotype of a
lymphoid blood cell or a T cell receptor idiotype of a lymphoid
blood cell, or a fragment, variant or derivative of said tumour
antigen; preferably survivin or a homologue thereof, an antigen
from the MAGE-family or a binding partner thereof or a fragment,
variant or derivative of said tumour antigen.
[0290] The composition may comprise further an pharmaceutically
acceptable carrier and/or pharmaceutically acceptable adjuvants as
defined herein. The composition may be used as a vaccine or for
treatment of a disease associated with cancer or tumour.
[0291] In the present invention, if not otherwise indicated,
different features of alternatives and embodiments may be combined
with each other. Furthermore, the term "comprising" shall not be
construed as meaning "consisting of", if not specifically
mentioned. However, in the context of the present invention, term
"comprising" may be substituted with the term "consisting of",
where applicable.
FIGURES
[0292] The following Figures are intended to illustrate the
invention further and shall not be construed to limit the present
invention thereto.
[0293] FIG. 1: shows the histone stem-loop consensus sequence
generated from metazoan and protozoan stem loop sequences (as
reported by Davila Lopez, M., & Samuelsson, T. (2008), RNA (New
York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 4001 histone
stem-loop sequences from metazoa and protozoa were aligned and the
quantity of the occurring nucleotides is indicated for every
position in the stem-loop sequence. The generated consensus
sequence representing all nucleotides present in the sequences
analyzed is given using the single-letter nucleotide code. In
addition to the consensus sequence, sequences are shown
representing at least 99%, 95% and 90% of the nucleotides present
in the sequences analyzed.
[0294] FIG. 2: shows the histone stem-loop consensus sequence
generated from protozoan stem loop sequences (as reported by Davila
Lopez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.),
14(1), 1-10. doi:10.1261/rna.782308). 131 histone stem-loop
sequences from protozoa were aligned and the quantity of the
occurring nucleotides is indicated for every position in the
stem-loop sequence. The generated consensus sequence representing
all nucleotides present in the sequences analyzed is given using
the single-letter nucleotide code. In addition to the consensus
sequence, sequences are shown representing at least 99%, 95% and
90% of the nucleotides present in the sequences analyzed.
[0295] FIG. 3: shows the histone stem-loop consensus sequence
generated from metazoan stem loop sequences (as reported by Davila
Lopez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.),
14(1), 1-10. doi:10.1261/rna.782308). 3870 histone stem-loop
sequences from metazoa were aligned and the quantity of the
occurring nucleotides is indicated for every position in the
stem-loop sequence. The generated consensus sequence representing
all nucleotides present in the sequences analyzed is given using
the single-letter nucleotide code. In addition to the consensus
sequence, sequences are shown representing at least 99%, 95% and
90% of the nucleotides present in the sequences analyzed.
[0296] FIG. 4: shows the histone stem-loop consensus sequence
generated from vertebrate stem loop sequences (as reported by
Davila Lopez, M., & Samuelsson, T. (2008), RNA (New York,
N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 1333 histone stem-loop
sequences from vertebrates were aligned and the quantity of the
occurring nucleotides is indicated for every position in the
stem-loop sequence. The generated consensus sequence representing
all nucleotides present in the sequences analyzed is given using
the single-letter nucleotide code. In addition to the consensus
sequence, sequences are shown representing at least 99%, 95% and
90% of the nucleotides present in the sequences analyzed.
[0297] FIG. 5: shows the histone stem-loop consensus sequence
generated from human (Homo sapiens) stem loop sequences (as
reported by Davila Lopez, M., & Samuelsson, T. (2008), RNA (New
York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 84 histone
stem-loop sequences from humans were aligned and the quantity of
the occurring nucleotides is indicated for every position in the
stem-loop sequence. The generated consensus sequence representing
all nucleotides present in the sequences analyzed is given using
the single-letter nucleotide code. In addition to the consensus
sequence, sequences are shown representing at least 99%, 95% and
90% of the nucleotides present in the sequences analyzed.
[0298] FIGS. 6 to 21: show mRNAs from in vitro transcription.
[0299] Given are the designation and the sequence of mRNAs obtained
by in vitro transcription. The following abbreviations are used:
[0300] ppLuc (GC): GC-enriched mRNA sequence coding for Photinus
pyralis luciferase [0301] ag: 3' untranslated region (UTR) of the
alpha globin gene [0302] A64: poly(A)-sequence with 64 adenylates
[0303] A120: poly(A)-sequence with 120 adenylates [0304] histoneSL:
histone stem-loop [0305] .alpha.CPSL: stem loop which has been
selected from a library for its specific binding of the
.alpha.CP-2KL protein [0306] PolioCL: 5' clover leaf from Polio
virus genomic RNA [0307] G30: poly(G) sequence with 30 guanylates
[0308] U30: poly(U) sequence with 30 uridylates [0309] SL:
unspecific/artificial stem-loop [0310] N32: unspecific sequence of
32 nucleotides [0311] NY-ESO-1 (G/C): GC-enriched mRNA sequence
coding for the human tumour antigen NY-ESO-1 [0312] Survivin(G/C):
GC-enriched mRNA sequence coding for the human tumour antigen
Survivin [0313] MAGE-C1 (G/C): GC-enriched mRNA sequence coding for
the human tumour antigen MAGE-C1 [0314] Within the sequences, the
following elements are highlighted: coding region (ORF) (capital
letters), ag (bold), histoneSL (underlined), further distinct
sequences tested (italic).
[0315] FIG. 6: shows the mRNA sequence of ppLuc(GC)-ag (SEQ ID NO:
43). [0316] By linearization of the original vector at the
restriction site immediately following the alpha-globin 3'-UTR
(ag), mRNA is obtained lacking a poly(A) sequence.
[0317] FIG. 7: shows the mRNA sequence of ppLuc(GC)-ag-A64 (SEQ ID
NO: 44). [0318] By linearization of the original vector at the
restriction site immediately following the A64 poly(A)-sequence,
mRNA is obtained ending with an A64 poly(A) sequence.
[0319] FIG. 8: shows the mRNA sequence of ppLuc(GC)-ag-histoneSL
(SEQ ID NO: 45). [0320] The A64 poly(A) sequence was replaced by a
histoneSL. The histone stem-loop sequence used in the examples was
obtained from Cakmakci et al. (2008). Molecular and Cellular
Biology, 28(3), 1182-1194.
[0321] FIG. 9: shows the mRNA sequence of
ppLuc(GC)-ag-A64-histoneSL (SEQ ID NO: 46). [0322] The histoneSL
was appended 3' of A64 poly(A).
[0323] FIG. 10: shows the mRNA sequence of ppLuc(GC)-ag-A120 (SEQ
ID NO: 47). [0324] The A64 poly(A) sequence was replaced by an A120
poly(A) sequence.
[0325] FIG. 11: shows the mRNA sequence of ppLuc(GC)-ag-A64-ag (SEQ
ID NO: 48). A second alpha-globin 3'-UTR was appended 3' of A64
poly(A).
[0326] FIG. 12: shows the mRNA sequence of
ppLuc(GC)-ag-A64-.alpha.CPSL (SEQ ID NO: 49). [0327] A stem loop
was appended 3' of A64 poly(A). The stem loop has been selected
from a library for its specific binding of the .alpha.CP-2KL
protein (Thisted et al., (2001), The Journal of Biological
Chemistry, 276(20), 17484-17496). .alpha.CP-2KL is an isoform of
.alpha.CP-2, the most strongly expressed .alpha.CP protein
(alpha-globin mRNA poly(C) binding protein) (Makeyev et al.,
(2000), Genomics, 67(3), 301-316), a group of RNA binding proteins,
which bind to the alpha-globin 3'-UTR (Chkheidze et al., (1999),
Molecular and Cellular Biology, 19(7), 4572-4581).
[0328] FIG. 13: shows the mRNA sequence of ppLuc(GC)-ag-A64-PolioCL
(SEQ ID NO: 50). [0329] The 5' clover leaf from Polio virus genomic
RNA was appended 3' of A64 poly(A).
[0330] FIG. 14: shows the mRNA sequence of ppLuc(GC)-ag-A64-G30
(SEQ ID NO: 51) [0331] A stretch of 30 guanylates was appended 3'
of A64 poly(A).
[0332] FIG. 15: shows the mRNA sequence of ppLuc(GC)-ag-A64-U30
(SEQ ID NO: 52) [0333] A stretch of 30 uridylates was appended 3'
of A64 poly(A).
[0334] FIG. 16: shows the mRNA sequence of ppLuc(GC)-ag-A64-SL (SEQ
ID NO: 53) [0335] A stem loop was appended 3' of A64 poly(A). The
upper part of the stem and the loop were taken from (Babendure et
al., (2006), RNA (New York, N.Y.), 12(5), 851-861). The stem loop
consists of a 17 base pair long, CG-rich stem and a 6 base long
loop.
