AT501628B1 - PROMOTER FOR EXPRESSION OF FOREIGN GENES IN NEURONAL CELLS - Google Patents

Description

2 AT 501 628 B1

The invention relates to a promoter for the expression of foreign genes in neuronal cells.

A promoter represents a regulatory start-up sequence important to gene expression within the genome of each organism; it determines if, how and to what extent the transcription of a gene into messenger RNA (mRNA) takes place. There are strong and weak promoters (ie, those that cause the formation of numerous or less numerous mRNA transcripts of the gene), as well as constitutive (constantly active), inducible (ie, those that control transcription depending on particular conditions) and tissue-specific promoters ,

The neuron-specific Thy1 promoter of the mouse has already been used several times to generate transgenic mice that express foreign genes in their central nervous system. Such mouse strains represent important instruments, in particular for the investigation of the molecular mechanisms of neurodegenerative diseases and for the development of related potential therapies.

The mThyl promoter is an atypical promoter without TATA box. It consists of two identical promoter nonamers, each followed by a short untranslated exon (exons 1a and 1b). Exon 1b is followed by intron A followed by the first translated exon (exon 2). This is followed by exons 3 and 4, each separated by another intron (introns B and C). This sequence (first promoter-nonamer up to and including exon 4) is flanked by untranslated regions (5 and 3 'UTRs), with the 3' UTR containing the poly A signal. It is believed that the 5 'UTR, exons 1a and 1b and intron A have regulatory properties that may also be responsible for neuron specific expression of the promoter (E. Spanopoulou et al., Mol. Cell. Biol 8: 3847-3856 and 1991; 11 (4): 2216-2228). The original mThyl promoter also displayed expression in the thymus in addition to neuronal expression. By deleting the region from exon 2 to exon 4, thymus expression could be eliminated (HA Ingraham and GA Evans, Mol. Cell Biol 1986; 6 (8): 2923-2931; M. Vidal et al., EMBO 1990; 9 (3): 833-840).

The mThyl deletion deletion promoter of exon 2-4 was and is used to direct transgene expression in the generation of transgenic animals to specifically express the corresponding transgene in the neurons. For example, transgenic mice expressing a mutant alpha-synuclein protein found in a rare hereditary form of Parkinson's disease under mThyl promoter control exhibit similar symptoms as 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 mutant forms of microtubule-associated protein tau are found (Gotz, J., et al., J. Biol. Chem. 2001; 276 (1): 529-34). Animal models of Alzheimer's disease are widely used in which amyloid precursor protein (APP, the precursor protein of beta-amyloid, which forms the so-called plaques in the brains of Alzheimer's patients.) Both normal amyloid (E.Massliah et al Rockenstein, J Neural Transm Suppl. 2000; 59: 175-83) as well as mutated amyloid from known hereditary forms of Alzheimer's dementia (J Davis et al., J. Biol. Chem. 2004; 279 (19): 20296-306 ) are expressed or overexpressed under Thy1 control.

However, the Thy1 promoter has significant disadvantages. The considerable size of the Thy1 promoter makes it difficult to handle for genetic engineering work, in particular for the installation of larger gene constructs. In view of the fact that the viral vectors which can be used for the generation of transgenic animals can only absorb limited amounts of foreign DNA, the promoter size considerably restricts the size of the gene to be transferred. Furthermore, the Thy1 promoter is often not strong enough to effect expression of the foreign gene in the transgenic animals to the desirable extent. 3 AT 501 628 B1

It was therefore the object of the invention to modify the known mThyl promoter structure so that a) the promoter size is reduced so that its use for genetic engineering facilitates and the transmission of larger foreign genes or gene fragments is made possible with stable vectors, b) the Expression of this foreign DNA is significantly increased in the transgenic mice thus generated and c) the expression of the transgene to be controlled by the promoter is neuron-specific.

It has now turned out that the object described above can be achieved by the construction of a genetically modified promoter which contains only certain (original or slightly modified) partial sequences of the mThyl promoter in combination with other regulatory elements.

The invention itself is characterized by the claimed features.

The invention relates to a promoter comprising a nucleic acid sequence derived from mammalian Thy-1 gene which effects the neuron-specific transcription of a heterologous, 3 'down-to-promoter nucleic acid sequence in both mammalian and non-mammalian cells, consisting of the sections (a ) of the mThyl 5'-untranslated region (UTR), the 5'-UTR upstream 20bp sequence and the sequence of the first nonameric promoter consisting of 9 nucleotides and flanking regions of the mouse Thy-1 gene (b) of the sequence of the exon 1a of the mouse Thy-1 gene (c) of the sequence of the second nonameric promoter consisting of 9 nucleotides of the mouse Thy-1 gene (d) of the sequence of the exon 1b of the mouse Thy-1 gene (s) of complete or partial sequence of the intron A of the mouse Thy-1 gene (f) of the sequence of a poly A signal (g) of the mammalian Thy-1 gene promoter sequence, which corresponds to that described under (a), (b), (c), (d), (e) and (f) described hybridized, the Pro when combined with a heterologous gene sequence, this neuron-specific transcribed in both mammalian and non-mammalian cells.

