U.S. patent application number 12/787755 was filed with the patent office on 2010-09-23 for pharmaceutical composition containing a stabilised mrna optimised for translation in its coding regions.
This patent application is currently assigned to CUREVAC GMBH. Invention is credited to INGMAR HOERR, STEVE PASCOLO, FLORIAN VON DER MULBE.
Application Number | 20100239608 12/787755 |
Document ID | / |
Family ID | 7687266 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100239608 |
Kind Code |
A1 |
VON DER MULBE; FLORIAN ; et
al. |
September 23, 2010 |
PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED
FOR TRANSLATION IN ITS CODING REGIONS
Abstract
The present invention relates to a pharmaceutical composition
containing an mRNA that is stabilised by sequence modifications in
the translated region and is optimised for the translation. The
pharmaceutical composition according to the invention is
particularly suitable as an inoculating agent as well as a
therapeutic agent for tissue regeneration. In addition a process is
described for determining sequence modifications that serve for the
stabilisation and translation optimisation of mRNA.
Inventors: |
VON DER MULBE; FLORIAN;
(STUTTGART, DE) ; HOERR; INGMAR; (TUBINGEN,
DE) ; PASCOLO; STEVE; (ZURICH, CH) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
CUREVAC GMBH
TUBINGEN
DE
|
Family ID: |
7687266 |
Appl. No.: |
12/787755 |
Filed: |
May 26, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10729830 |
Dec 5, 2003 |
|
|
|
12787755 |
|
|
|
|
PCT/EP02/06180 |
Jun 5, 2002 |
|
|
|
10729830 |
|
|
|
|
Current U.S.
Class: |
424/207.1 ;
424/204.1; 424/209.1; 424/218.1; 424/225.1; 424/227.1; 424/228.1;
424/230.1; 424/231.1; 514/3.7; 514/44R; 536/23.5; 536/23.72 |
Current CPC
Class: |
A61K 39/001184 20180801;
A61K 39/001194 20180801; C07K 14/245 20130101; C12N 2740/16034
20130101; A61K 39/001189 20180801; A61P 37/04 20180101; A61K 39/12
20130101; A61P 31/22 20180101; C12N 7/00 20130101; G16B 30/00
20190201; A61K 38/1816 20130101; A61K 39/001188 20180801; A61K
47/542 20170801; A61P 31/18 20180101; A61K 38/19 20130101; A61K
39/001156 20180801; A61K 39/00117 20180801; C12N 2310/334 20130101;
C12N 2760/16022 20130101; A61K 48/0075 20130101; A61P 31/04
20180101; C12N 2760/14122 20130101; A61P 31/20 20180101; A61K
39/0011 20130101; A61K 48/0066 20130101; A61P 43/00 20180101; C12N
2740/16022 20130101; A61K 39/001192 20180801; C12N 15/67 20130101;
C12N 2760/16071 20130101; A61K 39/001153 20180801; A61P 31/16
20180101; A61P 35/04 20180101; A61K 48/00 20130101; A61K 39/0258
20130101; C07K 14/005 20130101; C12N 2760/14134 20130101; A61K
38/28 20130101; A61K 39/001186 20180801; C12N 15/11 20130101; C12N
2770/24122 20130101; A61K 39/00 20130101; G16B 20/00 20190201; A61P
25/28 20180101; A61P 11/00 20180101; C07K 14/4727 20130101; C07K
14/4748 20130101; A61K 39/001197 20180801; C12N 2760/16034
20130101; C12N 2310/336 20130101; A61K 39/145 20130101; A61K
38/1735 20130101; A61K 38/193 20130101; A61P 31/12 20180101; A61P
35/00 20180101; A61K 39/21 20130101; A61K 2039/53 20130101; A61K
48/005 20130101; A61K 48/0083 20130101; A61P 31/00 20180101; Y02A
50/30 20180101; A61K 47/6455 20170801; A61P 31/14 20180101; A61K
39/001106 20180801; A61K 39/001191 20180801; C12N 2770/24134
20130101; A61K 38/19 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/207.1 ;
536/23.72; 536/23.5; 514/44.R; 424/204.1; 514/12; 424/228.1;
424/225.1; 424/227.1; 424/231.1; 424/230.1; 424/218.1;
424/209.1 |
International
Class: |
A61K 39/21 20060101
A61K039/21; C07H 21/02 20060101 C07H021/02; A61K 31/7105 20060101
A61K031/7105; A61K 39/12 20060101 A61K039/12; A61K 38/19 20060101
A61K038/19; A61K 39/29 20060101 A61K039/29; A61K 39/245 20060101
A61K039/245; A61K 39/145 20060101 A61K039/145; A61P 31/18 20060101
A61P031/18; A61P 31/12 20060101 A61P031/12; A61P 37/04 20060101
A61P037/04; A61P 31/14 20060101 A61P031/14; A61P 31/16 20060101
A61P031/16; A61P 31/20 20060101 A61P031/20; A61P 31/22 20060101
A61P031/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2001 |
DE |
10127283.9 |
Claims
1. Modified mRNA coding for at least one viral peptide or
polypeptide, characterised in that the G/C content of the region of
the modified mRNA coding for the peptide or polypeptide is
increased compared to the G/C content of the coding region of the
wild type mRNA coding for the peptide or polypeptide, and the
encoded amino acid sequence is unchanged as compared to the wild
type.
2. Modified mRNA according to claim 1, characterised in that the
G/C content of the region of the modified mRNA coding for the
peptide or polypeptide is increased by at least 7% points,
preferably at least 15% points, compared to the G/C content of the
coding region of the wild type mRNA coding for the peptide or
polypeptide.
3. Modified mRNA according to claim 1, characterised in that the
modified mRNA comprises a 5' cap structure and/or a poly-A tail of
at least 70 nucleotides and/or an IRES and/or a 5' stabilisation
sequence and/or a 3' stabilisation sequence.
4. Modified mRNA according to claim 1, characterised in that the
modified mRNA comprises at least one analogue of naturally
occurring nucleotides.
5. Modified mRNA according to claim 4, characterised in that the
analogue is selected from the group consisting of phosphorus
thioates, phosphorus amidates, peptide nucleotides,
methylphosphonates, 7-deazaguanosine, 5-methylcytosine and
inosine.
6. Modified mRNA according to claim 1, characterised in that the
viral antigen derives from the secreted form of a surface
antigen.
7. Modified mRNA according to claim 1, characterised in that the
mRNA codes for a surface antigen of a pathogenic viral germ.
8. Modified mRNA according to claim 1, characterised in that the
mRNA is associated with a cationic peptide or protein or is bound
thereto.
9. Modified mRNA according to claim 8, characterised in that the
cationic peptide or protein is selected from the group consisting
of protamine, poly-L lysine, and histones.
10. Modified mRNA according to claim 1, characterised in that the
polypeptide is a polyepitope of viral antigens.
11. Modified mRNA according to claim 1, characterised in that the
modified mRNA is a multicistronic mRNA.
12. Modified mRNA according to claim 1, characterised in that the
mRNA in addition codes for at least one cytokine.
13. Modified mRNA according to claim 1, characterised in that the
multicistronic RNA comprises more than one IRES sequence, wherein
the IRES sequences are in particular selected from picorna viruses
(e.g. FMDV), plague viruses (CFFV), polio viruses (PV), encephalo
myocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV),
hepatitis C viruses (HCV), classic swine fever viruses (CSFV),
murine leukemia virus (MLV), simian immunodefiency viruses (SIV),
or cricket paralysis viruses (CrPV).
14. Pharmaceutical composition characterized in that it contains a
modified mRNA according to claim 1 in combination with a
pharmaceutically acceptable carrier and/or vehicle.
15. Pharmaceutical composition according to claim 14, characterised
in that the pharmaceutical composition contains at least one immune
response stimulating adjuvant.
16. Pharmaceutical composition according to claim 14, which in
addition contains at least one cytokine.
17. A vaccine for inoculation against viral infectious diseases
comprising a modified mRNA according to claim 1.
18. Use of a pharmaceutical composition according to claim 17 for
the preparation of a vaccine for inoculation against infections
caused by AIDS, hepatitis A, B or C, Herpes, Herpes zoster, Dengue,
haemorrhagic infection, Yellow fever and influenza.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a Continuation of application Ser. No.
10/729,830 filed on Dec. 5, 2003, which is a continuation-in-part
of PCT Application No. PCT/EP02/06180 filed Jun. 5, 2002, which in
turn, claims priority from German Application 10127283.9, filed
Jun. 5, 2001. Applicants claim benefit under 35 U.S.C. .sctn.120 as
to the U.S. application and the PCT application and under 35 U.S.C.
.sctn.119 to the German application, and the disclosures of all of
said applications are incorporated herein by reference.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Sequence_Listing.sub.--22122.sub.--00009_CON2. The size of the text
file is 19 KB, and the text file was created on May 19, 2010.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a pharmaceutical
composition containing an mRNA that is stabilised by sequence
modifications in the translated region and is optimised for
translation. The pharmaceutical composition according to the
invention is suitable in particular as an inoculating agent and
also as a therapeutic agent for tissue regeneration. Furthermore a
process for determining sequence modifications that stabilise and
optimise mRNA translation is disclosed.
[0005] 2. Description of the Prior Art
[0006] Gene therapy and genetic vaccination are tools of molecular
medicine whose use in the treatment and prevention of diseases has
considerable potential. Both of these approaches are based on the
incorporation of nucleic acids into a patient's cells or tissue as
well as on the subsequent processing of the information coded by
the incorporated nucleic acids, i.e. the expression of the desired
polypeptides.
[0007] The conventional procedure involved in previous processes of
gene therapy and genetic vaccination is the use of DNA in order to
incorporate the required genetic information into the cell. In this
connection various processes for the incorporation of DNA into
cells have been developed, such as for example calcium phosphate
transfection, polyprene transfection, protoplast fusion,
electroporation, microinjection and lipofection, in which
connection lipofection in particular has proved to be a suitable
process.
[0008] A further process that has been suggested in particular in
the case of genetic vaccination involves the use of DNA viruses as
DNA vehicles. Because such viruses are infectious, a very high
transfection rate can be achieved when using DNA viruses as
vehicles. The viruses used are genetically altered so that no
functional infectious particles are formed in the transfected cell.
