Patent Application
20090025097
The present invention relates to a biological entity, notably a rat, carrying a regulator construct comprising a specific repressor gene and a responder construct comprising at least one segment corresponding to a short hairpin RNA (shRNA) or corresponding to complementary short interfering RNA (siRNA) strands or corresponding to miRNA, said at least one segment being under control of a promoter which contains an operator sequence corresponding to the repressor. The invention further relates to a method for preparing said biological entity and its use.
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 the two complementary siRNA strands using separate expression units both under the control of the U6 promoter (Lee, N. S. et al., 3. Nat. Biotechnol. 20(5):500-5 (2002); Du, Q. et al., Biochem. Biophys. Res. Commun. 325(1):243-9 (2004); Miyagishi, M. and Taira, K., Methods Mol. Biol.; 252:483-91 (2004)). The transgene based approach was further refined by the expression of short hairpin RNA (shRNA) molecules by a single transcription unit under the control of the U6 or H1 promoter (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 A. P. et al., Nature 418, 38-39 (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. (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).
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, H. et al., Nat. Biotechnol. 10, 1006-10 (2002)). WO 04/035782 discloses that an ubiquitous promoter driven shRNA construct provides for RNAi-mediated gene inhibition in multiple organs of the living organism. Further, an inducible gene expression system, e.g. a system based on the tetracycline dependent repressor, is suggested which allows temporary control of RNAi mediated gene silencing in transgenic cells lines and living organism. The configuration of said inducible systems as well as the choice of the repressor appeared critical with regard to the expression of inducible RNAi in multiple organs without background activity. However, since all experiments concerning inducible shRNA expression were performed in cultured cells in vitro, WO04/035782 does not allow a prediction whether such system is applicable for regulating body-wide transgene expression in a living animal (i.e. whether repression throughout development and tetracycline depend control of RNAi in different tissues does occur).
Temporary control of shRNA expression can be achieved by using engineered promoters containing a tetracycline operator (tetO) sequence (Ohkawa, J. and Taira, K., Hum. Gene Ther. 11(4):577-85 (2000)). The Tetracycline operator itself has no effect on shRNA expression. In the presence of the tetracycline repressor (tetR), however, transcription is blocked through binding of the repressor to the tetO sequence. De-repression is achieved by adding the inductor doxycycline, that causes the release of the TetR protein from the tetO site and allows transcription from the H1 promoter. Several attempts have been made to apply this strategy for the temporary control of antigens or shRNA expression in cultured cell lines (Ohkawa, J. and Taira, K., Hum. Gene Ther. 11(4):577-85 (2000); van de Wetering, M. et al., EMBO reports VOL 4, NO 6:609-615 (2003); Matsukura, 2003; Czauderna, F. et al., Nucleic Acids Res., 31(21):e127 (2003)). In these reports, the degree of doxycycline-inducible mRNA degradation was variable. In addition, background RNAi activity in the uninduced state was observed (van de Wetering, M. et al., EMBO reports VOL 4, No. 6:609-615 (2003)), indicating a limiting level of tetR expression in these cell lines.
WO 04/056964 describes the temporal control of shRNA expression in vitro using a codon-optimized tetracycline repressor. The system described in WO 04/056964 uses an engineered U6 promoter. A site-by-site comparison of the codon-optimized construct with the wildtype repressor, however, is lacking in WO 04/056964. Therefore, it is unclear whether codon optimization has any effect in the context of the particular shRNA construct used in this document. Furthermore, it is impossible to predict from the in vitro results presented in this document whether such system is applicable for regulating body-wide transgene expression in a living animal. WO 04/056964 furthermore describes the subcutaneous transplantation of transgenic cells, which were obtained by in vitro experiments, into nude mice. Again, these experiments just show the activity of shRNA constructs in a particular, transfected cell line, but not in different cell types or developmental stages of transgenic mice. The properties of such Doxycycline-responsive promoters for siRNA expression have so far not been tested in transgenic animals. In addition, the level of shRNA expression required for efficient RNAi has never been determined and, vice versa, it is unknown whether or to which extent a basal level of shRNA expression is tolerated without significant RNAi in the uninduced state of the system. It is therefore not obvious whether a tight control of RNAi can be achieved through Doxycycline inducible expression of shRNA transgenes in living animals.
Difficulties in expression of the lac repressor and tetR in transgenic animals have been attributed to their prokaryotic origin (Scrable & Stambrook, Genetics 147:297-304 (1997); Wells, D. J., Nucleic Acids Res., 27(11):2408-15 (1999); Urlinger, S. et al., Proc. Natl. Acad. Sci. USA 97(14): 7963-8 (2000)). Alteration of the coding region by changing unfrequently used codons and eliminating putative mammalian processing signals improved the expression of these sequences (Zhang et al., Gene 105:61-72 (1991); Anastassiadis, K. et al., Gene 298:159-72 (2002)). Scrable & Stambrook, Genetics 147:297-304 (1997) were able to show expression of a codon optimized lac repressor by Northern analysis in transgenic animals, but were unable to detect protein expression and failed to prove the activity of the repressor. Anastassiadis, K. et al. demonstrated improved regulatory properties of a VP16 domain fused to a codon-optimized tet-repressor in vitro. In this system, the VP16-tetR fusion protein activates a minimal promoter through binding tet-operator sequences upon induction with doxyxycline. The system therefore follows a different principle compared to transcriptional repression described in Ohkawa, J. and Taira, K., Hum. Gene Ther. 11(4):577-85 (2000); van de Wetering, M. et al., EMBO reports VOL 4, NO 6:609-615 (2003); Matsukura, 2003; Czauderna, F. et al., Nucleic Acids Res., 31(21):e127 (2003). Cronin, C. A. et al., Genes and Development 15:1506-1517 (2001) demonstrated that the expression of the lac repressor could only be achieved by an empirically combination of synthetic and wt parts of the repressor. No general prediction for transgene expression of bacterial genes in mice could be made, indicating that the codon optimization alone is not sufficient for improved transgene activity.
