Patent Application
20060218654
The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism.
RNA interference (RNAi) has been discovered some years ago as a tool for inhibition of gene expression (Fire, A. et al., Nature 391, 806-811 (1998)). It based on the introduction of double stranded RNA (dsRNA) molecules into cells, whereby one strand is complementary to the coding region of a target gene. Through pairing of the specific mRNA with the introduced RNA molecule, the mRNA is degraded by a cellular mechanism. Since long dsRNA provokes an interferon response in mammalian cells, the technology was initially restricted to organisms or cells showing no interferon response (Bass, B. L., Nature 411, 428-429 (2001)). The finding that short (<30 bp) interfering RNAs (siRNA) circumvent the interferon response extended the application to mammalian cells (Elbashir, S. M. et al., Nature 411, 494-498 (2001)).
Although RNAi in mice has been in principle demonstrated, the current technology does not allow performing systematic gene function analysis in vivo. So far the inhibition of gene expression has been achieved by injection of purified siRNA into the tail vain of mice (McCaffrey, A. P. et al., Nature 418, 38-39 (2002); Lewis, D. H. et al., Nature Genet. 32, 107-108 (2002)). Using this approach, gene inhibition is restricted to specific organs and persists only a few days. A further improvement of the siRNA technology is based on the intracellular transcription of short hairpin RNA (shRNA) molecules using gene expression vectors (see FIG. 1; Brummelkamp, T. R. et al., Science 296, 550-553 (2002); Paddison, P. J. et al, Genes Dev. 16, 948-958 (2002); Yu, J. Y., et al., Proc. Natl. Acad. Sci. USA 99, 6047-6052 (2002); Sui, G. et al., Proc. Natl. Acad. Sci. USA 99, 5515-5520 (2002); Paul, C. P. et al., Nature Biotechnol. 20, 505-508 (2002); Xia, H. et al., Nat. Biotechnol. 10, 1006-10 (2002); Jacque, J. M. et al., Nature 418(6896):435-8 (2002)). The activity of shRNA in mice has been demonstrated by McCaffrey et al., 2002 through injection of shRNA expression vectors into the tail vain. Again, gene inhibition was temporally and spatially restricted. Although these results demonstrate that the mechanism of shRNA mediated gene silencing is functional in mice, they do not clarify whether constitutive RNAi can be achieved in transgenic animals. Brummelkamp, T. R. et al., Science 296, 550-553 (2002), Paddison, P. J. et al., Genes Dev. 16, 948-958 (2002), Hemann, M. T. et al., Nat. Genet. 33(3):396-400 (2003); and Devroe, E. et al., BMC Biotechnol. 2(1):15 (2002) have shown the long-term inhibition of gene expression through stable integration of shRNA vectors in cultivated cell lines. These experiments included random integration of shRNA transgenes and screening for clones with appropriate siRNA expression, which is not applicable for testing of a large number of different shRNA transgenes in mice. Finally, several reports have demonstrated shRNA-mediated gene silencing in transgenic mice and rats (Hasuwa, H., et al., FEBS Lett. 532(1-2):227-30 (2002); Carmell, M. A. et al., Nat. Struct. Biol. 10(2):91-2 (2003); Rubinson, D. A. et al., Nat. Genet. 33(3):401-6 (2003); Kunath, T. et al., Nat. Biotechnol. (Apr. 7, 2003)). However, these experiments again included random integration of shRNA transgenes resulting in variable levels and patterns of shRNA expression. Thus, testing of ES cell clones or mouse lines with appropriate shRNA expression had been required, which is a laborious and time-consuming undertaking.
The in vivo validation of genes by RNAi mediated gene repression in a large scale setting requires the expression of siRNA at sufficiently high levels and with a predictable pattern in multiple organs. Targeted transgenesis provides the only approach to achieve reproducible expression of transgenes in the living organism (e.g. mammalians such as mice). It has been, however, questionable whether a single copy of a siRNA expression vector integrated into the genome would result in sufficiently high levels of siRNA required for RNAi-mediated gene inhibition in multiple organs of the living organism.