Despite this precautionary measure, however the risk of
uncontrolled propagation of the introduced therapeutic gene as well
as viral genes remains due to the possibility of recombination
events.
[0009] Normally DNA incorporated into a cell is integrated to a
certain extent into the genome of the transfected cell. On the one
hand this phenomenon can exert a desirable effect, since in this
way a long-lasting action of the introduced DNA can be achieved. On
the other hand the integration into the genome brings with it a
significant risk for gene therapy. Such integration events may, for
example, involve an insertion of the incorporated DNA into an
intact gene, which produces a mutation that interferes with or
completely ablates the function of the endogenous gene. As a result
of such integration events, enzyme systems that are important for
cellular viability may be switched off. Alternatively, there is
also the risk of inducing transformation of the transfected cell if
the integration site occurs in a gene that is critical for
regulating cell growth. Accordingly, when using DNA viruses as
therapeutic agents and vaccines, a carcinogenic risk cannot be
excluded. In this connection it should also be borne in mind that,
in order to achieve effective expression of the genes incorporated
into the cell, the corresponding DNA vehicles contain a strong
promoter, for example the viral CMV promoter. The integration of
such promoters into the genome of the treated cell may, however,
lead to undesirable changes in the regulation of the gene
expression in the cell.
[0010] A further disadvantage of the use of DNA as a therapeutic
agent or vaccine is the induction of pathogenic anti-DNA antibodies
in the patient, resulting in a potentially fatal immune
response.
[0011] In contrast to DNA, the use of RNA as a therapeutic agent or
vaccine is regarded as significantly safer. In particular, use of
RNA is not associated with a risk of stable integration into the
genome of the transfected cell. In addition, no viral sequences
such as promoters are necessary for effective transcription of RNA.
Beyond this, RNA is degraded rapidly in vivo. Indeed, the
relatively short half-life of RNA in circulating blood, as compared
to that of DNA, reduces the risks associating with developing
pathogenic anti-RNA antibodies. Indeed, anti-RNA antibodies have
not been detected to date. For these reasons RNA may be regarded as
the molecule of choice for molecular medicine therapeutic
applications.
[0012] However, some basic problems still have to be solved before
medical applications based on RNA expression systems can be widely
employed. One of the problems in the use of RNA is the reliable,
cell-specific and tissue-specific efficient transfer of the nucleic
acid. Since RNA is normally found to be very unstable in solution,
up to now RNA could not be used or used only very inefficiently as
a therapeutic agent or inoculating agent in the conventional
applications designed for DNA use.
[0013] Enzymes that break down RNA, so-called RNases
(ribonucleases), are responsible in part for the instability. Even
minute contamination by ribonucleases is sufficient to degrade down
RNA completely in solution. Moreover, the natural decomposition of
mRNA in the cytoplasm of cells is exquisitely regulated. Several
mechanisms are known which contribute to this regulation. The
terminal structure of a functional mRNA, for example, is of
decisive importance. The so-called "cap structure" (a modified
guanosine nucleotide) is located at the 5' end and a sequence of up
to 200 adenosine nucleotides (the so-called poly-A tail) is located
at the 3' end. The RNA is recognised as mRNA by virtue of these
structures and these structures contribute to the regulatory
machinery controlling mRNA regulation. In addition there are
further mechanisms that stabilise or destabilise RNA. Many of these
mechanisms are still unknown, although often an interaction between
the RNA and proteins appears to be important in this regard. For
example, an mRNA surveillance system has been described (Hellerin
and Parker, Annu. Rev. Genet. 1999, 33: 229 to 260), in which
incomplete or nonsense mRNA is recognised by specific feedback
protein interactions in the cytosol and is made accessible to
decomposition. Exonucleases appear to contribute in large measure
to this process.
[0014] Certain measures have been proposed in the prior art in
order to improve the stability of RNA and thereby enable its use as
a therapeutic agent or RNA vaccine.
[0015] In EP-A-1083232 a process for the incorporation of RNA, in
particular mRNA, into cells and organisms has been proposed in
order to solve the aforementioned problem of the instability of RNA
ex vivo. As described therein, the RNA is present in the form of a
complex with a cationic peptide or protein.
[0016] WO 99/14346 describes further processes for stabilising
mRNA. In particular modifications of the mRNA are proposed that
stabilise the mRNA species against decomposition by RNases. Such
modifications may involve stabilisation by sequence modifications,
in particular reduction of the C content and/or U content by base
elimination or base substitution. Alternatively, chemical
modifications may be used, in particular the use of nucleotide
analogues, as well as 5' and 3' blocking groups, an increased
length of the poly-A tail as well as the complexing of the mRNA
with stabilising agents, and combinations of the aforementioned
measures.
[0017] In U.S. Pat. No. 5,580,859 and U.S. Pat. No. 6,214,804 mRNA
vaccines and mRNA therapeutic agents are disclosed inter alia
within the scope of "transient gene therapy" (TGT). Various
measures are described therein for enhancing the translation
efficiency and mRNA stability that relate in particular to the
composition of the non-translated sequence regions.
[0018] Bieler and Wagner (in: Schleef (Ed.), Plasmids for Therapy
and Vaccination, Chapter 9, pp. 147 to 168, Wiley-VCH, Weinheim,
2001) report on the use of synthetic genes in combination with gene
therapy methods employing DNA vaccines and lentiviral vectors. The
construction of a synthetic gag-gene derived from HIV-1 is
described, in which the codons have been modified with respect to
the wild type sequence (alternative codon usage) in such a way as
to correspond to frequently used codons found in highly expressed
mammalian genes. In this way, in particular, the A/T content
compared to the wild type sequence was reduced. Moreover, the
authors found an increased rate of expression of the synthetic gag
gene in transfected cells. Furthermore, increased antibody
formation against the gag protein was observed in mice immunised
with the synthetic DNA construct. An increase in cytokine release
in vitro in the case of transfected spleen cells of such mice was
also observed. Finally, an induction of a cytotoxic immune response
in mice immunised with the gag expression plasmid was also found.
The authors of this article attribute the improved properties of
their DNA vaccine to a change in the nucleocytoplasmic transport of
the mRNA expressed by the DNA vaccine, which was due to the
optimised codon usage. The authors maintain that the effect of the
altered codon usage on the translation efficiency was only
slight.
SUMMARY OF THE INVENTION
[0019] The object of the present invention is to provide a new
system for gene therapy and genetic vaccination that overcomes the
disadvantages associated with the properties of DNA therapeutic
agents and DNA vaccines and that increases the effectiveness of
therapeutic agents based on RNA species.
[0020] This object is achieved by the embodiments of the present
invention characterised in the claims.
[0021] In particular, a modified mRNA, as well as a pharmaceutical
composition containing at least one modified mRNA of the present
invention and a pharmaceutically compatible carrier and/or vehicle
are provided. The modified mRNA encodes at least one biologically
active or antigenic peptide or polypeptide, wherein the sequence of
the mRNA comprises at least one modification as set forth herein
below as compared to the wild type mRNA. Such modifications may be
located in the region coding for the at least one peptide or
polypeptide, or in untranslated regions.
[0022] In one aspect, the G/C content of the region of the modified
mRNA coding for the peptide or polypeptide is increased relative to
that of the G/C content of the coding region of the wild type mRNA
coding for the peptide or polypeptide. The encoded amino acid
sequence, however, remains unchanged compared to the wild type
(i.e. silent with respect to the encoded amino acid sequence).
[0023] This modification is based on the fact that, for efficient
translation of an mRNA, the sequence of the region of the mRNA to
be translated is essential. In this connection the composition and
the sequence of the various nucleotides play an important role. In
particular sequences with an increased G (guanosine)/C (cytosine)
content are more stable than sequences with an increased A
(adenosine)/U (uracil) content. In accordance with the invention,
the codons are varied compared to the wild type mRNA, while
maintaining the translated amino acid sequence, so that they
contain increased amounts of G/C nucleotides. Since several
different codons can encode the same amino acid, due to degeneracy
of the genetic code, the codons most favourable for the stability
of the modified mRNA can be determined and incorporated
(alternative codon usage).
[0024] Depending on the amino acid to be coded by the modified
mRNA, various possibilities for modifying the mRNA sequence
compared to the wild type sequence are feasible. In the case of
amino acids that are encoded by codons that 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) do not require any alteration since no A or U
is present.
[0025] In the following cases the codons that contain A and/or U
nucleotides are altered by substituting other codons that code for
the same amino acids, but do not contain A and/or U. Examples
include: the codons for Pro, which may be changed from CCU or CCA
to CCC or CCG; the codons for Arg, which may be changed from CGU or
CGA or AGA or AGG to CGC or CGG; the codons for Ala, which may be
changed from GCU or GCA to GCC or GCG; the codons for Gly, which
may be changed from GGU or GGA to GGC or GGG.
[0026] In other cases, although A and/or U nucleotides may not be
eliminated from the codons, it is however possible to reduce the A
and U content by using codons that contain fewer A and/or U
nucleotides. For example: the codons for Phe, which may be changed
from UUU to UUC; the codons for Leu may be changed from UUA, CUU or
CUA to CUC or CUG; the codons for Ser, which may be changed from
UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr, which may
be changed from UAU to UAC; the stop codon UAA, which may be
changed to UAG or UGA; the codon for Cys, which may be changed from
UGU to UGC; the codon for His, which may be changed from CAU to
CAC; the codon for Gln, which may be changed from CAA to CAG; the
codons for Ile, which may be changed from AUU or AUA to AUC; the
codons for Thr, which may be changed from ACU or ACA to ACC or ACG;
the codon for Asn may be changed from AAU to AAC; the codon for
Lys, which may be changed from AAA to AAG; the codons for Val,
which may be changed from GUU or GUA to GUC or GUG; the codon for
Asp, which may be changed from GAU to GAC; the codon for Glu, which
may be changed from GAA to GAG.
[0027] In the case of the codons for Met (AUG) and Trp (UGG) there
is however no possibility of modifying the sequence.