The provision of an inducible system allowing tight temporal control of RNAi in multicellular organisms without background activity was highly desirable.
The rat is an excellent animal model for studying human diseases. To date, due to the lack of rats' germline embryonic stem cells this species has remained limited for genetic manipulation such as gene disruption. Trying to shut off genes in higher organisms using an RNAi, a natural phenomenon and a powerful tool for gene silencing, is widely used in the last years.
Recently, many scientific reports indicated successful mRNA silencing through embryonic stem cell transgenesis using DNA plasmids carrying shRNA cassette (Carmell, M. A., et al., Nat Struct Biol, 10(2):91-2 (2003); Saito, Y., et al., J Biol Chem, 280(52):42826-30 (2005); Seibler, J., et al., Nucleic Acids Res, 33(7):e67 (2005)1-3). Differently, difficulties of pronuclear shRNA application causing toxic effects and lack of germline transmission were reported (Carmell, M. A., et al., Nat Struct Biol, 10(2):91-2 (2003); Cao, W., et al., J Appl Genet, 46(2):217-25 (2005)). However, in 2006, the group under Garbers' leadership brought out one of the first reliable reports about stable and heritable shRNA gene knock down (KD) in rats using lentiviral DNA delivery. But still, the technological backdraw was the lack of germ-line transmission due to the mosaic transgene expression pattern observed in several founders (Dann, C. T., et al., Proc Natl Acad Sci USA, 103(30):11246-51 (2006)).
Nevertheless, up to date no shRNA knock down manipulation through pronuclear microinjection has been established in rat yet. Therefore, our aim was to develop fast, efficient and reversible gene knock down technology in this species using pronuclear transgenesis. To do this, we use tetracycline activation system based on wild type tetracycline repressor to induce expression of shRNA. Concerning common pharmaceutical interests we targeted the insulin receptor to produce a transgenic rat model falling insulin resistance. Here we show that inducible H1-tetO RNA polymerase III promoter maintaining a tight control over shRNA allows recovery of hyperglycemic rats back to the normal stage after doxycycline (DOX) cessation. Moreover, long lasted drug treatment of low DOX doses leads to chronic type II diabetes with typical renal damage of these rats and thus reflects symptoms seen in man.
Since now, silencing of any genes in rats is possible using this tetracycline inducible shRNA gene down regulation.
It was surprisingly found that a codon-optimized repressor gene, such as the tetracycline repressor gene, completely suppresses the activity of shRNA/siRNA/miRNA genes under the control of a particular promoter containing the corresponding operator, such as a tetO containing promoter, in transgenic animals. In contrast thereto the same configuration with the non codon-optimized tetracycline repressor gene showed a high degree of shRNA/siRNA/miRNA background activity in transgenic animals in the absence of doxycyclin induction. Thus, the present invention provides
(1) a biological entity selected from a vertebrate, a tissue culture derived from a vertebrate or one or more cells of a cell culture derived from a vertebrate, said biological entity carrying
(i) a responder construct comprising at least one segment corresponding to a short hairpin RNA (shRNA) or to complementary short interfering RNA (siRNA) strands or to miRNA, said at least one segment being under control of a ubiquitous promoter and said promoter containing an operator sequence being perfectly regulatable by a repressor; and
(ii) a regulator construct comprising a codon-optimized repressor gene, which provides for perfect regulation of the promoter containing the operator sequence of the responder construct;
(2) a method for preparing the biological entity as defined in (1) above or a method for constitutive and/or inducible gene knock down in a biological entity, which method comprises stably integrating (i) a responder construct as defined in (1) above, and (ii) a regulator construct as defined in (1) above into the genome of the biological entity; and
(3) the use of a biological entity as defined in (1) above for inducible gene knock down, and/or as a test system for pharmaceutical testing, and/or for gene target validation, and/or for gene function analysis.
The “biological entity” according to the present invention includes, but is not limited to, a vertebrate, a tissue culture derived from a vertebrate, or one or more cells of a cell culture derived from a vertebrate.
The term “vertebrate” according to the present invention relates to multi-cellular organisms such as mammals, e.g. non-human animals such as rodents (including mice, rats, etc.) and humans, or non-mammals, e.g. fish. Most preferred vertebrates are mice and fish.
“Tissue culture” according to the present invention refers to parts of the above-defined “vertebrates” (including organs and the like) which are cultured in vitro.
“Cell culture” according to the present invention includes cells isolated from the above-defined “vertebrates” which are cultured in vitro. These cells can be transformed (immortalized) or untransformed (directly derived from vertebrates; primary cell culture).
