Note: Descriptions are shown in the official language in which they were submitted.
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Promoter for the Expression of Forei2n Genes in Neuronal Cells
The invention relates to a promoter for the expression of foreign genes in
neuronal cells.
A promoter represents a regulatory starting sequence, important for the gene
expression, within the genome of any organism; it determines whether, how and
to what
extent the transcription of a gene takes place in messenger RNA (mRNA). There
are
"strong" and "weak" promoters (i.e., those that bring about the formation of
numerous or
less numerous mRNA transcripts of the gene), as well as constitutive
(constantly active),
inducible (i.e., those that control the transcription based on certain
conditions) and tissue-
specific promoters.
The neuron-specific Thyl promoter of the mouse was already used in multiple
ways to produce transgenic mice that express foreign genes in their central
nervous
system. Such mouse strains represent important instruments, in particular for
studying
molecular mechanisms of neurodegenerative diseases as well as for the
development of
these potential therapies.
The mThyl promoter is an atypical promoter without TATA-box. It is composed
of two identical promoter nonamers, in which in each case, a short, non-
translated Exon
follows (Exons la and Ib). The Intron A, which is followed by the first
translated Exon
(Exon 2), is connected to Exon lb. After that, the Exons 3 and 4 follow, in
each case
separated from one another by an additional Intron (Introns B and C). This
sequence
(first promoter-nonamer up to and including Exon 4) is flanked by non-
translated regions
(5'- and 3'-UTR), whereby the 3'-UTR contains the poly-A-signal. It is assumed
that the
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5'-UTR, the Exons 1 a and lb, as well as the Intron A have regulatory
properties that can
also be responsible for the neuron-specific expression of the promoter (E.
Spanopoulou et
al., Mol. Cell. Biol. 1988; 8: 3847-3856 and 1991; 11 (4): 2216-2228).
In addition to the neuronal expression, the original mThyl promoter also
showed
an expression in the thymus. The thymus expression could be excluded by the
deletion of
the range from Exon 2 to Exon 4 (H. A. Ingraham and G. A. Evans, Mol. Cell.
Biol.
1986; 6 (8): 2923-293 1; M. Vidal et al., EMBO 1990; 9 (3): 833-840).
The mThyl promoter with deletion of Exons 2-4 was and is used for control of
the transgenic expression in the generation of transgenic animals to express
the
corresponding transgene specifically in the neurons. Thus, for example,
transgenic mice
that express a mutated alpha-synuclein protein under mThyl-promoter control
found in a
rare congenital form of Parkinson's disease have similar symptoms to the
corresponding
patients (H. van der Putten et al., J Neurosci. 2000; 20 (16): 6021-9; B.
Sommer et al.,
Exp Gerontol. 2000; 35 (9-10): 1389-403). Similar murine animal models exist
for
certain human tauopathies, in which mutated forms of the protein Tau that is
associated
with the microtubule are found (J. Gotz et al., J Biol Chem. 2001; 276 (1):
529-34).
Animal models of Alzheimer's disease, in which amyloid precursor proteins
(APP, the
precursor protein of the beta-amyloid, which forms the so-called plaque in the
brains of
Alzheimer patients) are expressed or over-expressed under Thyl control, are
very
commonly used. In this case, both normal amyloid (E. Masliah and E.
Rockenstein, J
Neural Transm Suppl. 2000; 59: 175-83) and mutated amyloid can be formed from
known congenital forms of Alzheimer's dementia (J. Davis et al., J Biol Chem.
2004; 279
(19): 20296-306).
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The Thyl promoter has decisive drawbacks, however. The considerable size of
the Thyl promoter makes it difficult to handle for genetic engineering works,
in
particular for the incorporation of major gene constructs.
In view of the fact that the viral vectors that can also be used for the
generation of
transgenic animals can take up only limited amounts of foreign DNA, the
promoter size
considerably limits the size of the gene to be transferred. Also, the Thyl
promoter is
often not strong enough to produce in the transgenic animals an expression of
the foreign
gene to a desirable extent.
Therefore, the object according to the invention was to modify the known mThyl
promoter structure, such that
a) The promoter size is reduced such that its use for genetic engineering
works is
facilitated and the transfer of larger foreign genes or gene fragments with
stable vectors is made possible,
b) The expression of this foreign DNA in the thus produced transgenic mice is
increased significantly, and
c) The expression of the transgene to be controlled by the promoter is carried
out
in a neuron-specific manner.