[0028] The substitutions listed above may be used individually and
in all possible combinations in order to increase the G/C content
of a modified mRNA compared to the original sequence. Thus for
example all codons for Thr occurring in the original (wild type)
sequence can be altered to ACC (or ACG). Preferably, however,
combinations of the substitution possibilities given above are
employed, for example: substitution of all codons coding in the
original sequence for Thr to ACC (or ACG) and substitution of all
codons coding for Ser to UCC (or UCG or AGC); substitution of all
codons coding in the original sequence for Ile to AUC and
substitution of all codons coding for Lys to AAG and substitution
of all codons coding originally for Tyr to UAC; substitution of all
codons coding in the original sequence for Val to GUC (or GUG) and
substitution of all codons coding for Glu to GAG and substitution
of all codons coding for Ala to GCC (or GCG) and substitution of
all codons coding for Arg to CGC (or CGG); substitution of all
codons coding in the original sequence for Val to GUC (or GUG) and
substitution of all codons coding for Glu to GAG and substitution
of all codons coding for Ala to GCC (or GCG) and substitution of
all codons coding for Gly to GGC (or GGG) and substitution of all
codons coding for Asn to AAC; substitution of all codons coding in
the original sequence for Val to GUC (or GUG) and substitution of
all codons coding for Phe to UUC and substitution of all codons for
Cys to UGC and substitution of all codons coding for Leu to CUG (or
CUC) and substitution of all codons coding for Gln to CAG and
substitution of all codons encoding Pro to CCC (or CCG); etc.
[0029] Preferably the G/C content of the region of the modified
mRNA coding for the peptide or polypeptide is increased by at least
7%, more preferably by at least 15%, and particularly preferably by
at least 20% compared to the G/C content of the coded region of the
wild type mRNA encoding for the polypeptide.
[0030] In this connection it is particularly preferred to maximize
the G/C content of the modified mRNA as compared to that of the
wild type sequence. For some applications, it may be particularly
advantageous to maximise the G/C content of the modified mRNA in
the region encoding the at least one peptide or polypeptide.
[0031] In accordance with the invention, a further modification of
the mRNA comprised in the pharmaceutical composition of the present
invention is based on an understanding that the translation
efficiency is also affected by the relative abundance of different
tRNAs in various cells. A high frequency of so-called "rare" codons
in an RNA sequence, which are recognized by relatively rare tRNAs,
tends to decrease the translational efficiency of the corresponding
mRNA, whereas a high frequency of codons recognized by relatively
abundant rRNAs tends to enhance the translational efficiency of a
corresponding mRNA.
[0032] Thus, according to the invention, the modified mRNA (which
is contained in the pharmaceutical composition) comprises a region
coding for the peptide or polypeptide which is changed compared to
the corresponding region of the wild type mRNA so as to replace at
least one codon of the wild type sequence that is recognized by a
rare cellular tRNA with a codon recognized by an abundant cellular
tRNA, wherein the abundant and rare cellular tRNAs recognize the
same amino acid. In other words, the substituted codon in the
modified mRNA, which is recognized by a relatively frequent tRNA,
encodes the same amino acid as the wild type (unmodified)
codon.
[0033] Through such modifications, the RNA sequences are modified
so that codons are inserted/substituted that are recognized by
abundantly expressed cellular tRNAs. Modifications directed to
altering codon usage in a nucleic acid sequence to optimise
expression levels of polypeptides encoded therefrom are generally
referred to in the art as "codon optimisation."
[0034] Those tRNAs which are abundant or rare in a particular cell
are known to a person skilled in the art; see for example Akashi,
Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Each organism has a
preferred choice of nucleotide or codon usage to encode any
particular amino acid. Different species vary in their codon
preferences for translating mRNA into protein. The codon
preferences of a particular species in which a modified mRNA of the
present invention is to be expressed will, therefore, at least in
part dictate the parameters of codon optimisation for a nucleic
acid sequence.
[0035] By means of this modification, according to the invention
all codons of the wild type sequence that are recognized by a
relatively rare tRNA in a cell may in each case be replaced by a
codon that is recognized by a relatively abundant tRNA. As
described herein, however, the coding sequence of the peptide or
polypeptide is preserved. That is, a relatively abundant tRNA
species, which replaces a relatively rare tRNA species in a
modified mRNA of the invention, recognizes an amino acid identical
to that recognized by the rare tRNA species.
[0036] According to the invention, it is particularly preferred to
couple the sequential increase in the G/C fraction of a modified
mRNA (particularly, for example, a maximally modified G/C content),
with an increase in the number of codons recognized by abundant
tRNAs, wherein the amino acid sequence of the peptide or
polypeptide (one or more) encoded by the mRNA remains unaltered.
This preferred embodiment provides a particularly preferred mRNA
species, possessing properties of efficient translation and
improved stability. Such preferred mRNA species are well suited,
for example, for the pharmaceutical compositions of the present
invention.
[0037] Sequences of eukaryotic mRNAs frequently include
destabilising sequence elements (DSE) to which signal proteins can
bind and thereby regulate the enzymatic degradation of the mRNA in
vivo. Accordingly, for the further stabilisation of a modified mRNA
of the invention, which may be a component of a pharmaceutical
composition of the invention, one or more changes may be made in
the wild type mRNA sequence encoding the at least one peptide or
polypeptide, so as to reduce the number of destabilising sequence
elements present. In accordance with the invention, DSEs located
anywhere in an mRNA, including the coding region and in the
non-translated regions (3' and/or 5' UTR), may be mutated or
changed to generate a modified mRNA having improved properties.
[0038] Such destabilising sequences are for example AU-rich
sequences ("AURES") that occur in 3'-UTR regions of a number of
unstable mRNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83:
1670-1674). The RNA molecules contained in the pharmaceutical
composition according to the invention are therefore preferably
altered as compared to the wild type mRNA so as to reduce the
number of or eliminate these destabilising sequences. Such an
approach also applies to those sequence motifs recognised by
potential endonucleases. Such sequences include, for example,
GAACAAG, which is found in the 3'UTR of the gene encoding the
transferring receptor (Binder et al., EMBO J. 1994, 13: 1969-1980).
Sequence motifs recognized by endonucleases are also preferably
reduced in number or eliminated in the modified mRNA of the
pharmaceutical composition according to the invention.
[0039] Various methods are known to the person skilled in the art
that are suitable for the substitution of codons in the modified
mRNA according to the invention. In the case of relatively short
coding regions (that code for biologically active or antigenic
peptides), the whole mRNA may, for example, be chemically
synthesised using standard techniques.
[0040] Preferably, however, base substitutions are introduced using
a DNA matrix for the production of modified mRNA with the aid of
techniques routinely employed in targeted mutagenesis; see Maniatis
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 3.sup.rd Edition, Cold Spring Harbor, N.Y.,
2001.
[0041] In this method, a corresponding DNA molecule is therefore
transcribed in vitro for the production of the mRNA. This DNA
matrix has a suitable promoter, for example a T7 or SP6 promoter,
for in vitro transcription, followed by the desired nucleotide
sequence for the mRNA to be produced and a termination signal for
the in vitro transcription. According to the invention the DNA
molecule that forms the matrix of the RNA construct to be produced
is prepared as part of a plasmid replicable in bacteria, wherein
the plasmid is replicated or amplified during the course of
bacterial replication and subsequently isolated by standard
techniques. Plasmids suitable for use in the present invention
include, but are not limited to pT7Ts (GenBank Accession No.
U26404; Lai et al., Development 1995, 121: 2349-2360), the
pGEM.RTM. series, for example pGEM.RTM.-1 (GenBank Accession No.
X65300; from Promega) and pSP64 (GenBank-Accession No. X65327); see
also Mezei and Storts, Purification of PCR Products, in: Griffin
and Griffin (Eds.), PCR Technology: Current Innovation, CRC Press,
Boca Raton, Fla., 2001.
[0042] Thus, by using short synthetic DNA oligonucleotides that
comprise short single-strand transitions at the corresponding
cleavage sites, or by means of genes produced by chemical
synthesis, the desired nucleotide sequence can be cloned into a
suitable plasmid by molecular biology methods known to the person
skilled in the art (see Maniatis et al., above). The DNA molecule
is then excised from the plasmid, in which it may be present as a
single copy or multiple copies, by digestion with restriction
endonucleases.
[0043] The modified mRNA that is contained in the pharmaceutical
composition according to the invention may furthermore have a 5'
cap structure (a modified guanosine nucleotide). Examples of
suitable cap structures include, but are not limited to m7G(5')ppp
(5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
[0044] According to a further preferred embodiment of the present
invention the modified mRNA comprises a poly-A tail of at least 50
nucleotides, preferably at least 70 nucleotides, more preferably at
least 100 nucleotides and particularly preferably at least 200
nucleotides.
[0045] For efficient translation of the mRNA a productive binding
of the ribosomes to the ribosome binding site [Kozak sequence:
GCCGCCACCAUGG (SEQ ID NO: 13), the AUG forms the start codon] is
generally required. In this regard it has been established that an
increased A/U content around this site facilitates more efficient
ribosome binding to the mRNA.
[0046] In addition, it is possible to introduce one or more
so-called IRES ("internal ribosomal entry site") into the modified
mRNA. An IRES may act as the sole ribosome binding site, or may
serve as one of the ribosome binding sites of an mRNA. An mRNA
comprising more than one functional ribosome binding site may
encode several peptides or polypeptides that are translated
independently by the ribosomes ("multicistronic mRNA"). Examples of
IRES sequences that can be used according to the invention include
without limitation, those from picornaviruses (e.g. FMDV), pest
viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses
(ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses
(HCV), classical swine fever viruses (CSFV), murine leukemia virus
(MLV), simian immune deficiency viruses (SIV) or cricket paralysis
viruses (CrPV).
[0047] According to a further preferred embodiment of the present
invention the modified mRNA comprises in the 5' non-translated
and/or 3' non-translated regions stabilisation sequences that are
capable of increasing the half-life of the mRNA in the cytosol.
[0048] These stabilisation sequences may exhibit 100% sequence
homology with naturally occurring sequences that are present in
viruses, bacteria and eukaryotic cells, or may be derived from such
naturally occurring sequences (i.e., may comprise, e.g., mutations
substitutions, or deletions in these sequences). Stabilising
sequences that may be used in the present invention include, by way
of non-limiting example, the untranslated sequences (UTR) of the
.beta.-globin gene of Homo sapiens or Xenopus laevis. Another
example of a stabilisation sequence has the general formula
(C/U)CCAN.sub.xCCC(U/A)Py.sub.xUC(C/U)CC, which is contained in the
3'UTR of the very stable mRNAs that encode .alpha.-globin,
.alpha.-(I)-collagen, 15-lipoxygenase, or tyrosine hydroxylase (C.
F. Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410-2414).
Obviously such stabilisation sequences may be used individually or
in combination, as well as in combination with other stabilisation
sequences known to a person skilled in the art.
[0049] For the further stabilisation of the modified mRNA it is
preferred that the modified mRNA comprises at least one analogue of
a naturally occurring nucleotide. This approach is based on the
understanding that RNA-decomposing enzymes present in a cell
preferentially recognise RNA comprising naturally occurring
nucleotides as a substrate. The insertion of nucleotide analogues
into an RNA molecule, therefore, retards decomposition of the RNA
molecule so modified, whereas the effect of such analogs on
translational efficiency, particularly when inserted into the
coding region of the mRNA, may result in either an increase or
decrease in translation of the modified RNA molecule.
[0050] The following is a non-limiting list of nucleotide analogues
that can be used in accordance with the invention: phosphorus
amidates, phosphorus thioates, peptide nucleotides,
methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.
The preparation of such analogues is known to the person skilled in
the art, for example from U.S. Pat. No. 4,373,071, U.S. Pat. No.
4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S.
Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No.
4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S.
Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642.
According to the invention such analogues may be present in
non-translated and/or translated regions of the modified mRNA.
[0051] Furthermore the effective transfer of the modified mRNA into
the cells to be treated or into the organism to be treated may be
improved if the modified mRNA is associated with a cationic peptide
or protein, or is bound thereto. In particular in this connection
the use of protamine as polycationic, nucleic acid-binding protein
is particularly effective. It is also possible to use other
cationic peptides or proteins such as poly-L-lysine or histones.
Procedures for stabilising mRNA are described in EP-A-1083232,
whose relevant disclosure is incorporated herein in its
entirety.
[0052] For gene therapy applications, for example, wherein a
pharmaceutical composition of the invention is used, the modified
mRNA therein codes for at least one biologically active peptide or
polypeptide that is not formed or is only insufficiently or
defectively formed in the patient to be treated. Administration of
a modified mRNA encoding the at least one biologically active
peptide or polypeptide or a composition thereof to such a patient,
therefore, at least partially restores the expression and/or
activity of the at least one biologically active peptide or
polypeptide in the patient and thereby complements the patient's
genetic defect. The direct introduction of a normal, functional
gene into a living animal has been studied as a means for replacing
defective genetic information. In such studies, nucleic acid
sequences are introduced directly into cells of a living animal.
The following references pertain to methods for the direct
introduction of nucleic acid sequences into a living animal: Nabel
et al., (1990) Science 249:1285-1288; Wolfe et al., (1990) Science
247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Wolfe et
al. (1991) BioTechniques 11(4):474-485; and Felgner and Rhodes,
(1991) Nature 349:351-352, which are incorporated herein by
reference.
[0053] Accordingly, examples of polypeptides coded by a modified
mRNA of the invention include, without limitation, dystrophin, the
chloride channel, which is defectively altered in cystic fibrosis;
enzymes that are lacking or defective in metabolic disorders such
as phenylketonuria, galactosaemia, homocystinuria, adenosine
deaminase deficiency, etc.; enzymes that are involved in the
synthesis of neurotransmitters such as dopamine, norepinephrine and
GABA, in particular tyrosine hydroxylase and DOPA decarboxylase,
and .alpha.-1-antitrypsin, etc. Pharmaceutical compositions of the
invention may also be used to effect expression of cell surface
receptors and/or binding partners of cell surface receptors if the
modified mRNA contained therein encodes for such biologically
active proteins or peptides. Examples of such proteins that act in
an extracellular manner or that bind to cell surface receptors
include for example tissue plasminogen activator (TPA), growth
hormones, insulin, interferons, granulocyte-macrophage colony
stimulating factor (GM-CFS), and erythropoietin (EPO), etc. By
choosing suitable growth factors, the pharmaceutical composition of
the present invention may, for example, be used for tissue
regeneration. In this way diseases that are characterised by tissue
degeneration, for example neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, etc. and other
degenerative conditions, such as arthrosis, can be treated. In
these cases the modified mRNA, in particular that contained in the
pharmaceutical composition of the present invention, preferably
encodes, without limitation, a TGF-.beta. family member, EGF, FGF,
PDGF, BMP, GDNF, BDNF, GDF and neurotrophic factors such as NGF,
neutrophines, etc.
[0054] A further area of application of the present invention is
vaccination, i.e. the use of a modified mRNA for inoculation or the
use of a pharmaceutical composition comprising a modified mRNA as
an inoculating agent, or the use of a modified mRNA in the
preparation of the pharmaceutical composition for inoculation
purposes. Vaccination is based on introducing an antigen into an
organism or subject, in particular into a cell of the organism or
subject. In the context of the present invention, the genetic
information encoding the antigen is introduced into the organism or
subject in the form of a modified mRNA encoding the antigen. The
modified mRNA contained in the pharmaceutical composition is
translated into the antigen, i.e. the polypeptide or antigenic
peptide coded by the modified mRNA is expressed, and an immune
response directed against the polypeptide or antigenic peptide is
stimulated. For vaccination against a pathogenic organism, e.g., a
virus, a bacterium, or a protozoan, a surface antigen of such an
organism may be used as an antigen against which an immune response
is elicited. In the context of the present invention, a
pharmaceutical composition comprising a modified mRNA encoding such
a surface antigen may be used as a vaccine. In applications wherein
a genetic vaccine is used for treating cancer, the immune response
is directed against tumour antigens by generating a modified mRNA
encoding a tumour antigen(s), in particular a protein which is
expressed exclusively on cancer cells. Such a modified mRNA
encoding a tumour antigen may be used alone or as a component of a
pharmaceutical composition according to the invention, wherein
administration of either the modified mRNA or a composition thereof
results in expression of the cancer antigen(s) in the organism. An
immune response to such a vaccine would, therefore, confer to the
vaccinated subject a degree of protective immunity against cancers
associated with the immunizing cancer antigen. Alternatively, such
measures could be used to vaccinate a cancer patient with a
modified mRNA encoding a tumour antigen(s) expressed on the
patient's cancer cells so as to stimulate the cancer patient's
immune response to attack any cancer cells expressing the encoded
antigen.
[0055] In its use as a vaccine the pharmaceutical composition
according to the invention is suitable in particular for the
treatment of cancers (in which the modified mRNA codes for a
tumour-specific surface antigen (TSSA), for example for treating
malignant melanoma, colon carcinoma, lymphomas, sarcomas,
small-cell lung carcinomas, blastomas, etc. A non-limiting list of
specific examples of tumour antigens include, inter cilia, 707-AP,
AFP, ART-4, BAGE, .beta.-catenin/m, Bcr-abl, CAMEL, CAP-1, CASP-8,
CDC27/m, CDK4/m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE,
GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R1701, HPV-E7, HSP70-2M,
HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR/FUT, MAGE,
MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A,
NY-ESO-1, p190 minor bcr-abl, Pml/RAR.alpha., PRAMS, PSA, PSM,
RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, TEUAML1, TPI/m, TRP-1,
TRP-2, TRP-2/INT2 and WT1. In addition to the above application,
the pharmaceutical composition of the invention may be used to
treat infectious diseases, for example, viral infectious diseases
such as AIDS (HIV), hepatitis A, B or C, herpes, herpes zoster
(chicken pox), German measles (rubella virus), yellow fever, dengue
fever etc. (flavi viruses), flu (influenza viruses), haemorrhagic
infectious diseases (Marburg or Ebola viruses), bacterial
infectious diseases such as Legionnaires' disease (Legionella),
gastric ulcer (Helicobacter), cholera (Vibrio), E. coli infections,
staphylococcal infections, salmonella infections or streptococcal
infections, tetanus (Clostridium tetani), or protozoan infectious
diseases (malaria, sleeping sickness, leishmaniasis, toxoplasmosis,
i.e. infections caused by plasmodium, trypanosomes, leishmania and
toxoplasma). Preferably also in the case of infectious diseases the
corresponding surface antigens with the strongest antigenic
potential are encoded by the modified mRNA. With the aforementioned
genes of pathogenic vectors or organisms, in particular in the case
of viral genes, this is typically a secreted form of a surface
antigen. Moreover, according to the invention mRNAs preferably
coding for polypeptides are employed, because polypeptides
generally comprise multiple epitopes (polyepitopes). Polypeptides
comprising polyepitopes include but are not limited to, surface
antigens of pathogenic vectors or organisms, or of tumour cells,
preferably secreted protein forms.
[0056] Moreover, the modified mRNA according to the invention may
comprise in addition to the antigenic or therapeutically active
peptide or polypeptide, at least one further functional region that
encodes, for example, a cytokine that promotes the immune response
(e.g., a monokine, lymphokine, interleukin or chemokine, such as
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,
INF-.alpha., INF-.gamma., GM-CFS, LT-.alpha. or growth factors such
as hGH).
[0057] Furthermore, in order to increase immunogenicity, the
pharmaceutical composition according to the invention may contain
one or more adjuvants. The term "adjuvant" is understood in this
context to denote any chemical or biological compound that promotes
or augments a specific immune response. Various mechanisms may be
involved in this connection, depending on the various types of
adjuvants. For example, compounds that promote endocytosis of the
modified mRNA contained in the pharmaceutical composition by
dentritic cells (DC) form a first class of usable adjuvants. Other
compounds that activate or accelerate maturation of DC (for
example, lipopolysaccharides, TNF-.alpha. or CD40 ligand) comprise
a second class of suitable adjuvants. In general, any agent which
is recognized as a potential "danger signal" by the immune system
(LPS, GP96, oligonucleotides with the CpG motif) or cytokines such
as GM-CSF, may be used as an adjuvant. Co-administration of an
adjuvant enhances an immune response generated against an antigen
encoded by the modified mRNA. The aforementioned cytokines are
particularly preferred in this aspect. Other known adjuvants
include aluminium hydroxide, and Freund's adjuvant, as well as the
aforementioned stabilising cationic peptides or polypeptides such
as protamine. In addition, lipopeptides such as Pam3Cys are also
particularly suitable for use as adjuvants in the pharmaceutical
composition of the present invention; see Deres et al, Nature 1989,
342: 561-564.
[0058] The pharmaceutical composition according to the invention
comprises, in addition to the modified mRNA, a pharmaceutically
compatible carrier and/or a pharmaceutically compatible vehicle.
