Patent Grant
8530168
This application is the U.S. National Stage of International Application No. PCT/EP2008/005013, filed Jun. 20, 2008, which claims the benefit of European Patent Application No. 07 012 138.9, filed Jun. 21, 2007, each of which is hereby incorporated by reference.
The present application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 27, 2012, is named 50125163001.txt and is 329,640 bytes in size.
The present invention relates to a fusion protein comprising a Caspase domain or a functionally active variant thereof and a ligand binding domain of a nuclear hormone receptor, a nucleic acid coding for the fusion protein, a vector or cell comprising the nucleic acid, a method of producing the fusion protein, a non-human transgenic animal containing the nucleic acid, the use of the fusion protein for ligand-mediated induction of apoptosis of a cell, or for studying the function of a cell, tissue and/or organ or the use of a transgenic organism for studying the function of a cell at various developmental stages or as a disease model, a method for inducing apoptosis of a cell expressing a fusion protein or for identifying a ligand, or a medicament comprising a fusion protein, the nucleic acid, the vector or the cell, particularly for the treatment of cancer or for or after transplantation, particularly as safety mechanism.
One target of genetic and genomic research is focused on the elucidation of function of individual genes within cells and organisms. Many genes are active only in certain cells and thereby contribute to the complex organisation of the mammalian body composed of hundreds of different cell types. At the level of the whole organism not single genes or gene families interact but population of cell types exist and fulfill biological functions.
To investigate these cellular functions experimentally, mutant analysis is a powerful tool. Like genetic mutants that are used to study the function of individual genes and to create models of genetic disease it is desirable to be able to create mutants for specific cell types or populations of cells in order to study their functional role in vivo. This aspect is of particular interest for the creation of animal models of human degenerative diseases that are characterized by the loss of specific cell populations, e.g. the loss of dopaminergic neurons in Parkinson's disease, or to mimic the damage of specific organs like heart or liver.
Furthermore, cells taken from a donor individual or cells grown in in vitro cultures can be transplanted or transferred into a recipient for research or therapeutic purposes. Upon cell transfer it is desirable to be able to ablate specifically all or some of the transplanted cells either to study the functions these cells fulfill in the recipient body or to enhance the safety of cell therapy if the transplanted cells thread the recipient by e.g. tumorigenesis or a graft versus host reaction (Cohen, et al., Immunol Today, 20, 172-176 (1999)) (Cohen, et al., Leuk Lymphoma, 34, 473-480 (1999)) (Cohen, et al., Hum Gene Ther, 10, 2701-2707 (1999) (Berger, et al., Blood, 103, 1261-1269 (2004)).
Moreover, in a cancer therapy termed suicide gene therapy tumor cells are equipped with an expression vector for a gene that allows to destroy these cells upon administration of a specific drug (Hurwitz, et al., Hum Gene Ther, 10, 441-448 (1999)) (Fillat, et al., Curr Gene Ther, 3, 13-26 (2003)) (Niculescu-Duvaz and Springer, Mol Biotechnol, 30, 71-88 (2005)) (Portsmouth, et al., Mol Aspects Med, 28, 4-41 (2007)).
In conclusion, it is an important aspect of biological and medical research to be able to manipulate the cellular composition of an organism such as the mammalian body. Ideally, methods would be available that enable cell ablation in a specific and also in a timed manner and that are safe, simple and universally applicable to all cell types and organs of the mammalian body.
Over the last two decades a variety of genetic methods has been developed to ablate selected cells in the body, mostly using the mouse as a model organism. These strategies can be classified into non-inducible methods that cannot be regulated from outside and lead to preprogrammed cell death during development and into inducible methods that employ initially innocent transgenes that are able mediate cell death upon administration of an inducer molecule.
The various methods are further distinguished by the biochemical mechanisms that lead to cell death, i.e. either by the accumulation of toxic products and necrotic cell death or by the use of endogenous pathways that lead to programmed cell death through apoptosis. The innate immune system reacts differently to cells that underwent pathological (necrosis) or physiological cell death (apoptosis) such that the clearance of necrotic cells is associated with proinflammatory responses of phagocytic macrophages (Cocco and Ucker, Mol Biol Cell, 12, 919-930 (2001)) (Krysko, et al., Apoptosis, 11, 1709-1726 (2006)). Therefore, the latter method is most appropriate to model disease processes that involve apoptotic cell death.
The strategies for non-inducible cell ablation in mice have used transgenes that employ the cell type specific expression of toxic proteins like the diphtheria toxin A chain (Breitman, et al., Science, 238, 1563-1565 (1987)) (Breitman, et al., Mol Cell Biol, 10, 474-479 (1990))(Kaur, et al., Development, 105, 613-619 (1989)) or Ricin (Landel, et al., Genes Dev, 2, 1168-1178 (1988)). This method was later refined such that the expression of diphtheria toxin can be controlled by the activity of Cre recombinase. In such double transgenic mice Cre recombinase is expressed from a cell type specific promoter while the diphtheria toxin transgene is under control of an ubiquitous active promoter but toxin expression occurs only upon Cre mediated deletion of an inhibitory DNA segment (Brockschnieder, et al., Mol Cell Biol, 24, 7636-7642 (2004)) (Brockschnieder, et al., Genesis, 44, 322-327 (2006)) (Ivanova, et al., Genesis, 43, 129-135 (2005)). Non-inducible cell ablation strategies rely solely on the activity of cell type specific promoter region, the activity of which cannot be further influenced in vivo. Thus, cell ablation occurs upon the initial activation of the utilised promoter region during embryonic development.
To gain also control on the timing of cell ablation a variety of inducible ablation strategies has been developed. Two of these methods are based on the transgenic expression of prokaryotic enzymes that modify specific prodrugs into cytotoxic derivates. The prodrugs are not recognised by mammalian enzymes. Thus, the cells expressing the prokaryotic enzyme are only killed upon the administration of the specific prodrug.
The use of a thymidine kinase derived from Herpes simplex virus (HSV-tk) enables to kill HSV-tk expressing, dividing cells by the administration of Ganciclovir (GANC) (Sofroniew, et al., Brain Res, 835, 91-95 (1999)) (Visnjic, et al., J Bone Miner Res, 16, 2222-2231 (2001)) (Rindi, et al., Development, 126, 4149-4156 (1999)) (Tian, et al., Am J Pathol, 163, 789-801 (2003)) (Ito, et al., Nat Med, 11, 1351-1354 (2005)) (Dancer, et al., Gene Ther, 10, 1170-1178 (2003)) (Lalancette-Hebert, et al., J Neurosci, 27, 2596-2605 (2007)) (Zhang, et al., Febs J, 272, 2207-2215 (2005)). GANC is phosphorylated only by HSV-tk and then blocks DNA replication leading to the death of mitotic cells. Postmitotic, resting cells cannot be ablated with the HSV-tk/GANC system.
The use of the Nitroreductase (NTR) gene derived from E. coli enables to kill NTR expressing cells by the administration of the prodrug CB1954 (Clark, et al., Gene Ther, 4, 101-110 (1997)) (Cui, et al., Glia, 34, 272-282 (2001)) (Isles, et al., J Neurobiol, 47, 183-193 (2001)) (Gusterson, et al., Recent Results Cancer Res, 163, 31-45 (2003)). The cytotoxic derivative leads to the formation of interstrand DNA crosslinks which are poorly repaired by the cells. The NTR system is independent of the cell cycle and can be applied to non-dividing cells (Grove, et al., Cancer Res, 63, 5532-5537 (2003)). The prodrug CB1954, however, has evolved from cancer therapy and a significant bystander effect has been observed because of local spread of the activated prodrug that leads to the death of neighbored cells (Bridgewater, et al., Hum Gene Ther, 8, 709-717 (1997)) (Nishihara, et al., Anticancer Res, 18, 1521-1525 (1998)). While this effect is beneficial for cancer therapy it diminishes the utility of the NTR system for specific cell ablation.
In another inducible approach cells that express a receptor for diphtheria toxin (DTR) from a cell type specific transgene can be killed by the in vivo administration of diphtheria toxin A chain (DTA) (Buch, et al., Nat Methods, 2, 419-426 (2005)) (Chang and Yang, Sci STKE, 2003, PL1 (2003)) (Stoneman, et al., Circ Res, (2007)). DTA is toxic upon internalisation that is mediated by the transgenic DTR.
Besides the use of toxins or enzymes that lead to cytotoxic products two methods for inducible cell ablation have been developed that exploit endogenous cellular mechanisms of programmed cell death.
In the system described by Takebayashi (Takebayashi, et al., Cancer Res, 56, 4164-4170 (1996)) the transmembrane and intracellular domain of the Fas death receptor (amino acid 135-305) has been fused N-terminally to the ligand binding domain of the rat estrogen receptor. This fusion protein was constitutively expressed in L929 cells known to be sensitive to Fas-mediated apoptosis. From studies with wildtype estrogen receptor it has been found that upon ligand administration the ER domain undergoes a conformational change that leads to the dissociation of bound heat shock proteins and receptor dimerisation. The administration of estradiol to Fas-ER expressing L929 cells, T-lymphocytes or HeLa cells leads to cell death by apoptosis (Takebayashi, et al., Cancer Res, 56, 4164-4170 (1996)) (Kawaguchi, et al., Cancer Lett, 116, 53-59 (1997)) (Kametaka, et al., Cancer Sci, 94, 639-643 (2003)).
In a variation of this method the non-modified ER domain was replaced by a mutant murine ER ligand binding domain (amino acids 287-599) that harbours a single amino acid exchange (G525R). This mutation leads to a strongly reduced affinity to estradiol but the receptor can still be activated by 4-OH-tamoxifen. This Fas-ER(G525R) fusion protein was tested in the mouse cell line L929 (Kodaira, et al., Jpn J Cancer Res, 89, 741-747 (1998)). The Fas-ER method uses the extrinsic CD95 apoptosis pathway to induce cell death. Since this pathway is restricted in vivo largely to cells of the immune system (Krammer, Nature, 407, 789-795 (2000)) most other cell types in the body may be unresponsive to Fas-ER fusion proteins.