Most siRNA expression vectors are based on polymerase III dependent (Pol III) promoters (U6 or H1) that allow the production of transcripts carrying only a few non-homologous bases at their 3′ ends. It has been shown that the presence of non-homologous RNA at the ends of the shRNA stretches lower the efficiency of RNAi mediated gene silencing (Xia et al., Nat. Biotechnol. 10, 1006-10 (2002)). However, it has been unpredictable for a person skilled in the art which genomic region within the living organism promotes an appropriate expression pattern of the Pol III promoter driven shRNA constructs required for RNAi-mediated gene inhibition in multiple organs of the living organism.
For the temporal control of RNAi mediated gene silencing in transgenic cells lines and living organism, a tight system for inducible siRNA expression is needed. Inducible gene expression systems based on the tetracycline dependent repressor are known. It is, however, not obvious whether a stably integrated, single copy configuration of these systems can be created that allows inducible RNAi in multiple organs without background activity.
It has now been found that the modification of murine embryonic stem (ES) cells by the introduction of shRNA expression vectors through targeted transgenesis allows the inhibition of gene expression in mice generated from these modified ES cells. The invention thus provides
(1) the use of an expression vector comprising a short hairpin RNA (shRNA) construct under control of a ubiquitous promoter for preparing an agent for constitutive and/or inducible gene knock down (hereinafter also referred to as “RNAi mediated inhibition of gene expression”) in a living organism, in tissue culture or in cells of a cell culture;
(2) a method for constitutive and/or inducible gene knock down in a living organism, in tissue culture or in cells of a cell culture which comprises stably integrating an expression vector as defined in (1) above into the genome of the living organism, in the genome of the tissue culture or into the genome of the cells of the cell culture;
(3) a living organism, tissue culture or cell culture having stably integrated, preferably at a polymerase II dependent locus of the living organism, tissue culture or cells of the cell culture, an expression vector as defined in (1) above; and
(4) an expression vector as defined in (1) above.
The agent of embodiment (1) is suitable for in vivo and in vitro applications.
The living organisms in embodiments (1) to (3) above are preferably vertebrates. In embodiments (2) and (3) the living organisms are more preferably non-human vertebrates. The tissue cultures and the cells of the cell cultures of (1) to (3) above are preferably derived from vertebrates. The method of embodiment (2) includes in vivo and in vitro reaction schemes.
A: Firefly luciferase activity in mice in the absence (delta shFluc, black bars) or presence of the U6- (U6-shFluc, white bars) and H1-shRNA transgenes (H1-shFluc, grey bars), respectively. All mice carried the firefly and the Renilla luciferase transgenes. Relative values of Firefly luciferase activity in different organs are given as indicated. All values of Fluc activity were normalized by using the Rluc activity for reference.
B: Efficiency of shRNA-mediated repression of firefly luciferase expression in mice. Percentages of U6- (white bars) and H1-shRNA mediated (grey bars) repression of Firefly luciferase activity are shown. ND: not determined.
The term “living organisms” according to the present invention relates to multi-cell organisms which can be living vertebrates (hereinafter shortly referred to as “vertrebrates”) such as mammals (e.g. non-human animals such as rodents (including mice and rats, etc.; and humans) or non-mammals (e.g. fish) or can be invertebrates such as insects or worms, or can be plants (higher plants, algi or fungi). Most preferred living organisms are vertebrates, in particular mice and fish.
“Tissue culture” according to the present invention refers to parts of the above defined living organisms (including organs and the like) which are cultured in vitro.
“Cell culture” according to the present invention includes cells isolated from the above defined living organisms which are cultured in vitro. These cells can be transformed (immortalized) or untransformed (directly derived from living organisms; primary cell culture).