Appropriate methods for achieving a suitable formulation and
preparation of the pharmaceutical composition according to the
invention are described in "Remington's Pharmaceutical Sciences"
(Mack Pub. Co., Easton, Pa., 1980), which is herein incorporated by
reference in its entirety. For parenteral administration suitable
carriers include for example sterile water, sterile saline
solutions, polyalkylene glycols, hydrogenated naphthalene and in
particular biocompatible lactide polymers, lactide/glycolide
copolymers or polyoxyethylene/polyoxypropylen-e copolymers.
Compositions according to the invention may contain fillers or
substances such as lactose, mannitol, substances for the covalent
coupling of polymers such as for example polyethylene glycol to
inhibitors according to the invention, complexing with metal ions
or incorporation of materials in or on special preparations of
polymer compound, such as for example polylactate, polyglycolic
acid, hydrogel or on liposomes, microemulsions, microcells,
unilamellar or multilamellar vesicles, erythrocyte fragments or
spheroplasts. The respective modifications of the compositions are
chosen depending on physical properties such as, for example,
solubility, stability, bioavailability or degradability. Controlled
or constant release of the active component according to the
invention in the composition includes formulations based on
lipophilic depot substances (for example fatty acids, waxes or
oils). Coatings of substances or compositions according to the
invention containing such substances, namely coatings with polymers
(for example poloxamers or poloxamines), are also disclosed within
the scope of the present invention. Moreover substances or
compositions according to the invention may contain protective
coatings, for example protease inhibitors or permeability
enhancers. Preferred carriers are typically aqueous carrier
materials, in which water for injection (WFI) or water buffered
with phosphate, citrate or acetate, etc., is used, and the pH is
typically adjusted to 5.0 to 8.0, preferably 6.0 to 7.0. The
carrier or the vehicle will in addition preferably contain salt
constituents, for example sodium chloride, potassium chloride or
other components that for example make the solution isotonic. In
addition the carrier or the vehicle may contain, besides the
aforementioned constituents, additional components such as human
serum albumin (HSA), polysorbate 80, sugars or amino acids.
[0059] The concentration of the modified mRNA in such formulations
may therefore vary within a wide range from 1 .mu.g to 100 mg/ml.
The pharmaceutical composition according to the invention is
preferably administered parenterally, for example intravenously,
intraarterially, subcutaneously or intramuscularly to the patient.
It is also possible to administer the pharmaceutical composition
topically or orally.
[0060] The invention thus also provides a method for the treatment
of the aforementioned medical conditions or an inoculation method
for the prevention of the aforementioned conditions, which
comprises the administration of the pharmaceutical composition
according to the invention to a subject or patient, in particular a
human patient.
[0061] A typical regimen for preventing, suppressing, or treating a
pathology related to a viral, bacterial, or protozoan infection,
may comprise administration of an effective amount of a vaccine
composition as described herein, administered as a single
treatment, or repeated as enhancing or booster dosages, over a
period up to and including between one week and about 24 months, or
any range or value therein.
[0062] According to the present invention, an "effective amount" of
a vaccine composition is one that is sufficient to achieve a
desired biological effect. It is understood that nature and manner
of the administration and the effective dosage may be determined by
a medical practitioner based on a number of variables including the
age, sex, health, and weight of the recipient, the medical
condition to be treated and its stage of progression, the kind of
concurrent treatment, if any, frequency of treatment, and the
nature of the desired outcome. The ranges of effective doses
provided below are not intended to limit the invention, but are
provided as representative preferred dose ranges. However, the most
preferred dosage will be tailored to the individual subject, as is
understood and determinable by one of skill in the art, without
undue experimentation. See, e.g., Berkow et al., eds., The Merck
Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman et
al., eds., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y.,
(1990); Avery's Drug Treatment: Principles and Practice of Clinical
Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD.,
Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology,
Little, Brown and Co., Boston, Mass. (1985); and Katzung, ed. Basic
and Clinical Pharmacology, Fifth Edition, Appleton and Lange,
Norwalk, Conn. (1992), which references and references cited
therein, are entirely incorporated herein by reference.
[0063] The present invention relates to the use of genetic material
(e.g., nucleic acid sequences) as immunizing agents. In one aspect,
the present invention relates to the introduction of exogenous or
foreign modified DNA or RNA molecules into an individual's tissues
or cells, wherein these molecules encode an exogenous protein
capable of eliciting an immune response to the protein. The
exogenous nucleic acid sequences may be introduced alone or in the
context of an expression vector wherein the sequences are operably
linked to promoters and/or enhancers capable of regulating the
expression of the encoded proteins. The introduction of exogenous
nucleic acid sequences may be performed in the presence of a cell
stimulating agent capable of enhancing the uptake or incorporation
of the nucleic acid sequences into a cell. Such exogenous nucleic
acid sequences may be administered in a composition comprising a
biologically compatible or pharmaceutically acceptable carrier. The
exogenous nucleic acid sequences may be administered by a variety
of means, as described herein, and well known in the art.
[0064] Such methods may be used to elicit immunity to a pathogen,
absent the risk of infecting an individual with the pathogen. The
present invention may be practiced using procedures known in the
art, such as those described in PCT International Application
Number PCT/US90/01515, wherein methods for immunizing an individual
against pathogen infection by directly injecting polynucleotides
into the individual's cells in a single step procedure are
presented.
[0065] In one aspect, the present invention relates to methods for
eliciting immune responses in an individual or subject which can
protect the individual from pathogen infection. Accordingly,
genetic material that encodes an immunogenic protein is introduced
into a subject's cells either in vivo or ex vivo. The genetic
material is expressed by these cells, thereby producing immunogenic
target proteins capable of eliciting an immune response. The
resulting immune response is broad based and involves activation of
the humoral immune response and both arms of the cellular immune
response.
[0066] This approach is useful for eliciting a broad range of
immune responses against a target protein. Target proteins may be
proteins specifically associated with pathogens or the individual's
own "abnormal" or infected cells. Such an approach may be used
advantageously to immunize a subject against pathogenic agents and
organisms such that an immune response against a pathogen protein
provides protective immunity against the pathogen. This approach is
particularly useful for protecting an individual against infection
by non-encapsulated intracellular pathogens, such as a virus, which
produce proteins within the host cells. The immune response
generated against such proteins is capable of eliminating infected
cells with cytotoxic T cells (CTLs).
[0067] The immune response elicited by a target protein produced by
vaccinated cells in a subject is a broad-based immune response
which includes B cell and T cell responses, including CTL
responses. It has been observed that target antigen produced within
the cells of the host are processed intracellularly into small
peptides, which are bound by Class I MHC molecules and presented in
the context of Class I on the cell surface. The Class I MHC-target
antigen complexes are capable of stimulating CD8.sup.+T cells,
which are predominantly CTLs. Notably, genetic immunization
according to the present invention is capable of eliciting CTL
responses (killer cell responses).
[0068] The CTL response is crucial in protection against pathogens
such as viruses and other intracellular pathogens which produce
proteins within infected cells. Similarly, the CTL response can be
utilized for the specific elimination of deleterious cell types,
which may express aberrant cell surface proteins recognizable by
Class I MHC molecules.
[0069] The genetic vaccines of the present invention may be
administered to cells in conjunction with compounds that stimulate
cell division and facilitate uptake of genetic constructs. This
step provides an improved method of direct uptake of genetic
material. Administration of cell stimulating compounds results in a
more effective immune response against the target protein encoded
by the genetic construct.
[0070] According to the present invention, modified DNA or mRNA
that encodes a target protein is introduced into the cells of an
individual where it is expressed, thus producing the target
protein. The modified DNA or RNA may be operably linked to
regulatory elements (e.g., a promoter) necessary for expression in
the cells of the individual. Other elements known to skilled
artisans may also be included in genetic constructs of the
invention, depending on the application.
[0071] As used herein, the term "genetic construct" refers to the
modified DNA or mRNA molecule that comprises a nucleotide sequence
which encodes the target protein and which may include initiation
and termination signals operably linked to regulatory elements
including a promoter and polyadenylation signal (for modified DNA)
capable of directing expression in the cells of the vaccinated
individual. As used herein, the term "expressible form" refers to
gene constructs which contain the necessary regulatory elements
operably linked to a coding sequence of a target protein, such that
when present in the cell of the individual, the coding sequence is
expressed. As used herein, the term "genetic vaccine" refers to a
pharmaceutical preparation that comprises a genetic construct.
[0072] The present invention provides genetic vaccines, which
include genetic constructs comprising DNA or RNA which encode a
target protein. As used herein, the term "target protein" refers to
a protein capable of eliciting an immune response. The target
protein is an immunogenic protein derived from the pathogen or
undesirable cell-type, such as an infected or transformed cell. In
accordance with the invention, target proteins may be
pathogen-associated proteins or tumour-associated proteins. The
immune response directed against the target protein protects the
individual against the specific infection or disease with which the
target protein is associated. For example, a genetic vaccine
comprising a modified DNA or RNA molecule that encodes a
pathogen-associated target protein is used to elicit an immune
response that will protect the individual from infection by the
pathogen.
[0073] DNA and RNA-based vaccines and methods of use are described
in detail in several publications, including Leitner et al. (1999,
Vaccines 18:765-777), Nagashunmugam et al. (1997, AIDS 11:
1433-1444), and Fleeton et al. (2001, J Infect Dis 183:1395-1398)
the entire contents of each of which is incorporated herein by
reference.
[0074] In order to test expression, genetic constructs can be
tested for expression levels in vitro using cells maintained in
culture, which are of the same type as those to be vaccinated. For
example, if the genetic vaccine is to be administered into human
muscle cells, muscle cells grown in culture such as solid muscle
tumor cells of rhabdomyosarcoma may be used as an in vitro model
for measuring expression levels. One of ordinary skill in the art
could readily identify a model in vitro system which may be used to
measure expression levels of an encoded target protein.
[0075] In accordance with the invention, multiple inoculants can be
delivered to different cells, cell types, or tissues in an
individual. Such inoculants may comprise the same or different
nucleic acid sequences of a pathogenic organism. This allows for
the introduction of more than a single antigen target and maximizes
the chances for developing immunity to the pathogen in a vaccinated
subject.
