Capping prone RNA polymerase enzymes and their applications
09540671 ยท 2017-01-10
Assignee
Inventors
Cpc classification
A61P25/18
HUMAN NECESSITIES
C12P19/34
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61P25/14
HUMAN NECESSITIES
C12Y201/01056
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P7/04
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
International classification
C12P19/34
CHEMISTRY; METALLURGY
Abstract
The invention provides a chimeric enzyme comprising at least one catalytic domain of a RNA triphosphatase, at least one catalytic domain of a guanylyltransferase, at least one catalytic domain of a N.sup.7-guanine methyltransferase, and at least one catalytic domain of a DNA-dependant RNA polymerase. The invention also provides pharmaceutical composition comprising said chimeric enzyme and uses of said chimeric enzyme.
Claims
1. A hetero-oligomeric enzyme comprising: a catalytic domain of a RNA triphosphatase; a catalytic domain of a guanylyltransferase; a catalytic domain of a N7-guanine methyltransferase; and a catalytic domain of a DNA-dependent RNA polymerase, wherein the catalytic domain of a DNA-dependent RNA polymerase is linked covalently or non-covalently with the catalytic domain of a RNA triphosphatase, the catalytic domain of a guanylyltransferase, or the catalytic domain of a N7-guanine methyltransferase, and wherein the hetero-oligomeric enzyme is non-natural.
2. The hetero-oligomeric enzyme according to claim 1, wherein the catalytic domain of a N7-guanine methyltransferase and the catalytic domain of a DNA-dependent RNA polymerase are included in a monomer.
3. The hetero-oligomeric enzyme according to claim 1, wherein the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase are included in a monomer.
4. The hetero-oligomeric enzyme according to claim 1, wherein the catalytic domain of a RNA triphosphatase, the catalytic domain of a guanylyltransferase and the catalytic domain of a DNA-dependent RNA polymerase are included in a monomer.
5. The hetero-oligomeric enzyme according to claim 1, further comprising a domain that enhances the activity of at least one catalytic domain of the hetero-oligomeric enzyme.
6. The hetero-oligomeric enzyme according to claim 1, wherein at least one of the catalytic domain of a N7-guanine methyltransferase, the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase is a catalytic domain of the vaccinia mRNA capping enzyme.
7. The hetero-oligomeric enzyme according to claim 1, comprising: a first monomer comprising a catalytic domain of a N7-guanine methyltransferase and a catalytic domain of a DNA-dependent RNA polymerase, and a second monomer comprising a catalytic domain of a RNA triphosphatase and a catalytic domain of a guanylyltransferase, wherein the catalytic domain of a N7-guanine methyltransferase is a catalytic domain of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase are catalytic domains of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and wherein the hetero-oligomeric enzyme further comprises a 31-kDa subunit encoded by the vaccinia virus D12L gene.
8. The hetero-oligomeric enzyme according to claim 1, comprising: a first monomer comprising a catalytic domain of a N7-guanine methyltransferase, and a second monomer comprising a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, and a catalytic domain of a DNA-dependent RNA polymerase, wherein the catalytic domain of a N7-guanine methyltransferase is a catalytic domain of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase are catalytic domains of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and wherein the hetero-oligomeric enzyme further comprises a 31-kDa subunit encoded by the vaccinia virus D12L gene.
9. A method for producing an RNA molecule with a 5-terminal m.sup.7GpppN cap, comprising contacting a DNA sequence encoding the RNA molecule with the hetero-oligomeric enzyme according to claim 1, wherein the DNA sequence is operatively linked to a promoter for the catalytic domain of a DNA-dependent RNA polymerase.
10. A hetero-oligomeric enzyme comprising: a monomer comprising a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, and a catalytic domain of a N7-guanine methyltransferase; and a complex comprising a catalytic domain of a DNA-dependent RNA polymerase, wherein the monomer and the complex are linked covalently or non-covalently, and wherein the hetero-oligomeric enzyme is non-natural.
11. The hetero-oligomeric enzyme according to claim 10, comprising a complex comprising a catalytic domain of a bacterial DNA-dependent RNA polymerase.