The invention further relates to a nucleic acid construct consisting of the sections (a) of the mThyl 5'-untranslated region (UTR), the 5-UTR upstream 20 bp sequence and the sequence of the first nonamere promoter consisting of 9 nucleotides and flanking regions of the mouse Thy -1 gene (b) of the sequence of exon 1a of the mouse Thy-1 gene (c) of the sequence of the second nonameric promoter consisting of 9 nucleotides of the mouse Thy-1 gene (d) of the sequence of exon 1b of the Mouse Thy-1 gene (s) of the complete or partial sequence of the intron A of the mouse Thy-1 gene (f) of the sequence of a poly A signal (g) of the mammalian Thy-1 gene promoter sequence which with the sequence described under (a), (b), (c), (d), (e) and (f), wherein the promoter sequence, when combined with a heterologous gene sequence, this neuron specific in both mammalian and also transcribed in non-mammalian cells.

Advantageous embodiments of this nucleic acid construct are disclosed according to claims 3 to 7. 4 AT 501 628 B1

The invention further relates to a plasmid comprising a nucleic acid construct according to claim 2.

Furthermore, the invention relates to isolated cells or cell lines, which comprises either a promoter according to claim 1, or a nucleic acid construct according to any one of claims 2 to 7.

The invention also relates to a transgenic non-human animal comprising a promoter according to claim 1 or a nucleic acid construct according to any one of claims 2 to 7.

The invention further relates to a method for gene expression in neuronal and non-neuronal cells, wherein the transformation / transfection of the cells is carried out with a vector comprising a nucleic acid construct according to one of claims 2 to 7.

Further advantageous embodiments of this method are disclosed in claims 16 to 18.

The invention further relates to the use of a vector comprising a nucleic acid construct according to one of claims 2 to 7 for the preparation of injection solutions for the treatment of neuronal dysfunctions such as stroke, ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, brain and spinal cord trauma, Amyotrophic Lateral Sclerosis.

Further advantageous uses of this vector are disclosed according to claims 20 and 21.

The invention further relates to a kit for the expression of recombinant gene products comprising isolated cells or cell lines according to any one of claims 9 to 11.

Furthermore, the invention relates to a medicament comprising a promoter according to claim 1 or a nucleic acid construct according to any one of claims 2 to 7. The invention is explained in more detail below with reference to experimental data.

Starting from the mThyl expression cassette (Moechars et al., 1996, EMBO J. 15 (6), 126574) which has an initial size of about 8.2 kb and which contains an Xho IB linker instead of the mThyl exons 2 to 4 with the help of restriction endonucleases larger deletions and then made smaller insertions, so that in the end a only 1.6 kb promoter cassette was formed.

In the first step, the mThyl expression cassette was cleaved with Sma I, thereby removing the region adjacent to the 5 = end of the mThyl 5 UTR. The resulting construct, into which 3 = terminal of the mThyl intron A the eGFP reporter gene (encoded for a green fluorescent -resulting protein, which is harmless for the mouse and serves as a fluorescence-microscopic marker), was transfected into two neuronal cell lines ( SH-SY5Y and Neuro-2A) for promoter activity (eGFP expression).

In a second step, the sequence was digested with the restriction enzymes Nco I and Nde I, thereby removing portions of the mThyl intron A, the controls eGFP, the mThyl 3 = -UTR with the mThyl poly AB signal and the 3 = flanking mThyl sequence regions were. Instead, a sequence consisting of the eGFP sequence and the late SV40 poly A signal A was ligated with the reduced mThyl promoter sequence. The aforementioned steps are shown in Fig. 1, the correlation diagram mThyl gene sequence (A) / mThyl promoter cassette (B) / mTUB promoter cassette (C). Above the respective figure, the mThyl portion is labeled, below the non-mThyl portion.

The resulting sequence, excluding the eGFP gene sequence, was termed the mTUB promoter (mouse Thy1 Usable for Brain expression, sequence see Fig. 3). Again, the functionality was achieved by transfection into SH-SY5Y and neuro-2A cells and control of eGFP expression compared to eGFP constructs under the control of the unchanged mThyl promoter (s) for human platelet Derived Growth Factor (hPDGF) and cytomegalovirus (CMV), as shown below.