A cell ablation method that utilises ubiquitously expressed components of the intrinsic apoptosis pathways was first described by MacCorkle (MacCorkle, et al., Proc Natl Acad Sci USA, 95, 3655-3660 (1998)). For this method a domain of the FK506 binding protein FKBP was fused to the N-terminus of Caspase-1 or Caspase-3 and expressed in human Jurkat T cell lymphoma cells. Upon administration of dimeric FK506 (FK1012; Pruschy, et al., Chem Biol, 1, 163-172 (1994)), a chemical inducer of dimerisation (CID), the fusion proteins undergo oligomerisation and lead to cell death by apoptosis. This system was further developed by the fusion of one or more modified FKBP domains (Fv) to the N-terminus of Fas, Bax, Caspase-1, -3, -8 and -9 (Fan, et al., Hum Gene Ther, 10, 2273-2285 (1999)) (Hou and Hsu, Am J Physiol Heart Circ Physiol, 289, H477-487 (2005)). The Fv domain can be dimerised by the FK1012 analogs AP1903 (Fan, et al., Hum Gene Ther, 10, 2273-2285 (1999)) or AP20187 (Chang, et al., J Biol Chem, 278, 16466-16469 (2003)) that exhibit a higher affinity to the modified Fv domain than to the wildtype FKBP. However, FK506 and analogs that bind to FKBP exhibit a strong immunosuppressive action in vivo (Bierer, et al., Curr Opin Immunol, 5, 763-773 (1993)). The CID apoptosis system has been used for the ablation of transplanted endothelial cells in vivo that were transduced with a viral vector expressing a Fv-Caspase-9 fusion protein (Nor, et al., Gene Ther, 9, 444-451 (2002)) and to demonstrate suicide gene therapy of prostate cancer cells with a viral vector expressing a Fv-Caspase-1 protein (Shariat, et al., Cancer Res, 61, 2562-2571 (2001)). This system was further used in transgenic mice expressing a Fv-Caspase-3 fusion protein in hepatocytes as a model of inducible liver injury (Mallet, et al., Nat Biotechnol, 20, 1234-1239 (2002)) and in transgenic mice expressing a Fv-Caspase-8 fusion protein in adipocytes to create a model of inducible lipoatrophy (Pajvani, et al., Nat Med, 11, 797-803 (2005)).
Although great efforts have been undertaken to derive systems that allow inducible cell ablation in the mammalian body the existing technologies have severe limitations that limit their practical use:
In contrast to the diversity of biological and medical research application of inducible apoptosis systems for mammalian cells, very limited efforts have been made to optimise inducible apoptosis techniques towards a universal use in mammals. Alternative apoptosis induction systems of different ligand specificity could further enhance the flexibility of cell and tissue engineering in vivo.
The reason for this unsatisfying situation is readily explained by a number of requirements that should be fulfilled—at least in part—by a universally useful inducible apoptosis system in mammals:
Therefore, it was an object of the present invention to provide an alternative fusion protein providing inducible apoptosis and preferably avoiding one or more of the above limitations. Particularly, the object to be solved by the invention of the present application is the provision of an inducible apoptosis system alternative to the Casp-FKBP and the Fas-ER systems, which has a different ligand binding domain or a different apoptosis inducing domain. Such an alternative inducible apoptosis system is particularly desirable for all those applications which require universal activation in any organ and any cell type of the mammalian body, including the brain.
Surprisingly, this object has been solved by a fusion protein comprising a Caspase domain or a functionally active variant thereof and a ligand binding domain of a nuclear hormone receptor or a functionally active variant thereof.
Given the limited knowledge on the protein biochemistry of steroid receptors and the molecular mechanisms of apoptosis, it is presently not possible to rationally design biological active and inducible apoptosis-inducing fusion proteins. In particular, it has not been described that a protein such as a Caspase that naturally requires proteolytic processing to develop enzymatic activity or that a protein that acts as a protease could be successfully fused with the ligand binding domain of a nuclear hormone receptor (LBD) into a ligand inducible fusion protein. In particular, upon fusion of a Caspase domain with a steroid receptor LBD it was unpredictable whether such a fusion protein developed biological activity since the molecular mechanism of Caspase activation are essentially unknown. For the only described example of the fusion of an apoptosis-related molecule, the Fas receptor, with the ER(T) LBD mutant, it is important to note that the intracytoplasmic domain of Fas receptor does not act as a protease and it has been found that a simple fusion of this domain with ER(T) is biologically inactive. Biological activity could be only detected in a fusion protein that also included the transmembrane region of the Fas receptor (Takebayashi, et al., Cancer Res, 56, 4164-4170 (1996)) such that it is unlikely that this fusion protein becomes only activated by heat shock protein dissociation or induced dimerisation but rather by a third, yet unknown mechanism. With regard to the above described Caspase fusion proteins with one or more FKBP-derived dimeriser (CID) domains it has been found that the forced oligomerisation leads to Caspase activation but the underlying mechanism remains unknown. In this system the CID domains have been fused onto the N-terminal end of Caspases or Caspase domains. Fusion proteins with dimeriser domains onto the C-terminal end of Caspases or Caspase domains have not been described and it is unknown whether such molecules would develop biological activity. The expression of a newly designed Caspase fusion protein in mammalian cells can be in general a difficult task since the two fusion protein domains may not acquire their native conformation during translation. Furthermore, the three-dimensional structure of such a fusion protein may be inappropriate for the interaction of a pair of Caspase domains, for the proteolytic activity of activated Caspase or for the ligand induced activation of the fusion partner. In addition, a newly designed fusion protein may exhibit a short half-life or form aggregates that lead to its rapid degradation by the proteasome machinery, or the fusion protein mRNA exhibits a short half-life or may contain cryptic splice sites.
Inventors could now show that fusion proteins comprising a Caspase domain, particularly a domain of Caspase 8 or 9, and a ligand binding domain of a nuclear hormone receptor, particularly mutant mammalian estrogen receptor ER(T2), expressed in mammalian cells induced apoptosis in these cells upon exposure to a ligand for that ligand binding domain of a nuclear hormone receptor, particularly the synthetic ligand 4-hydroxy-tamoxifen. It was proven that either full length Caspase or a functionally active fragment thereof can be used within the fusion protein.
Quantitative analysis of apoptosis upon ligand administration using the cells transiently expressing the fusion protein of the invention revealed that observed cell death in combination with the expression of the fusion proteins is a specific effect. In particular, the inventors provide first evidence for three highly efficient Caspase-ER(T2) fusion proteins: myrCasp8-ER(T2), Casp8-ER(T2) and Casp9full-ER(T2) (see Examples).
Also the stable genomic integration of active ER(T2) fusion proteins confirmed the results obtained for transient expression, namely the ability of the fusion protein to induce 4-OH-tamoxifen-dependent cell death in stably transfected cells.
Taken together, inventors have demonstrated for the first time that fusion constructs of Caspase domains and ligand binding domains of nuclear hormone receptor provide a highly efficient system to conditionally ablate mammalian cells. Moreover, since Caspases, particularly Caspase 8 and Caspase 9, are ubiquitously expressed in mammalian tissues and are both involved in different pathways of apoptosis, the potential universal application of nuclear hormone receptor fusions to Caspases, such as Caspase 8 or Caspase 9, for inducible cell ablation is of commercial relevance in biotechnology.
The present invention is the first disclosure of a protein that naturally requires proteolytic processing to develop enzymatic activity and that a fusion protein comprising a protein that acts as a protease, could be successfully fused with a steroid receptor LBD into a ligand inducible fusion protein.
The resulting Caspase nuclear hormone receptor ligand binding domain fusion proteins allow the highly efficient induction of Caspase activation leading to apoptosis in mammalian cells upon administration of a ligand that binds to the ligand binding domain.
The improved inducible apoptosis system of the present invention provides a universal apoptosis system for use in mammalian cells and organisms that allows to study the biological function of selected cells or a cell type in the mammalian body and thereby the creation of a wide range of animal models of human diseases. This apoptosis system further allows to remove transplanted cells that contain a fusion protein expression vector from the body of a recipient upon induction or to destroy tumor cells that were transduced or transfected with a fusion protein expression vector.
Accordingly, in a first aspect the present invention relates to a fusion protein comprising
Thus, the present invention enables the highly efficient modification of the cellular composition of the mammalian body by cell type-specific, inducible apoptosis. Said process possesses the following advantages over current technology:
In a preferred embodiment of the invention upon exposure to a ligand of the ligand binding domain of a nuclear hormone receptor, the fusion protein is capable of inducing apoptosis in a cell, preferably a eukaryotic cell, expressing the fusion protein.
Therefore, the first component of the fusion protein is a Caspase domain that is any domain of a Caspase capable of inducing apoptosis or a functionally active variant thereof.
Caspases are central components of the machinery for apoptosis. Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of multicellular organisms (Jacobson, et al., Cell, 88, 347-354 (1997)). In humans, both excessive and insufficient apoptosis can lead to severe pathological consequences. Suppression of the apoptotic machinery causes autoimmune diseases and is a hallmark of cancer (Hanahan and Weinberg, Cell, 100, 57-70 (2000)) (Thompson, Science, 267, 1456-1462 (1995). On the other hand, abnormal upregulation of apoptosis contributes to neurological disorders (Yuan and Yankner, Nature, 407, 802-809 (2000)).
Fourteen distinct mammalian Caspases have been identified so far; at least 7 of these play important roles during apoptosis (Shi, Mol Cell, 9, 459-470 (2002)), namely Caspases 2, 3, 6, 7, 8, 9 and 10.