12. The hetero-oligomeric enzyme according to claim 10, comprising a complex comprising a catalytic domain of the DNA-dependent RNA polymerase of Escherichia coli.
13. A method for producing an RNA molecule with a 5-terminal m.sup.7GpppN cap, comprising contacting a DNA sequence encoding the RNA molecule with the hetero-oligomeric enzyme according to claim 10, wherein the DNA sequence is operatively linked to a promoter for the catalytic domain of a DNA-dependent RNA polymerase.
Description
BRIEF DESCRIPTION OF DRAWINGS
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(16) The present invention will be explained in detail with examples in the following, but the technical scope of the present invention is not limited to these examples.
EXAMPLES
Example 1
Example of Active Monomeric Chimeric Enzyme Np868R-T7RNAP
(17) I. Plasmids
(18) One plasmid has been synthesized, which encode for fusions between NP868R, the mRNA capping enzyme of the African Swine Fever Virus, and the wild-type phage DNA-dependent RNA polymerase of the bacteriophage T7. The capping enzyme was fused to the amino-terminal end of the T7 RNA polymerase via a (Gly.sub.3Ser).sub.4 linker. The pNP868R-T7RNAP plasmid was used to assess the activity of the encoded enzyme by a firefly luciferase gene reporter expression assay (
(19) The plasmid encoding for the NP868R-T7RNAP and T7RNAP (T7 RNA polymerase) enzymes were synthesized in four steps by GeneArt AG (Regensburg, Germany). The protein sequence encoded by pT7RNA plasmid corresponds to SEQ ID NO:19. The protein sequence encoded by pNP868R-T7RNAP plasmid corresponds to SEQ ID NO:20. Firstly, a DNA fragment containing the T7 RNA polymerase promoter and the multiple cloning site (MCS) was removed from the pCMV-Script plasmid (Stratagene, La Jolla, Calif. USA). Secondly, a cassette was introduced in the pCMV-Script plasmid between its CMV promoter and its SV40 polyadenylation signal. This cassette consisted of the Lac operator stem-loop (Gilbert and Maxam 1973), a MCS, a poly[A]-tract, and a T class-I hairpin terminator signal (Lyakhov, He et al. 1997). Thirdly, the Kozak consensus sequence for initiation of translation (Kozak 1987), followed by the entire open-reading frame (ORF) of the NP868R-T7RNAP or T7RNAP enzymes were assembled from synthetic oligonucleotides using a PCR-based method, cloned and fully sequence verified. The ORF of the NP868R-T7RNAP (SEQ ID NO:21) and of the ORF of the T7RNAP (SEQ ID NO:22) were optimized by altering for preferred codon usage, removing of cis-acting elements such as cryptic splice sites and poly(A) signals, as well as improving mRNA stability by removal of direct repeats and secondary structure elements. Fourthly, the entire ORFs of each NP868R-T7RNAP or T7RNAP were subcloned in the MCS of the cassette, resulting in the pNP868R-T7RNAP plasmid and the pT7RNAP plasmid. As a consequence of the construction strategy, two additional amino-acids (Glu, Phe) were added immediately downstream to the ATG of the Kozak sequence, two other were added immediately upstream to the (Gly.sub.3Ser).sub.4 linker (Gly, Pro), and two immediately downstream to this linker (Leu, Glu) of the NP868R-T7RNAP enzyme. Finally, the pNP868R-T7RNAP and pT7RNAP plasmids had the following design (
(20) Two plasmids encoding for the firefly luciferase reporter gene were synthesized by Eurofins/MWG/Operon (Ebersberg, Germany). The pET-22b(+)RNAPp-Luciferase plasmid (named pT7p-Luciferase thereafter) was designed to assay the activity of the chimeric enzyme according to the invention. A test sequence was introduced in the pET-22b(+) backbone (Novagen, San Diego, Calif. USA), which consisted of an array of RNA polymerase promoters (T7, T3 and SP6 phage RNAP promoters, followed by the E. coli ribosomal rrnD1 promoter), a Lac operator stem-loop sequence, the entire ORF of the firefly luciferase, a poly[A]-track, a hepatitis-D ribozyme encoding sequence for RNA auto-cleavage (Conzelmann and Schnell 1994; Garcin, Pelet et al. 1995; Bridgen and Elliott 1996; Schurer, Lang et al. 2002; Walker, Avis et al. 2003) and the T terminator for phage RNA polymerase transcription (
(21) II. Cell Culture and Transfection
(22) The Human Embryonic Kidney 293 cells (HEK-293, ATCC CRL 1573) were grown at 37 C. with 5% CO2. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 3.97 mM L-alanyl-L-glutamine (substituted on a molar equivalent basis for L-glutamine), 10% fetal bovine serum (FBS), 1% non-essential amino-acids, 1% penicillin and streptomycin, and 0.2% fungizone.