Promoter properties may be different in vitro and in vivo. For example, the intrinsic neuron-specific mThyl promoter after transfection of the hamster ovary cell line CHO-K1 with a mThyl / eGFP construct a strong expression of the eGFP reporter gene (unpublished own data). On the one hand to be able to characterize the mTUB promoter according to the invention comprehensively and on the other hand to solve 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. Appendix 3 shows fluorescence micrographs of brain slices of this mouse strain. In the neurons of the transgenic mouse strain a clear eGFP expression could be detected. Sections through various organs of the transgenic mouse strain showed no eGFP-positive cells other than eGFP-positive nerve cells. It can be clearly demonstrated that the mTUB promoter expresses the gene under its control neuron-specific.

The mTUB-eGFP transgenic mouse strain was generated as follows, whereby for microinjection the promoter-gene construct was incorporated into an insulator cassette (chicken-β-globin insulator sequences) which should facilitate the expression and protect against foreign regulation (plasmid pC2xlNS, JSW-Research). 1. Preparation and Preparation of the Vector for Microinjection: Starting plasmid = pmTUB-eGFP (JSW-Research) Digestion with Not I, Pvu I, and Nde I to Obtain the Promoter Insert Construct. MTUB-eGFP construct has Not I or nd / ends; Nde I tail is filled in with polymerase I Klenow fragment

• Incorporation of the mTUB-eGFP construct into the pC2xlNS insulator vector 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, (see Fig 2: construct before linearization) • Gelelution of the linear construct 'Insulator-mTUB-eGFP-insulator A (7280 bp) 2. Microinjection of the linear construct For microinjection, the linear insulator-mTUB-eGFP insulator construct was constructed from a 0.8% TAE Agarose gel without ethidium bromide isolated, purified and diluted with microinjection buffer so far that per DNA injection volume 1000 DNA molecules were present. The injection was carried out according to the standard protocol (Brem et al., 1985). 3 weeks old hormone-treated female CB6F1 mice were mated in advance with C57BL / 6 males. The resulting fertilized CB6F1 (B6) oocytes were removed and cultured until two clear pronuclei were visible. The purified DNA construct was then injected into the pronucleus followed by overnight culture to the 2-cell stage. The two-cell embryos were then implanted into pseudopregnant Foster mice. After 18-19 days the boys were born.

The control gene eGFP was used to characterize promoter properties and transgene expression as quickly and easily as possible. The eGFP gene can be replaced by any other neuron-specific gene to be expressed (see Figure 3, sequence of the mTUB promoter cassette).

The functional regions are assigned to the following nucleotide regions in the sequence.

Claims (24)