Caspases involved in apoptosis are generally divided into two categories, the initiator Caspases, which include without limitation Caspase 1, 8, 9, and 10, and the effector Caspases which include without limitation Caspase 3, 6, and 7.
An initiator Caspase is in general characterised by an extended N-terminal prodomain (>90 amino acids) important for its function, whereas an effector Caspase contains 20-30 residues in its prodomain sequence.
Caspases are produced in cells as catalytically inactive zymogens and must undergo proteolytic activation during apoptosis. The activation of an effector Caspase (e.g. Caspase 3) is performed by an initiator Caspase (e.g. Caspase 8 or 9) through cleavage at specific internal aspartate residues that separate the large and small subunits. The initiator Caspases are autoactivated; as this activation triggers a cascade of downstream Caspase activation, it is tightly regulated and requires the assembly of a multicomponent complex termed apoptosome (Bao and Shi, Cell Death Differ, 14, 56-65 (2007)). The initiator Caspases contain one of two protein-protein interaction motifs, the CARD (Caspase recruitment domain) or the DED (death effector domain). These motifs interact with similar motifs present on oligomerized adaptor proteins, bringing multiple initiator Caspase molecules into close proximity and facilitating their autoactivation (Shi, Mol Cell, 9, 459-470 (2002)).
However, the exact mechanisms by which the initiator Caspases are activated by the apoptosome remain elusive. Several models have been proposed: i) the induced proximity model summarizes the general process of initiator Caspase activation, ii) the proximity-driven dimerisation model describes how initiator Caspases respond to induced proximity, iii) the induced conformation model posits that the activated conformation for the active site of a initiator Caspase is attained through direct interaction with the apoptosome or through homo-oligomerization facilitated by the apoptosome (Bao and Shi, Cell Death Differ, 14, 56-65 (2007)). The functional Caspase unit is a homodimer, with each monomer comprising a large 20 kDa and a small 10 kDA subunit. Homodimerization is mediated by hydrophobic interactions, with 6 antiparallel beta-strands from each catalytic subunit forming a single contiguous 12-stranded beta-sheet. Several alpha-helices and short beta-strands are located on either side of the central beta-sheet, giving rise to a globular fold. The active sites, formed by four protruding loops from the scaffold, are located at two opposite ends of the beta-sheet (Shi, Mol Cell, 9, 459-470 (2002)). Once activated the effector Caspases are responsible for the proteolytic cleavage of a broad spectrum of cellular targets, leading ultimately to cell death.
In accordance with the present invention the first component of the fusion protein may also be a functionally active variant of a Caspase domain. Functional active variants are obtainable by changing the sequence of the Caspase domain as defined herein and are characterized by having a biological activity similar to that displayed by the Caspase domain from which it is derived, including the ability to induce apoptosis. Ability to induce apoptosis of a variant can be determined e.g. as described in the Examples, i.e. by producing a fusion protein as described in Example 1, wherein the variant is to be substituted for the Caspase domain, expressing the fusion protein and determining apoptosis in response to e.g. 4-OH-tamoxifen as described in Example 2 or 3.
Alternatively, the activity of a functionally active variant can be determined in vitro by the cleavage of chromophore-conjugated synthetic peptide substrates that mimic the cleavage site for the respective Caspase. Suitable tests, which can be used in order to determine activity of a variant are described in the art (see e.g. Kohler et al., J Immunol Methods, 265, 97-110 (2002)) (Thornberry et al., J Biol Chem, 272, 17907-11 (1997)).
The variant of an Caspase is functionally active in the context of the present invention, if the activity of the fragment amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the Caspase without sequence alteration.
A variant of the above Caspases in accordance with the present invention relates to a mutant of the respective original (viz. wild-type) Caspase having a Caspase activity as defined above (e.g. at least about 50% of said wild-type Caspase). Variants include truncated forms of the Caspase (such as N- or C-terminal truncated Caspase proteins), deletion-type mutants (where one or more amino acid residues or segments having more than one continuous amino acid residue have been deleted from the primary sequence of the wildtype Caspase), replacement-type mutants (where one or more amino acid residues or segments of the primary sequence of the wildtype Caspase have been replaced with alternative amino acid residues or segments), or the addition of signal peptides that alter intracellular localisation (where e.g. the myristoylation signal sequence GSSKSKPKDPSQR (SEQ ID NO: 82) have been added to the Caspase N-terminus) or combinations thereof.
In one embodiment of the present invention the Caspase domain or functionally active variant thereof may be a fragment. The fragment is characterized by being derived from a naturally occurring Caspase as defined below by one or more amino acid deletions. The deletion(s) may be C-terminally, N-terminally and/or internally. Preferably the fragment is obtained by at most 100, more preferably by at most 50, even more preferably at most 30, still more preferably at most 10, most preferably 1, 2, 3, 4 or 5 deletion(s).
The functional active fragment may be also characterized by its sequence homology to the wild type domain. Accordingly, in one preferred embodiment of the invention the functional active fragment consists of at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, most preferably 99% of any naturally occurring Caspase, e.g. those listed above. The functional active fragment as defined above may be derived from the peptide by one or more amino acid deletions. The deletions may be C-terminally, N-terminally and/or internally.
In another preferred embodiment of the invention the Caspase domain is a functionally active variant of a Caspase, wherein the variant is derived from any naturally occurring Caspase, e.g. those listed above, by one or more amino acid deletion(s), addition(s) and/or substitution(s) and preferably wherein the variant has at least 50% sequence identity to a naturally occurring Caspase domain. In a more preferred embodiment the functional active variant has a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, most preferably 99% to any naturally occurring Caspase, e.g. those listed above.
The percentage of sequence identity can be determined e.g. by sequence alignment. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms have been described e.g. in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 or Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448, 1988.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Variants of any naturally occurring Caspase, e.g. those listed above, are typically characterized using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of at least 35 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
As noted above, the functionally active variant of a Caspase is obtained by sequence alterations in the sequence of the Caspase, wherein the variant retains the function of the Caspase (see above). The term “functionally active variant” includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants.
However, if the variant is obtained from a Caspase by one or more substitution(s) conservative substitution(s) is/are preferred. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. In one embodiment, one conservative substitution is included in the variant. In another embodiment, two conservative substitutions or less are included in the peptide. In a further embodiment, three conservative substitutions or less are included in the variant.
Examples of conservative amino acid substitutions include, but are not limited to, the following: Ala→Ser; Arg→Lys; Asn→Gln or His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→; His→Asn or Gln; Ile→Leu or Val; Leu→Ile or Val; Lys→Arg or Gln or Asn; Met→Leu or Ile; Phe→Met or Leu or Tyr; Ser→Thr; Thr→Ser; Trp→Tyr; Tyr→Trp or Phe; Val→Ile or Leu, wherein the amino acid mentioned first (before the arrow) indicates the original amino acid without substitution and the second amino acid(s) (after the arrow) indicate(s) the amino acid to be substituted for the respective first amino acid.
In case of one or more amino acid addition(s), these may result for the cloning of the Caspase or functionally active variant thereof, e.g. due to the use of particular restriction sites, and may or may not alter (increase or decrease) the activity of the Caspase. Alternatively, amino acids may be added in order to achieve a desired result, e.g. addition of a tag to provide for convenient purification.
Caspase proteins which can be used in the Caspase domain of the fusion protein of the present invention include, but are not limited to, a certain type of apoptosis inducing proteases belonging to the mammalian families of initiator and effector Caspases (Ho and Hawkins, Febs J, 272, 5436-5453 (2005)) (Bao and Shi, Cell Death Differ, 14, 56-65 (2007)) (Shi, Mol Cell, 9, 459-470 (2002)). These families include Caspase-3 (the amino acid sequences of said murine and human Caspase are shown in SEQ ID NOS: 58 and 59, respectively), Caspase-7 (the amino acid sequences of said murine and human Caspase are shown in SEQ ID NOS: 60 and 61, respectively), Caspase-6 (the amino acid sequences of said murine and human Caspase are shown in SEQ ID NOS: 62 and 63, respectively), Caspase-8 (the amino acid sequences of said murine and human Caspase are shown in SEQ ID NOS: 64 and 65, respectively), Caspase-10 (the amino acid sequence of said human Caspase is shown in SEQ ID NO: 66), Caspase-9 (the amino acid sequences of said murine and human Caspase are shown in SEQ ID NOS: 67 and 68, respectively), Caspase-2 (the amino acid sequences of said murine and human Caspase are shown in SEQ ID NOS: 69 and 70, respectively), Caspase-12 (the amino acid sequence of said murine Caspase is shown in SEQ ID NO: 71), and the like, or mutants thereof. Other vertebrate Caspases known in the art are also applicable.
Preferably, in the context of the present invention the Caspase domain is a Caspase or functionally active variant thereof selected from the group consisting of Caspase-2, Caspase-3, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, and Caspase-12, or functionally active variant thereof, most preferably Caspase-8 or Caspase-9 or a functionally active variant thereof. More preferably, the Caspase is a mammalian Caspase, especially a murine or human Caspase, still more preferably selected from the group consisting of Caspase-2, Caspase-3, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, and Caspase-12, most preferably Caspase-8 or Caspase-9, especially a murine or human Caspase-8 or Caspase-9. Preferred examples of sequences of Caspases are those of SEQ ID NO: 58 to 71, especially of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67 or SEQ ID NO: 68.
In another preferred embodiment of the invention the Caspase domain of the fusion protein as defined above comprises or consists of the amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67 or SEQ ID NO: 68; or functionally active variants, particularly fragments thereof.
Most preferably, in the fusion protein of the invention the Caspase domain is preferably a murine Caspase-9 having the amino acid sequence shown in SEQ ID NO: 67 or a N-terminal truncated form thereof, or a murine Caspase-8 having the amino acid sequence shown in SEQ ID NO: 64 or a modified Caspase-8 that is fused with a myristoylation signal sequence at the N-terminus. Suitable truncated forms of the Caspase-9 comprise amino acid residues 92 to 454 of SEQ ID NO: 67; suitable modified Caspase-8 having an N-terminal fusion with a myristoylation signal peptide comprise the sequence GSSKSKPKDPSQR (SEQ ID NO: 82).