(23) The day before transfection, HEK-293 cells were plated in 24 well plates at densities of approximately 810.sup.4 cells per well. One hour prior to transfection, the medium was changed to fresh complete medium without antibiotics. Transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif. USA) according to manufacturer's recommendations. In brief, plasmid DNA diluted in Opti-MEM I reduced serum medium (Invitrogen, Carlsbad, Calif. USA) and mixed with Lipofectamine 2000, were added to the cell medium. Following transfection, cells were incubated up to 120 hours prior to testing for luciferase and SEAP gene reporter expression.
(24) Cells were co-transfected with the pT7RNAP or pNP868R-T7RNAP (0.4 g DNA/well and 1 L/well Lipofectamine 2000), together with the pT7p-Luciferase reporter plasmid (0.4 g DNA/well and 1 L/well Lipofectamine 2000). A series of other transfection conditions were used as negative controls and included: (a) the same co-transfection as before, except that the pT7p-Luciferase was digested by the BamHI restriction enzyme, which disrupts the physical connection between the luciferase ORF and the T7 promoter, (b) the pNP868R-T7RNAP or pT7RNAP plasmids alone, (c) the pT7p-Luciferase reporter plasmid digested or not alone, (d) the transfection reagent alone (i.e. Lipofectamine 2000). Cells were also transfected with the pORF-eSEAP plasmid (InvivoGen, San Diego, Calif.; used to normalize for transfection efficacy), as well as with the pCMV-T7RNAP plasmid (used as an active comparator).
(25) III. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(26) The firefly luciferase luminescence was assayed with the Luciferase Assay System according to manufacturer's recommendations (Promega, Madison Wis. USA). In brief, HEK-293 cells were lysed in Cell Culture Lysis Reagent (CCLR) lysis buffer, and then centrifuged at 12,000g for 2 minutes at 4 C. Luciferase Assay Reagent (Promega; 100 l/well) was added to supernatant (20 l/well). Luminescence readout was taken on a luminometer reader (Fluostar; BMG Labtech, Offenburg Germany) according to the manufacturer's instructions.
(27) The expression of pORF-eSEAP plasmid was used to normalize for transfection efficiency. This plasmid encodes for the secreted placental alkaline phosphatase (SEAP), which was assayed for enzymatic activity in cell culture medium using the Quanti-Blue colorimetric enzyme assay kit (InvivoGen, San Diego, Calif.) at selected time points. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU; relative light units), then divided by SEAP absorbance to normalize for transfection efficacy (OD, optic density) ratio.
(28) IV. Statistical Analysis
(29) All statistical analyses were performed using Student's t two-tailed test adjusted by Holm-Bonferroni correction for multiple testing, if appropriate. A p-value of less than 0.05 was regarded as being significant.