6 AT 501 628 B1 sen 1 - 222: 223-291: 292 - 345: 537 - 545: 592 - 733: 346- 1378: 1379- 1392 1393-2112 2113-2130 2131 -2350 3'-part of rnThyl 5 ' UTR; 1. nonameric mThyl promoter and flanking regions, mThyl exon 1a; 2. nonameric mThyl promoter; mThyl exon 1b; 5 'part of the mThyl intron A; Polylinker I; Controls eGFP (not part of the disclosure); Polylinker II; SV40 late poly A signal. The following table shows a comparison of the mTUB promoter according to the invention with the unchanged murine Thy1 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 transfected with the eGFP reporter gene under control of the respective promoter and the transfection efficiency (TE in%) and the mean fluorescence intensity (MFI in arbitrary units), both measured by FacScan, compared (Transfection efficiency in percent = (fluorescent cells / non-fluorescent cells) x 100). mTUB-eGFP (TE / MFI) mThyl-eGFP (TE / MFI) hPDGF-eGFP (TE / MFI) CMV-eGFP (TE / MFI) SH-SY5Y 5% / 13 Not measurable / Not measurable 0.2% / 16 10% / 110 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 in terms of both transfection efficiency and expression intensity. In SH-SY5Y cells, mTUB achieves a 25-fold higher transfection efficiency than the hPDGF promoter at approximately the same average signal intensity, in Neuro-2A cells a 1.5-fold higher transfection efficiency than the hPDGF promoter at approximately 6-fold average signal intensity. The transfection efficiency is comparable to that of the CMV promoter, with significantly higher average signal intensity (in Neuro-2A). Fig. 4 shows fluorescence microscopic images of grafts of the transgenic mouse strain mTUB-eGFP-tg compared to the corresponding non-transgenic strain C57B16 ntg. The cerebral sections clearly show the eGFP-expressing neurons of the transgenic mouse compared to the eGFP-negative brain section of the non-transgenic mouse. Claims 1. A promoter comprising a mammalian Thy-1 gene-derived nucleic acid sequence which effects neuron-specific transcription of a heterologous 3 'down-to-promoter nucleic acid sequence in both mammalian and non-mammalian cells, consisting of the portions (a ) of the mThyl 5'-untranslated region (UTR), the 5'-UTR upstream 20bp sequence and the sequence of the first nonameric promoter consisting of 9 nucleotides and flanking regions of the mouse Thy-1 gene (b) of the sequence of the exon 1a of the mouse Thy-1 gene (c) the sequence of the second nonameric promoter consisting of 9 nucleotides of the mouse Thy-1 gene 7 AT 501 628 B1 (d) of the sequence of the exon 1b of the mouse Thy-1 Gene (s) of the complete or partial sequence of the intron A of the mouse Thy-1 gene (f) of the sequence of a poly A signal (g) of the mammalian Thy-1 gene promoter sequence which is identical to that described under (a) , (b), (c), (d), (e) and (f) described hybridi hybridi wherein the promoter sequence, when combined with a heterologous gene sequence, transcribes this neuron-specific in both mammalian and non-mammalian cells. 2. Nucleic acid construct consisting of the sections (a) of the mThyl 5-untranslated region (UTR), the 5'-UTR upstream 20 bp sequence and the sequence of the first nonamere promoter consisting of 9 nucleotides and flanking regions of the mouse Thy-1 Gene (b) of the sequence of the exon 1a of the mouse Thy-1 gene (c) of the sequence of the second nonameric promoter consisting of 9 nucleotides of the mouse Thy-1 gene (d) of the sequence of the exon 1b of the mouse Thy -1-gene (s) of the complete or partial sequence of the intron A of the mouse Thy-1 gene (f) of the sequence of a poly A signal (g) of the mammalian Thy-1 gene promoter sequence, which corresponds to the (a), (b), (c), (d), (e) and (f) hybridized, wherein the promoter sequence, when combined with a heterologous gene sequence, this neuron specific in both mammalian and non-mammalian Mammalian cells transcribed. A nucleic acid construct comprising an expression cassette comprising the portions (a), (b), (c), (d), (e), (f) and (g) characterized as claimed in claim 2, and a heterologous nucleic acid sequence existing between the portions (e) and (f) are positioned and operatively associated with them. 4. Nucleic acid construct according to claim 3, characterized in that the section (g) is a therapeutic gene sequence. 5. Nucleic acid construct according to claim 3, characterized in that the heterologous nucleic acid sequence between the sections (e) and (f) encodes a protein. 6. 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 and a vector. 7. Nucleic acid construct according to claim 6, characterized in that the vector is a virus or derived from a virus. 8. A plasmid comprising a nucleic acid construct according to claim 2. An isolated cell or cell line comprising a promoter according to claim 1. 10. An isolated cell or cell line comprising a nucleic acid construct according to claim 2. 11. An isolated cell or cell line comprising a nucleic acid construct according to one of claims 3 to 7. 12. A transgenic non-human animal comprising a promoter according to claim 1. 13. A transgenic non-human animal comprising a nucleic acid construct according to claim 2. 14. Transgenic non-human animal comprising a nucleic acid construct according to one of the claims 8 to 50 of claim 1. 15. A method for gene expression in neuronal and non-neuronal cells, which comprises the transformation / transfection of the cells with a vector comprising a nucleic acid construct according to one of claims 2 to 7. 16. The method according to claim 15, characterized in that the vector for the gene transport is mixed with a polymeric carrier substance. 17. The method according to claim 16, characterized in that a cationic polymer and / or a lipid is (are) used as the polymeric carrier substance. 18. The method according to claim 16, characterized in that is used as a polymeric carrier polyethyleneimine. 19. Use of a vector comprising a nucleic acid construct according to one of claims 2 to 7 for the preparation of injection solutions for the treatment of neuronal dysfunctions, such as stroke, ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, brain and spinal cord trauma, amyotrophic lateral sclerosis. 20. Use of a vector comprising a nucleic acid construct according to one of claims 2 to 7 for the preparation of injection solutions for the treatment of neurogenic disorders. 21. Use according to claim 19 or 20, characterized in that the injection solutions are suitable for injecting into the central nervous system or in the cerebrospinal fluid. 22. Kit for the expression of recombinant gene products comprising isolated cells or cell lines according to one of claims 9 to 11. 23. A pharmaceutical composition comprising a promoter according to claim 1. 24. A pharmaceutical composition comprising a nucleic acid construct according to any one of claims 2 to 7. In addition, 3 sheets drawings