[0336] FIG. 17: shows ppLuc(GC)-ag-A64-N32 (SEQ ID NO: 54) [0337]
By linearization of the original vector at an alternative
restriction site, mRNA is obtained with 32 additional nucleotides
following poly(A).
[0338] FIG. 18: shows the mRNA sequence of NY-ESO-1(GC)-ag-A64-C30
(SEQ ID NO: 55)
[0339] FIG. 19: shows the mRNA sequence of
NY-ESO-1(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 56)
[0340] FIG. 20: shows the mRNA sequence of
Survivin(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 57)
[0341] FIG. 21: shows the mRNA sequence of
MAGE-C1(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 58)
[0342] FIG. 22: shows that the combination of poly(A) and histoneSL
increases protein expression from mRNA in a synergistic manner.
[0343] The effect of poly(A) sequence, histoneSL, and the
combination of poly(A) and histoneSL on luciferase expression from
mRNA was examined. Therefore different mRNAs were electroporated
into HeLa cells. Luciferase levels were measured at 6, 24, and 48
hours after transfection. Little luciferase is expressed from mRNA
having neither poly(A) sequence nor histoneSL. Both a poly(A)
sequence or the histoneSL increase the luciferase level. Strikingly
however, the combination of poly(A) and histoneSL further strongly
increases the luciferase level, manifold above the level observed
with either of the individual elements, thus acting
synergistically. Data are graphed as mean RLU.+-.SD (relative light
units.+-.standard deviation) for triplicate transfections. Specific
RLU are summarized in Example 14.2.
[0344] FIG. 23: shows that the combination of poly(A) and histoneSL
increases protein expression from mRNA irrespective of their order.
[0345] The effect of poly(A) sequence, histoneSL, the combination
of poly(A) and histoneSL, and their order on luciferase expression
from mRNA was examined. Therefore different mRNAs were lipofected
into HeLa cells. Luciferase levels were measured at 6, 24, and 48
hours after the start of transfection. Both an A64 poly(A) sequence
or the histoneSL give rise to comparable luciferase levels.
Increasing the length of the poly(A) sequence from A64 to A120 or
to A300 increases the luciferase level moderately. In contrast, the
combination of poly(A) and histoneSL increases the luciferase level
much further than lengthening of the poly(A) sequence. The
combination of poly(A) and histoneSL acts synergistically as it
increases the luciferase level manifold above the level observed
with either of the individual elements. The synergistic effect of
the combination of poly(A) and histoneSL is seen irrespective of
the order of poly(A) and histoneSL and irrespective of the length
of poly(A) with A64-histoneSL or histoneSL-A250 mRNA. Data are
graphed as mean RLU.+-.SD for triplicate transfections. Specific
RLU are summarized in Example 14.3.
[0346] FIG. 24: shows that the rise in protein expression by the
combination of poly(A) and histoneSL is specific. [0347] The effect
of combining poly(A) and histoneSL or poly(A) and alternative
sequences on luciferase expression from mRNA was examined.
Therefore different mRNAs were electroporated into HeLa cells.
Luciferase levels were measured at 6, 24, and 48 hours after
transfection. Both a poly(A) sequence or the histoneSL give rise to
comparable luciferase levels. The combination of poly(A) and
histoneSL strongly increases the luciferase level, manifold above
the level observed with either of the individual elements, thus
acting synergistically. In contrast, combining poly(A) with any of
the other sequences is without effect on the luciferase level
compared to mRNA containing only a poly(A) sequence. Thus, the
combination of poly(A) and histoneSL acts specifically and
synergistically. Data are graphed as mean RLU.+-.SD for triplicate
transfections. Specific RLU are summarized in Example 14.4.
[0348] FIG. 25: shows that the combination of poly(A) and histoneSL
increases protein expression from mRNA in a synergistic manner in
vivo. [0349] The effect of poly(A) sequence, histoneSL, and the
combination of poly(A) and histoneSL on luciferase expression from
mRNA in vivo was examined. Therefore different mRNAs were injected
intradermally into mice. Mice were sacrificed 16 hours after
injection and Luciferase levels at the injection sites were
measured. Luciferase is expressed from mRNA having either a
histoneSL or a poly(A) sequence. Strikingly however, the
combination of poly(A) and histoneSL strongly increases the
luciferase level, manifold above the level observed with either of
the individual elements, thus acting synergistically. Data are
graphed as mean RLU.+-.SEM (relative light units.+-.standard error
of mean). Specific RLU are summarized in Example 14.5.
[0350] FIG. 26: shows that the combination of poly(A) and histoneSL
increases NY-ESO-1 protein expression from mRNA. [0351] The effect
of poly(A) sequence and the combination of poly(A) and histoneSL on
NY-ESO-1 expression from mRNA was examined. Therefore different
mRNAs were electroporated into HeLa cells. NY-ESO-1 levels were
measured at 24 hours after transfection by flow cytometry. NY-ESO-1
is expressed from mRNA having only a poly(A) sequence. Strikingly
however, the combination of poly(A) and histoneSL strongly
increases the NY-ESO-1 level, manifold above the level observed
with only a poly(A) sequence. Data are graphed as counts against
fluorescence intensity. Median fluorescence intensities (MFI) are
summarized in Example 14.6.
[0352] FIG. 27: shows that the combination of poly(A) and histoneSL
increases the level of antibodies elicited by vaccination with
mRNA. [0353] The effect of poly(A) sequence and the combination of
poly(A) and histoneSL on the induction of anti NY-ESO-1 antibodies
elicited by vaccination with mRNA was examined. Therefore C57BL/6
mice were vaccinated intradermally with different mRNAs complexed
with protamine. The level of NY-ESO-1-specific antibodies in
vaccinated and control mice was analyzed by ELISA with serial
dilutions of sera. Anti NY-ESO-1 IgG2a[b] is induced by mRNA having
only a poly(A) sequence. Strikingly however, the combination of
poly(A) and histoneSL strongly increases the anti NY-ESO-1 IgG2a[b]
level, manifold above the level observed with only a poly(A)
sequence. Data are graphed as mean endpoint titers. Mean endpoint
titers are summarized in Example 14.7.
EXAMPLES
[0354] The following Examples are intended to illustrate the
invention further and shall not be construed to limit the present
invention thereto.
1. Generation of Histone-Stem-Loop Consensus Sequences
[0355] Prior to the experiments, histone stem-loop consensus
sequences were determined on the basis of metazoan and protozoan
histone stem-loop sequences. Sequences were taken from the
supplement provided by Lopez et al. (Davila Lopez, M., &
Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.
