AT501628A1 - PROMOTER FOR EXPRESSION OF FOREIGN GENES IN NEURONAL CELLS - Google Patents

Description

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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 certain 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. Gemntol 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 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) are expressed and overexpressed under Thy1 control are widely used. Both normal amyloid (E. Masliah and E. Rockenstein, J. Neural Transm Suppl. 2000; 59: 175-83) and mutated amyloid from known hereditary forms of Alzheimer's disease (J Davis et al., J Biol Chem 2004; 279 (19): 20296-306). • 9 • · 2

However, the Thy1 promoter has significant disadvantages. The considerable size of the Thy1 promoter makes it difficult for genetic engineering work, in particular for the installation of larger gene constructs, difficult to handle.

In view of the fact that the viral vectors which can also 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.

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 been found that the object described above can be achieved by the construction of a genetically engineered promoter which contains only certain (original or slightly altered) partial sequences of the mThyt promoter in combination with other regulatory elements.

The invention itself is characterized by the claimed features.

Starting from the mThyl expression cassette (Moechars et al., 1996, EMBO J. 15 (6), 1265-74), which has an initial size of about 8.2 kb, and which instead of the mThyl exons 2 to 4 has an Xho I linker With the help of restriction endonucleases, larger deletions were set and then smaller inserts were made, so that at the end a 1.6 kb promoter cassette was formed.

In the first step, the mThyl expression cassette was cleaved with Sma I, thereby removing the 5'-end adjacent to the mThyl 5 UTR. The resulting construct, into which the eGFP reporter gene (coded for a green fluorescent protein, which is harmless for the mouse and serves as a fluorescence-microscopic marker) 3 'to the end of the mThyl intron A, 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, whereby 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 regions were removed. Instead, a sequence consisting of the eGFP sequence and the "late SV40 poly A signal" 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). Once again, 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 ···· ··· ··········· The unchanged mThyl promoter or promoters for human Platelet-Derived Growth Factor (hPDGF) and cytomegalovirus (CMV), as shown later, are confirmed.

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 expression and protect against foreign regulation (plasmid pC2xlNS, JSW-Research). 1. Prepare and prepare the vector for microinjection: Starting plasmid = pmTUB-eGFP (JSW-Research) Digest with Not /, Pvu I and Nde I to obtain the promoter-insert construct mTUB-eGFP construct has emergency / 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) • 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 so that there were 1000 DNA molecules per injection volume. 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 DIMA construct was then injected into the pronucleus followed by obstruction 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 FIG. 3, sequence of the mTUB promoter cassette).

The functional regions are assigned to the following nucleotide regions in the sequence: 1-222 223-291 292-345 537-545 592-733 346-1378 1379-1392 1393-2112 2113-2130 2131-2350

3-memberedmethyl 5'-UTR 1. nonameramer mThyl promoter and flanking regions mThyl exon 1a 2. nonameramer mThyl promoter mThyl exon 1b

5 'part of the mThyl intron A polylinker I

Control gene eGFP (not part of the patent)

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 nontransgenic strain C57BI6 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 (25)

···· ··· ········································································································································································································································· A mammalian-derived nucleic acid sequence which effects neuron-specific transcription of a heterologous, 3 'downstream promoter nucleic acid sequence in both mammalian and non-mammalian cells. 2. Nucleic acid sequence consisting of the sections (a) of the rnlhyl 5 '- "untranslated region" (UTR), the 5'-UTR upstream 20bp comprehensive 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 exon la of the mouse Thy-1 gene. (c) the sequence of the second nonameric promoter consisting of 9 nucleotides of the mouse Thy-1 gene (d) the sequence of the exon lb of the mouse Thy-1 gene (e) 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 of (a), (b), (c), (d), ( e) and (f), wherein the promoter sequence, when combined with a heterologous gene sequence, transcribes this neuron-specific in both mammalian and non-mammalian cells. A nucleic acid construct comprising an expression cassette comprising the portions (a), (b), (c), (d), (e), (f) and (g) characterized according to claim 2 and a heterologous nucleic acid Sequence positioned between and operatively associated with portions (e) and (f). 4. Nucleic acid construct according to claim 3, characterized in that the section (g) is a therapeutic gene sequence. 5. nucleic acid construct comprising the nucleic acid sequence according to claim 2 as part of a plasmid. 6. Nucleic acid construct according to claim 3, characterized in that the heterologous nucleic acid sequence between the sections (e) and (f) encodes a protein. 7. nucleic acid construct comprising a vector and a nucleic acid molecule with the nucleic acid sequence according to claim 2. 8. Nucleic acid construct according to claim 7, characterized in that the vector is a virus or derived from a virus. An isolated cell or cell line comprising the promoter of claim 1. 10. An isolated cell or cell line comprising the nucleic acid sequence according to claim 2. 11. An isolated cell or cell line comprising a nucleic acid construct according to any one of claims 3 to 8. 12. A transgenic non-human animal comprising the promoter of claim 1. 13. A transgenic non-human animal comprising the nucleic acid sequence of claim 2. • • • • • • • • • • • • • • • 2 14. A transgenic non-human animal comprising a nucleic acid construct according to any one of claims 3 to claim 8. 15. A method for gene expression in neuronal and non-neuronal cells, which comprises the transformation / transfection of the cells with a vector comprising the nucleic acid sequence according to claim 2 or a nucleic acid construct according to one of claims 3 to 8. 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 is used as a polymeric carrier to a cationic polymer and / or a lipid (are). 18. The method according to claim 16, characterized in that is used as a polymeric carrier polyethyleneimine. 19. Use of a vector comprising the nucleic acid sequence of claim 2 or a nucleic acid construct according to any one of claims 3 to 8 for the preparation of injection solutions, ijwelehe) for injecting into the central nervous system or in the cerebrospinal fluid. 20. Use of a vector comprising the nucleic acid sequence according to claim 2 or a nucleic acid construct according to one of claims 3 to 8 for the preparation of injection solutions for the treatment of neuronal dysfunctions, such as stroke, ischaemia, epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease , Brain and spinal cord trauma, Amyotrophic lateral sclerosis. 21. Use of a vector comprising the nucleic acid sequence according to claim 2 or a nucleic acid construct according to any one of claims 3 to 8 for the preparation of injection solutions for the treatment of neurogenic disorders. 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 protein which is encoded by the nucleic acid sequence according to claim 2. 25. A pharmaceutical composition comprising a nucleic acid construct according to one of claims 3 to 8. JSW Research Forschungslabor GmbH represented by: .BEER iNreto i. Can Dungier Ä PATENTANWÄLTE DIP * ---------- by: DIPL.-ING. Dipl.-Ing. Dr. techn. (ID No .: 419)