The second component of the fusion protein of the invention is the ligand binding domain of a nuclear hormone receptor (LBD) or a functionally active variant thereof. The LBD is located in the carboxyl-terminal half of the receptor, consists in general of about 300 amino acids.
It is noted that the ligand binding domain of a nuclear hormone receptor (LBD) or a functionally active variant thereof can be activated upon binding of a ligand to the LBD. In accordance with the present invention, the Caspase activity of the fusion protein in a cell is significantly higher in the presence of ligand as compared to its activity in the absence of ligand for the LBD or variant thereof.
A “significantly higher activity” in accordance with the present invention refers to an increase in death rate of at least 25%, preferably at least 50%, more preferably at least 75, most preferably at least 90%. The death rate may be determined as detailed in Example 2 or 3, wherein the respective fusion protein is to be used instead of those used for the Examples and the ligand is to be chosen in accordance with the LBD. For example, the death rate may be determined within 3 days in the presence of the ligand, e.g. 4-OH-Tamoxifen at a 10−6 molar (or lower) concentration to a MEF5 cell clone that expresses a stably integrated Caspase-ER(T2) fusion protein expression vector.
Fusion proteins including wild type and mutant steroid receptors have already been generated to regulate the activity of the fusion partner by natural or synthetic ligands (Picard, Curr Opin Biotechnol, 5, 511-515 (1994)). It is believed that the heat shock proteins that are bound to the ligand binding domain of a nuclear hormone receptor (LBD) and that dissociate upon ligand binding keep the fusion partner inactive in the absence of ligand (Picard, Curr Opin Biotechnol, 5, 511-515 (1994)). This inhibition could occur either by sterical hindrance or by partial unfolding of the protein structure. The exact mechanism of this phenomenon has not been resolved such that a predictable construction of regulated, inducible LBD fusion proteins is not possible (Picard, Curr Opin Biotechnol, 5, 511-515 (1994)).
Therefore, the derivation of new fusion proteins is presently rather an empirical approach than a theoretical approach. The only parameter known that might be important for the regulability of a fusion protein is the length of the connecting region between the fusion partner and the LBD (Picard, Curr Opin Biotechnol, 5, 511-515 (1994)). If the spacing is rather long the fusion partner may be active even in the absence of ligand but if the distance is too short the activity of the fusion partner can be completely abolished even in the presence of ligand. A second, intrinsic property of LBD fusion proteins is the presence of a hidden nuclear localisation function that becomes active only upon ligand binding such that fusion proteins usually accumulate in the nucleus.
Furthermore, it has been found that the wildtype estrogen receptor LBD forms a dimer upon binding of the natural ligand estradiol or the synthetic ligand 4-OH-Tamoxifen (Kumar and Chambon, Cell, 55, 145-156 (1988)), other synthetic ligands like ICI 164384 do not induce dimerisation (Fawell, et al., Cell, 60, 953-962 (1990)). The exact mechanism of the dimerisation process is not characterised but point mutants that are unable to dimerise have been described. These mutations fall into the same LBD subdomain that is also important for the specificity of ligand binding such that mutants that exhibit altered ligand binding properties may be also affected for dimerisation (Fawell, et al., Proc Natl Acad Sci USA, 87, 6883-6887 (1990)). For fusion partner proteins that act as monomer a forced dimerisation would be an unwanted but unavoidable effect, however, for the described inducible fusion proteins (see below) dimerisation does not severely interfere with protein function.
Inducible fusion proteins with steroid receptor LBD's have generated with the following functional types of proteins: transcription factors like E2F1, STAT6 and Ets (Agger, et al., Oncogene, 24, 780-789 (2005))(Kamogawa, et al., J Immunol, 161, 1074-1077 (1998)) (Pelczar, et al., Biochem Biophys Res Commun, 239, 252-256 (1997)), protein kinases like Raf or Btk (Samuels, et al., Mol Cell Biol, 13, 6241-6252 (1993)) (Tomlinson, et al., BMC Immunol, 2, 4 (2001)), oncogenes like Myc or Rel (Madruga, et al., Immunobiology, 202, 394-407 (2000)) (Littlewood, et al., Nucleic Acids Res, 23, 1686-1690 (1995)), and DNA recombinases like Cre recombinase (Metzger, et al., Proc Natl Acad Sci USA, 92, 6991-6995 (1995)) or FLP recombinase (Hunter, et al., Genesis, 41, 99-109 (2005)). The fusion of steroid receptor LBD's with proteins that require further processing to become active or that directly act as a protease has not been described.
In the context of the present invention a LBD of any nuclear hormone receptor may be used. Nuclear hormones receptors are a class of protein molecules found within the interior of cells that are responsible for sensing the presence of hormones and certain other molecules. Nuclear receptors have the ability to directly bind to DNA and regulate the expression of adjacent genes. The regulation of gene expression by nuclear receptors is ligand dependent. In other words, nuclear receptors normally are only active in the presence of ligand. More specifically, ligand binding to a nuclear receptor results in a conformational change in the receptor which in turn activates the receptor resulting in e.g. up-regulate of gene expression. Examples of nuclear hormone receptors of the estrogen-like receptor family, which is preferred, include without limitation estrogen receptor (estrogen receptor-α, estrogen receptor-β, an estrogen related receptor, a ketosteroid receptor such as the glucocorticoid receptor, the mineralocorticoid receptor, the progesterone receptor or the androgen receptor. Alternative receptor subfamilies are thyroid hormone receptor-like, retinoid X receptor-like, nerve growth factor IB-like, steroidogenic factor-like and germ cell nuclear factor-like receptors.
Regulated fusion proteins have particularly been constructed with the LBD of the well characterised estrogen receptor (ER), either in its wildtype form that responds to the natural ligand estradiol or by the use of receptor mutants that exhibit strongly reduced estradiol binding but can be activated by synthetic ligands like 4-OH-Tamoxifen. The use of such ER LBD mutants may be of importance for in vivo applications in mammals, if spontaneous activation of a fusion partner by naturally occurring estrogens is to be avoided.
The first mutant of this type was a murine ER LBD that contains a single point mutation replacing a glycine by an arginine residue at position 525 of the mouse or the analogous position 521 of the human estrogen receptor (ER(T)) (Brocard, et al., Proc Natl Acad Sci USA, 94, 14559-14563 (1997)) (Danielian, et al., Mol Endocrinol, 7, 232-240 (1993)). However, as compared to the wildtype ER LBD this mutation also lowers the affinity of the mutant LBD to the synthetic ligand 4-OH-Tamoxifen. A new human ER LBD mutant harbouring three point mutations (G400V/M543A/L544A, named ER(T2)) was described by Feil (Feil, et al., Biochem Biophys Res Commun, 237, 752-757 (1997)) and it was found in fusion with Cre recombinase to act in transgenic mice 10-fold more sensitive to 4-OH-Tamoxifen induction as compared to the ER(T) mutant (Indra, et al., Nucleic Acids Res, 27, 4324-4327 (1999)). The ER(T) (G525) mutant has been shown to dimerise with the ligand 4-OH-Tamoxifen (Danielian, et al., Mol Endocrinol, 7, 232-240 (1993)); for the triple mutant ER(T2) dimerisation has not been characterised.
Besides the estrogen receptor occasionally other steroid receptor mutants have been used to derive inducible fusion proteins, i.e. a mutant human progesterone receptor (Wang, et al., Proc Natl Acad Sci USA, 91, 8180-8184 (1994)) (Kellendonk, et al., Nucleic Acids Res, 24, 1404-1411 (1996)) (Kellendonk, et al., J Mol Biol, 285, 175-182 (1999)) a mutant glucocorticoid receptor (Brocard, et al., Nucleic Acids Res, 26, 4086-4090 (1998)) and a mutant androgen receptor (Kaczmarczyk and Green, Nucleic Acids Res, 31, e86 (2003)). Modified receptors are also described in US 2003/109683.
LBDs modified to only bind a compound selected from the group consisting of non-natural ligands, anti-hormones and non-native ligands are preferred.
The most frequently used ER fusion protein is Cre-ER(T2) consisting of Cre recombinase in fusion with the ER(T2) LBD in combination with the inducer 4-OH-Tamoxifen for in vitro or Tamoxifen for in vivo application. Cre-ER(T2) is frequently used in transgenic mice as a system that enables inducible DNA recombination in vivo. In such Cre-ER(T2) transgenic mice the fusion protein is expressed from a cell type specific promoter and allows the inactivation of a modified endogenous gene that has been flanked with two Cre (loxP) recognition sites. Inducible recombination with this system has been demonstrated in vivo for a variety of cell types and peripheral organs (Indra, et al., Nucleic Acids Res, 27, 4324-4327 (1999)) (Kuhbandner, et al., Genesis, 28, 15-22 (2000)) (Vooijs, et al., EMBO Rep, 2, 292-297 (2001)) (Minamino, et al., Circ Res, 88, 587-592 (2001)) (Sohali, et al., Circ Res, 89, 20-25 (2001)) (Imai, et al., Proc Natl Acad Sci USA, 98, 224-228 (2001)) (Bex, et al., J Urol, 168, 2641-2644 (2002)) (Bosenberg, et al., Genesis, 44, 262-267 (2006)) (Guo, et al., Genesis, 32, 8-18 (2002)) (Hayashi and McMahon, Dev Biol, 244, 305-318 (2002)). Important for applications to the central nervous system is the performance of the CreER(T2) system which has been shown for neurons and glia cells in the brain of adult mice (Weber, et al., Eur J Neurosci, 14, 1777-1783 (2001)) (Leone, et al., Mol Cell Neurosci, 22, 430-440 (2003)) (Doerflinger, et al., Genesis, 35, 63-72 (2003)) (Hirriinger, et al., Glia, 54, 11-20 (2006)) (Mori, et al., Glia, 54, 21-34 (2006)) (Zhao, et al., Genesis, 44, 364-371 (2006)). For in vivo applications usually Tamoxifen is used as inducing compound upon intraperitoneal, subcutaneous or oral administration. Tamoxifen is metabolised in the liver into the ER(T2) ligand 4-OH-Tamoxifen. The pharmacology of Tamoxifen in rodents and man is well established and it is used clinically for the therapy of female breast cancer (Fromson, et al., Xenobiotica, 3, 711-714 (1973)) (Fromson, et al., Xenobiotica, 3, 693-709 (1973)) (Etgen, Horm Behav, 13, 97-112 (1979)) (Furr and Jordan, Pharmacol Ther, 25, 127-205 (1984)) (Grainger and Metcalfe, Nat Med, 2, 381-385 (1996)) (Buckley and Goa, Drugs, 37, 451-490 (1989)).