It has now turned out that the above-described object can be achieved by the
design of a promoter that is altered by genetic engineering, which contains
only certain
(original or slightly altered) partial sequences of the mThyl promoter in
combination
with other regulatory elements.
The invention relates to a promoter that comprises a nucleic acid sequence
that is
derived from Thy-1 mammals and that brings about the neuron-specific
transcription of a
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heterologous nucleic acid sequence that is located 3'- downward from the
promoter both
in mammal and in non-mammal cells.
The invention furthermore relates to a nucleic acid sequence that consists of
the
sections
(a) The mThyl 5'-"untranslated region" (UTR), the 20bp sequence that is
located in
front of 5'-UTR, and the sequence of the first nonameric promoter that
consists of
9 nucleotides and flanking regions of the mouse-Thy-1- gene
(b) The sequence of the Exon 1 a of the mouse-Thy-1 gene
(c) The sequence of the second nonameric promoter that consists of 9
nucleotides of
the mouse-Thy-1 gene
(d) The sequence of the Exon lb of the mouse-Thy-1 gene
(e) The complete or partial sequence of the Intron A of the mouse-Thy-1 gene
(f) The sequence of a poly A signal
(g) The mammal-Thy-l-gene-promoter sequence, which hybridizes with the
sequence that is described under (a), (b), (c), (d), (e) and (f), whereby the
promoter sequence, if it is combined with a heterologous gene sequence,
transcribes the latter in a neuron-specific manner both in mammal cells and in
non-mammal cells.
The invention furthermore relates to a nucleic acid construct, comprising an
expression cassette, which contains the sections (a), (b), (c), (d), (e), (f)
and (g)
characterized according to claim 2 as well as a heterologous nucleic acid
sequence, which
is positioned between the sections (e) and (f) and is associated operatively
with the latter.
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The invention furthermore relates to advantageous configurations of this
nucleic acid
construct as they are disclosed according to claims 4 to 8.
The invention relates to an isolated cell or cell line that comprises the
promoter
according to claim 1.
The invention also relates to an isolated cell or cell line that comprises the
nucleic
acid sequence according to claim 2. In an advantageous way, this isolated cell
or cell line
comprises a nucleic acid construct with the features according to claims 3 to
8.
The invention relates to a transgenic non-human animal, comprising the
promoter
according to claim 1.
The invention also relates to a transgenic non-human animal, comprising the
nucleic acid sequence according to claim 2. In this case, in an advantageous
way, the
transgenic non-human animal comprises a nucleic acid construct according to
one of
claims 3 to claim 8.
The invention relates to a process for gene expression in neuronal and non-
neuronal cells, which contains the transformation/transfection of the cells
with a vector
that comprises the nucleic acid sequence according to claim 2 or a nucleic
acid construct
according to one of claims 3 to 8.
The invention relates to advantageous configurations of this process with the
features according to claims 16 to 18.
The invention relates to the use of a vector that comprises the nucleic acid
sequence according to claim 2 or a nucleic acid construct according to one of
claims 3 to
8 for the production of injection solutions, which are suitable for injection
into the central
nervous system or into the cerebrospinal fluid.
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The invention relates to the use of a vector that comprises the nucleic acid
sequence according to claim 2 or a nucleic acid construct according to one of
claims 3 to
8 for the production of injection solutions for treating neuronal disorders,
such as stroke,
ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's
disease, brain
and spinal cord trauma, and amyotrophic lateral sclerosis.
The invention relates to the use of a vector that comprises the nucleic acid
sequence according to claim 2 or a nucleic acid construct according to one of
claims 3 to
8 for the production of injection solutions for treating neurogenetic
disorders.
The invention relates to a kit for the expression of recombinant gene products
that
comprise isolated cells or cell lines according to one of claims 9 to 11.
The invention relates to a pharmaceutical agent that comprises a promoter
according to claim 1.
The invention also relates to a pharmaceutical agent that comprises a protein,
which is coded by the nucleic acid sequence according to claim 2.
The invention furthermore relates to a pharmaceutical agent that comprises a
nucleic acid construct according to one of claims 3 to 8.