(30) V. Gene Reporter Expression Assay
(31) A firefly luciferase reporter luminescence assay was used to assess the translatability of mRNA generated by the chimeric enzyme according to the invention or T7RNAP enzyme. The co-transfection of the pT7RNAP and pT7p-Luciferase plasmids triggered low but detectable luciferase expression signal in comparison to cells co-transfected with the pT7RNAP/BamHI-digested version of the pT7p-Luciferase plasmid (which therefore demonstrate that luciferase gene reporter expression is driven by the phage T7 promoter;
(32) The pNP868R-T7RNAP plasmid was cotransfected with the pT7p-Luciferase plasmid and tested under same conditions as above. At peak, approximately 23-fold higher luciferase expression signal was observed with pNP868R-T7RNAP/pT7p-Luciferase than with the pT7RNAP/pT7p-Luciferase plasmids (
(33) In summary, the activity of the chimeric NP868R-T7RNAP enzyme according to the invention encoded by the pNP868R-T7RNAP plasmid has been demonstrated using a firefly luciferase reporter luminescence assay. The specificity of the present findings is supported by a series of controls, which suggest that both the mRNA capping and DNA-dependent RNA polymerase enzymatic activities of the NP868R-T7RNAP enzyme are retained when expressed in HEK-293 cells.
(34) VI. Gene Reporter Expression Assay in Alpha-Amanitin Treated Cells
(35) To further demonstrate that the transcription by pNP868R-T7RNAP is dependent of its phage DNA-dependent T7 RNA polymerase moiety, gene transfection assays were also performed in -amanitin treated cells. Alpha-amanitin is a specific inhibitor of the nuclear RNA polymerase II (Jacob, Sajdel et al. 1970; Kedinger, Gniazdowski et al. 1970; Lindell, Weinberg et al. 1970), which binds its Rpb1 subunit (Bushnell, Cramer et al. 2002). In contrast, alpha-amanitin has no effect on transcription by the phage T7 RNA polymerase which was used to engineer the NP868R-T7RNAP chimeric enzyme according to the invention (Kupper, McAllister et al. 1973; Engleka, Lewis et al. 1998).
(36) To initiate the expression of the NP868R-T7RNAP enzyme, which is driven by the RNA polymerase II-dependent CMV promoter, cells were transfected with the pNP868R-T7RNAP 24 hours before addition of -amanitin to cell medium (at concentrations ranging from 0 to 20g/ml) and a second transfection with the pT7p-Luciferase plasmid (
(37) As expected, -amanitin nearly completely abolished firefly luciferase gene reporter expression of pCMV-Luciferase transfected cells (
(38) The present findings, therefore confirms that the transcription by NP868R-T7RNAP enzyme depends of the enzymatic activity of its phage T7 DNA-dependent RNA polymerase moiety.
(39) VII. Immunofluorescence
(40) The subcellular distribution of the NP868R-T7RNAP enzyme was investigated by indirect immunofluorescence. HEK-293 cells were plated in 24 well plates at 810.sup.4 cells/well, on poly-L-lysine coated coverslips (BD BioCoat; Bioscience, Mississauga, ON USA), then transfected as previously described. Six and 24-hours after transfection, cells were washed in phosphate buffered saline (PBS), and then fixed in 4% paraformaldehyde for 15 minutes. After fixation, cells were washed with PBS, and then permeabilised for 30 minutes in PBS containing 5% goat serum (Invitrogen), 0.1% Triton X-100 and 0.02% sodium azide. Cells were incubated overnight at 4 C. with the mouse monoclonal antibody raised against T7 RNA Polymerase (1:200, Novagen). After extensive washing with PBS, cells were incubated for 3 hours at room temperature with fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse IgG (Sigma-Aldrich). Cell nuclei were stained with 4-6-Diamidino-2-phenylindole (DAPI) for 5 minutes. Cells were then washed and mounted in the anti-fade medium Mowiol 4-88 (Calbiochem, Gibbstown, N.J. USA). Cells were analyzed by using an epifluorescence microscope with appropriate filters.
(41) As expected, a weak but detectable FITC signal was observed at both 6 and 24-hours in the cytoplasm of cells transfected with the pNP868R-T7RNAP plasmid, while their nuclei were stained by DAPI.
(42) VIII. Cell Viability, Cytotoxicity and Apoptosis Assays
(43) The ApoTox-Glo Triplex Assay (Promega, Madison Wis.) was used to investigate whether the expression of the NP868R-T7RNAP enzyme impair viability, or induce toxicity or apoptosis of transfected cells. Two protease activities were assayed by fluorescence: one is a marker of cell viability (i.e. the peptide substrate GF-AFC), and the other is a marker of cytotoxicity (i.e. the peptide substrate bis-AAF-R110). Apoptosis was assayed by the luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity.