[0076] According to the invention, the genetic vaccine may be
introduced in vivo into cells of an individual to be immunized or
ex vivo into cells of the individual which are re-implanted after
incorporation of the genetic vaccine. Either route may be used to
introduce genetic material into cells of an individual. As
described herein above, preferred routes of administration include
intramuscular, intraperitoneal, intradermal, and subcutaneous
injection. Alternatively, the genetic vaccine may be introduced by
various means into cells isolated from an individual. Such means
include, for example, transfection, electroporation, and
microprojectile bombardment. These methods and other protocols for
introducing nucleic acid sequences into cells are known to and
routinely practiced by skilled practitioners. After the genetic
construct is incorporated into the cells, they are re-implanted
into the individual. Prior to re-implantation, the expression
levels of a target protein encoded by the genetic vaccine may be
assessed. It is contemplated that otherwise non-immunogenic cells
that have genetic constructs incorporated therein can be implanted
into autologous or heterologous recipients.
[0077] The genetic vaccines according to the present invention
comprise about 0.1 to about 1000 micrograms of nucleic acid
sequences (i.e., DNA or RNA). In some preferred embodiments, the
vaccines comprise about 1 to about 500 micrograms of nucleic acid
sequences. In some preferred embodiments, the vaccines comprise
about 25 to about 250 micrograms of nucleic acid sequences. Most
preferably, the vaccines comprise about 100 micrograms nucleic acid
sequences.
[0078] The genetic vaccines according to the present invention are
formulated according to the mode of administration to be used. One
having ordinary skill in the art can readily formulate a genetic
vaccine that comprises a genetic construct. In cases where
intramuscular injection is the chosen mode of administration, for
example, an isotonic formulation is generally used. As described in
detail herein above, additives for isotonicity can include sodium
chloride, dextrose, mannitol, sorbitol and lactose. Isotonic
solutions such as phosphate buffered saline are preferred.
Stabilizers can include gelatin and albumin.
[0079] In some embodiments of the invention, the individual is
administered a series of vaccinations to produce a comprehensive
immune response. According to this method, at least two and
preferably four injections are given over a period of time. The
period of time between injections may include from 24 hours apart
to two weeks or longer between injections, preferably one week
apart. Alternatively, at least two and up to four separate
injections may be administered simultaneously to different parts of
the body.
[0080] While this disclosure generally discusses immunization or
vaccination in the context of prophylactic methods of protection,
the terms "immunizing" or "vaccinating" are meant to refer to both
prophylactic and therapeutic methods. Thus, a method for immunizing
or vaccinating includes both methods of protecting an individual
from pathogen challenge, as well as methods for treating an
individual suffering from pathogen infection. Accordingly, the
present invention may be used as a vaccine for prophylactic
protection or in a therapeutic manner; that is, as a reagent for
immunotherapeutic methods and preparations.
[0081] The amount of a modified nucleic acid sequence generated
using the methods of the invention which provides a therapeutically
effective dose in the treatment of a patient with, for example,
cancer or a pathogen-related disorder can be determined by standard
clinical techniques based on the present description. In addition,
in vitro assays may optionally be employed to help identify optimal
dosage ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the
seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each subject's
circumstances. However, suitable dosage ranges for intravenous
administration are generally directed to achieve a concentration of
about 20-500 micrograms of polypeptide encoded by the modified
nucleic acid per kilogram body weight. Suitable dosage ranges for
intranasal administration are generally directed to achieve a
concentration of about 0.01 pg to 1 mg of polypeptide encoded by
the modified nucleic acid per kg body weight. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0082] The compositions comprising the modified nucleic acid
molecules of the invention can be administered for prophylactic
and/or therapeutic treatments. In therapeutic applications,
compositions are administered to a patient already suffering from a
hyperproliferative disorder (such as, e.g., cancer) in an amount
sufficient to cure or at least partially arrest the symptoms of the
disease and its complications. An amount adequate to accomplish
this is defined as a "therapeutically effective amount or dose."
Amounts effective for this use will depend on the severity of the
disease and the weight and general state of the patient.
[0083] Compositions comprising modified nucleic acid molecules of
the invention can be administered alone, or in combination, and/or
in conjunction with known therapeutic agents/compounds used for the
treatment of a patient with a particular disorder. For the
treatment of a patient with cancer, for example, a composition
comprising at least one modified nucleic acid of the invention
which encodes a tumour antigen, may be used in conjunction with one
or more known cancer therapeutics, such as those described in the
Physicians' Desk Reference, 54.sup.th Edition (2000) or in Cancer:
Principles & Practice of Oncology, DeVita, Jr., Hellman, and
Rosenberg (eds.) 2nd edition, Philadelphia, Pa.: J. B. Lippincott
Co., 1985, wherein standard treatment protocols and dosage
formulations are presented.
[0084] In addition a method is also provided for determining how to
modify the sequence of an mRNA so as to generate a modified mRNA
having altered properties, which may be used alone or in a
pharmaceutical composition of the invention. In this connection,
and in accordance with the invention, the modification of an RNA
sequence is carried out with two different optimisation objectives:
to maximize G/C content, and to maximize the frequency of codons
that are recognized by abundantly expressed tRNAs. In the first
step of the process a virtual translation of an arbitrary RNA (or
DNA) sequence is carried out in order to generate the corresponding
amino acid sequence. Starting from the amino acid sequence, a
virtual reverse translation is performed that provides, based on
degeneracy of the genetic code, all of the possible choices for the
corresponding codons. Depending on the required optimisation or
modification, corresponding selection lists and optimisation
algorithms are used for choosing suitable codons. The algorithms
are executed on a computer, normally with the aid of suitable
software. In accordance with the present invention, a suitable
software program comprises a source code of Appendix I. Thus, the
optimised mRNA sequence is generated and can be output, for
example, with the aid of a suitable display device and compared
with the original (wild type) sequence. The same also applies with
regard to the frequency of the individual nucleotides. The changes
compared to the original nucleotide sequence are preferably
emphasised. Furthermore, according to a preferred embodiment,
naturally occurring stable sequences are incorporated therein to
produce an RNA stabilised by the presence of natural sequence
motifs. A secondary structural analysis may also be performed that
can analyse, on the basis of structural calculations, stabilising
and destabilising properties or regions of the RNA.
[0085] Also encompassed by the present invention are modified
nucleic acid sequences generated using the above computer-based
method. Exemplary modified nucleic acid sequences of the invention
include SEQ ID NOs: 3-7, 10 and 11. The present invention also
includes pharmaceutical compositions of modified nucleic acid
sequences of the invention, including SEQ ID NOs: 3-7, 10 and
11.
BRIEF DESCRIPTION OF THE DRAWINGS
[0086] FIG. 1 shows wild type sequences and modified sequences for
the influenza matrix protein.
[0087] FIG. 1A (SEQ ID NO: 1) shows the wild type gene and FIG. 1B
(SEQ ID NO: 2) shows the amino acid sequence derived therefrom
(1-letter code). FIG. 1C (SEQ ID NO: 3) shows a gene sequence
coding for the influenza matrix protein, whose G/C content is
increased as compared to that of the wild type sequence. FIG. 1D
(SEQ ID NO: 4) shows the sequence of a gene that codes for a
secreted form of the influenza matrix protein (including an
N-terminal signal sequence), wherein the G/C content of the
sequence is increased relative to that of the wild type sequence.
FIG. 1E (SEQ ID NO: 5) shows an mRNA coding for the influenza
matrix protein, wherein the mRNA comprises stabilising sequences
not present in the corresponding wild type mRNA. FIG. 1F (SEQ ID
NO: 6) shows an mRNA coding for the influenza matrix protein that
in addition to stabilising sequences also contains an increased G/C
content. FIG. 1G (SEQ ID NO: 7) likewise shows a modified mRNA that
codes for a secreted form of the influenza matrix protein and
comprises, as compared to the wild type, stabilising sequences and
an elevated G/C content. In FIG. 1A and FIGS. 1C to 1G the start
and stop codons are shown in bold type. Nucleotides that are
changed relative to the wild type sequence of FIG. 1A are shown in
capital letters in 1C to 1G.
[0088] FIG. 2 shows wild type sequences and modified sequences
according to the invention that encode for the tumour antigen
MAGE1.
[0089] FIG. 2A (SEQ ID NO: 8) shows the sequence of the wild type
gene and FIG. 2B (SEQ ID NO: 9) shows the amino acid sequence
derived therefrom (3-letter code). FIG. 2C (SEQ ID NO: 10) shows a
modified mRNA coding for MAGE1, whose G/C content is increased as
compared to the wild type. FIG. 2D (SEQ ID NO: 11) shows the
sequence of a modified mRNA encoding MAGE1, in which the codon
usage has been optimised as frequently as possible with respect to
the tRNA present in the cell and to the coding sequence in
question. Start and stop codons are shown in each case in bold
type.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0090] The following examples describe the invention in more detail
and in no way are to be construed as restricting the scope
thereof.
Example 1
[0091] As an exemplary embodiment of the process for determining
the sequence of a modified mRNA according to the invention, a
computer program was established that modifies the nucleotide
sequence of an arbitrary mRNA in such a way as to maximise the G/C
content of the nucleic acid, and maximise the presence of codons
recognized by abundant tRNAs present in a particular cell(s). The
computer program is based on an understanding of the genetic code
and exploits the degenerative nature of the genetic code. By this
means a modified mRNA having desirable properties is obtained,
wherein the amino acid sequence encoded by the modified mRNA is
identical to that of the unmodified mRNA sequence. Alternatively,
the invention may encompass alterations in either the G/C content
or codon usage of an mRNA to produce a modified mRNA.
[0092] The source code in Visual Basic 6.0 (program development
environment employed: Microsoft Visual Studio Enterprise 6.0 with
Servicepack 3) is given in the Appendix I.
Example 2
[0093] An RNA construct with a sequence of the lac-Z gene from E.
coli optimised with regard to stabilisation and translational
efficiency was produced with the aid of the computer program of
Example 1. A G/C content of 69% (compared to the wild type sequence
of 51%; C. F. Kalnins et al., EMBO J. 1983, 2(4): 593-597) was
achieved in this manner. Through the synthesis of overlapping
oligonucleotides that comprise the modified sequence, the optimised
sequence was produced according to methods known in the art. The
terminal oligonucleotides have the following restriction cleavage
sites: at the 5' end an EcoRV cleavage site, and at the 3' end a
BglII cleavage site. The modified lacZ sequence was incorporated
into the plasmid pT7Ts (GenBank Accession No. U26404; C. F. Lai et
al., see above) by digestion with EcoRV/BglII. pT7Ts contains
untranslated region sequences from the .beta.-globin gene of
Xenopus laevis at the 5' and 3' ends. The plasmid was cleaved with
the aforementioned restriction enzymes to facilitate insertion of
the modified lacZ sequence having compatible 5' and 3' termini.