doi:10.1261/rna.782308), who identified a large number of natural
histone stem-loop sequences by searching genomic sequences and
expressed sequence tags. First, all sequences from metazoa and
protozoa (4001 sequences), or all sequences from protozoa (131
sequences) or alternatively from metazoa (3870 sequences), or from
vertebrates (1333 sequences) or from humans (84 sequences) were
grouped and aligned. Then, the quantity of the occurring
nucleotides was determined for every position. Based on the tables
thus obtained, consensus sequences for the 5 different groups of
sequences were generated representing all nucleotides present in
the sequences analyzed. In addition, more restrictive consensus
sequences were also obtained, increasingly emphasizing conserved
nucleotides
2. Preparation of DNA-Templates
[0355] [0356] A vector for in vitro transcription was constructed
containing a T7 promoter followed by a GC-enriched sequence coding
for Photinus pyralis luciferase (ppLuc(GC)), the center part of the
3' untranslated region (UTR) of alpha-globin (ag), and a poly(A)
sequence. The poly(A) sequence was immediately followed by a
restriction site used for linearization of the vector before in
vitro transcription in order to obtain mRNA ending in an A64
poly(A) sequence. mRNA obtained from this vector accordingly by in
vitro transcription is designated as "ppLuc(GC)-ag-A64". [0357]
Linearization of this vector at alternative restriction sites
before in vitro transcription allowed to obtain mRNA either
extended by additional nucleotides 3' of A64 or lacking A64. In
addition, the original vector was modified to include alternative
sequences. In summary, the following mRNAs were obtained from these
vectors by in vitro transcription (mRNA sequences are given in
FIGS. 6 to 17):
TABLE-US-00004 [0357] (SEQ ID NO: 43) ppLuc(GC)-ag (SEQ ID NO: 44)
ppLuc(GC)-ag-A.sub.64 (SEQ ID NO: 45) ppLuc(GC)-ag-histoneSL (SEQ
ID NO: 46) ppLuc(GC)-ag-A.sub.64-histoneSL (SEQ ID NO: 47)
ppLuc(GC)-ag-A.sub.120 (SEQ ID NO: 48) ppLuc(GC)-ag-A.sub.64-ag
(SEQ ID NO: 49) ppLuc(GC)-ag-A.sub.64-aCPSL (SEQ ID NO: 50)
ppLuc(GC)-ag-A.sub.64-PolioCL (SEQ ID NO: 51)
ppLuc(GC)-ag-A.sub.64-G.sub.30 (SEQ ID NO: 52)
ppLuc(GC)-ag-A.sub.64-U.sub.30 (SEQ ID NO: 53)
ppLuc(GC)-ag-A.sub.64-SL (SEQ ID NO: 54)
ppLuc(GC)-ag-A.sub.64-N.sub.32
[0358] Furthermore DNA plasmid sequences coding for the tumour
antigens NY-ESO-1, Survivin and MAGE-C1 were prepared accordingly
as described above. [0359] In summary, the following mRNAs were
obtained from these vectors by in vitro transcription (mRNA
sequences are given in FIGS. 18 to 21):
TABLE-US-00005 [0359] (SEQ ID NO: 55)
NY-ESO-.sub.1(GC)-ag-A.sub.62-C.sub.30 (SEQ ID NO: 56)
NY-ESO-.sub.1(GC)-ag-A.sub.62-C.sub.30-histoneSL (SEQ ID NO: 57)
Survivin(GC)-ag-A.sub.62-C.sub.30-histoneSL (SEQ ID NO: 58)
MAGE-C.sub.1(GC)-ag-A.sub.64-C.sub.30-histoneSL
3. In Vitro Transcription
[0360] The DNA-template according to Example 2 was linearized and
transcribed in vitro using T7-Polymerase. The DNA-template was then
digested by DNase-treatment. All mRNA-transcripts contained a
5'-CAP structure obtained by adding an excess of
N7-Methyl-Guanosine-5'-Triphosphate-5'-Guanosine to the
transcription reaction. mRNA thus obtained was purified and
resuspended in water. 4. Enzymatic Adenylation of mRNA [0361] Two
mRNAs were enzymatically adenylated: [0362] ppLuc(GC)-ag-A64 and
ppLuc(GC)-ag-histoneSL. [0363] To this end, RNA was incubated with
E. coli Poly(A)-polymerase and ATP (Poly(A) Polymerase Tailing Kit,
Epicentre, Madison, USA) following the manufacturer's instructions.
mRNA with extended poly(A) sequence was purified and resuspended in
water. The length of the poly(A) sequence was determined via
agarose gel electrophoresis. Starting mRNAs were extended by
approximately 250 adenylates, the mRNAs obtained are designated as
ppLuc(GC)-ag-A300 and ppLuc(GC)-ag-histoneSL-A250, respectively. 5.
Luciferase Expression by mRNA Electroporation [0364] HeLa cells
were trypsinized and washed in opti-MEM. 1.times.10.sup.5 cells in
200 .mu.l of opti-MEM each were electroporated with 0.5 .mu.g of
ppLuc-encoding mRNA. As a control, mRNA not coding for ppLuc was
electroporated separately. Electroporated cells were seeded in
24-well plates in 1 ml of RPMI 1640 medium. 6, 24, or 48 hours
after transfection, medium was aspirated and cells were lysed in
200 .mu.l of lysis buffer (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10%
glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF). Lysates were
stored at -20.degree. C. until ppLuc activity was measured. 6.
Luciferase Expression by mRNA Lipofection [0365] HeLa cells were
seeded in 96 well plates at a density of 2.times.10.sup.4 cells per
well. The following day, cells were washed in opti-MEM and then
transfected with 0.25 .mu.g of Lipofectin-complexed ppLuc-encoding
mRNA in 150 .mu.l of opti-MEM. As a control, mRNA not coding for
ppLuc was lipofected separately. In some wells, opti-MEM was
aspirated and cells were lysed in 200 .mu.l of lysis buffer 6 hours
after the start of transfection. In the remaining wells, opti-MEM
was exchanged for RPMI 1640 medium at that time. In these wells,
medium was aspirated and cells were lysed in 200 .mu.l of lysis
buffer 24 or 48 hours after the start of transfection. Lysates were
stored at -20.degree. C. until ppLuc activity was measured.
7. Luciferase Measurement
[0365] [0366] ppLuc activity was measured as relative light units
(RLU) in a BioTek SynergyHT plate reader at 5 seconds measuring
time using 50 .mu.l of lysate and 200 .mu.l of luciferin buffer (25
mM Glycylglycin, pH 7.8 (NaOH), 15 mM MgSO.sub.4, 2 mM ATP, 75
.mu.M luciferin). Specific RLU were calculated by subtracting RLU
of the control RNA from total RLU. 8. Luciferase Expression by
Intradermal mRNA Injection (Luciferase Expression In Vivo) [0367]
Mice were anaesthetized with a mixture of Rompun and Ketavet. Each
ppLuc-encoding mRNA was injected intradermally (0.5 .mu.g of mRNA
in 50 .mu.l per injection). As a control, mRNA not coding for ppLuc
was injected separately. 16 hours after injection, mice were
sacrificed and tissue collected. Tissue samples were flash frozen
in liquid nitrogen and lysed in a tissue lyser (Qiagen) in 800
.mu.l of lysis buffer (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10%
glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF). Subsequently
samples were centrifuged at 13500 rpm at 4.degree. C. for 10
minutes. Lysates were stored at -80.degree. C. until ppLuc activity
was measured (see 7. luciferase measurement). 9. NY-ESO-1
Expression by mRNA Electroporation [0368] HeLa cells were
trypsinized and washed in opti-MEM. 2.times.10.sup.5 cells in 200
.mu.l of opti-MEM were electroporated with 10 .mu.g of
NY-ESO-1-encoding mRNA. Cells from three electroporations were
combined and seeded in a 6-well plate in 2 ml of RPMI 1640 medium.
24 hours after transfection, cells were harvested and transferred
into a 96 well V-bottom plate (2 wells per mRNA). Cells were washed
with phosphate buffered saline (PBS) and permeabilized with 200
.mu.l per well of Cytofix/Cytoperm (Becton Dickinson (BD)). After
15 minutes, cells were washed with PERM/WASH.RTM. buffer (BD).
Then, cells were incubated for 1 hour at room temperature with
either mouse anti-NY-ESO-1 IgG1 or an isotype control (20
.mu.g/ml). Cells were washed twice with PERM/WASH.RTM. buffer
again. Next, cells were incubated for 1 hour at 4.degree. C. with a
1:500 dilution of Alexa-647 coupled goat-anti-mouse IgG. Finally,
cells were washed twice with PERM/WASH.RTM. buffer. Cells were
resuspended in 200 .mu.l of buffer (PBS, 2% FCS, 2 mM EDTA, 0.01%
sodium azide). NY-ESO-1 expression was quantified by flow cytometry
as median fluorescence intensity (MFI). 10. Induction of Anti
NY-ESO-1 Antibodies by Vaccination with mRNA [0369] C57BL/6 mice
were vaccinated intradermally with NY-ESO-1-encoding mRNA complexed
with protamine (5 times in 14 days). Control mice were treated with
buffer. The level of NY-ESO-1-specific antibodies in vaccinated and
control mice was analyzed 8 days after the last vaccination by
ELISA: 96 well ELISA plates (Nunc) were coated with 100 .mu.l per
well of 10 .mu.g/ml recombinant NY-ESO-1 protein for 16 hours at
4.degree. C. Plates were washed two times with wash buffer (PBS,
0.05% TWEEN.RTM. 20 non-ionic detergent). To block unspecific
binding, plates were then incubated for 2 hours at 37.degree. C.
with blocking buffer (PBS, 0.05% TWEEN.RTM. 20 non-ionic detergent,
1% BSA). After blocking, 100 .mu.l per well of serially diluted
mouse sera were added and incubated for 4 hours at room
temperature. Plates were then washed three times with wash buffer.