(44) Cell culture and transfections were performed as previously, except that HEK-293 cells were plated in 96-well plates at densities ranging of 1.210.sup.4 cells per well. Cells were transfected with the pNP868R-T7RNAP plasmid, the pT7RNAP plasmid, or the transfection reagent only. ApoTox-Glo Triplex Assay was performed according to manufacturer's recommendations. In brief, at selected time points, the viability/cytotoxicity reagent containing both GF-AFC Substrate and bis-AAF-R110 substrates were added to the wells and incubated for 30 minutes at 37 C., before fluorescence assessment at two different wavelength sets for viability and cytotoxicity. The caspase reagent was then added to all wells, and luminescence was measured after 30 minutes incubation at room temperature. Statistical analysis was performed as above. Cell viability, cytotoxicity and apoptosis levels were expressed as the luminescence/fluorescence signal in studied cells subtracted for luminescence/fluorescence in untreated cells.
(45) As previously reported (Patil, Rhodes et al. 2004), the cell viability, cytotoxicity and apoptosis were significantly impaired in cells treated with the transfection reagent (i.e. Lipofectamine 2000) as compared to untreated cells (
(46) In conclusion, no obvious difference in cytotoxicity, cell viability, and apoptosis of the NP868R-T7RNAP enzyme can be demonstrated in comparison to T7RNAP, which has no recognized capping enzymatic activity.
Example 2
Examples of Active Monomeric Chimeric ENZYMES NP868R-T3RNAP and NP868R-SP6RNAP
(47) Two other types of monomeric chimeric enzymes according to the invention have been generated, which consist of NP868R, the monomeric mRNA capping enzyme of the African Swine Fever Virus, fused to the amino-terminal end of the wild type T3 or SP6 monomeric bacteriophage DNA-dependent RNA polymerases, via the flexible linker (Gly.sub.3Ser).sub.4.
(48) I. Methods
(49) The sequences used to generate said monomeric chimeric enzymes were assembled from synthetic oligonucleotides using a PCR-based method, cloned and fully sequence verified. These sequences were subcloned in the pCMV-Script plasmid containing the subcloning cassette previously described. Finally, all the plasmids used for expression had the similar design: CMV IE1 promoter/enhancer promoter, Kozak sequence followed by the ORFs, poly[A]-track, T terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal (
(50) As a consequence of the subcloning strategy, amino-acids were added immediately downstream to the ATG of the Kozak sequence encoded by the plasmids (Glu-Phe-Leu-Glu for pT3RNAP and pSP6RNAP; Glu-Phe for pNP868R-T3RNAP and pNP868R-SP6RNAP). In addition, two amino-acids were added immediately upstream (Gly-Pro for pNP868R-T3RNAP and pNP868R-SP6RNAP) or downstream to the (Gly.sub.3Ser).sub.4 linker (Leu-Glu for pNP868R-T3RNAP and pNP868R-SP6RNAP).
(51) HEK-293 cells were grown as previously described in 24-wells plates and transfected using the Lipofectamine 2000 reagent, and the appropriate plasmids (0.4 g DNA/well, plus 1 L/well lipofectamine 2000, per transfected plasmid). The firefly luciferase luminescence was assayed as previously described using the pT7p-Luciferase (which also contains both the T3 and SP6 promoters) and the Luciferase Assay System. The expression of pORF-eSEAP plasmid was used to normalize for transfection efficacy as previously described. Statistical analyses were performed using Student's t two-tailed test adjusted by Holm-Bonferroni correction for multiple testing, if appropriate. A p-value of less than 0.05 was regarded as being statistically significant.
(52) II. Results
(53) As shown in
(54) These results demonstrate the activity of different types of monomeric chimeric enzymes according to the invention.