[0094] The pT7Ts-lac-Z construct was propagated in bacteria and
purified by phenol-chloroform extraction. 2 .mu.g of the construct
were transcribed in vitro using methods known to a skilled artisan
and the modified mRNA was produced.
Example 3
[0095] The gene for the influenza matrix protein (wild type
sequence, see FIG. 1A; derived amino acid sequence, see FIG. 1B)
was optimised with the aid of the computer program according to the
invention of Example 1. The G/C-rich sequence variant shown in FIG.
1C (SEQ ID NO: 3) was thereby formed. A G/C-rich sequence coding
for a secreted form of the influenza matrix protein, which includes
an N-terminal signal sequence was also determined (see FIG. 1D; SEQ
ID NO: 4). The secreted form of the influenza matrix protein has
the advantage of increased immunogenicity as compared to that of
the non-secreted form.
[0096] Corresponding mRNA molecules were designed starting from the
optimised sequences. The mRNA for the influenza matrix protein,
optimised with regard to G/C content and codon usage, was
additionally provided with stabilising sequences in the 5' region
and 3' region (the stabilisation sequences derive from the 5'-UTRs
and 3'-UTRs of the .beta.-globin-mRNA of Xenopus laevis;
pT7Ts-Vektor in C. F. Lai et al., see above). See also FIG. 1E; SEQ
ID NO: 5, which includes only stabilising sequences and 1F; SEQ ID
NO: 6, which includes both increased G/C content and stabilising
sequences. The mRNA coding for the secreted form of the influenza
matrix protein was likewise also sequence optimised in the
translated region and provided with the aforementioned stabilising
sequences (see FIG. 1G; SEQ ID NO: 7).
Example 4
[0097] The mRNA encoding the tumour antigen MAGE1 was modified with
the aid of the computer program of Example 1. The sequence shown in
FIG. 2C (SEQ ID NO: 10) was generated in this way, and has a 24%
higher G/C content (351 G, 291 C) as compared to the wild type
sequence (275 G, 244 C). In addition, by means of alternative codon
usage, the wild type sequence was improved with regard to
translational efficiency by substituting codons corresponding to
tRNAs that are more abundant in a target cell (see FIG. 2D; SEQ ID
NO: 11). The G/C content was likewise raised by 24% by the
alternative codon usage.
Sequence CWU 1
1
131774DNAInfluenza virusInfluenza matrix wildtype gene (for
comparison) 1agatctaaag atgagtcttc taaccgaggt cgaaacgtac gttctctcta
tcatcccgtc 60aggccccctc aaagccgaga tcgcacagag acttgaagat gtctttgcag
ggaagaacac 120cgatcttgag gttctcatgg aatggctaaa gacaagacca
atcctgtcac ctctgactaa 180ggggatttta ggatttgtgt tcacgctcac
cgtgcccagt gagcgaggac tgcagcgtag 240acgctttgtc caaaatgccc
ttaatgggaa cggggatcca aataacatgg acaaagcagt 300taaactgtat
aggaagctca agagggagat aacattccat ggggccaaag aaatctcact
360cagttattct gctggtgcac ttgccagttg tatgggcctc atatacaaca
ggatgggggc 420tgtgaccact gaagtggcat ttggcctggt atgtgcaacc
tgtgaacaga ttgctgactc 480ccagcatcgg tctcataggc aaatggtgac
aacaaccaac ccactaatca gacatgagaa 540cagaatggtt ttagccagca
ctacagctaa ggctatggag caaatggctg gatcgagtga 600gcaagcagca
gaggccatgg aggttgctag tcaggctagg caaatggtgc aagcgatgag
660aaccattggg actcatccta gctccagtgc tggtctgaaa aatgatcttc
ttgaaaattt 720gcaggcctat cagaaacgaa tgggggtgca gatgcaacgg
ttcaagtgaa ctag 7742252PRTInfluenza virus 2Met Ser Leu Leu Thr Glu
Val Glu Thr Tyr Val Leu Ser Ile Ile Pro1 5 10 15Ser Gly Pro Leu Lys
Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30Ala Gly Lys Asn
Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr 35 40 45Arg Pro Ile
Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60Thr Leu
Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75
80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala
85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly
Ala 100 105 110Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala
Ser Cys Met 115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr
Thr Glu Val Ala Phe 130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln
Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val
Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175Asn Arg Met Val
Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190Ala Gly
Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln 195 200
205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser
210 215 220Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln
Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe
Lys 245 2503775DNAArtificial SequenceDescription of Artificial
Sequence Influenza matrix gene with increased G/C-content
3agatctaaag atgagcctgc tgaccgaggt ggagacctac gtgctgagca tcatccccag
60cggccccctg aaggccgaga tcgcccagag gctggaggac gtgttcgccg gcaagaacac
120cgacctggag gtgctgatgg agtggctgaa gaccaggccc atcctgagcc
ccctgaccaa 180gggcatcctg ggcttcgtgt tcaccctgac cgtgcccagc
gagcgcggcc tgcagcgccg 240ccgcttcgtg cagaacgccc tgaacggcaa
cggcgacccc aacaacatgg acaaggccgt 300gaagctgtac aggaagctga
agagggagat caccttccac ggcgccaagg agatcagcct 360gagctacagc
gccggcgccc tggccagctg catgggcctg atctacaaca ggatgggcgc
420cgtgaccacc gaggtggcct tcggcctggt gtgcgccacc tgcgagcaga
tcgccgacag 480ccagcaccgc agccacaggc agatggtgac caccaccaac
cccctgatca ggcacgagaa 540caggatggtg ctggccagca ccaccgccaa
ggccatggag cagatggccg gcagcagcga 600gcaggccgcc gaggccatgg
aggtggccag ccaggccagg cagatggtgc aggccatgag 660gaccatcggc
acccacccca gcagcagcgc cggcctgaag aacgacctgc tggagaacct
720gcaggcctac cagaagcgca tgggcgtgca gatgcagcgc ttcaagtgaa ctagt
7754844DNAArtificial SequenceDescription of Artificial Sequence
Influenza matrix gene for secreted form (with N-terminal signal
sequence) with increased G/C-content 4agatctaaag atggccgtca
tggccccccg caccctggtg ctgctgctga gcggcgccct 60ggccctgacc cagacctggg
ctagcctgct gaccgaggtg gagacctacg tgctgagcat 120catccccagc
ggccccctga aggccgagat cgcccagagg ctggaggacg tgttcgccgg
180caagaacacc gacctggagg tgctgatgga gtggctgaag accaggccca
tcctgagccc 240cctgaccaag ggcatcctgg gcttcgtgtt caccctgacc
gtgcccagcg agcgcggcct 300gcagcgccgc cgcttcgtgc agaacgccct
gaacggcaac ggcgacccca acaacatgga 360caaggccgtg aagctgtaca
ggaagctgaa gagggagatc accttccacg gcgccaagga 420gatcagcctg
agctacagcg ccggcgccct ggccagctgc atgggcctga tctacaacag
480gatgggcgcc gtgaccaccg aggtggcctt cggcctggtg tgcgccacct
gcgagcagat 540cgccgacagc cagcaccgca gccacaggca gatggtgacc
accaccaacc ccctgatcag 600gcacgagaac aggatggtgc tggccagcac
caccgccaag gccatggagc agatggccgg 660cagcagcgag caggccgccg
aggccatgga ggtggccagc caggccaggc agatggtgca 720ggccatgagg
accatcggca cccaccccag cagcagcgcc ggcctgaaga acgacctgct
780ggagaacctg caggcctacc agaagcgcat gggcgtgcag atgcagcgct
tcaagtgaac 840tagt 8445942RNAArtificial SequenceDescription of
Artificial Sequence Influenza matrix mRNA with stabilisation
sequences 5gcuuguucuu uuugcagaag cucagaauaa acgcucaacu uuggcagauc
uaaagaugag 60ucuucuaacc gaggucgaaa cguacguucu cucuaucauc ccgucaggcc
cccucaaagc 120cgagaucgca cagagacuug aagaugucuu ugcagggaag
aacaccgauc uugagguucu 180cauggaaugg cuaaagacaa gaccaauccu
gucaccucug acuaagggga uuuuaggauu 240uguguucacg cucaccgugc
ccagugagcg aggacugcag cguagacgcu uuguccaaaa 300ugcccuuaau
gggaacgggg auccaaauaa cauggacaaa gcaguuaaac uguauaggaa
360gcucaagagg gagauaacau uccauggggc caaagaaauc ucacucaguu
auucugcugg 420ugcacuugcc aguuguaugg gccucauaua caacaggaug
ggggcuguga ccacugaagu 480ggcauuuggc cugguaugug caaccuguga
acagauugcu gacucccagc aucggucuca 540uaggcaaaug gugacaacaa
ccaacccacu aaucagacau gagaacagaa ugguuuuagc 600cagcacuaca
gcuaaggcua uggagcaaau ggcuggaucg agugagcaag cagcagaggc
660cauggagguu gcuagucagg cuaggcaaau ggugcaagcg augagaacca
uugggacuca 720uccuagcucc agugcugguc ugaaaaauga ucuucuugaa
aauuugcagg ccuaucagaa 780acgaaugggg gugcagaugc aacgguucaa
gugaacuagu gacugacuag cccgcugggc 840cucccaacgg gcccuccucc
ccuccuugca ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 9426942RNAArtificial
SequenceDescription of Artificial Sequence Influenza matrix mRNA