Possible methods of implementing the invention can be represented as follows:
Starting from the mThyl-expression cassette (Moechars et al. 1996, EMBO J.
15(6), 126574), which has an original size of about 8.2 kb and which contains
an Xho I
linker instead of the mThyl Exons 2 to 4, major deletions were put in place
with the aid
of restriction endonucleases, and then minor insertions were performed, so
that at the end,
a promoter cassette that was only 1.6 kb was produced.
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Starting from the mThyl -expression cassette (Moechars et al. 1996, EMBO J.
15(6), 126574), which has an original size of about 8.2 kb and which instead
of the
mThyl Exons 2 to 4 contains an Xho I linker, major deletions were put into
place with
the aid of restriction endonucleases and then minor insertions were performed,
so that at
the end, a promoter cassette that was only 1.6 kb was produced.
In the first step, the mThyl -expression cassette was cleaved with Sma I, and
thus
the area adjoining the mThyl 5'-UTR on the 5'-end was removed. The construct
that
was produced, in which the eGFP reporter gene (coded for a Green Fluorescent
Protein,
which is harmless to mice and serves as a fluorescence-microscopic marker) was
integrated 3'-terminally from mThyl Intron A, was studied by means of
transfection in
two neuronal cell lines (SH-SY5Y and Neuro-2A) in promoter activity (eGFP
expression).
In a second step, the sequence was digested with the restriction enzymes Nco I
and Nde I, by which parts of the mThyl Intron A, the control gene eGFP, the
mThyl 3'-
UTR together with the mThyl Poly A signal and the 3' flanking mThyl-sequence
ranges
were removed. Instead of this, a sequence that consists of the eGFP sequence
and the
"late SV40 Poly A Signal" was ligated with the reduced mThyl-promoter
sequence. The
above-mentioned steps are depicted in Fig. 1, the correlation diagram mThyl-
gene
sequence (A) / mThyl promoter cassette (B) / mTUB promoter cassette (C). The
mThyl
part is labeled above the respective figure, and the non-mThyl part is labeled
below it.
The thus produced sequence, excluding the eGFP-gene sequence, was referred to
as an mTUB-promoter (mouse Thyl Usable for Brain expression; for sequence, see
Fig.
3). Also here, the functionality was confirmed by means of transfection in SH-
SY5Y and
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neuro-2A cells and control of the eGFP expression in comparison to eGFP design
under
the control of the unaltered mThyl promoter or the promoters for human
Platelet-Derived
Growth Factor (human PDGF) and cytomegalovirus (CMV), as shown below.
Promoter properties can be different in vitro and in vivo. Thus, e.g., the
inherent
neuron-specific mThyl promoter produces a strong expression of the eGFP
reporter gene
after transfection of the hamster ovarian cell line CHO-Kl with an mThyl/eGFP
construct. On the one hand, to be able to characterize broadly the promoter
mTUB
according to the invention, and, on the other hand, to achieve the second part
of the
object according to the invention (neuron specificity), a transgenic mouse
strain (mTUB-
eGFP-tg) was generated, which expresses eGFP under the control of the mTUB
promoter. Figure 4 shows fluorescence micrographies of brain sections of this
mouse
strain. In the neurons of the transgenic mouse strain, a clear eGFP expression
could be
detected. Sections through the most varied organs of the transgenic mouse
strain showed
no eGFP-positive body cells except for eGFP-positive nerve cells. It can
clearly be
shown that the mTUB promoter expresses the gene, which is under its control,
in a
neuron-specific manner.
The mTUB-eGFP-transgenic mouse strain was generated as follows, whereby for
microinjection, the promoter-gene construct was incorporated in an insulator
cassette
(egg-p-globin insulator sequences), which is to facilitate the expression and
protect from
foreign regulation (plasmid pC2xINS, JSW Research).