The “responder construct” and the “regulator construct” according to the invention of the present application are suitable for stable integration into the “vertebrates” or into cells of the cell culture, e.g. by homologous recombination, recombinase mediated cassette exchange (hereinafter “RMCE”) reaction, or random integration. The vector(s) for integration of the constructs into the vertebrates by homologous recombination preferably contain(s) homologous sequences suitable for targeted integration at a defined locus, preferably at a polymerase II or III dependent locus of the living organisms or cells of the cell culture. Such polymerase II or III dependent loci include, but are not limited to, the Rosa26 locus (the murine Rosa26 locus being depicted in SEQ ID NO:11), collagen, RNA polymerase, actin, and HPRT. Homologous sequences suitable for integration into the murine Rosa26 locus are shown in SEQ ID Nos:6 and 7.
The responder construct contains at least one ubiquitous promoter which controls the expression of the at least one segment corresponding to a short hairpin RNA (shRNA), or to complementary short interfering RNA (siRNA) strands, or to “miRNA”, i.e., small RNAs (20-25 nucleotides in length) that are function in repressing mRNA translation or in mRNA degradation within a cell and that are processed from long, single stranded RNA sequences that fold into hairpin structures (in the following shortly referred to as “shRNA segment”, “siRNA segment” and “miRNA segment”, respectively). Thus, said segment is under control of a ubiquitous promoter, wherein said promoter contains at least one operator sequence, by which said promoter is perfectly and ubiquitously regulatable by a repressor. The segment corresponding to the shRNA, siRNA and miRNA are preferably comprised of DNA.
The regulator construct may also contain ubiquitous promoter(s) (constitutive, inducible or the like). Preferably the ubiquitous promoter of the regulator and/or responder construct is selected from polymerase I, II and III dependent promoters, most preferably is a polymerase II or III dependent promoter including, but not limited to, a CMV promoter, a CAGGS promoter (see nucleotides 3231-4860 of SEQ ID NO:1), 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 of the “responder construct” contains an operator sequence allowing for “perfect regulation” by a corresponding repressor. “Perfect regulation” and “perfectly regulatable” within the meaning of the invention means that it permits control of the expression to an extent that no significant background activity is determined in the biological entity. This means that the suppression of the expression of the shRNA/siRNA/miRNA is controlled by a rate of at least 70%, preferably by a rate of at least 90%, more preferably by at least 95%, even more preferably by at least 98%, and most preferably by 100%. Suitable operator sequences are such operator sequences, which render the promoter susceptible to regulation by the corresponding codon-optimized repressor gene present within the regulator construct, including, but not limited to, tetO, GalO, lacO, etc.
The responder construct may further contain functional sequences selected from splice acceptor sequences (such as a splice acceptor of adenovirus (see nucleotides 1129-1249 of SEQ ID NO:1), etc.), polyadenylation sites (such as synthetic polyadenylation sites (see nucleotides 2995-3173 of SEQ ID NO:1), the polyadenylation site of human growth hormones (see nucleotides 4977-5042 of SEQ ID NO:1), or the like), selectable marker sequences (such as the neomycin phosphotransferase gene of E. coli transposon, etc.), recombinase recognition sequences (such as loxP, FRT, etc), and so on.
Particularly preferred responder constructs carry a Pol III dependent promoter (inducible H1 or the like) containing tetO (for H1-tetO see nucleotides 4742-4975 of SEQ ID NO:3), and the at least one shRNA segment or siRNA segment. Particularly preferred regulator constructs carry a polymerase II (Pol II) dependent promoter (CMV, CAGGS or the like) and the codon optimized repressor gene tet.
In case shRNA segments are utilized within the responder construct, the responder construct preferably comprises at least one shRNA segment having a nucleotide (e.g. DNA) sequence of the structure A-B-C or C-B-A. In case siRNA segments are utilized within the responder construct, the responder construct preferably comprises at least two DNA segments A and C or C and A, wherein each of said at least two segments is under the control of a separate promoter as defined above (such as the Pol III promoter including inducible U6, H1 or the like). In the above segments
The above shRNA, siRNA and miRNA segments may further comprise stop and/or polyadenylation sequences.
Suitable siRNA sequences for the knockdown of a given target gene are well known in the art (e.g. the particular siRNA sequences mentioned in Lee N. S. et al., J. Nat. Biotechnol. 20(5):500-5 (2002) gcctgtgcctcttcagctacc (SEQ ID NO:12) and gcggagacagcgacgaagagc (SEQ ID NO:13) and in Du, Q. et al., Nucl. Acids Res. 21; 33(5):1671-7 (2005) cttattggagagagcacga (SEQ ID NO:14)) or can readily be determined by the skilled artisan.
Suitable miRNA sequences for the knockdown of a given target gene are known in the art and include hsa-mir-30a MI0000088 (GCGACUGUAAACAUCCUCGACUGGAA-GCUGUGAAGCCACAGAUGGGCUUUCAGUCGGAUGUUUGCAGCUGC; SEQ ID NO:237) and the corresponding processed miRNA hsa-miR-30a MIMAT0000087 (UGUAAACA-UCCUCGACUGGAAG; SEQ ID NO:238), hsa-mir-155 MI0000681 (CUGUUAAUGCUA-AUCGUGAUAGGGGUUUUUGCCUCCAACUGACUCCUACAUAUUAGCAUUAACAG; SEQ ID NO:239) and the corresponding processed miRNA hsa-miR-155 MIMAT0000646 (UUAAUGCUAAUCGUGAUAGGGGU; SEQ ID NO:240), and hsa-mir-29a MI0000087 (AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUC GGUUAU; SEQ ID NO:241) and the corresponding processed miRNA hsa-miR-29a MIMAT0000086 (UAGCACCAUCUGAAAUCGGUUA; SEQ ID NO:242).