with increased G/C-content and stabilisation sequences 6gcuuguucuu
uuugcagaag cucagaauaa acgcucaacu uuggcagauc uaaagaugag 60ccugcugacc
gagguggaga ccuacgugcu gagcaucauc cccagcggcc cccugaaggc
120cgagaucgcc cagaggcugg aggacguguu cgccggcaag aacaccgacc
uggaggugcu 180gauggagugg cugaagacca ggcccauccu gagcccccug
accaagggca uccugggcuu 240cguguucacc cugaccgugc ccagcgagcg
cggccugcag cgccgccgcu ucgugcagaa 300cgcccugaac ggcaacggcg
accccaacaa cauggacaag gccgugaagc uguacaggaa 360gcugaagagg
gagaucaccu uccacggcgc caaggagauc agccugagcu acagcgccgg
420cgcccuggcc agcugcaugg gccugaucua caacaggaug ggcgccguga
ccaccgaggu 480ggccuucggc cuggugugcg ccaccugcga gcagaucgcc
gacagccagc accgcagcca 540caggcagaug gugaccacca ccaacccccu
gaucaggcac gagaacagga uggugcuggc 600cagcaccacc gccaaggcca
uggagcagau ggccggcagc agcgagcagg ccgccgaggc 660cauggaggug
gccagccagg ccaggcagau ggugcaggcc augaggacca ucggcaccca
720ccccagcagc agcgccggcc ugaagaacga ccugcuggag aaccugcagg
ccuaccagaa 780gcgcaugggc gugcagaugc agcgcuucaa gugaacuagu
gacugacuag cccgcugggc 840cucccaacgg gcccuccucc ccuccuugca
ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa 94271011RNAArtificial SequenceDescription
of Artificial Sequence Influenza matrix mRNA coding for secreted
form with increased G/C-content and stabilisation sequences
7gcuuguucuu uuugcagaag cucagaauaa acgcucaacu uuggcagauc uaaagauggc
60cgucauggcc ccccgcaccc uggugcugcu gcugagcggc gcccuggccc ugacccagac
120cugggccagc cugcugaccg agguggagac cuacgugcug agcaucaucc
ccagcggccc 180ccugaaggcc gagaucgccc agaggcugga ggacguguuc
gccggcaaga acaccgaccu 240ggaggugcug auggaguggc ugaagaccag
gcccauccug agcccccuga ccaagggcau 300ccugggcuuc guguucaccc
ugaccgugcc cagcgagcgc ggccugcagc gccgccgcuu 360cgugcagaac
gcccugaacg gcaacggcga ccccaacaac auggacaagg ccgugaagcu
420guacaggaag cugaagaggg agaucaccuu ccacggcgcc aaggagauca
gccugagcua 480cagcgccggc gcccuggcca gcugcauggg ccugaucuac
aacaggaugg gcgccgugac 540caccgaggug gccuucggcc uggugugcgc
caccugcgag cagaucgccg acagccagca 600ccgcagccac aggcagaugg
ugaccaccac caacccccug aucaggcacg agaacaggau 660ggugcuggcc
agcaccaccg ccaaggccau ggagcagaug gccggcagca gcgagcaggc
720cgccgaggcc auggaggugg ccagccaggc caggcagaug gugcaggcca
ugaggaccau 780cggcacccac cccagcagca gcgccggccu gaagaacgac
cugcuggaga accugcaggc 840cuaccagaag cgcaugggcg ugcagaugca
gcgcuucaag ugaacuagug acugacuagc 900ccgcugggcc ucccaacggg
cccuccuccc cuccuugcac caaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 10118940DNAHomo
sapiensMAGE1 wildtype-gene (for comparison) 8catcatgtct cttgagcaga
ggagtctgca ctgcaagcct gaggaagccc ttgaggccca 60acaagaggcc ctgggcctgg
tgtgtgtgca ggctgccacc tcctcctcct ctcctctggt 120cctgggcacc
ctggaggagg tgcccactgc tgggtcaaca gatcctcccc agagtcctca
180gggagcctcc gcctttccca ctaccatcaa cttcactcga cagaggcaac
ccagtgaggg 240ttccagcagc cgtgaagagg aggggccaag cacctcttgt
atcctggagt ccttgttccg 300agcagtaatc actaagaagg tggctgattt
ggttggtttt ctgctcctca aatatcgagc 360cagggagcca gtcacaaagg
cagaaatgct ggagagtgtc atcaaaaatt acaagcactg 420ttttcctgag
atcttcggca aagcctctga gtccttgcag ctggtctttg gcattgacgt
480gaaggaagca gaccccaccg gccactccta tgtccttgtc acctgcctag
gtctctccta 540tgatggcctg ctgggtgata atcagatcat gcccaagaca
ggcttcctga taattgtcct 600ggtcatgatt gcaatggagg gcggccatgc
tcctgaggag gaaatctggg aggagctgag 660tgtgatggag gtgtatgatg
ggagggagca cagtgcctat ggggagccca ggaagctgct 720cacccaagat
ttggtgcagg aaaagtacct ggagtaccgg caggtgccgg acagtgatcc
780cgcacgctat gagttcctgt ggggtccaag ggccctcgct gaaaccagct
atgtgaaagt 840ccttgagtat gtgatcaagg tcagtgcaag agttcgcttt
ttcttcccat ccctgcgtga 900agcagctttg agagaggagg aagagggagt
ctgagcatga 9409308PRTHomo sapiensTumor antigen MAGE1 protein
sequence 9Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala
Leu Glu1 5 10 15Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala
Ala Thr Ser 20 25 30Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu
Val Pro Thr Ala 35 40 45Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly
Ala Ser Ala Phe Pro 50 55 60Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln
Pro Ser Glu Gly Ser Ser65 70 75 80Ser Arg Glu Glu Glu Gly Pro Ser
Thr Ser Cys Ile Leu Glu Ser Leu 85 90 95Phe Arg Ala Val Ile Thr Lys
Lys Val Ala Asp Leu Val Gly Phe Leu 100 105 110Leu Leu Lys Tyr Arg
Ala Arg Glu Pro Val Thr Lys Ala Glu Met Leu 115 120 125Glu Ser Val
Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe Gly 130 135 140Lys
Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys Glu145 150
155 160Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly
Leu 165 170 175Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro
Lys Thr Gly 180 185 190Phe Leu Ile Ile Val Leu Val Met Ile Ala Met
Glu Gly Gly His Ala 195 200 205Pro Glu Glu Glu Ile Trp Glu Glu Leu
Ser Val Met Glu Val Tyr Asp 210 215 220Gly Arg Glu His Ser Ala Tyr
Gly Glu Pro Arg Lys Leu Leu Thr Gln225 230 235 240Asp Leu Val Gln
Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp Ser 245 250 255Asp Pro
Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala Glu 260 265
270Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala Arg
275 280 285Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg
Glu Glu 290 295 300Glu Glu Gly Val30510939RNAArtificial
SequenceDescription of Artificial Sequence MAGE1 mRNA with
increased G/C-content 10augagccugg agcagcgcag ccugcacugc aagccggagg
aggcgcugga ggcgcagcag 60gaggcgcugg gccuggucug cguccaggcg gcgacgagca
gcagcagccc gcugguccug 120ggcacgcugg aggagguccc gacggcgggc
agcacggacc cgccgcagag cccgcagggc 180gcgagcgcgu ucccgacgac
gaucaacuuc acgcgccagc gccagccgag cgagggcagc 240agcagccgcg
aggaggaggg cccgagcacg agcugcaucc uggagagccu guuccgcgcg
300gucaucacga agaaggucgc ggaccugguc ggcuuccugc ugcugaagua
ccgcgcgcgc 360gagccgguca cgaaggcgga gaugcuggag agcgucauca
agaacuacaa gcacugcuuc 420ccggagaucu ucggcaaggc gagcgagagc
cugcagcugg ucuucggcau cgacgucaag 480gaggcggacc cgacgggcca
cagcuacguc cuggucacgu gccugggccu gagcuacgac 540ggccugcugg
gcgacaacca gaucaugccg aagacgggcu uccugaucau cguccugguc
600augaucgcga uggagggcgg ccacgcgccg gaggaggaga ucugggagga
gcugagcguc 660auggaggucu acgacggccg cgagcacagc gcguacggcg
agccgcgcaa gcugcugacg 720caggaccugg uccaggagaa guaccuggag
uaccgccagg ucccggacag cgacccggcg 780cgcuacgagu uccugugggg
cccgcgcgcg cuggcggaga cgagcuacgu caagguccug 840gaguacguca
ucaaggucag cgcgcgcguc cgcuucuucu ucccgagccu gcgcgaggcg
900gcgcugcgcg aggaggagga gggcgucuga gcgugauga 93911939RNAArtificial
SequenceDescription of Artificial Sequence MAGE1 mRNA with
alternative use of codon 11augagccugg agcagcgcag ccugcacugc
aagcccgagg aggcccugga ggcccagcag 60gaggcccugg gccuggugug cgugcaggcc
gccaccagca gcagcagccc ccuggugcug 120ggcacccugg aggaggugcc
caccgccggc agcaccgacc ccccccagag cccccagggc 180gccagcgccu
uccccaccac caucaacuuc acccgccagc gccagcccag cgagggcagc
240agcagccgcg aggaggaggg ccccagcacc agcugcaucc uggagagccu
guuccgcgcc 300gugaucacca agaagguggc cgaccuggug ggcuuccugc
ugcugaagua ccgcgcccgc 360gagcccguga ccaaggccga gaugcuggag
agcgugauca agaacuacaa gcacugcuuc 420cccgagaucu ucggcaaggc
cagcgagagc cugcagcugg uguucggcau cgacgugaag 480gaggccgacc
ccaccggcca cagcuacgug cuggugaccu gccugggccu gagcuacgac
540ggccugcugg gcgacaacca gaucaugccc aagaccggcu uccugaucau
cgugcuggug 600augaucgcca uggagggcgg ccacgccccc gaggaggaga
ucugggagga gcugagcgug 660auggaggugu acgacggccg cgagcacagc
gccuacggcg agccccgcaa gcugcugacc 720caggaccugg ugcaggagaa
guaccuggag uaccgccagg ugcccgacag cgaccccgcc 780cgcuacgagu
uccugugggg cccccgcgcc cuggccgaga ccagcuacgu gaaggugcug
840gaguacguga ucaaggugag cgcccgcgug cgcuucuucu uccccagccu
gcgcgaggcc 900gcccugcgcg aggaggagga gggcguguga gccugauga
939127RNAArtificial SequenceDescription of Artificial Sequence
Sequence motif recognizable for an endonuclease contained in the
3'UTR-segment of the gene coding for the transferrin receptor (see
p.10 of description) 12gaacaag 71313RNAArtificial
SequenceDescription of Artificial Sequence Kozak sequence, ribosome
binding site (see p. 12 of description) 13gccgccacca ugg 13
* * * * *