1. Production and Preparation of the Vector for Microinjection:
0 Starting plasmid = pmTUB-eGFP (JSW Research)
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= Digestion with Not I, Pvu I and Nde I for obtaining the promoter-insert
construct
= mTUB-eGFP construct has Not I- or Nde I-ends; Nde I-end is made up
with polymerase I Klenow fragments
= Incorporation of the mTUB-eGFP construct in the insulator vector
pC2xINS that is opened with Not I and Pml I
= Sequencing of the cloning transitions
= Removal of the vector sequences (pKO Backbone) and linearization of the
insulator-mTUB-eGFP-insulator construct with Mlu I,
= Gel elution of the linear construct "insulator-mTUB-eGFP insulator"
(7280 bp)
2. Microinjection of the Linear Construct
For the microinjection, the linear insulator-mTUB-eGFP-insulator construct
was isolated from a 0.8% TAE-agarose gel without ethidium bromide,
purified and diluted with microinjection buffer to the extent that 1000 DNA
molecules were present per injection volume. The injection was carried out
according to standard protocol (Brem et al., 1985). To this end, 3-week-old
hormone-treated female CB6F1 mice were paired with C57BL/6 males in the
preliminary area. The resulting fertilized CB6F1 (B6) oocytes were removed
and cultivated until two clear pronuclei were visible. The purified DNA
construct was then injected into the pronucleus, followed by an overnight
cultivation until the two-cell stage was achieved. The two-cell embryos were
CA 02604241 2007-10-12
then implanted in pseudo-pregnant foster mice. After 18-19 days, the babies
were born.
The control gene eGFP was used to be able to characterize promoter properties
and transgene expression as quickly and simply as possible. The eGFP gene can
be
replaced by any other gene that is to be expressed in a neuron-specific manner
(see Fig. 3,
sequence of the mTUB promoter cassette).
In this case, the functional areas are correlated to the following nucleotide
ranges
in the sequence:
1- 222: 3'-Part of the mThyl 5'-UTR
223 - 291: 1. Nonameric mThyl promoter and flanking regions
292 - 345: mThyl Exon 1 a
537 - 545: 2. Nonameric mThyl promoter
592 - 733: mThyl Exon lb
346 - 1378: 5'-Part of mThyl-Intron A
1379 - 1392: Polylinker I
1393 - 2112: Control gene eGFP (not part of the disclosure)
The coding sequence, which extends from nucleotide 1393 to
2112, stands for the reporter gene eGFP, which is not part of the
disclosure. The late SV40 poly A sequence, which can be replaced
by any other poly A sequence, follows the reporter gene sequence.
2113 - 2130: Polylinker II
2131 - 2350: Late SV40 Poly A signal
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The table below shows a comparison of the mTUB promoter according to the
invention with the unaltered murine Thyl promoter, as well as the promoters of
the
human Platelet-Derived Growth Factor (hPDGF) and the cytomegalovirus (CMV).
Both
SH-SY5Y and neuro-2A cells were transfixed with the eGFP-reporter gene under
control
of the respective promoter, and the transfection efficiency (TE in %) as well
as the mean
fluorescence intensity (MFI in random units), both measured by means of
FacScan, were
compared (transfection efficiency in power = (fluorescent cells/non-
fluorescent cells) x
100).
mTUB-eGFP mThyl-eGFP hPDGF-eGFP CMV-eGFP
(TE-MFI) (TE / MFI) (TE / MFI) (TE / MFI)
SH-SY5Y 5% / 13 Not 0.2% / 16 10% / 110
Measurable/Not
Measurable
Neuro-2A 79% / 293 9% / 25 52% / 46 86% / 201
Thus, the mTUB promoter in both neuronal cell lines is far superior to the
mThyl
promoter both with respect to the transfection efficiency and the expression
intensity. In
SH-SY5Y cells, mTUB achieves 25 x-higher transfection efficiency than the
hPDGF
promoter with approximately the same average signal intensity, and in neuro-2A
cells,
mTUB achieves 1.5 x-higher transfection efficiency than the hPDGF promoter in
the case
of approximately 6 x average signal intensity. The transfection efficiency is
comparable
to that of the CMV promoter in the case of clearly higher average signal
intensity (in
Neuro-2A).
In Fig. 4, fluorescence-microscopic images of sections of the brain of the
transgenic mouse strain mTUB-eGFP-tg are shown in comparison to the
corresponding
non-transgenic strain C57BI6 ntg. In the brain sections, the eGFP-expressing
neurons of
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the transgenic mouse are discerned quite clearly in comparison to the eGFP-
negative
brain section of the non-transgenic mouse.