Alternative nuclear receptor ligand binding domains (LBD) which can be used as the LBD domain of the fusion protein of the present invention belong to the nuclear receptor superfamily of proteins (Mangelsdorf, et al., Cell, 83, 835-839 (1995)) and include, but are not limited to, steroid hormone receptors, vitamin-A and -D receptors and retinoic receptors. The structure of the LBD of nuclear steroid receptors consists of a conserved arrangement of a series of 11-12 alpha-helices closely folded in a similar manner (Kumar and Thompson, Steroids, 64, 310-319 (1999)).
Preferably, in the context of the present invention the nuclear hormone receptor is selected from the group consisting of an estrogen receptor, a progesterone receptor, a glucocorticoid receptor, an androgen receptor and a functionally active variant thereof, particularly an estrogen receptor or a functionally active variant thereof as defined above. More preferably, the receptor is a mammalian nuclear hormone receptor, especially a human nuclear hormone receptor, still more preferably selected from the group consisting of an estrogen receptor, a progesterone receptor, a glucocorticoid receptor, an androgen receptor and a functionally active variant thereof. Examples of those include Estrogen receptors (the amino acid sequence of said mouse estrogen receptor alpha is shown in SEQ ID NO: 72, of which the LBD comprises the residues 355-547 and the sequence of said human estrogen receptor alpha is shown in SEQ ID NO: 73, of which the LBD comprises the residues 351-543), Progesterone receptors (the amino acid sequence of said mouse progesterone receptor is shown in SEQ ID NO: 74 and the sequence of said human progesterone receptor is shown in SEQ ID NO: 75, of which the LBD comprises the residues 641-891 or 641-933), Glucocorticoid receptors (the amino acid sequence of said murine glucocorticoid receptor is shown in SEQ ID NO: 76, and the sequence of said human glucocorticoid receptor is shown in SEQ ID NO: 77, of which the LBD comprises the residues 500-777), Androgen receptors (the amino acid sequence of said murine androgen receptor is shown in SEQ ID NO: 78, and the sequence of said human androgen receptor is shown in SEQ ID NO: 79), and the like, or mutants thereof. Other nuclear hormone and steroid receptor LBDs known in the art are also applicable. However, preferred examples of sequences of nuclear hormone receptor are those of SEQ ID NO: 72 to 80, especially SEQ ID NO: 80.
As detailed above, ligands to the LBD are needed in order to activate the LBD and induce apoptosis of a cell, in which the fusion protein is expressed.
Specific examples of compounds which bind the ligand binding domain include 5-alpha-pregnane-3,20-dione; 11[beta]-(4-dimethylaminophenyl)-17[beta]-hydroxy-17[alpha]-ropinyl-4,9-estradiene-3-one; 11[beta]-(4-dimethylaminophenyl)-17[alpha]-hydroxy-17[beta]-(3-hydroxypropyl)-13 [alpha]-methyl-4,9-gonadiene-3-one; 11[beta]-(4-acetylphenyl)-17[beta]-hydroxy-17[alpha]-(1-propinyl)-4,9-estradiene-3-one; 11 [beta]-(4-dimethylaminophenyl)-17[beta]-hydroxy-17[alpha]-(3-hydroxy-1(Z)-propenyl-estra-4,9-diene-3-one; (7[beta], 11[beta], 17 [beta])-11-(4-dimethylaminophenyl)-7-methyl-4′,5′-dihydrospiro[ester-4,9-diene-17,2′(3′H)-furan]-3-one; (11[beta],14[beta], 17[alpha])-4′,5′-dihydro-11-(4-dimethylaminophenyl)-[spiroestra-4,9-diene-17,2′(3′H)-furan]-3-one; Raloxifen, Naloxifen, 4-OH-tamoxifen or ICI 164384.
Particularly for mutant LBD the following ligands may be used in order to induce apoptosis:
In accordance with the present invention the second component of the fusion protein may also be a functionally active variant of a LBD as defined above. Functional active variants are obtainable by changing the sequence of the LBD as defined herein and are characterized by having a biological activity similar to that displayed by the LBD from which it is derived, including the ability to be activated upon binding of a ligand. Ability to be activated can be determined e.g. as described in the Examples, i.e. by producing a fusion protein as described in Example 1, wherein the variant is to be substituted for the LBD, expressing the fusion protein and comparing apoptosis in presence and absence of a ligand for the respective LBD, e.g. 4-OH-tamoxifen, as described in Example 2 or 3. The variant of a LBD is functionally active in the context of the present invention, if the activity of the fragment amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the LBD without sequence alteration.
Alternatively, the ligand binding affinity of the wild-type LBD and the mutant LBD may be determined. The ligand binding affinity of such mutants can be determined by incubation of the LBD with radioactive labeled ligand at various concentrations in the presence or absence of unlabelled ligand and subsequent measurement of bound labeled ligand, e.g. as described by Smith and Sestili, Clin Chem 26, 543-50 (1980)). A functionally active variant of the above LBD in accordance with the present invention relates to a mutant of the respective original (viz. wild-type) LBD having a ligand binding affinity to the natural or a synthetic ligand of at least 0.1%, preferably at least 1%, more preferably at least 10% of that of said wild-type LBD.
Variants include truncated forms of the LBD (such as N- or C-terminal truncated LBD proteins), deletion-type mutants (where one or more amino acid residues or segments having more than one continuous amino acid residue have been deleted from the primary sequence of the wildtype LBD), replacement-type mutants (where one or more amino acid residues or segments of the primary sequence of the wildtype LBD have been replaced with alternative amino acid residues or segments), or the addition of signal peptides that alter intracellular localisation, or combinations thereof.
In one embodiment of the present invention the LBD or functionally active variant thereof may be a fragment. The fragment is characterized by being derived from a naturally occurring LBD as defined below by one or more amino acid deletions. The deletion(s) may be C-terminally, N-terminally and/or internally. Preferably, the fragment is obtained by at most 100, more preferably by at most 50, even more preferably at most 30, still more preferably at most 10, most preferably 1, 2, 3, 4 or 5 deletion(s).
The functionally active fragment may be also characterized by other structural similarity. Accordingly, in one preferred embodiment of the invention the functional active fragment consists of at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, most preferably 99% of any naturally occurring LBD, e.g. those listed above. The functional active fragment as defined above may be derived from the peptide by one or more amino acid deletions. The deletions may be C-terminally, N-terminally and/or internally. For progesterone receptor variants c terminal mutations are preferred, as those are more likely to effect reduction or abolishment of ligand binding.
In another preferred embodiment of the invention the LBD is a functionally active variant of a LBD, wherein the variant is derived from any naturally occurring LBD, e.g. those listed above, by one or more amino acid deletion(s), addition(s) and/or substitution(s) and preferably wherein the variant has at least 50% sequence identity to a naturally occurring LBD. In a more preferred embodiment the functional active variant has a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, most preferably 99% to any naturally occurring LBD, e.g. those listed above.
The percentage of sequence identity can be determined as described above in connection with the variant of a Caspase.
As noted above, the functionally active variant of a LBD is obtained by sequence alterations in the sequence of the LBD, wherein the variant retains the function of the LBD (see above). The term “functionally active variant” includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants.
However, if the variant is obtained from a LBD by one or more substitution(s) conservative substitution(s) is/are preferred. Conservative substitutions are as detailed above.
In case of one or more amino acid addition(s), these may result for the cloning of the LBD or functionally active variant thereof, e.g. due to the use of particular restriction site, and may or may not alter (increase or decrease) the activity of the LBD. Alternatively, amino acids may be added in order to achieve a desired result, e.g. addition of a tag to provide for convenient purification.
As detailed above, particularly for in vivo application receptor mutants are preferred which may not be activated by the respective naturally occurring ligand in order to avoid induction of apoptosis upon the presence of the ligand in the body of the respective animal. In accordance with this, the above specified mutants are preferred in the context of the present invention. Particularly, estrogen receptors with single point mutation replacing a glycine by an arginine residue at position 525 of the mouse or the analogous position 521 of the human estrogen receptor (ER(T)) or with three point mutation (G400V/M543A/L544A, named ER(T2)) are preferred. Alternatives are the above specified mutant human progesterone receptor, the mutant glucocorticoid receptor and a mutant androgen receptor as well as any other mutant nuclear hormone receptor known to the skilled person.
In a very preferred embodiment of the invention the functionally active variant of the nuclear hormone receptor is the human estrogen receptor LBD mutant ER(T2) (residues 282-595 of the human estrogen receptor as published in: Feil, et al., Biochem Biophys Res Commun, 237, 752-757 (1997)) with amino acid exchanges from Glycine to Valine at position 400 (G400V), from Methionine to Alanine at position 543 (M543A), from Leucine to Alanine at position 544 and having the amino acid sequence shown in SEQ ID NO: 80.