Next, 100 .mu.l per well of biotinylated rat anti-mouse IgG2a[b]
detection antibody (BD Biosciences) diluted 1:600 in blocking
buffer was allowed to bind for 1 hour at room temperature. Plates
were washed again three times with wash buffer, followed by
incubation for 30 minutes at room temperature with 100 .mu.l per
well of horseradish peroxidase-coupled streptavidin. After four
washes with wash buffer, 100 .mu.l per well of
3,3',5,5'-tetramethylbenzidine (Thermo Scientific) was added. Upon
the resulting change in color 100 .mu.l per well of 20% sulfuric
acid was added. Absorbance was measured at 405 nm. 11. Induction of
Anti Survivin Antibodies by Vaccination with mRNA [0370] C57BL/6
mice were vaccinated intradermally with Survivin-encoding mRNA
complexed with protamine (5 times in 14 days). Control mice were
treated with buffer. The level of Survivin-specific antibodies in
vaccinated and control mice was analyzed 8 days after the last
vaccination by ELISA: 96 well ELISA plates (Nunc) were coated with
100 .mu.l per well of 10 .mu.g/ml recombinant Survivin protein for
16 hours at 4.degree. C. Plates were washed two times with wash
buffer (PBS, 0.05% TWEEN.RTM. 20 non-ionic detergent). To block
unspecific binding, plates were then incubated for 2 hours at
37.degree. C. with blocking buffer (PBS, 0.05% TWEEN.RTM. 20
non-ionic detergent, 1% BSA). After blocking, 100 .mu.l per well of
serially diluted mouse sera were added and incubated for 4 hours at
room temperature. Plates were then washed three times with wash
buffer. Next, 100 .mu.l per well of biotinylated rat anti-mouse
IgG2a[b] detection antibody (BD Biosciences) diluted 1:600 in
blocking buffer was allowed to bind for 1 hour at room temperature.
Plates were washed again three times with wash buffer, followed by
incubation for 30 minutes at room temperature with 100 .mu.l per
well of horseradish peroxidase-coupled streptavidin. After four
washes with wash buffer, 100 .mu.l per well of
3,3',5,5'-tetramethylbenzidine (Thermo Scientific) was added. Upon
the resulting change in color 100 .mu.l per well of 20% sulfuric
acid was added. Absorbance was measured at 405 nm. 12. Induction of
Anti MAGE-C1 Antibodies by Vaccination with mRNA [0371] C57BL/6
mice were vaccinated intradermally with MAGE-C1-encoding mRNA
complexed with protamine (5 times in 14 days). Control mice were
treated with buffer. The level of MAGE-C1-specific antibodies in
vaccinated and control mice was analyzed 8 days after the last
vaccination by ELISA: 96 well ELISA plates (Nunc) were coated with
100 .mu.l per well of 10 .mu.g/ml recombinant MAGE-C1 protein for
16 hours at 4.degree. C. Plates were washed two times with wash
buffer (PBS, 0.05% TWEEN.RTM. 20 non-ionic detergent). To block
unspecific binding, plates were then incubated for 2 hours at
37.degree. C. with blocking buffer (PBS, 0.05% TWEEN.RTM. 20
non-ionic detergent, 1% BSA). After blocking, 100 .mu.l per well of
serially diluted mouse sera were added and incubated for 4 hours at
room temperature. Plates were then washed three times with wash
buffer. Next, 100 .mu.l per well of biotinylated rat anti-mouse
IgG2a[b] detection antibody (BD Biosciences) diluted 1:600 in
blocking buffer was allowed to bind for 1 hour at room temperature.
Plates were washed again three times with wash buffer, followed by
incubation for 30 minutes at room temperature with 100 .mu.l per
well of horseradish peroxidase-coupled streptavidin. After four
washes with wash buffer, 100 .mu.l per well of
3,3',5,5'-tetramethylbenzidine (Thermo Scientific) was added. Upon
the resulting change in color 100 .mu.l per well of 20% sulfuric
acid was added. Absorbance was measured at 405 nm.
13. Detection of an Antigen-Specific Cellular Immune Response (T
Cell Immune Response) by ELI SPOT:
[0371] [0372] C57BL/6 mice are vaccinated intradermally with
MAGE-C1 encoding mRNA complexed with protamine (5 times in 14
days). Control mice are treated with buffer. 1 week after the last
vaccination mice are sacrificed, the spleens are removed and the
splenocytes are isolated. The splenocytes are restimulated for 7
days in the presence of peptides from the above antigen (peptide
library) or coincubated with dendritic cells generated from bone
marrow cells of native syngeneic mice, which are electroporated
with mRNA coding for the antigen. To determine an antigen-specific
cellular immune response INFgamma secretion was measured after
re-stimulation. For detection of INFgamma a coat multiscreen plate
(Millipore) is incubated overnight with coating buffer 0.1 M
carbonate-bicarbonate buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3,
8.4 g/l NaHCO.sub.3) comprising antibody against INF.gamma. (BD
Pharmingen, Heidelberg, Germany). Stimulators and effector cells
are incubated together in the plate in the ratio of 1:20 for 24 h.
The plate is washed with 1.times.PBS and incubated with a
biotin-coupled secondary antibody. After washing with
1.times.PBS/0.05% TWEEN.RTM. 20 non-ionic detergent, the substrate
(5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid
Substrate System from Sigma Aldrich, Taufkirchen, Germany) is added
to the plate and the conversion of the substrate could be detected
visually.
14. Results
14.1 Histone Stem-Loop Sequences:
[0373] In order to characterize histone stem-loop sequences,
sequences from metazoa and protozoa (4001 sequences), or from
protozoa (131 sequences) or alternatively from metazoa (3870
sequences), or from vertebrates (1333 sequences) or from humans (84
sequences) were grouped and aligned. Then, the quantity of the
occurring nucleotides was determined for every position. Based on
the tables thus obtained, consensus sequences for the 5 different
groups of sequences were generated representing all nucleotides
present in the sequences analyzed. Within the consensus sequence of
metazoa and protozoa combined, 3 nucleotides are conserved, a T/U
in the loop and a G and a C in the stem, forming a base pair.
Structurally, typically a 6 base-pair stem and a loop of 4
nucleotides is formed. However, deviating structures are common: Of
84 human histone stem-loops, two contain a stem of only 5
nucleotides comprising 4 base-pairs and one mismatch. Another human
histone stem-loop contains a stem of only 5 base-pairs. Four more
human histone stem-loops contain a 6 nucleotide long stem, but
include one mismatch at three different positions, respectively.
Furthermore, four human histone stem-loops contain one wobble
base-pair at two different positions, respectively. Concerning the
loop, a length of 4 nucleotides seems not to be strictly required,
as a loop of 5 nucleotides has been identified in D.
dzscoideum.
[0374] In addition to the consensus sequences representing all
nucleotides present in the sequences analyzed, more restrictive
consensus sequences were also obtained, increasingly emphasizing
conserved nucleotides. In summary, the following sequences were
obtained: [0375] (Cons): represents all nucleotides present [0376]
(99%): represents at least 99% of all nucleotides present [0377]
(95%): represents at least 95% of all nucleotides present [0378]
(90%): represents at least 90% of all nucleotides present
[0379] The results of the analysis of histone stem-loop sequences
are summarized in the following Tables 1 to 5 (see also FIGS. 1 to
5):
TABLE-US-00006 TABLE 1 Metazoan and protozoan histone stem-loop
consensus sequence: (based on an alignment of 4001 metazoan and
protozoan histone stem-loop sequences) (see also FIG. 1) < <
< < < < .cndot. .cndot. .cndot. # A 2224 1586 3075 2872
1284 184 0 13 12 9 1 47 59 0 # T 172 188 47 205 19 6 0 569 1620 199
3947 3830 3704 4001 # C 1557 2211 875 918 2675 270 0 3394 2342 3783
51 119 227 0 # G 25 16 4 6 23 3541 4001 25 27 10 2 5 11 0 Cons N*
N* N N N N G N N N N N N T 99% H* H* H H V V G Y Y Y Y H H T 95% M*
H* M H M S G Y Y Y T T Y T 90% M* M* M M M S G Y Y C T T T T
.cndot. > > > > > > # A 675 3818 195 1596 523 0
14 3727 61 771 2012 2499 # T 182 1 21 15 11 0 179 8 64 557 201 690
# C 3140 7 50 31 16 4001 3543 154 3870 2636 1744 674 # G 4 175 3735
2359 3451 0 265 112 4 37 43 138 Cons N N N N N C N N N N* N* N* 99%
H R V V R C B V H H* N* N* 95% M A R R R C S M C H* H* H* 90% M A G
R R C S A C H* M* H*
TABLE-US-00007 TABLE 2 Protozoan histone stem-loop consensus
sequence: (based on an alignment of 131 protozoan histone stem-loop
sequences) (see also FIG. 2) < < < < < < .cndot.