Suitable shRNA sequences for the knock down of a 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 artisan.
The “regulator construct” comprises a repressor gene, which provides for perfect regulation of the operators of the responder construct. In particular, the repressor gene encodes a repressor, i.e. a molecule acting on the operator of the promoter to therewith inhibit (down-regulate) the expression of the shRNA/siRNA/miRNA. Suitable repressor genes include codon-optimized repressors (i.e., repressor genes where the codon usage is adapted to the codon usage of vertebrates), including, but not limited to, a codon-optimized tet repressor, a codon-optimized Gal repressor, a codon-optimized lac repressor and variants thereof. Particularly preferred is the codon optimized tet repressor, most preferred a codon-optimized tet repressor having the sequence of nucleotides 5149 to 5916 of SEQ ID NOs:2 or 3.
Embodiment (2) of the invention pertains to a method for preparing the biological entity as defined hereinbefore and to a method for constitutive and/or inducible gene knock down in a biological entity, which stably integrating
(i) the responder construct as defined hereinbefore, and
(ii) a regulator construct as defined hereinbefore into the genome of the biological entity.
In particular the method comprises subsequent or contemporary integration of the responder construct, and the regulator construct into the genome of vertebrate cells. In case of (non-human) mammals the constructs are preferably integrated into embryonic stem (ES) cells of said mammals.
Various methods are applicable for the integration of the constructs.
A first integration method is the so called “homologous recombination” which utilizes an integration vector comprising the functional nucleotide sequence to be integrated and DNA sequences homologous to the integration site, where said homologous DNA sequences flank the functional nucleotide sequence. In a particular preferred embodiment of the invention, both, the responder construct and the regulator construct are integrated by homologous recombination on the same or different allel(s).
A second integration method is the RMCE reaction, which comprises the steps of
(i) modifying a starting cell by introducing an acceptor DNA which integrates into the genome of the starting cell (e.g. by homologous recombination), and wherein the acceptor DNA comprises two mutually incompatible recombinase recognition sites (RRSs), and introducing into such modified cell;
(ii) a donor DNA comprising the same two mutually incompatible RRSs contained in the acceptor DNA by utilizing an integration vector comprising a functional DNA sequence flanked by the RRSs; and
(iii) a recombinase which catalyzes recombination between the RRSs of the acceptor and donor.
In a preferred embodiment of the invention the integration of at least one of the responder construct and the regulator construct is effected by RMCE reaction. Details of the first and second method, in particular for integration at the murine Rosa26 locus are discussed in detail in applicant's WO 2004/063381, the disclosure of which is herewith incorporated by reference. For the integration at the murine Rosa26 locus (the sequence thereof being depicted in SEQ ID NO:11) by homologous recombination, the integration vector caries homologous flanking sequences of 0.2 to 20 kB, preferably 1 to 8 kB length. Suitable sequences include, but are not limited to, the sequences depicted in SEQ ID NOs:6 and 7.
A third integration method is the so-called “random transgenesis” where an integration vector is randomly integrated into the genome of the cell. By pronucleus injection of the linearized vector one or more copies of the DNA-fragment integrates randomly into the genome of the mouse embryo. The resulting founder lines have to be characterized for the expression of the transgene (Palmiter, R. D. and Brinster, R. L., Annu. Rev. Genet. 20:465-499 (1986)). Hasuwa H. et al. FEBS Lett. 532(1-2):227-230 (2002) used this technology for the generation of siRNA expressing mice and rats.
Particularly preferred in the invention is that the integration vector (in all three integration methods discussed above) carries both, the responder construct and the regulator construct.
The preparation of the vertebrate is hereinafter further described by reference to the mouse and rat system. This shall, however, not be construed as limiting the invention. The preferred method for producing a shRNA in a mouse and rat (and also mouse or rat tissue and cells derived from such mouse and rat) that expresses the codon optimized repressor protein comprising the steps of:
The inducible gene knock-down according to embodiments (2) and (3) of the invention moreover comprises the step of administering a suitable inducer compound to the biological entity (in particular the mouse or rat) or ceasing the administering of the inducer compound to therewith induce or cease the expression of the respective shRNA/siRNA/miRNA.
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 and rats);
(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.
Plasmid construction: All plasmid constructs were generated by standard DNA cloning methods.