The expression vector according to the invention of the present application is suitable for stable integration into the living organism or into cells of the cell culture (vectors for transient integration are also contemplated). It is moreover preferred that it contains homologous sequences suitable for targeted integration at a defined locus, preferably at a polymerase II dependent locus of the living organisms or cells of the cell culture. Such polymerase II dependent loci include, but are not limited to, Rosa26 locus (the murine Rosa26 locus being depicted in SEQ ID NO:1), collagen, RNA polymerase, actin and HPRT.
The expression vector may further contain functional sequences selected from splice acceptor sequences (such as a splice acceptor of adenovirus, etc.), polyadenylation sites (such as synthetic polyadenylation sites, the polyadenylation site of human growth hormones, or the like), selectable marker sequences (such as the neomycin phosphotransferase gene of E. coli transposon, etc.), etc.
The ubiquitous promoter in the vector according to the invention is preferably selected from polymerase I, II and III dependent promoters, preferably is a polymerase II or III dependent promoter including, but not limited to, a CMV promoter, a CAGGS promoter, a snRNA promoter such as U6, a RNAse P RNA promoter such as H1, a tRNA promoter, a 7SL RNA promoter, a 5 S rRNA promoter, etc.
The ubiquitous promoter can be a constitutive promoter, or can be an inducible promoter. Suitable inducible promoters are the above-mentioned polymerase I, II and III dependent promoters containing an operator sequence including, but not limited to, tet, Gal4, lac, etc.
The expression vector of the invention is suitable for the following particularly preferred approaches (for constitutive and inducible expression):
The ShRNA within the vector of the invention preferably comprises
(I) at least one DNA segment A-B-C wherein
A is a is to 35, preferably 19 to 29 bp DNA sequence being at least 95%, preferably 100% complementary to the gene to be knocked down (e.g. firefly luciferase, p53, etc.);
B is a spacer DNA sequence having 5 to 9 bp forming the loop of the expressed RNA hair pin molecule, and
C is a 15 to 35, preferably 19 to 29 bp DNA sequence being at least 85% complementary to the sequence A; and
(II) a stop and/or polyadenylation sequence.
Suitable shRNA sequences for the knock down of given target gene are well known in the art (see e.g. the particular shRNA sequences mentioned in Tables 1 and 2 below) or can readily be determined by the skilled artesian.
HOMO SAPIENS
A preferred embodiment of the method (2) of the invention concerns the following steps:
The vector according to embodiment (4) of the invention is suitable for stable or transient integration. Said vector is suitable for gene transfer.
The technology of the present application provides for the following advantages:
(i) A stable and body wide inhibition of gene expression by generating transgenic animals (such as mice).
(ii) A reversible inhibition of gene expression using the inducible constructs.
The invention is furthermore described by the following examples which are, however, not to be construed so as to limit the invention.
Methods
Plasmid construction: All plasmid constructs were generated by standard DNA cloning methods. The U6- and the H1-promoter fragments were amplified from human genomic DNA. The Renilla luciferase (Rluc) and firefly luciferase (Fluc) coding regions (Promega) were placed 3′ of the adenovirus splice acceptor site (Friedrich and Soriano, Genes Dev. 9:1513-23 (1991); see SEQ ID NOs: 3, 5 and 7) to facilitate transcription from the endogenous rosa26 promoter. For homologous recombination in embryonic stem cells, the expression cassettes were flanked by homology regions for the Rosa26 locus (see SEQ ID NO: 2).
Cell culture: Culture and targeted mutagenesis of F1 (C57BL/6×129Sv/Ev) ES cells were carried out as previously described (Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). A Laboratory Manual. In Manipulating the Mouse Embryo. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289.). For Cre-mediated deletion, ES cells were transiently transfected with 5 μg of the cre expression plasmid pCAGGScre (SEQ ID NO: 18). 24 h after transfection, 1000 ES cells were plated on a 10 cm dish. Individual clones were isolated and analyzed for cre-mediated recombination by Southern blot using standard protocols.