.cndot. .cndot. .cndot. > > > > > > # A 52 32 71
82 76 13 0 12 12 9 1 46 3 0 75 82 53 79 20 0 4 94 17 35 74 56 # T
20 32 37 21 8 3 0 21 85 58 86 70 65 131 28 1 17 13 10 0 15 7 31 32
30 28 # C 45 59 20 25 38 0 0 86 8 54 42 13 58 0 27 2 6 31 10 131
112 5 82 58 30 40 # G 14 8 3 3 9 115 131 12 26 10 2 2 5 0 1 46 55 8
91 0 0 25 1 6 7 7 Cons N* N* N N N D G N N N N N N T N N N N N C H
N N N* N* N* 99% N* N* N N N D G N N N B N N T H V N N N C H N H N*
N* N* 95% N* N* H H N R G N N N Y H B T H R D N N C Y D H H* N* N*
90% N* H* H H V R G N D B Y H Y T H R D H N C Y R H H* H* H*
TABLE-US-00008 TABLE 3 Metazoan histone stem-loop consensus
sequence: (based on an alignment of 3870 (including 1333 vertebrate
sequences) metazoan histone stem-loop sequences) (see also FIG. 3)
< < < < < < .cndot. .cndot. .cndot. # A 2172 1554
3004 2790 1208 171 0 1 0 0 0 1 56 0 # T 152 156 10 184 11 3 0 548
1535 141 3861 3760 3639 3870 # C 1512 2152 855 893 2637 270 0 3308
2334 3729 9 106 169 0 # G 11 8 1 3 14 3426 3870 13 1 0 0 3 6 0 Cons
N* N* N N N N G N B Y Y N N T 99% H* H* M H M V G Y Y Y T Y H T 95%
M* M* M M M S G Y Y C T T Y T 90% M* M* M M M S G Y Y C T T T T
.cndot. > > > > > > # A 600 3736 142 1517 503 0
10 3633 44 736 1938 2443 # T 154 0 4 2 1 0 164 1 33 525 181 662 # C
3113 5 44 0 6 3870 3431 149 3788 2578 1714 634 # G 3 129 3680 2351
3360 0 265 87 3 31 36 131 Cons N V N D N C N N N N* N* N* 99% H R V
R R C B V M H* H* N* 95% M A G R R C S M C H* H* H* 90% M A G R R C
S A C H* M* H*
TABLE-US-00009 TABLE 4 Vertebrate histone stem-loop consensus
sequence: (based on an alignment of 1333 vertebrate histone
stem-loop sequences) (see also FIG. 4) < < < < <
< .cndot. .cndot. .cndot. # A 661 146 1315 1323 920 8 0 1 0 0 0
1 4 0 # T 63 121 2 2 6 2 0 39 1217 2 1331 1329 1207 1333 # C 601
1062 16 6 403 1 0 1293 116 1331 2 0 121 0 # G 8 4 0 2 4 1322 1333 0
0 0 0 3 1 0 Cons N* N* H N N N G H Y Y Y D N T 99% H* H* M A M G G
Y Y C T T Y T 95% H* H* A A M G G C Y C T T Y T 90% M* M* A A M G G
C T C T T T T .cndot. > > > > > > # A 441 1333 0
1199 21 0 1 1126 26 81 380 960 # T 30 0 1 0 1 0 2 1 22 91 91 12 # C
862 0 2 0 0 1333 1328 128 1284 1143 834 361 # G 0 0 1330 134 1311 0
2 78 1 18 28 0 Cons H A B R D C N N N N* N* H* 99% H A G R R C C V
H N* N* M* 95% M A G R G C C V C H* H* M* 90% M A G R G C C M C Y*
M* M*
TABLE-US-00010 TABLE 5 Homo sapiens histone stem-loop consensus
sequence: (based on an alignment of 84 human histone stem-loop
sequences) (see also FIG. 5) < < < < < < .cndot.
.cndot. .cndot. .cndot. > > > > > > # A 10 17 84
84 76 1 0 1 0 0 0 1 0 0 12 84 0 65 3 0 0 69 5 0 10 64 # T 8 6 0 0 2
2 0 1 67 0 84 80 81 84 5 0 0 0 0 0 0 0 4 25 24 3 # C 62 61 0 0 6 0
0 82 17 84 0 0 3 0 67 0 1 0 0 84 84 5 75 57 44 17 # G 4 0 0 0 0 81
84 0 0 0 0 3 0 0 0 0 83 19 81 0 0 10 0 2 6 0 Cons N* H* A A H D G H
Y C T D Y T H A S R R C C V H B* N* H* 99% N* H* A A H D G H Y C T
D Y T H A S R R C C V H B* N* H* 95% H* H* A A M G G C Y C T T T T
H A G R G C C V M Y* N* M* 90% H* M* A A A G G C Y C T T T T M A G
R G C C R M Y* H* M*
[0380] Wherein the used abbreviations were defined as followed:
TABLE-US-00011 abbreviation Nucleotide bases remark G G Guanine A A
Adenine T T Thymine U U Uracile C C Cytosine R G or A Purine Y T/U
or C Pyrimidine M A or C Amino K G or T/U Keto S G or C Strong (3H
bonds) W A or T/U Weak (2H bonds) H A or C or T/U Not G B G or T/U
or C Not A V G or C or A Not T/U D G or A or T/U Not C N G or C or
T/U or A Any base * present or not Base may be present or not
14.2 the Combination of Poly(A) and histoneSL Increases Protein
Expression from mRNA in a Synergistic Manner. [0381] To investigate
the effect of the combination of poly(A) and histoneSL on protein
expression from mRNA, mRNAs with different sequences 3' of the
alpha-globin 3'-UTR were synthesized: mRNAs either ended just 3' of
the 3'-UTR, thus lacking both poly(A) sequence and histoneSL, or
contained either an A64 poly(A) sequence or a histoneSL instead, or
both A64 poly(A) and histoneSL 3' of the 3'-UTR.
Luciferase-encoding mRNAs or control mRNA were electroporated into
HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours
after transfection (see following Table 6 and FIG. 22).
TABLE-US-00012 [0381] TABLE 6 RLU at RLU at RLU at mRNA 6 hours 24
hours 48 hours ppLuc(GC)-ag-A64-histoneSL 466553 375169 70735
ppLuc(GC)-ag-histoneSL 50947 3022 84 ppLuc(GC)-ag-A64 10471 19529
4364 ppLuc(GC)-ag 997 217 42
[0382] Little luciferase was expressed from mRNA having neither
poly(A) sequence nor histoneSL. Both a poly(A) sequence or the
histoneSL increased the luciferase level to a similar extent.
Either mRNA gave rise to a luciferase level much higher than did
mRNA lacking both poly(A) and histoneSL. Strikingly however, the
combination of poly(A) and histoneSL further strongly increased the
luciferase level, manifold above the level observed with either of
the individual elements. The magnitude of the rise in luciferase
level due to combining poly(A) and histoneSL in the same mRNA
demonstrates that they are acting synergistically. [0383] The
synergy between poly(A) and histoneSL was quantified by dividing
the signal from poly(A)-histoneSL mRNA (+/+) by the sum of the
signals from histoneSL mRNA (-/+) plus poly(A) mRNA (+/-) (see
following Table 7).
TABLE-US-00013 [0383] TABLE 7 RLU at RLU at RLU at A64 histoneSL 6
hours 24 hours 48 hours + + 466553 375169 70735 - + 50947 3022 84 +
- 10471 19529 4364 Synergy 7.6 16.6 15.9
[0384] The factor thus calculated specifies how much higher the
luciferase level from mRNA combining poly(A) and histoneSL is than
would be expected if the effects of poly(A) and histoneSL were
purely additive. The luciferase level from mRNA combining poly(A)
and histoneSL was up to 16.6 times higher than if their effects
were purely additive. This result confirms that the combination of
poly(A) and histoneSL effects a markedly synergistic increase in
protein expression. 14.3 the Combination of Poly(A) and histoneSL
Increases Protein Expression from mRNA Irrespective of their Order.
[0385] The effect of the combination of poly(A) and histoneSL might
depend on the length of the poly(A) sequence and the order of
poly(A) and histoneSL. Thus, mRNAs with increasing poly(A) sequence
length and mRNA with poly(A) and histoneSL in reversed order were
synthesized: Two mRNAs contained 3' of the 3'-UTR either an A120 or
an A300 poly(A) sequence. One further mRNA contained 3' of the
3'-UTR first a histoneSL followed by an A250 poly(A) sequence.
Luciferase-encoding mRNAs or control mRNA were lipofected into HeLa
cells. Luciferase levels were measured at 6, 24, and 48 hours after
the start of transfection (see following Table 8 and FIG. 23).
TABLE-US-00014 [0385] TABLE 8 RLU at RLU at RLU at mRNA 6 hours 24
hours 48 hours ppLuc(GC)-ag-histoneSL-A250 98472 734222 146479
ppLuc(GC)-ag-A64-histoneSL 123674 317343 89579
ppLuc(GC)-ag-histoneSL 7291 4565 916 ppLuc(GC)-ag-A300 4357 38560
11829 ppLuc(GC)-ag-A120 4371 45929 10142 ppLuc(GC)-ag-A64 1928
26781 537
[0386] Both an A64 poly(A) sequence or the histoneSL gave rise to
comparable luciferase levels. In agreement with the previous
experiment did the combination of A64 and histoneSL strongly
increase the luciferase level, manifold above the level observed
with either of the individual elements. The magnitude of the rise
in luciferase level due to combining poly(A) and histoneSL in the
same mRNA demonstrates that they are acting synergistically. The
synergy between A64 and histoneSL was quantified as before based on
the luciferase levels of A64-histoneSL, A64, and histoneSL mRNA
(see following Table 9). The luciferase level from mRNA combining
A64 and histoneSL was up to 61.7 times higher than if the effects
of poly(A) and histoneSL were purely additive.