Basic rosa26 targeting vector: A 129 SV/EV-BAC library (Incyte Genomics) was screened using a probe against exon2 of the Rosa26 locus (amplified from mouse genomic DNA using Rscreen1s (GACAGGACAGTGCTTGTTTAAGG; SEQ ID NO:4) and Rscreen1as (TGACTACACAATATTGCTCGCAC; SEQ ID NO:5)). Out of the identified BACclone a 11 kb EcoRV subfragment was inserted into the HindIII site of pBS. Two fragments (a 1 kb SacII/XbaI- and a 4 kb XbaI-fragment; see SEQ ID NOs:6 and 7) were used as homology arms and inserted into a vector containing a FRT-flanked neomycin resistance gene or hygromycin resistance gene to generate the basic Rosa26 targeting vectors. The splice acceptor site (SA) from adenovirus (Friedrich, G. and Soriano, P., Genes Dev., 5:1513-23 (1991)) was inserted as PCR-fragment (amplified using the oligonucleotides ATACCTGCAGGGGTGACCTGCACGTCTAGG (SEQ ID NO:15) and ATACCTGCAGGAGTACTGGAAAGACCGCGAAG (SEQ ID NO:16)) between the 5′ arm and the FRT flanked neomycin resistance gene or the FRT flanked hygromycin resistant gene. The Renilla luciferase (Rluc) and firefly luciferase (Fluc) coding regions (Promega) were placed 3′ of the SA site (Friedrich, G. and Soriano, P., Genes Dev. 9:1513-23 (1991); see SEQ ID NOs:1, 2 and 3)) to facilitate transcription from the endogenous rosa26 promoter.
Insertion of transgenes into the targeting vector: All subsequently described transgenes were inserted 3′ of the Renilla luciferase (Rluc) or firefly luciferase genes. The H1-promoter fragments were amplified from human genomic DNA (using the oligonucleotides AACTATGGCCGGCCGAAGAACTCGTCAAGAAGGCG (SEQ ID NO: 17) and TATGGTACCGTTTAAACGCGGCCGCAAATTTFATTAGAGC (SEQ ID NO:18)) and the tet-operator sequences was placed 3′ of the TATA-box. 3′ of the H1-promoter with the tet-operator sequence a Fluc-specific shRNA was inserted by BbsI/AscI using annealed oligonucleotides forming the sequence aggattccaattcagcgggagccacct gatgaagcttgatcgggtggctctcgctgagttggaatccattttttt (SEQ ID NO:8; Paddison, P. J. et al., Genes Dev. 16:948-58 (2002)). The codon optimized tet-repressor was PCR amplified from pBS-hTA+nls (Anastassiadis, K. et al., Gene 298:159-72 (2002)) using the oligonucleotides atcgaattcaccatgtccagactgg (sense; SEQ ID NO:9), ataggatccttaagagccagactca catttcagc (antisense; SEQ ID NO:10)) and inserted 3′ of the CAGGS promoter.
Vector 1 (SEQ ID NO:1) contains the following elements in 5′ to 3′ orientation: 5′ homology region for murine rosa26 locus (nucleotides 24-1079), adenovirus splice acceptor site (nucleotides 1129-1249), firefly luciferase (nucleotides 1325-2977), synthetic polyA (2995-3173), CAGGS promoter (nucleotides 3231-4860), synthetic intron (nucleotides 4862-5091), coding region of the wt tet repressor (nucleotides 5148-5750), synthetic polyA (nucleotides 5782-5960), FRT-site (nucleotides 6047-6094), PGK-hygro-polyA (nucleotides 6114-8169), FRT-site, 3′ homology region for rosa26 locus (nucleotides 8312-12643), PGK-Tk-polyA (nucleotides 12664-14848).
Vector 2 (SEQ ID NO:2) contains the following elements in 5′ to 3′ orientation: 5′ homology region for rosa26 locus (nucleotides 24-1102), adenovirus splice acceptor site (nucleotides 1129-1249), firefly luciferase (nucleotides 1325-2977), synthetic polyA (nucleotides 2995-3173), CAGGS promoter (nucleotides 3231-4860), synthetic intron (nucleotides 4862-5091), coding region of the codon optimized tet repressor (nucleotides 5149-5916), synthetic polyA (nucleotides 5946-6124), FRT-site (nucleotides 6211-6258), PGK-hygro-polyA (nucleotides 6278-8333), FRT-site, 3′ homology region for rosa26 locus (nucleotides 8476-12807), PGK-Tk-polyA (nucleotides 12828-15012).
Vector 3 (SEQ ID NO:3) contains the following elements in 5′ to 3′ orientation: 5′ homology region for rosa26 locus (nucleotides 31-2359), adenovirus splice acceptor site (nucleotides 2409-2529), Renilla luciferase (nucleotides 2605-3540), synthetic polyA (nucleotides 3558-3736), hgH-polyA (nucleotides 3769-4566), loxP-site (nucleotides 4587-4620), H1-tetO (nucleotides 4742-4975), shRNA (nucleotides 4977-5042), TTTTTT, loxP-site (nucleotides 5056-5089), FRT-site (nucleotides 5105-5152), PGK-hygro-polyA (nucleotides 5165-6974), FRT-site (nucleotides 6982-7029), 3′ homology region for rosa26 locus (nucleotides 7042-11373), PGK-Tk-polyA (nucleotides 11394-13578).
Cell culture: Culture and targeted mutagenesis of ES cells were carried out as described in Hogan, B. et al., A Laboratory Manual. In Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., pp. 253-289 (1994) with ES cell lines derived from F1 embryos. Cre-mediated deletion has been performed for the deletion of the shRNA part of the constructs to generate the control mice without knockdown. Therefore 5 μg of a cre-expressing construct has been electroporated and the following day 1000 cells were plated at a 10 cm dish. The developing clones were isolated and screened by southern for cre-mediated deletion of the shRNA responder construct.