Example 3
Examples of Active Dimeric and Trimeric Chimeric Enzymes
(55) Different types of active oligomeric chimeric enzymes according to the invention have been generated as shown in
(56) I. Methods
(57) The sequences used to generate the chimeric enzymes were assembled from synthetic oligonucleotides using a PCR-based method, cloned and fully sequence verified. These sequences were subcloned in the pCMV-Script plasmid with the subcloning cassette previously described. Finally, all the plasmids used for expression had the similar design: CMV IE1 promoter/enhancer promoter, Kozak sequence followed by the open-reading frames (ORFs), poly[A]-track, T terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal (
(58) As a consequence of the subcloning strategy, two amino-acids were added immediately downstream to the ATG of the Kozak sequence of some plasmids (Leu-Glu for pT7RNAP; Glu-Phe for pNP868R, pD1R, pD12L, pD1R-T7RNAP, and pD12L-T7RNAP), immediately downstream to the leucine-zipper sequences (Leu-Glu for pEE.sub.1234L-T7RNAP; Glu-Phe for pRR.sub.1234L-NP868R, pRR.sub.1234L-D1R and pRR.sub.1234L-D12L), and at the carboxyl-terminal end of some encoded proteins (Gly-Pro for pNP868R, pRR.sub.1234L-NP868R, pD1R, pD12L, pRR.sub.1234L-D1R and pRR.sub.1234L-D12L). In addition, two amino-acids were added immediately upstream (Gly-Pro for pD1R-T7RNAP and pD12L-T7RNAP) or downstream to the (Gly.sub.3Ser).sub.4 linker (Leu-Glu for pD1R-T7RNAP and pD12L-T7RNAP).
(59) As previously described, the Human Embryonic Kidney 293 cells (HEK-293) were grown in 24-wells plates. HEK-293 cells were transfected using the lipofectamine 2000 reagent, and the appropriate plasmids (0.4 g DNA/well, plus 1 L/well lipofectamine 2000, per transfected plasmid) as previously described. The firefly luciferase luminescence was assayed as previously described using the pET-22b(+)T7RNAPp-Luciferase reporter plasmid (designated pT7p-Luciferase thereafter) and the Luciferase Assay System. The expression of pORF-eSEAP plasmid was used to normalize the transfection efficacy as previously described.
(60) Gene reporter expression was expressed as the luciferase luminescence in studied condition subtracted by the luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Statistical analyses were performed using Student's t two-tailed test adjusted by Holm-Bonferroni correction for multiple testing, if appropriate. A p-value of less than 0.05 was regarded as being statistically significant.
(61) II. Results
(62) II.1 Heterodimeric RR.sub.1234L-NP868R/EE.sub.1234L-T7RNAP Chimeric Enzyme
(63) The activity of the heterodimeric enzyme RR.sub.1234L-NP868R/EE.sub.1234L-T7RNA chimeric enzyme (encoded by pRR.sub.1234L-pNP868R and pEE.sub.1234L-T7RNAP plasmids, respectively) has been demonstrated. This heterodimeric enzyme is generated by non-covalent linkage between the monomeric African Swine Fever Virus mRNA capping enzyme pNP868R and the monomeric T7 RNA polymerase, via the EE.sub.1234L and RR.sub.1234L leucine-zippers (
(64) As expected, the transfection of the plasmid encoding for the African Swine Fever Virus mRNA capping enzyme alone with or without leucine-zipper sequences (i.e. pRR.sub.1234L-NP868R and pNP868R, respectively) do not induce any detectable luciferase reporter gene expression (
(65) The HEK293 cells co-transfected with the pRR.sub.1234L-NP868R and pEE.sub.1234L-T7RNA plasmids (encoding for NP868R and T7RNAP with leucine-zippers), together with the reporter pT7p-luciferase plasmid, show strong luciferase reporter gene expression signal, which is 87% to that of pCMV-T7RNAP plasmid (non-statistically significant difference, Student's t-test; (
(66) These results demonstrate the activity of heterodimeric chimeric enzymes according to the invention and that the non-covalent linkage between NP868R and T7RNAP by leucine-zippers increases significantly the expression of the gene reporter driven by said chimeric enzymes.