TABLE-US-00015 [0386] TABLE 9 RLU at RLU at RLU at A64 histoneSL 6
hours 24 hours 48 hours + + 123674 317343 89579 - + 7291 4565 916 +
- 1928 26781 537 Synergy 13.4 10.1 61.7
[0387] In contrast, increasing the length of the poly(A) sequence
from A64 to A120 or to A300 increased the luciferase level only
moderately (see Table 8 and FIG. 19). mRNA with the longest poly(A)
sequence, A300, was also compared to mRNA in which a poly(A)
sequence of similar length was combined with the histoneSL,
histoneSL-A250. In addition to having a long poly(A) sequence, the
order of histoneSL and poly(A) is reversed in this mRNA relative to
A64-histoneSL mRNA. The combination of A250 and histoneSL strongly
increased the luciferase level, manifold above the level observed
with either histoneSL or A300. Again, the synergy between A250 and
histoneSL was quantified as before comparing RLU from
histoneSL-A250 mRNA to RLU from Moo mRNA plus histoneSL mRNA (see
following Table 10). The luciferase level from mRNA combining A250
and histoneSL was up to 17.0 times higher than if the effects of
poly(A) and histoneSL were purely additive.
TABLE-US-00016 [0387] TABLE 10 RLU at RLU at RLU at histoneSL
A250/A300 6 hours 24 hours 48 hours + + 98472 734222 146479 + -
7291 4565 916 - + 4357 38560 11829 Synergy 8.5 17.0 11.5
[0388] In summary, a highly synergistic effect of the combination
of histoneSL and poly(A) on protein expression from mRNA has been
demonstrated for substantially different lengths of poly(A) and
irrespective of the order of poly(A) and histoneSL. 14.4 the Rise
in Protein Expression by the Combination of Poly(A) and histoneSL
is Specific [0389] To investigate whether the effect of the
combination of poly(A) and histoneSL on protein expression from
mRNA is specific, mRNAs with alternative sequences in combination
with poly(A) were synthesized: These mRNAs contained 3' of A64 one
of seven distinct sequences, respectively. Luciferase-encoding
mRNAs or control mRNA were electroporated into HeLa cells.
Luciferase levels were measured at 6, 24, and 48 hours after
transfection (see following Table 11 and FIG. 24).
TABLE-US-00017 [0389] TABLE 11 RLU at RLU at RLU at mRNA 6 hours 24
hours 48 hours ppLuc(GC)-ag-A64-N32 33501 38979 2641
ppLuc(GC)-ag-A64-SL 28176 20364 874 ppLuc(GC)-ag-A64-U30 41632
54676 3408 ppLuc(GC)-ag-A64-G30 46763 49210 3382
ppLuc(GC)-ag-A64-PolioCL 46428 26090 1655 ppLuc(GC)-ag-A64-aCPSL
34176 53090 3338 ppLuc(GC)-ag-A64-ag 18534 18194 989
ppLuc(GC)-ag-A64-histoneSL 282677 437543 69292
ppLuc(GC)-ag-histoneSL 27597 3171 0 ppLuc(GC)-ag-A64 14339 48414
9357
[0390] Both a poly(A) sequence or the histoneSL gave rise to
comparable luciferase levels. Again, the combination of poly(A) and
histoneSL strongly increased the luciferase level, manifold above
the level observed with either of the individual elements, thus
acting synergistically. In contrast, combining poly(A) with any of
the alternative sequences was without effect on the luciferase
level compared to mRNA containing only a poly(A) sequence. Thus,
the combination of poly(A) and histoneSL increases protein
expression from mRNA in a synergistic manner, and this effect is
specific. 14.5 The combination of poly(A) and histoneSL increases
protein expression from mRNA in a Synergistic Manner In Vivo.
[0391] To investigate the effect of the combination of poly(A) and
histoneSL on protein expression from mRNA in vivo,
Luciferase-encoding mRNAs with different sequences 3' of the
alpha-globin 3'-UTR or control mRNA were injected intradermally
into mice: mRNAs contained either an A64 poly(A) sequence or a
histoneSL instead, or both A64 poly(A) and histoneSL 3' of the
3'-UTR. Luciferase levels were measured at 16 hours after injection
(see following Table 12 and FIG. 25).
TABLE-US-00018 [0391] TABLE 12 RLU at mRNA 16 hours
ppLuc(GC)-ag-A64-histoneSL 38081 ppLuc(GC)-ag-histoneSL 137
ppLuc(GC)-ag-A64 4607
[0392] Luciferase was expressed from mRNA having either a histoneSL
or a poly(A) sequence. Strikingly however, the combination of
poly(A) and histoneSL further strongly increased the luciferase
level, manifold above the level observed with either of the
individual elements. The magnitude of the rise in luciferase level
due to combining poly(A) and histoneSL in the same mRNA
demonstrates that they are acting synergistically. [0393] The
synergy between poly(A) and histoneSL was quantified by dividing
the signal from poly(A)-histoneSL mRNA (+/+) by the sum of the
signals from histoneSL mRNA (-/+) plus poly(A) mRNA (+/-) (see
following Table 13).
TABLE-US-00019 [0393] TABLE 13 RLU at A64 histoneSL 16 hours + +
38081 - + 137 + - 4607 Synergy 8.0
[0394] The factor thus calculated specifies how much higher the
luciferase level from mRNA combining poly(A) and histoneSL is than
would be expected if the effects of poly(A) and histoneSL were
purely additive. The luciferase level from mRNA combining poly(A)
and histoneSL was 8 times higher than if their effects were purely
additive. This result confirms that the combination of poly(A) and
histoneSL effects a markedly synergistic increase in protein
expression in vivo. 14.6 the Combination of Poly(A) and histoneSL
Increases NY-ESO-1 Protein Expression from mRNA. [0395] To
investigate the effect of the combination of poly(A) and histoneSL
on protein expression from mRNA, NY-ESO-1-encoding mRNAs with
different sequences 3' of the alpha-globin 3'-UTR were synthesized:
mRNAs contained either an A64 poly(A) sequence or both A64 poly(A)
and histoneSL 3' of the 3'-UTR. NY-ESO-1-encoding mRNAs were
electroporated into HeLa cells. NY-ESO-1 levels were measured at 24
hours after transfection by flow cytometry (see following Table 14
and FIG. 26).
TABLE-US-00020 [0395] TABLE 14 MFI at 24 hours mRNA anti-NY-ESO-1
isotype control NY-ESO-1(GC)-ag-A64-histoneSL 15600 1831
NY-ESO-1(GC)-ag-A64 1294 849
[0396] NY-ESO-1 was expressed from mRNA having only a poly(A)
sequence. Strikingly however, the combination of poly(A) and
histoneSL strongly increased the NY-ESO-1 level, manifold above the
level observed with only a poly(A) sequence. 14.7 the Combination
of Poly(A) and histoneSL Increases the Level of Antibodies Elicited
by Vaccination with mRNA. [0397] To investigate the effect of the
combination of poly(A) and histoneSL on the induction of antibodies
elicited by vaccination with mRNA, C57BL/6 mice were vaccinated
intradermally with protamine-complexed, NY-ESO-1-encoding mRNAs
with different sequences 3' of the alpha-globin 3'-UTR. mRNAs
contained either an A64 poly(A) sequence or both A64 poly(A) and
histoneSL 3' of the 3'-UTR. The level of NY-ESO-1-specific
antibodies in vaccinated and control mice was analyzed by ELISA
with serial dilutions of sera (see following Table 15 and FIG.
27).
TABLE-US-00021 [0397] TABLE 15 mRNA mean IgG2a[b] endpoint titer
NY-ESO-1(GC)-ag-A64-histoneSL 763 NY-ESO-1(GC)-ag-A64 20
[0398] Anti NY-ESO-1 IgG2a[b] was induced by mRNA having only a
poly(A) sequence. Strikingly however, the combination of poly(A)
and histoneSL strongly increased the anti NY-ESO-1 IgG2a[b] level,
manifold above the level observed with only a poly(A) sequence.