The preferred ligands for the activation of the ER(T2) mutant LBD are non-steroidal antiestrogens, the most preferred ligand is 4-OH-Tamoxifen (chemical name: [trans-1-(4β-dimethylaminoethoxyphenyl) 1,2-diphenylbut-1-ene]). Alternatively, ICI 164384 may be used.
Within the fusion protein the binding domain of the nuclear hormone receptor ligand may be linked to the N-terminal or C-terminal of the Caspase domain; however, preferably the ligand binding domain of the nuclear hormone receptor is linked to the C-terminal of the Caspase domain. The ligand binding domain of the nuclear hormone receptor may be either linked to the Caspase domain directly or via a linker. Preferably, the linker is composed of amino acids.
In one embodiment of the invention the linker consists of 1 to 100 amino acid residues, preferably 1 to 40 amino acids, more preferably 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Especially, the linker consists essentially of neutral amino acids. An exemplary linker is composed of the glycine, alanine and/or valine or particularly consists of the sequence Ala-Asp-Gln (as shown in SEQ ID NO: 20).
An alternative linker comprises a part of the nuclear hormone receptor D and F domain sequences flanking the ligand binding domain (Metzger, et al., Proc Natl Acad Sci USA, 92, 6991-6995 (1995)).
In addition to the components listed and specified above, the fusion protein may comprise further components such as a myristoylation signal sequence such as GSSKSKPKDPSQR (SEQ ID NO: 82), a subcellular targeting signal like a nuclear localization sequence or any other suitable sequence such as a tag, e.g. for purification. Other suitable sequences are known to the skilled persons and it is within the knowledge of the skilled person to select and combine further sequences with the components of the fusion protein as defined above.
Myristoylation is an irreversible, post-translational protein modification found in animals, plants, fungi and viruses. In this protein modification a myristoyl group (derived from myristic acid) is covalently attached via an amide bond to the alpha-amino group of an N-terminal glycine residue of a nascent polypeptide. The modification is catalyzed by the enzyme N-myristoyltransferase, and occurs most commonly on glycine residues exposed during co-translational N-terminal methionine removal. Myristoylation plays a vital role in membrane targeting and signal transduction in plant responses to environmental stress. A myristoylation signal sequence may be included in order to increase the local concentration of the fusion protein at membranes which might lead to faster and more efficient induction of apoptosis.
A nuclear localization signal sequence provides for the active transport into the nucleus of eukaryotic cells. Such a signal peptide domain preferably has a length of 5 to 74, preferably 7 to 15 amino acid residues. More preferably, the signal peptide domain comprises a segment of 6 amino acid residues wherein at least 2 amino acid residues, preferably at least 3 amino acid residues are positively charged basic am9no acids. Basic amino acids include, but are not limited to, lysine, arginine, and histidine.
Highly preferred fusion proteins of the present invention comprise the amino acid sequence for a CASP8-ER(T2) protein shown in SEQ ID NO: 20 and a myrCASP8-ER(T2) protein shown in SEQ ID NO: 23 (suitable DNA sequence coding for said fusion proteins being shown in SEQ ID NO: 19 and 22, respectively), and the amino acid sequence for a CASP9full-ER(T2) protein shown in SEQ ID NO: 29 and a CASP9trunc-ER(T2) protein shown in SEQ ID NO: 32, (a suitable DNA sequence coding for said fusion protein being shown in SEQ ID NO: 28 and 31, respectively), and the amino acid sequence for a CASP3-ER(T2) protein shown in SEQ ID NO: 37 (a suitable DNA sequence coding for said fusion protein being shown in SEQ ID NO: 36).
Most preferred fusion proteins consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32 or SEQ ID NO: 37. Especially preferred fusion proteins consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32 or SEQ ID NO: 37.
A further subject of the invention relates to a nucleic acid coding for the fusion protein of the invention, wherein the nucleic acid may be comprised in a vector. The term nucleic acid includes DNA and RNA such as genomic DNA, cDNA and mRNA, or combinations thereof. The nucleic acid may comprise—in addition to the sequence coding for the fusion protein—further sequences e.g. required for the transcription and/or translation of the nucleic acid coding for the fusion protein. This may include a promoter, enhancer, transcription and/or translation initiation and/or termination sequences, selection markers, sequences protecting or directing the RNA or fusion protein within the cell. The selection and combination of these sequences is within the knowledge of the person skilled in the art and may be selected in accordance with the cell the nucleic acid or fusion protein is intended for. Sequences of preferred nucleic acids are identified in SEQ ID NO: 18, 21, 27, 30 and 35.
The term “vector” as used herein refers to a construction comprised of genetic material designed to direct transformation of a targeted cell. A vector contains multiple genetic elements positionally and sequentially oriented with other necessary elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary translated in the transfected cells. The term vector as used herein can refer to nucleic acid, e.g., DNA derived from a plasmid, cosmid, phagemid or bacteriophage, into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular proteins. The term “plasmid” as used herein refers to a construction comprised of extrachromosomal genetic material, usually of a circular duplex of DNA which can replicate independently of chromosomal DNA. The plasmid does not necessarily replicate.
The vector can contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector molecule can confer some well-defined phenotype on the host organism which is either selectable or readily detected. The vector may have a linear or circular configuration. The components of a vector can contain but is not limited to a DNA molecule incorporating: (1) DNA; (2) a sequence encoding the fusion protein of the invention; and (3) regulatory elements for transcription, translation, RNA processing, RNA stability, and replication.
The purpose of the vector is to provide expression of a nucleic acid sequence in cells or tissue. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence. Expression products may be proteins, polypeptides, or RNA. The nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous, constitutive, or regulated. The vector can also be used as a prokaryotic element for replication of plasmid in bacteria and selection for maintenance of plasmid in bacteria.
In one embodiment the vector comprises the following elements linked sequentially at an appropriate distance to allow functional expression: a promoter, a 5′ mRNA leader sequence, a translation initiation site, a nucleic acid cassette containing the sequence of the fusion protein to be expressed, a 3′ mRNA untranslated region, and a polyadenylation signal sequence. As used herein the term “expression vector” refers to a DNA vector that contains all of the information necessary to produce a recombinant protein in a heterologous cell.
In addition, the term “vector” as used herein can also include viral vectors. A “viral vector” in this sense is one that is physically incorporated in a viral particle by the inclusion of a portion of a viral genome within the vector, e.g., a packaging signal, and is not merely DNA or a located gene taken from a portion of a viral nucleic acid. Thus, while a portion of a viral genome can be present in a vector of the present invention, that portion does not cause incorporation of the vector into a viral particle and thus is unable to produce an infective viral particle.
A vector as used herein can also include DNA sequence elements which enable extra-chromosomal (episomal) replication of the DNA. Vectors capable of episomal replication are maintained as extra-chromosomal molecules and can replicate. These vectors are not eliminated by simple degradation but continue to be copied. These elements may be derived from a viral or mammalian genome. These provide prolonged or “persistent” expression.
Examples of vectors are pCAG (see Examples), pBR322, the pUC series, pBluescript, pTZ, pSP and pGEM. The components of the nucleic acid or of the vector are selected in such a way that the nucleic acid is expressed and the fusion protein is produced by the target cell.
Another subject of the invention relates to a cell comprising the nucleic acid and/or vector of the present invention.
“Cells” and “eukaryotic cells” according to the present invention include cells isolated from the below defined living organism and cultured in vitro. These cells can be transformed (immortalized) or untransformed (directly derived from the living organism; primary cell culture).
“Microorganism” according to the present invention relates to prokaryotes (e.g. E. coli) and eukaryotic microorganisms (e.g. yeasts).
The “organisms” according to the present invention are multi-cell organisms and can be vertebrates such as mammals (humans and non-human animals including rodents such as mice or rats) or non-mammals (e.g. fish), or can be invertebrates such as insects or worms, or can be plants (higher plants, algae or fungi). Most preferred living organisms are mice and humans.
The term “mammal” as used in the context of the present invention includes non-human mammals and humans.
The cell can be any suitable cell, especially a eukaryotic cell, for example a fungal, plant or animal cell. Cell lines of these cells are also included. Preferably, it is a mammalian cell, especially a murine or human cell or cell line. Examples of such mammalian cells are HEK 293 cells, CHO cells, HeLa cells, CaCo cells, NIH 3T3 cells or mouse embryonic fibroblast cell line MEF5/N9 (see Examples). Examples of insect cells are SF9, drosophila, butterfly and bee cells. The cell may also be a cell line, which is particularly useful for studying apoptosis or identification of ligand for the LBD. The present invention also provides stable cell lines transformed with the plasmids of the present invention.
Another subject of the invention relates to a method for producing the fusion protein of the invention comprising
A cell of the invention as defined above comprising the nucleic acid and/or vector of the present invention and having been obtained from a cell line stably expressing the fusion protein of the invention or by transfection or transformation as defined above, may be grown and propagated in cell culture.
Cells that are cultured directly from an animal or person are known as primary cells. With the exception of some derived from tumors, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability.
An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types and it is within the knowledge of the skilled person to select a suitable cell line.
For cultivation cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37° C., 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrient components. Antibiotics can also be added to the growth media. Amongst the common manipulations carried out on culture cells are media changes and passaging cells. However, selection of suitable conditions is known to the skilled person. However, for producing the fusion protein the culturing is preferably carried out in the absence of a ligand for the LBD, which would induce apoptosis of the cells and therefore, be counterproductive.
If necessary, the fusion protein can also be isolated from the cells. If a sufficient amount of the fusion protein has been secreted into the medium (e.g. due to suitable secretory signal sequences), this can be separated from the cells, e.g. by removing the supernatant medium. Otherwise it may be necessary to disrupt the cells. This can be effected for example by lysis of the cells e.g. by means of ultrasound or hypotonic medium. To remove insoluble components, the sample obtained can for example be centrifuged, especially at 10000×g to 15000×g, and the supernatant obtained can be used.