(67) II.2 Heterodimeric D1R/D12L-T7RNAP and D12L/D1R-T7RNAP Chimeric Enzymes
(68) The activity of other types of heterodimeric chimeric enzyme has also been demonstrated, using the vaccinia mRNA capping enzyme.
(69) By itself, the vaccinia mRNA capping enzyme is a heterodimer consisting of: (i) a 95 kDa subunit encoded by the vaccinia virus D1R gene (genomic sequence ID# NC_006998.1; GeneID#3707562; UniProtKB/Swiss-Prot ID# YP_232988.1), designated hereafter as D1R, which has RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine methyltransferase enzymatic activities (Cong and Shuman 1993; Niles and Christen 1993; Higman and Niles 1994; Mao and Shuman 1994; Gong and Shuman 2003), (ii) and a 31-kDa subunit encoded by the vaccinia virus D12L gene (genomic sequence ID# NC_006998.1; GeneID#3707515; UniProtKB/Swiss-Prot ID#YP_232999.1), designated hereafter as D12L, which has no intrinsic enzymatic activity, but enhances drastically the RNA N7-guanine methyltransferase activity of the D1R subunit (Higman, Bourgeois et al. 1992; Higman, Christen et al. 1994; Mao and Shuman 1994).
(70) D1R or D12L were fused to the amino-terminal end of the T7 RNA polymerase, via the (Gly.sub.3Ser).sub.4 linker (encoded by D1R-T7RNAP or D12L-T7RNAP, respectively). When co-expressed, each fusion proteins, together with the other vaccinia mRNA capping enzyme subunit (encoded by pD12L or D1R, respectively), generate two different heterodimeric chimeric enzymes designated as D12L/D1R-T7RNAP and D1R/D12L-T7RNAP, respectively (
(71) These results demonstrate the activity of different types of heterodimeric chimeric enzymes according to the invention and that covalent linkage between the subunits of the vaccinia mRNA capping enzyme and the T7RNAP stimulates significantly the gene reporter expression. As also expected, in presence of the reporter pT7p-luciferase plasmid, the expression of D1R and/or D12L without T7 RNA polymerase induces virtually no detectable luciferase expression.
(72) II.3 Heterotrimeric D12L/RR.sub.1234L-D1R/EE.sub.1234L-T7RNAP and D1R/RR.sub.1234L-D12L/EE.sub.1234L-T7RNAP Chimeric Enzymes
(73) The activity of heterotrimeric chimeric enzyme has also been demonstrated.
(74) The basic RR.sub.1234L leucine zipper was fused to the amino-terminal ends of either the D1R or D12L subunits of the vaccinia virus mRNA capping enzyme (encoded by pRR.sub.1234L-D1R and RR.sub.1234L-D12L, respectively), while the complementary acidic EE.sub.1234L leucine-zipper was added to the amino-terminal end of T7 RNA polymerase (encoded by the pEE.sub.1234L-T7RNA plasmid). The co-expression of pEE.sub.1234L-T7RNAP, together with either pRR.sub.1234L-D1R or pRR.sub.1234L-D12L, plus the other vaccinia mRNA capping enzyme subunit (pD12L and pD1R plasmids, respectively), therefore generate two different heterotrimeric CCPP enzymes, designated as D12L/RR.sub.1234L-D1R/EE.sub.1234L-T7RNAP and D1R/RR.sub.1234L-D12L/EE.sub.1234L-T7RNAP, respectively (
(75) The T7 RNA polymerase displayed 7-fold higher luciferase gene reporter signal when coexpressed with the D1R/D12L subunits of the vaccinia virus mRNA capping enzyme than in their absence. These results are therefore in line with those obtained by the vaccinia virus/bacteriophage RNAP hybrid expression system, in which the translatability of uncapped T7 transcripts is increased by the expression of the vaccinia mRNA capping enzyme provided by a recombinant virus (Fuerst, Niles et al. 1986; Fuerst, Earl et al. 1987; Elroy-Stein, Fuerst et al. 1989; Fuerst, Fernandez et al. 1989; Fuerst and Moss 1989; Elroy-Stein and Moss 1990).