Sequence CWU 1
1
58116RNAartificialhistone stem-loop sequence according to formula
(Ic) metazoan and protozoan histone stem-loop consensus sequence
without stem bordering elementsmisc_feature(1)..(1)n is selected
from a nucleotide selected from A, U, T, G and C, or a nucleotide
analogue thereofmisc_feature(3)..(8)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(10)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(16)..(16)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
1ngnnnnnnun nnnncn 16226RNAartificialhistone stem-loop sequence
according to formula (IIc) metazoan and protozoan histone stem-loop
consensus sequence with stem bordering
elementsmisc_feature(1)..(6)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(8)..(13)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(15)..(19)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(21)..(26)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
2nnnnnngnnn nnnunnnnnc nnnnnn 26316RNAartificialhistone stem-loop
sequence according to formula (Id) without stem bordering
elementsmisc_feature(1)..(1)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(3)..(8)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(10)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(16)..(16)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
3ncnnnnnnun nnnngn 16426RNAartificialhistone stem-loop sequence
according to formula (IId) with stem bordering
elementsmisc_feature(1)..(6)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(8)..(13)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(15)..(19)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(21)..(26)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
4nnnnnncnnn nnnunnnnng nnnnnn 26516RNAartificialhistone stem-loop
sequence according to formula (Ie) protozoan histone stem-loop
consensus sequence without stem bordering
elementsmisc_feature(3)..(8)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(10)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
5dgnnnnnnun nnnnch 16626RNAartificialhistone stem-loop sequence
according to formula (IIe) protozoan histone stem-loop consensus
sequence with stem bordering elementsmisc_feature(1)..(5)n is
selected from a nucleotide selected from A, U, T, G and C, or a
nucleotide analogue thereofmisc_feature(8)..(13)n is selected from
a nucleotide selected from A, U, T, G and C, or a nucleotide
analogue thereofmisc_feature(15)..(19)n is selected from a
nucleotide selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(22)..(26)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
6nnnnndgnnn nnnunnnnnc hnnnnn 26716RNAartificialhistone stem-loop
sequence according to formula (If) metazoan histone stem-loop
consensus sequence without stem bordering
elementsmisc_feature(1)..(1)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(3)..(3)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(7)..(8)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(10)..(10)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(12)..(12)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(14)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(16)..(16)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
7ngnbyynnun vndncn 16826RNAartificialhistone stem-loop sequence
according to formula (IIf) metazoan histone stem-loop consensus
sequence with stem bordering elementsmisc_feature(1)..(6)n is
selected from a nucleotide selected from A, U, T, G and C, or a
nucleotide analogue thereofmisc_feature(8)..(8)n is selected from a
nucleotide selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(12)..(13)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(15)..(15)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(17)..(17)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(19)..(19)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(21)..(26)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
8nnnnnngnby ynnunvndnc nnnnnn 26916RNAartificialhistone stem-loop
sequence according to formula (Ig) vertebrate histone stem-loop
consensus sequence without stem bordering
elementsmisc_feature(1)..(1)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(8)..(8)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(16)..(16)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
9nghyyydnuh abrdcn 161026RNAartificialhistone stem-loop sequence
according to formula (IIg) vertebrate histone stem-loop consensus
sequence with stem bordering elementsmisc_feature(1)..(2)n is
selected from a nucleotide selected from A, U, T, G and C, or a
nucleotide analogue thereofmisc_feature(4)..(6)n is selected from a
nucleotide selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(13)..(13)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(21)..(25)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
10nnhnnnghyy ydnuhabrdc nnnnnh 261116RNAartificialhistone stem-loop
sequence according to formula (Ih) humane histone stem-loop
consensus sequence (Homo sapiens) without stem bordering elements
11dghycudyuh asrrcc 161226RNAartificialhistone stem-loop sequence
according to formula (IIh) human histone stem-loop consensus
sequence (Homo sapiens) with stem bordering
elementsmisc_feature(1)..(1)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(25)..(25)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
12nhaahdghyc udyuhasrrc cvhbnh 261316DNAartificialhistone stem-loop
sequences (without stem-bordering elements) according to formula
(Ic) 13vgyyyyhhth rvvrcb 161416DNAartificialhistone stem-loop
sequences (without stem-bordering elements) according to formula
(Ic) 14sgyyyttytm arrrcs 161516DNAartificialhistone stem-loop
sequences (without stem-bordering elements) according to formula
(Ic) 15sgyycttttm agrrcs 161616DNAartificialhistone stem-loop
sequences (without stem-bordering elements) according to formula
(Ie)misc_feature(3)..(5)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(7)..(8)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(12)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
16dgnnnbnnth vnnnch 161716DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula
(Ie)misc_feature(3)..(5)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(13)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
17rgnnnyhbth rdnncy 161816DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula
(Ie)misc_feature(3)..(3)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(14)..(14)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
18rgndbyhyth rdhncy 161916DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (If)
19vgyyytyhth rvrrcb 162016DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (If)
20sgyycttytm agrrcs 162116DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (If)
21sgyycttttm agrrcs 162216DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (Ig)
22ggyycttyth agrrcc 162316DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (Ig)
23ggcycttytm agrgcc 162416DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (Ig)
24ggctcttttm agrgcc 162516DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (Ih)
25dghyctdyth asrrcc 162616DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (Ih)
26ggcyctttth agrgcc 162716DNAartificialhistone stem-loop sequences
(without stem-bordering elements) according to formula (Ih)
27ggcycttttm agrgcc 162826DNAartificialhistone stem-loop sequence
(with stem bordering elements) according to formula
(IIc)misc_feature(25)..(26)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue thereof
28hhhhvvgyyy yhhthrvvrc bvhhnn 262926DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula (IIc)
29mhmhmsgyyy ttytmarrrc smchhh 263026DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula (IIc)
30mmmmmsgyyc ttttmagrrc sachmh 263126DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula
(IIe)misc_feature(1)..(5)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(8)..(10)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(12)..(13)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(17)..(19)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(22)..(22)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(24)..(26)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
31nnnnndgnnn bnnthvnnnc hnhnnn 263226DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula
(IIe)misc_feature(1)..(2)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(5)..(5)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(8)..(10)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(18)..(19)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(25)..(26)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
32nnhhnrgnnn yhbthrdnnc ydhhnn 263326DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula
(IIe)misc_feature(1)..(1)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(8)..(8)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(19)..(19)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
33nhhhvrgndb yhythrdhnc yrhhhh 263426DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula
(IIf)misc_feature(26)..(26)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue thereof
34hhmhmvgyyy tyhthrvrrc bvmhhn 263526DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula (IIf)
35mmmmmsgyyc ttytmagrrc smchhh 263626DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula (IIf)
36mmmmmsgyyc ttttmagrrc sachmh 263726DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula
(IIg)misc_feature(24)..(25)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue thereof
37hhmamggyyc ttythagrrc cvhnnm 263826DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula (IIg)
38hhaamggcyc ttytmagrgc cvchhm 263926DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula (IIg)
39mmaamggctc ttttmagrgc cmcymm 264026DNAartificialhistone stem-loop
sequence (with stem bordering elements) according to formula
(IIh)misc_feature(1)..(1)n is selected from a nucleotide selected
from A, U, T, G and C, or a nucleotide analogue
thereofmisc_feature(25)..(25)n is selected from a nucleotide
selected from A, U, T, G and C, or a nucleotide analogue thereof
40nhaahdghyc tdythasrrc cvhbnh 264126DNAartificialhistone stem-loop
sequence (with stem bordering
elements) according to formula (IIh)misc_feature(25)..(25)n is
selected from a nucleotide selected from A, U, T, G and C, or a
nucleotide analogue thereof 41hhaamggcyc tttthagrgc cvmynm
264226DNAartificialhistone stem-loop sequence (with stem bordering
elements) according to formula (IIh) 42hmaaaggcyc ttttmagrgc crmyhm
26431747RNAartificialmRNA sequence of ppLuc(GC)-ag 43gggagaaagc
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 1740auagauc 1747441806RNAartificialmRNA
sequence of ppLuc(GC)-ag-A64 44gggagaaagc 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 1800aaaaaa 1806451772RNAartificialmRNA sequence of
ppLuc(GC)-ag-histoneSL 45gggagaaagc 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
1740auagaucuca aaggcucuuu ucagagccac ca 1772461835RNAartificialmRNA
sequence of ppLuc(GC)-ag-A64-histoneSL 46gggagaaagc 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 1800aaaaaaugca ucaaaggcuc uuuucagagc cacca
1835471869RNAartificialmRNA sequence of ppLuc(GC)-ag-A120
47gggagaaagc 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 1740auagaucuaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaa
1869481858RNAartificialmRNA sequence of ppLuc(GC)-ag-A64-ag
48gggagaaagc 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 1800aaaaaaugca uccugcccga
ugggccuccc aacgggcccu ccuccccucc uugcaccg
1858491894RNAartificialmRNA sequence of ppLuc(GC)-ag-A64-aCPSL
49gggagaaagc 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 1800aaaaaaugca ucaauuccua
cacgugaggc gcugugauuc ccuauccccc uucauucccu 1860auacauuagc
acagcgccau ugcauguagg aauu 1894501909RNAartificialmRNA sequence of
ppLuc(GC)-ag-A64-PolioCL 50gggagaaagc 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
1800aaaaaaugca ucaauucuaa aacagcucug ggguuguacc caccccagag
gcccacgugg 1860cggcuaguac uccgguauug cgguacccuu guacgccugu
uuuagaauu 1909511841RNAartificialmRNA sequence of
ppLuc(GC)-ag-A64-G30 51gggagaaagc 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
1800aaaaaaugca uggggggggg gggggggggg gggggggggg g
1841521841RNAartificialmRNA sequence of ppLuc(GC)-ag-A64-U30
52gggagaaagc 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 1800aaaaaaugca uuuuuuuuuu
uuuuuuuuuu uuuuuuuuuu u 1841531857RNAartificialmRNA sequence of
ppLuc(GC)-ag-A64-SL 53gggagaaagc 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
1800aaaaaaugca uuauggcggc cguguccacc acggauauca ccguggugga cgcggcc
1857541838RNAartificialppLuc(GC)-ag-A64-N32 54gggagaaagc 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 1800aaaaaaugca ucccccucua gacaauugga auuccaua
183855747RNAartificialmRNA sequence of NY-ESO-1(GC)-ag-A64-C30
55gggagaaagc uuaccaugca ggccgagggc cgcggcaccg gcggcucgac cggcgacgcc
60gacgggcccg gcggcccggg caucccggac ggcccgggcg ggaacgcggg cggcccgggc
120gaggccggcg ccaccggcgg gcggggcccg cggggcgccg gcgccgcccg
ggcgagcggc 180cccggcgggg gcgccccgcg gggcccgcac ggcggcgccg
ccagcggccu gaacgggugc 240ugccggugcg gcgcccgcgg cccggagagc
cggcuccugg aguucuaccu ggccaugccg 300uucgcgaccc cgauggaggc
cgagcuggcc cggcggagcc uggcccagga cgccccgccg 360cugcccgugc
cgggcgugcu ccugaaggag uucacgguga gcggcaacau ccugaccauc
420cggcugaccg ccgcggacca ccggcagcug cagcugucga ucagcagcug
ccuccagcag 480cugagccugc ugauguggau cacccagugc uuccugccgg
uguuccuggc ccagccgccc 540agcggccagc gccggugacc acuaguuaua
agacugacua gcccgauggg ccucccaacg 600ggcccuccuc cccuccuugc
accgagauua auaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaauauu cccccccccc cccccccccc
720cccccccccc ucuagacaau uggaauu 74756761RNAartificialmRNA sequence
of NY-ESO-1(GC)-ag-A64-C30- histoneSL 56gggagaaagc uuaccaugca
ggccgagggc cgcggcaccg gcggcucgac cggcgacgcc 60gacgggcccg gcggcccggg
caucccggac ggcccgggcg ggaacgcggg cggcccgggc 120gaggccggcg
ccaccggcgg gcggggcccg cggggcgccg gcgccgcccg ggcgagcggc
180cccggcgggg gcgccccgcg gggcccgcac ggcggcgccg ccagcggccu
gaacgggugc 240ugccggugcg gcgcccgcgg cccggagagc cggcuccugg
aguucuaccu ggccaugccg 300uucgcgaccc cgauggaggc cgagcuggcc
cggcggagcc uggcccagga cgccccgccg 360cugcccgugc cgggcgugcu
ccugaaggag uucacgguga gcggcaacau ccugaccauc 420cggcugaccg
ccgcggacca ccggcagcug cagcugucga ucagcagcug ccuccagcag
480cugagccugc ugauguggau cacccagugc uuccugccgg uguuccuggc
ccagccgccc 540agcggccagc gccggugacc acuaguuaua agacugacua
gcccgauggg ccucccaacg 600ggcccuccuc cccuccuugc accgagauua
auaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaugca uccccccccc cccccccccc 720cccccccccc
ccaaaggcuc uuuucagagc caccaggaau u 76157646RNAartificialmRNA
sequence of Survivin(GC)-ag-A64-C30- histoneSL 57gggagaaagc
uuaccauggg cgcccccacc cugccgccgg ccuggcagcc guuccucaag 60gaccaccgca
ucucgaccuu caagaacugg ccguuccugg agggcugcgc gugcaccccg
120gagcggaugg ccgaggccgg cuucauccac ugccccaccg agaacgagcc
ggaccuggcc 180cagugcuucu ucugcuucaa ggagcuggag ggcugggagc
cggacgacga cccgaucgag 240gagcacaaga agcacagcag cggcugcgcc
uuccugagcg ugaagaagca guucgaggag 300cugacgcucg gggaguuccu
gaagcuggac cgggagcggg ccaagaacaa gaucgcgaag 360gagaccaaca
acaagaagaa ggaguucgag gagaccgcca agaaggugcg gcgggccauc
420gagcagcugg ccgccaugga cugaccacua guuauaagac ugacuagccc
gaugggccuc 480ccaacgggcc cuccuccccu ccuugcaccg agauuaauaa
aaaaaaaaaa aaaaaaaaaa 540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaugcauccc cccccccccc 600cccccccccc ccccccccaa
aggcucuuuu cagagccacc agaauu 646581813RNAartificialmRNA sequence of
MAGE-C1(GC)-ag-A64-C30- histoneSL 58gggagaaagc uuaccaugca
guccccgcug cagggcgagg aguuccagag cucccugcag 60agccccgugu ccaucugcag
cuccagcacc cccuccagcc ucccgcagag cuuccccgag 120uccagccagu
ccccccccga gggcccgguc cagagccccc ugcacucccc gcagagcccc
180ccggagggga ugcacuccca gagcccccug cagucccccg agagcgcccc
cgagggcgag 240gacucccuca gcccgcugca gaucccccag uccccgcugg
agggggagga cagccucucc 300agccugcacu ucccccaguc cccgcccgag
ugggaggaca gccugagccc ccuccacuuc 360ccccaguucc cgccccaggg
cgaggacuuc caguccagcc ugcagucccc cgugagcauc 420ugcuccagcu
ccacgagccu gucccucccc cagagcuucc cggagucccc ccagagcccg
480cccgaggggc cggcgcaguc cccccugcag cgccccguga gcuccuucuu
cagcuacacc 540cuggccuccc uccugcagag cucccacgag agcccgcaga
gcccgcccga gggccccgcc 600caguccccgc ugcagagccc cgucuccagc
uuccccucca gcaccuccag cucccucagc 660caguccagcc ccguguccag
cuucccgucc agcaccucca gcucccugag caagagcucc 720cccgagagcc
cccugcaguc ccccgugauc agcuucucca gcuccacgag ccucuccccg
780uucagcgagg aguccagcuc ccccgucgac gaguacacca gcuccagcga
cacccugcug 840gaguccgaca gccucaccga cuccgagagc cugaucgaga
gcgagccccu guucaccuac 900acgcucgacg agaaggugga cgagcuggcc
cgguuccugc uccugaagua ccaggugaag 960cagcccauca ccaaggccga
gaugcugacc aacgucaucu cccgcuacac cggcuacuuc 1020ccggugaucu
uccggaaggc gcgcgaguuc aucgagaucc ucuucgggau cagccugcgg
1080gagguggacc ccgacgacuc cuacgucuuc gugaacacgc uggaccucac
cagcgagggc 1140ugccuguccg acgagcaggg gaugagccag aaccgccugc
ucauccugau ccuguccauc 1200aucuucauca agggcaccua cgccagcgag
gaggucaucu gggacgugcu cuccgggauc 1260ggcgugcggg ccggccgcga
gcacuucgcc uucggggagc cccgggagcu gcugaccaag 1320gucugggugc
aggagcacua ccucgaguac cgcgaggugc ccaacagcuc cccgccccgg
1380uacgaguucc uguggggccc ccgcgcccac agcgagguca ucaagcggaa
ggugguggag 1440uuccuggcga ugcucaagaa cacggucccc aucaccuucc
cguccagcua caaggacgcc 1500cugaaggacg uggaggagcg ggcccaggcc
aucaucgaca ccaccgacga cuccacggcc 1560accgagagcg cguccagcuc
cgugaugagc cccagcuucu ccagcgagug accacuaguu 1620auaagacuga
cuagcccgau gggccuccca acgggcccuc cuccccuccu ugcaccgaga
1680uuaauaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1740aaaaaaaaau gcaucccccc cccccccccc cccccccccc
cccccaaagg cucuuuucag 1800agccaccaga auu 1813
* * * * *