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 pseudo-pregnant females using standard protocols (Hogan, B. et al. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. 253-289 (1994)).
Preparation and application of doxycycline: 2 mg doxycycline (Sigma, D-9891) was solved in 1 liter H2O with 10% Sucrose. This solution was given in drinking bottles of mice and prepared freshly every 3 days.
Luciferase measurement in organs: Organs were homogenized at 4° C. in lysis buffer (0.1 M KH2PO4, 1 mM DTT, 0.1% Triton® X-100) using a tissue grinder. Spin for 5 min 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.
Discussion: The coding regions of the wt (Gossen and Bujard, PNAS. 89: 5547-5551;
Mice were fed for 10 days with drinking water in the presence or absence of 2 μg/ml Doxycycline.
Vector construction: The following shRNA sequences were cloned 3′ of the H1-tet promoter (SEQ ID NO:222, nucleotides 158-391) followed by five thymidines.
The resulting vectors were named pIR1-pIR6. For example the sequence of pIR5 (SEQ ID NO:222) contains the shRNA IR5 (SEQ ID NO:222, nucleotides 393-440 and SEQ ID NO:228).
Rosa26/CAGGS-tetR/Insulin-receptor-shRNA exchange vector (
Cell culture: Cultures of ES cells were carried out as described in Hogan, B. et al., A Laboratory Manual. In Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., pp. 253-289 (1994) with ES cell lines derived from F1 embryos. Transfection of Art4.12 ES cells containing the FRT/F3 configuration with the pIR5-tet (SEQ ID NO:223) exchange vector has been described in Seibler et al., Nucleic Acids Res. 2005 Apr. 14; 33(7):e67 and PCT/EP05/053245.
Doxycycline induction of ES cells: Cells were treated with 1 μg/ml doxycycline (Doxycycline Hyclate, Sigma D-9891) for 48 h and medium was changed every day.
Transient transfections of muscle cells: C2C12 myoblasts were grown at 37° C. in an atmosphere of 5% CO2 in Dulbecco 's modified Eagle 's medium (DMEM) containing 10%/0 fetal calf serum (FCS), 4500 mg/l glucose and 1× non-essential amino acids. Transfection studies were carried out with 1.35×105 cells plated on a 6-well plate. Cells were transfected 2.5 μg DNA (1.25 μg GFP-vector and 1.25 μg of one of the pIR1-6 vectors). DNA was mixed with 10 μl Lipofectamin (Invitrogen, #18324-111) and 200 μl Optimem (Gibco BRL, #51985-026) and incubated for 45 min at RT. For transfection, cells were washed with 1×PBS and incubated for 5 h in 2 ml starving medium, containing the Optimen-DNA-Solution. After 5 h medium DMEM with 20% FCS was added to the cells. 24 h after transfection cells were washed with 1×PBS and fixed with methanol for 3 min, washed with 1×PBS and dried. Cells were stained with DAPI in Vectashield (Vector). Cells were analyzed for GFP expression and transfection efficiency.
Mice: All mice were kept in the animal facility at Artemis Pharmaceuticals GmbH in micro-isolator cages (Tecniplast Sealsave). B6D2F1 Mice for the generation of tetraploid blastocysts were obtained from Harlan, N L.
Production of ES mice by tetraploid embryo complementation: The production of mice by tetraploid embryo complementation was essentially performed as described in Eggan et al., Proc Natl Acad Sci USA, 98, 6209-6214.
Doxycycline treatment: 2 mg/ml doxycycline (Doxycycline Hyclate, Sigma D-9891) was dissolved in water with 10% sucrose, 20 μg/ml doxycycline was dissolved in water with 1% sucrose and 2 μg/ml doxycycline was dissolved in water with 0.1% sucrose. The doxycycline solutions were freshly made every second day and kept dark.
Protein isolation: Cells were lysed in Protein extraction buffer containing 1% Triton® X-100, 0.1% SDS, 10 mM Tris-HCl pH 7.4, 1.25 mM Tris Base, 10 mM EDTA, 50 mM NaCl, 50 mM NaF, 50 μg Aprotinin protein concentration was measured using the Warburg formula.
Western Blot Proteins were fractionated on a 10% SDS-Page gel and semi-dry blotted for 30 min with 200 mA. Primary antibodies against Insulin receptor and AKT were from Santa Cruz and Cell Signaling Technology. IR antibody was diluted 1:200 and AKT 1:1000 in 2% milk powder (MP) in TBS. Second antibody was goat anti-rabbit IgG (whole molecule)-peroxidase (Sigma, #A6154-1mL), diluted 1:1000 in 2% MP/TBS used with ECL reagents (Amersham, #RPN 2105).
RNA isolation: Total RNA was isolated with peqGOLD TriFast (peqLab, #30-2020) using 2.5 ml for a confluent grown 10 cm plate. Cells were centrifuged for 15 min at 13000 rpm, 4° C. Supernatant was transferred in a new siliconized 2 ml Eppendorf tube and 0,3× volume Chloroform was added to the supernatant. The solution was mixed and centrifuged for 15 min at 13000 rpm, 4° C. The supernatant was transferred into a new siliconized 1.5 ml tube and was precipitated with the same volume of isopropanol. RNA was dissolved in DEPC-H2O.