For induction, cells were cultivated in cell culture medium containing 1 μg/ml Doxycycline (Sigma) for 2 days.
For transient transfection of cells, mixtures of 350 ng supercoiled plasmid DNA pre-complexed with the FuGene6 transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) were added to the cells in 250 μl medium, according to the manufacturers protocol. Each plasmid mixture contained 50 ng of the beta-galactosidase expression vector CMV-lacZ (SEQ ID NO: 17), 100 ng of CAGGS-Fluc (SEQ ID NO: 15), 100 ng of the tet inducible H1shRNA vector (SEQ ID NO: 11, 12 or 13), and pUC19 to a total amount of 350 ng DNA. For each sample to be tested four individual wells were transfected. One day after the addition of the DNA preparations, the cell culture medium was exchanged.
Luciferase measurement: Stably transfected cells: 25.000 cells were plated on a 24 well plate and analyzed 48 h later. Luciferase activity was detected using the Dual-Glo™ Luciferase Assay System (Promega) and a Lumat LB 9507 (EG&G Berthold) according to the manufacturers protocols.
Transiently transfected cells: The cells were lysed 48 hours after transfection with 100 μl/well of lysate reagent supplemented with protease inhibitors (β-galactosidase reporter gene assay kit, Roche Diagnostics). The lysates were centrifuged and 20 μl were used to determine the μ-galactosidase activities using the β-galactosidase reporter gene assay (Roche Diagnostics) according to the manufacturers protocol in a Lumat LB 9507 luminometer (Berthold). To measure Luciferase activity, 20 μl of lysate was diluted into 250 μl assay buffer (50 mM glycylglycin, 5 mM MgCl2, 5 mM ATP) and the relative light units (RLU) were counted in a Lumat LB 9507 luminometer after addition of 100 μl of a 1 mM Luciferin (Roche Diagnostics) solution.
Generation of chimeric mice: Recombinant ES cells were injected into blastocysts from Balb/C mice and chimeric mice were obtained upon transfer of blastocysts into pseudopregnant females using standard protocols (Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., 253-289 (1994)).
Luciferase measurement in organs: Organs were homogenized at 4° C. in lysis buffer (0.1M KH2PO4, 1 mM DTT, 0.1% Triton® X-100) using a tissue grinder. Spin for 5′ at 2000×g (4° C.) to pellet debris and assay supernatant for Luc activities using the Dual Luciferase Assay (Promega, Inc.) according to the manufacturer protocol.
The firefly luciferase gene along with a splice acceptor sequence (Friedrich and Soriano, Genes Dev. 9:1513-23 (1991)) was inserted into the first allele of the rosa26 locus by homologous recombination in ES cells. The shRNA expression cassettes under the control of the H1 or the U6 promoter and a Renilla luciferase gene were inserted into the second allele of the rosa26 locus by homologous recombination. The Renilla luciferase gene was used as a reference to normalize the values of firefly luciferase activity.
Deleting the loxP-flanked shRNA expression cassette by transient transfection of a cre expression plasmid generated the negative control.
The shRNA expression cassette under the control of the U6 promoter containing tet-operator sequences, and a Renilla luciferase gene were inserted into the first allele of the rosa26 locus (
NIH3T3 cells were transiently transfected with constructs expressing the luciferase and tet repressor genes (SEQ ID NOs:15 and 16) together with the shRNA constructs under the control of the H1 promoter containing tet-operator sequences (sequence ID NOs:11, 12 and 13;
To examine the activity of the Rosa26/U6- and H1-shRNA transgenes in vivo, recombinant ES cells were injected into diploid blastocysts and chimeric mice were obtained upon transfer of blastocysts into pseudopregnant females.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02023283.1 | Oct 2002 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/11233 | 10/10/2003 | WO | 2/27/2006 |
| Number | Date | Country | |
|---|---|---|---|
| 60467814 | May 2003 | US | |
| 60485969 | Jul 2003 | US |