Still another subject of the invention relates to a non-human transgenic organism, preferably a non-human transgenic mammal, containing the nucleic acid and/or vector of the present invention.
In general, transgenic animals of the invention exhibit an expression of fusion protein of the invention, optionally tissue-specifically e.g. by using a tissue-specific promoter; therefore, they are very suitable, for example for studying the function of a cell, tissue and/or organ, e.g. at various developmental stages. Preference is given to using transgenic mice. Other examples of a non-human mammal according to the invention is a rat, a guinea pig, a rabbit, a cow, a goat, a sheep, a horse, a pig, a dog, a cat or a monkey.
The transgenic non-human animal may be produced by a series of techniques known to the skilled person. For example, the method may comprise the following steps:
The methods for introducing blastocytes are known to the skilled person. The blastocyte can, for example, be introduced by injection (Hogan, B., Beddington, R., Constantini, F. and Lacy, E., A laboratory Manual (1994), Cold Spring Harbor Laboratory Press).
A transgenic non-human mammal can be identified, for example, by extracting genomic DNA from the transgenic non-human mammal, for example from the tail of a mouse. In a subsequent PCR (polymerase chain reaction), use is made of primers which specifically recognize the transgene for the nucleic acid according to the invention. Integration of the transgene can be detected in this way.
Another possibility for effecting the identification is by means of southern blotting. In this method, genomic DNA is transferred to a membrane and detected using DNA probes, for example radioactively labeled DNA probes, which are specific for the sought-after transgene.
Methods for producing a transgenic non-human mammal according to the invention by means of regenerating a non-human stem cell, oocyte, precursor cell or immortalized cell to give a transgenic non-human animal, in particular transgenic mice, are known to the skilled person from DE 196 25 049 and the U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,625,122; U.S. Pat. No. 5,698,765; U.S. Pat. No. 5,583,278 and U.S. Pat. No. 5,750,825, and encompass transgenic animals which can be produced, for example, by directly injecting expression vectors according to the invention into embryos or spermatocytes or by transfecting expression vectors into embryonic stem cells (Polites and Pinkert: DNA Microinjection and Transgenic Animal Production, pages 15-68 in Pinkert, 1994: Transgenic Animal Technology: A Laboratory Handbook, Academic Press, San Diego, USA; Houdebine 1997, Harwood Academic Publishers, Amsterdam, The Netherlands; Doetschman: Gene Transfer in Embryonic Stem Cells, pages 115-146 in Pinkert, 1994, see above; Wood: Retrovirus-Mediated Gene Transfer, pages 147-176 in Pinkert, 1994, see above; Monastersky: Gene Transfer Technology: Alternative Techniques and Applications, pages 177-220 in Pinkert, 1994, see above).
A transgenic non-human mammal according to the invention can also be prepared by directly injecting a nucleic acid according to the invention into the pronucleus of a non-human mammal.
A large number of methods for preparing transgenic animals, in particular transgenic mice, are also known to the skilled person from, inter alia, WO 98/36052, WO 01/32855, DE 196 25 049, U.S. Pat. No. 4,736,866, U.S. Pat. No. 5,625,122, U.S. Pat. No. 5,698,765, U.S. Pat. No. 5,583,278 and U.S. Pat. No. 5,750,825 and encompass transgenic animals which can be produced, for example, by directly injecting vectors according to the invention into embryos or spermatocytes or by transfecting vectors or nucleic acids into embryonic stem cells (Polites and Pinkert, in Pinkert, (1994) Transgenic animal technology, A Laboratory Handbook, Academic Press, London, UK, pages 15 to 68; Doetschmann, in Pinkert, 1994, see above, pages 115 to 146).
A further subject of the invention relates to the use of the fusion protein according to any the invention for ligand-mediated induction of apoptosis of a cell, preferably a eukaryotic cell, more preferably a mammal cell, especially a human cell. In patent systems not allowing claims directed to therapeutic and/or diagnostic methods of the human or animal body, these therapeutic and/or diagnostic methods are excluded; however, e.g. research methods are included. The fusion protein e.g. may be used for studying the function of a cell, tissue and/or organ. Also the transgenic non-human organism according to the invention may be used for studying the function of a cell at various developmental stages or as a disease model.
The inducible apoptosis system of the invention provides a universal cell ablation system for use in mammalian cells and organisms that allows to study the biological function of selected cells or a cell type in the mammalian body and thereby the creation of a wide range of animal models of human diseases. Such an inducible apoptosis system is particularly desirable for all those applications which require its universal activation in any organ and any cell type of the mammalian body, including the brain. It is to be noted that the non-human transgenic animal can be used to study the function of cells, e.g. in mice, by inducible apoptosis. For this purpose the coding region of the fusion proteins of the invention may be combined with suitable sequences as detailed above, for example a cell type specific promoter region, and the fusion protein transgene be inserted into the mouse germline by pronuclear injection or other methods known in the art. The administration of ligand that activates the Caspase activity of the fusion protein leads to the complete or partial ablation of the selected target cells in fusion protein transgenic mice at any chosen ontogenetic stage. The prespecified temporal and/or spatial restricted cell death is of particular use to model neurodegenerative diseases in mice and to develop model therapies that compensate for the loss of cellular functions through enhanced regeneration or through cell transfer. Alternatively to the use of germline transgenic mice, the fusion protein coding region can be combined with a cell type specific or ubiquitous active promoter region and inserted into the genome of a viral vector that is used to transduce the fusion protein expression unit locally into somatic tissues of embryonic, juvenile or adult mice.
Another subject of the invention relates to a method for inducing apoptosis of a cell expressing a fusion protein according to the invention, the method comprising
Particularly, the method may be used in order to treat a patient as described below. Alternatively, the method may be an in vitro method, allowing studying apoptosis in a cell, identifying new ligands for LBD etc. as described within the present description of the invention.
Another subject of the invention relates to a method for identifying a ligand to a ligand binding domain of a nuclear hormone receptor or a functionally active variant thereof, the method comprising
In another application of the inducible apoptosis system of the invention further allows to identify a ligand to a LBD. Substances deemed to be potential agonists or antagonists of a LBD may be screened for substances that inhibit or induce Caspase activity using cells according to the invention. Cells expressing the fusion protein of the invention may be cultured in the presence of a substance. If the substances induced apoptosis, it is identified as an agonistic ligand of the respective LBD of the fusion protein.
If it is screened for an antagonistic ligand of the LBD, the substance is tested in the presence of an agonistic ligand for the LBD. If the substance is capable of inhibiting apoptosis induced by the agonistic ligand (i.e. a ligand capable of inducing apoptosis of a cell upon binding to the LBD of a fusion protein of the invention), it is identified as antagonistic ligand. Ligands, particularly agonistic ligands, may be chosen in accordance with the above description. The selection of suitable conditions for the methods for identifying a ligand is within the knowledge of the skilled person and/or may be chosen in accordance with Examples 2 and 3.
Furthermore, the in vivo efficacy of such compounds can be assessed in transgenic mice of the invention that express the fusion protein of the invention upon coadministration of a test substance and optionally an agonistic ligand.
Still another subject of the invention relates to a medicament comprising a fusion protein according to the invention, a nucleic acid according to the invention, a vector according to the invention and/or a cell according to the invention.
The medicament of the invention may be used for the treatment of a disease requiring enhancement of apoptosis, particularly for the treatment of cancer or for or after transplantation, particularly as safety mechanism.
The inducible apoptosis system of the invention further allows for specifically ablating cells such as cancer cells or transplanted cells that contain a fusion protein, nucleic acid or vector of the invention from the body of a recipient by the administration of a ligand that activates the fusion protein.
For this purpose, all or a selected population of cells that are transferred into a recipient for therapeutic purposes (e.g. bone marrow transplantation, neurons or keratinocytes grown in in vitro cultures) can e.g. be transduced with a viral fusion protein expression vector or other methods known in the art. In this context, the fusion protein of the invention may be used as a safety mechanism in case that the transplanted cells or their progeny thread the recipient by e.g. tumorigenesis or a graft versus host reaction.
In another application the inducible apoptosis system of the invention further allows to destroy tumor cells by suicide gene therapy e.g. upon transduction or transfection of these cells with a fusion protein expression vector and ligand administration.
The medicament of the present invention may encompass pharmaceutically acceptable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the (poly)peptides herein disclosed. In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Also encompassed by the present inventions are methods for treating or preventing the diseases specified herein by administering to a patient an effective amount of a fusion protein of the invention, a nucleic acid coding of the invention, a vector comprising the nucleic acid and/or a cell of the invention.
The medicament may be administered to a subject in need thereof, preferably mammals, and still more preferably humans, in any conventional manner, including oral, intranasal, intramuscular, intra-lymph node, intradermal, intraperitoneal, subcutaneous, and combinations thereof, but most preferably through local administration, such as local injection.
Nucleic acid delivery compositions and methods are known to those of skill in the art. The nucleic acid of the invention may be employed in the methods of this invention or in the compositions described herein as DNA sequences, either administered as naked DNA, associated with a pharmaceutically acceptable carrier or comprised in a vector. The nucleic may be administered therapeutically or as a safety mechanism e.g., by injection.
An “effective amount” of a medicament may be calculated as that amount capable of exhibiting an in vivo effect, e.g. preventing or ameliorating a sign or symptoms. Such amounts may be determined by one of skill in the art. Preferably, such a composition is administered directly to the intended site of action, e.g. directly into a tumor. However, it may also be formulated to be administered by any other suitable route. The selection of the route of delivery and dosage of such therapeutic compositions are within the skill of the art.
Treatment in the context of the present invention refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
The present invention is further illustrated by the following Figures and Examples which are, however, not to be construed as limiting the scope of the invention.
These constructs were planned to be used as a positive control.