Northern Blot: 30 μg RNA were fractionated on a 15% denaturating polyacrylamid gel and blotted on a nylon membrane with an ampacity of 3.3 mA/cm2 for 35 min. The RNA was cross-linked to the membrane using UV-light and incubation at 80° C. for 30 min. The membrane was incubated for 2 h in 10 ml prehybridisation solution and labeled with a radioactive probe specific for the used shRNA. 10 U T4-Polynukleotid-kinase (NEB) and 10 μCi γ-[32P]-ATP (10 U μCi/μl) were used for labeling of the radioactive probe.
To investigate the potential of the Doxycycline (Dox) inducible shRNA expression system in vivo, the insulin receptor (IR) gene was chosen as a well-characterized target involved in glucose homeostasis and the development of Diabetes mellitus. Six different shRNA sequences directed against the IR mRNA (SEQ ID NO:221) were tested in the IR expressing muscle cell line C2C12. shRNA coding regions were cloned into a H1 expression vector (pIR1-6) and transiently transfected into C2C12 cells using lipofection. Western blot analysis of protein extracts derived from transfected cells revealed a significant RNAi activity of shRNA constructs pIR5 and pIR6, leading to a >80% reduction of IR expression (
The RMCE strategy (Seibler et al., Nucleic Acids Res. 2005 Apr. 14; 33(7):e67) was subsequently used for targeted insertion shRNA sequence #IR-5 under the control of the H1tet promoter along with a constitutive expression cassette of the codon optimized tet-repressor (SEQ. ID NO:222;
Mice were generated by injection of recombinant ES cell clones into tetraploid blastocysts (Eggan K. (2001) Proc Natl Acad Sci USA 98, 6209-6214.). Approximately six completely ES cell derived mice were obtained from 100 transferred blastocysts into pseudo-pregnant mothers. ShRNA transgenic mice were fed with 2 mg/ml doxycycline in the drinking water for 5 d and the degree of knockdown was detected at the protein level in liver and heart. Western blot analysis revealed a near complete removal of IR in Doxycycline treated animals, whereas the IR expression in untreated controls remained unaltered (
Insertion of transgenes into the targeting vector: All subsequently described transgenes were inserted 3′ of the Renilla luciferase (Rluc) of the basic rosa26 targeting vector described in Example 1. The U6-promoter fragments were amplified from human genomic DNA (using the oligonucleotides ATCGGGATCCAGTGGAAAGAC GCGCAGG (SEQ ID NO:230) and GCTCTAGAAGACCACTTTCTCTATCACTGATAGGGAG ATATATAAAGCCAAGAAATCGA (SEQ ID NO:231)) and the tet-operator sequences was placed 3′ of the TATA-box resulting in the U6-promoter with the tet-operator sequence (U6-tet promoter; SEQ ID NO:232). 3′ of the U6-tet promoter a Fluc-specific shRNA was inserted by BbsI/XbaI using annealed oligonucleotides forming the sequence gggattccaattcagcgggagccacctgatgaagcttgatcgggtggctctcgctgagttggaatc cattttttt (SEQ ID NO:233; Paddison, P. J. et al., Genes Dev. 16:948-58 (2002)). The resulting vector 4 (SEQ ID NO:234) contains the following elements in 5′ to 3′ orientation: 5′ homology region for rosa26 locus (nucleotides 25-1103), adenovirus splice acceptor site (nucleotides 1130-1250), Renilla luciferase (nucleotides 1326-2261), synthetic polyA (nucleotides 2279-2457), hgH-polyA (nucleotides 2490-3287), loxP-site (nucleotides 3308-3341), U6-tetO (nucleotides 3408-3671), shRNA (nucleotides 3672-3740), TTTTTT, loxP-site (nucleotides 3758-3791), FRT-site (nucleotides 3807-3854), PGK-hygro-polyA (nucleotides 3867-5676), FRT-site (nucleotides 5684-5731), 3′ homology region for rosa26 locus (nucleotides 5744-10075), PGK-Tk-polyA (nucleotides 10096-12280).