(76) A strong luciferase gene reporter signal was shown in HEK-293 cells expressing either the D1R/RR.sub.1234L-D12L/EE.sub.1234L-T7RNAP or the D12L/RR.sub.1234L-D1R/EE.sub.1234L-T7RNAP CCPP enzymes, in presence of the reporter pT7p-luciferase plasmid (
(77) These results demonstrate the activity of heterotrimeric chimeric enzymes according to the invention and that the non-covalent linkage between any of the subunits of the vaccinia mRNA capping enzyme and the T7 RNA polymerase increases significantly the gene reporter expression.
(78) III. Conclusion
(79) These present results show the activity of different types of heterodimeric and heterotrimeric chimeric enzymes according to the invention, generated by covalent or non-covalent linkage.
(80) The present results also provide evidences that covalent or non-covalent linkage between the different catalytic domain of the chimeric enzyme and in particular between capping enzymes and RNA polymerases allows the optimization of the gene reporter expression by the chimeric enzymes.
Example 4
Stimulation of Luciferase Reporter Gene Expression by Silencing Sequences Against the Cellular RNA Polymerase II and Capping Enzyme
(81) I. Methods
(82) HEK-293 cells were grown as previously described in 24-wells plates and transfected using the Lipofectamine 2000 reagent, and the appropriate concentration of siRNA (Qiagen; Hilden, Germany) and plasmids (0.4 g DNA/well, plus 1 L/well lipofectamine 2000, per transfected plasmid). The NP868R-SP6 chimeric enzyme, which has strong demonstrated activity, was used in the present experiment. The firefly luciferase luminescence was assayed as previously described using the pT7p-Luciferase (which also contains both the T3 and SP6 promoters) and the Luciferase Assay System. The expression of pORF-eSEAP plasmid was used to normalize for transfection efficacy as previously described.
(83) Four siRNA that target the human POLR2A (NCBI Gene ID#5430; mRNA sequence ID# NM_000937.4; NCBI protein sequence ID# NP_000928.1) were used: SI04364381 (mRNA sequence 1255-1275: CAGCGGTTGAAGGGCAAGGAA (SEQ ID NO:43)), SI04369344 (mRNA sequence 830-850: ATGCGGAATGGAAGCACGTTA (SEQ ID NO:44)), SI04250162 (mRNA sequence 2539-2559: ATGGTCGTGTCCGGAGCTAAA (SEQ ID NO:45)), and SI04354420 (mRNA sequence 4896-4916: CAGCGGCTTCAGCCCAGGTTA (SEQ ID NO:46)).
(84) In addition, four siRNA that target the human RNGTT (Gene ID#8732; mRNA sequence ID# NM_003800.3; NCBI protein sequence ID# NP_003791.3) were used: SI00055986 (mRNA sequence 3187-3207: ATGGATTTAAAGGGCGGCTAA (SEQ ID NO:47)), SI03021508 (mRNA sequence 430-450: TTCAAGGTTCTATGACCGAAA (SEQ ID NO:48)), SI00055972 (mRNA sequence 2530-2550: CAGGGTTGTTAAGTTGTACTA (SEQ ID NO:49)) and SI00055979 (mRNA sequence 4132-4152: TACCATCTGCAGTATTATAAA (SEQ ID NO:50)).
(85) II. Results
(86) In a first series of experiments, the effects of four POLR2A siRNA and four RNGTT siRNA were tested at 25 nM final concentration (
(87) The POLR2A SI04369344 and the RNGTT SI00055972 siRNA, which have show the highest stimulation rate, were selected for a second series of experiments. Expression of the luciferase reporter gene driven by NP868R-SP6RNAP was assayed in presence of siRNA at concentrations ranging from 0 to 100 nM (
(88) III. Conclusion
(89) The present findings demonstrate that the silencing of the cellular transcription and post-transcriptional machineries by siRNA stimulate the reporter gene expression driven by the NP868R-SP6RNAP chimeric enzyme.
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