The U6-tet promoter construct (SEQ ID NO:232) was tested using a dual reporter system consisting of firefly luciferase (Fluc) as a test substrate and Renilla reniformis luciferase (Rluc) as a reference (
The relative firefly luciferase activity was determined in different organs of animals carrying the shRNA construct together with the luciferase- and tetR-transgenes. Upon induction with doxycycline, expression of the shRNA under the control of the engineered U6 promoter resulted in repression of firefly luciferase activity in most organs, ranging between 20-90% gene silencing (
Generation of shRNA transgenic rats: The tetracycline system based DNA construct carrying shRNA cassette against the InsR was designed by 3. Seibler (Seibler, J. et al., Nucleic Acids Res 35(7):e54 (2007)). The tetO sequence was inserted 3′ of the TATA box of the human H1-promoter H1-tet controlling the InsR-shRNA. Downstream of the shRNA cassette was inserted codon optimized TetR driven by the CAGGS promoter (Seibler, J. et al., ibid.; see SEQ ID NO:222 and
To test both transgenic lines TetO14 and TetO29 for TetR and shRNA expression the animals were treated with 2 mg/ml DOX in the drinking water for 4 d. By a specific Ribonuclease Protection Assay (RPA) shRNA expression of both treated lines in several tissues was confirmed: muscle, liver, brown adipose tissue (BAT), white adipose tissue (WAT), kidney, heart and brain. No shRNAs were detectable in untreated transgenic rats (
Downregulation of InsR was assayed with Western blot analysis, which monitored an efficient gene silencing in both transgenic lines, when treated with DOX (
Glucose and Insulin: During the DOX treatment (2 mg/ml) blood was taken from the tail-vein of rats to measure blood glucose and plasma insulin. Drastic increases of these parameters were detected after three days of DOX treatment in TetO29 rats and one day later also in TetO14 rats (
Blood glucose levels became 3 fold higher than in control animals. Correspondingly, the plasma insulin level was enhanced for more than 7 fold (
Insulin Signaling: First, an insulin sensitivity test was performed to check whether glucose levels in the InsR knock down rats can be affected by insulin injection. The blood glucose was measured before and 15 min after i.p. injection of insulin (10 U/kg) or saline as a control. Insulin led to a significant decrease in glucose in control animals (WT DOX+ and TetO29 DOX−) but not in the treated transgenic rats (
For further studies, the intracellular signaling attempt of the InsR in rats acutely treated with insulin was determined. The phosphorylation of the Akt protein was analyzed, a Ser/Thr kinase activated through the cascade of reactions initiated by the InsR after insulin binding. Western blotting analyses of proteins from WAT, BAT and skeletal muscle showed stronger phosphorylation of Akt after insulin injection in all control rats. In contrast, no or very weak Akt phosphorylation was seen in DOX treated transgenic rats (
Reversibility of knockdown: Next, it was tested whether the InsR knock down was reversible. Different DOX doses (20 mg/kg, 2 mg/kg and 0.5 mg/kg) were employed in three groups of female TetO29 rats. Once glucose levels between 250 and 300 mg/dl were reached in the treated transgenic rats, DOX was withdrawn from their drinking water. Despite cessation of the drug blood glucose increased further in all tested groups until reaching a plateau (350 mg/dl-450 mg/dl) and, dependent on the given dose, stayed stable for 1-2 weeks. After that, the increased glucose levels slowly returned back to normal level in all examined groups (
In parallel to the blood glucose level, drinking level increased dose-dependently in all DOX treated transgenic rats and returned to normal level after drug withdrawal (data not shown).
These data show that the tetracycline inducible system used in these rats to shRNA mediated gene knock down is completely reversible after cessation of DOX.
Chronic diabetes type II model: In order to establish a novel chronic model of type II diabetes mellitus, a group of TetO29 rats was treated daily with 0.5 mg/kg of DOX solution (0.5 mg/ml) containing 1% sucrose. When blood glucose reached 300 mg/dl this dose was changed to unlimited daily drinking of the 1 μg/ml DOX solution (in 1% sucrose). This dosage was maintained for the duration of the study which lasted 40 days. The long term treatment with these low DOX doses resulted in a slow enhancement of the blood glucose levels and drinking volume in transgenic rats (
Moreover, a slight progressive loss of body weight was observed in the chronically diabetic rats (
In the chronically treated rats also a high expression of shRNA and near complete down regulation of InsR in the liver was detected (data not shown).
Chronic diabetes mellitus leads to damage of kidney, heart, vessels and retina. In order to test whether such pathologies appear in our chronic model we collected urine to estimate the daily urinary output and albumin excretion. Measurements were carried out once weekly in the last 3 weeks of the study. Our analyses showed significant polyuria of chronically treated TetO29 rats in the last 2 weeks of the treatment (week 5 and 6) compared to the non treated TetO29 group (
To determine the renal damage of recovered TetO rats after DOX cessation, the same set of tests was performed. Interestingly, total urine volume was significantly higher compared to untreated TetO29 group (data not shown). Albumin excretion was slightly, but not significantly increased (data not shown). In spite of reversible shRNA activation these data show that high drug doses may lead to irreversible diabetic damages.)
Lack of toxicity: The complete reversibility of the phenotype after DOX withdrawal was already a support against a toxic effect of the shRNA expression. Nevertheless, we tested whether shRNA expression triggers interferon (IFN) response in acute or chronically treated TetO rats. For this purpose western blotting was used to detect PKR, an interferon-inducible Ser/Thr specific protein kinase. No PKR upregulation was detected in all tested tissues, such as BAT, WAT and brain, after acute high dose treatment with DOX as well as in the liver after chronic low dose treatment (
We further checked for alteration in the biogenesis of natural pre-microRNA. Using RPA we did not observe any alterations in the expression of the endogenous mir-122 in the line of transgenic rats after long term shRNA induction by low dose DOX treatment of TetO29 rats (
| Number | Date | Country | Kind |
|---|---|---|---|
| 05105076.3 | Jun 2005 | EP | regional |
| 06110759.5 | Mar 2006 | EP | regional |
This application is a continuation-in-part of U.S. application Ser. No. 11/912,451, filed on Oct. 24, 2007, which is a 371 of PCT/EP06/063001, filed Jun. 8, 2006, which claims foreign priority benefit under 35 U.S.C. § 119 of the European Patent Application No. 05105076.3 filed Jun. 9, 2005 and European Patent Application No. 06110759.5 filed Mar. 7, 2006.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11912451 | US | |
| Child | 12118025 | US |