All cotransfections were performed with a fixed amount of the reporter plasmid pCMV-β-gal-pA and 50 ng or 100 ng of the expression plasmids pCAG-Bax-ER(T2) (sample 4), pCAG-ER(T2)-Bax (sample 5), pCAG-Casp8-ER(T2) (sample 6), pCAG-myrCasp8-ER(T2) (sample 7), pCAG-Casp9full-ER(T2) (sample 8), pCAG-Casp9trunc-ER(T2) (sample 9), pCAG-Casp3-ER(T2) (sample 10), pCAG-Casp3-ED4ER(T2) (sample 11). Negative control: transfection with pCAG-Cre-ER(T2) (sample 1). Planned positive controls: transfection with the plasmids pCAG-HAFas-ER(T2) (sample 2) or pCAG-MFas-ER(T2) (sample 3).
The vertical rows show the mean values and standard deviation of “Relative light units” (RLU) obtained from cell lysates with the assay for β-galactosidase activity and the relative β-galactosidase activity of the cells transfected with various ER(T2) fusion constructs as compared to the negative control without tamoxifen defined as 1.1.
Applications of an inducible cell ablation tool by using the chimeric system of Caspase-FKBP and Fas-ER have been efficiently demonstrated in several studies.
However, this system does not provide universal usage in any organ and any cell type of the mammalian body, including the brain. Here, we investigated an alternative inducible apoptosis system designed for universal application in mammalian cells and tissues, including the brain. To test the activity of different chimeric genes, we fused either a Caspase-8, -9, -3, or Bax domain to the ligand binding domain of the mutant mammalian estrogen receptor ER(T2) which is unable to bind estrogen yet retains high affinity for a synthetic ligand, 4-hydroxy-tamoxifen (Tm). Upon administration of the ligand which induces dimerisation of the ER(T2) domain, apoptosis should be directed by the activated Caspase or Bax domain. To compare and quantify the activity of the different CASP/Bax-ER(T2) expression vectors, these constructs were transiently introduced into the mouse fibroblast cell line MEF5/N9 together with a reporter vector encoding β-galactosidase.
A. Plasmid Constructions:
For the construction of pCAG-Cre-ER(T2)-bpA a 2.3 kb fragment coding for the Cre-ER(T2) fusion protein (Feil, et al., Biochem Biophys Res Commun, 237, 752-757 (1997)) was isolated from pSS1 (SEQ ID NO: 1; R. Kühn, unpublished) by digestion with PacI, PvuII and ScaI and ligated into pCAG-C31Int(NLS)-bpA (R. Kühn, unpublished; SEQ ID NO: 2), opened with SphI and PacI. pCAG-Cre-ER(T2)-bpA (SEQ ID NO: 3;
The expression vectors pCAG-HAFas-ER(T2) and pCAG-MFas-ER(T2) were constructed as following: mouse Fas insert encoding amino acids 135 to 305 for the transmembrane and intracellular domain of the Fas receptor was amplified by PCR (Phusion High-fidelity PCR kit, New England Biolabs) from the plasmid pDNR-LIB-Fas (RZPD GmbH, Berlin, Germany, clone ID: IRALp962N1053Q). To generate the 574 bp HAFas fragment, the oligonucleotides fasIER-A (SEQ ID NO: 6), containing the influenza virus hemagglutinin epitope, YPYDVPDYA (SEQ ID NO: 81), and fasER-B (SEQ ID NO: 7) were used. The 602 bp MFas variant was obtained by using the oligonucleotides fas2ER-A (SEQ ID NO: 8), containing a signal sequence for cell membrane targeting, and fasER-B. Both PCR fragments were flanked by a 5′ PacI site and a 3′ SalI site. The ends of the PCR products were digested with the respective enzymes and ligated into plasmid pCAG-Cre-ER(T2)-bpA, opened with PacI and XhoI, giving rise to the expression vectors pCAG-HAFas-ER(T2) (SEQ ID NO: 9) and pCAG-MFas-ER(T2) (SEQ ID NO: 10). pCAG-HAFas-ER(T2) (
Construction of the expression plasmids pCAG-Casp8-ER(T2)-bpA and pCAG-myrCasp8-ER(T2)-bpA: mouse Caspase 8 insert was PCR amplified from the plasmid pCMV-SPORT6-Casp8 (RZPD GmbH, Berlin, Germany, clone ID: IRAVp968E1193D). The 814 bp Casp8 PCR fragment was generated by using the oligonucleotides Casp8ER-A (SEQ ID NO: 15) and Casp8ER-B (SEQ ID NO: 16). The 853 bp myrCasp8 variant, containing a N-terminal myristoylation sequence motif, was obtained by using the oligonucleotides myrCasp8ER-A (SEQ ID NO: 17) and Casp8ER-B. Both PCR products were digested with PacI and XhoI and inserted into the PacI/XhoI cloning site of pCAG-Cre-ER(T2)-bpA, giving rise to the expression vectors pCAG-Casp8-ER(T2)-bpA and pCAG-myrCasp8-ER(T2)-bpA. pCAG-Casp8-ER(T2)-bpA (SEQ ID NO: 18;
The expression vectors pCAG-Casp9full-ER(T2)-bpA and pCAG-Casp9trunc-ER(T2)-bpA for the Caspase 9 ER(T2) fusion protein and a Caspase-activation recruitment domain (CARD) deleted form, referred to as truncated Caspase 9 ER(T2) fusion protein, were constructed as following: mouse Caspase 9 insert was PCR amplified from the plasmid pXY-Asc-Casp9 (RZPD GmbH, Berlin, Germany, clone ID: IRAVp968D06114D) by using the oligonucleotides Casp9fullER-A (SEQ ID NO: 24) and Casp9ER-B (SEQ ID NO: 25) for generating the 1.3 kb Casp9full fragment. To obtain the 1.1 kb truncated variant Casp9trunc, the oligonucleotides Casp9truncER-A (SEQ ID NO: 26) and Casp9ER-B were used. The PCR products were digested with PacI/SalI and ligated into the PacI/XhoI cloning site of pCAG-Cre-ER(T2)-bpA, giving rise to the expression vectors pCAG-Casp9full-ER(T2)-bpA and pCAG-Casp9trunc-ER(T2)-bpA. pCAG-Casp9full-ER(T2)-bpA (SEQ ID NO: 27;
Construction of the expression plasmids pCAG-Casp3-ER(T2)-bpA and pCAG-Casp3-ED4ER(T2)-bpA: mouse Caspase 3 insert was amplified from the plasmid pFLCI-Casp3 (Open Biosystems, Huntsville, USA, clone ID: EMM1002-7378750). The 856 bp PCR fragment was obtained by using the oligonucleotides Casp3ER-A (SEQ ID NO: 33) and Casp3ER-B (SEQ ID NO: 34) and digested with PacI/XhoI. To generate the expression vector pCAG-Casp3-ER(T2)-bpA, the digested PCR product was inserted into pCAG-Cre-ER(T2), opened with PacI/XhoI. pCAG-Casp3-ER(T2)-bpA (SEQ ID NO: 35;
Generation of the expression plasmid pCAG-Bax-ER(T2)-bpA occurred by PCR amplifying mouse Bax from the plasmid pCMV-SPORT6-bax (RZPD GmbH, Berlin, Germany, clone ID: IRAVp968B0795D) with the oligonucleotides BaxER-A (SEQ ID NO: 44) and BaxER-B (SEQ ID NO: 45). The 606 bp PCR product was digested with PacI/XhoI and ligated into the PacI/XhoI site of pCAG-Cre-ER(T2)-bpA. pCAG-Bax-ER(T2)-bpA (SEQ ID NO: 46;
B. Cell Culture and Transient Transfections
The transformed mouse embryonic fibroblast cell line MEF5/N9 (Schwenk et al., Nucleic Acids Research, 26(6) 1427-1432 (1998)) was obtained from Michel Aguet (University of Zurich, Switzerland). The cells were grown in DMEM medium (Life Technologies) supplemented with 2 mM glutamate and 10% fetal calf serum at 37° C., 5% CO2 in humid atmosphere and passaged upon trypsinization. One day before transient transfection 4·104 cells were plated into a 24-well plate (Falcon). For the transient transfection of MEF5/N9 cells with plasmids, each well received into 500 μl of medium a total amount of 150 ng or 200 ng supercoiled plasmid DNA complexed before with the FuGene6 transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol. Each 150 ng or 200 ng DNA preparation (
Results:
To assess the efficacy of a novel approach for inducible cell death, we generated different fusion genes of Caspases or bax and the mutant mammalian estrogen receptor ER(T2), which dimerizes upon administration of the synthetic ligand 4-hydroxy-tamoxifen. We first constructed the plasmid pCAG-Cre-ER(T2) (see
To investigate the cell death effect of the different ER(T2) fusion constructs quantitatively, we performed transient cotransfections of the ER(T2) fusion protein expression vectors together with a fixed amount of a reporter plasmid coding for β-galactosidase in MEF5/N9 fibroblast cells. Transfected cells were treated 12 hours after transfection with 4-OH-tamoxifen at a concentration of 10−8 M. Following 48 hours of 4-OH-tamoxifen induction, the cells from the various samples were lysed and the activity of β-galactosidase in the lysates was determined by a chemiluminescence assay and expressed in “Relative Light Units” (RLU) (
To determine the relative activity of the tested ER(T2) fusion constructs inducing apoptosis in the presence of 4-OH-tamoxifen, the RLU values of β-galactosidase activity were divided individually for each sample by the RLU value obtained from the sample which received the plasmid pCAG-Cre-ER(T2) but was not treated with 4-OH-tamoxifen (defined as negative control). The relative activity of the tested Caspase- or bax-ER(T2) fusion proteins was then compared to the negative control for 4-OH-tamoxifen-dependent cell death defined as an activity of 1.
Taken together, in the transient transfection system shown in
Using the transient transfection system of Example 1, we identified Caspase-ER(T2) fusion constructs that exhibit 4-OH-tamoxifen-dependent activity. However, the high copy number of expression plasmids following transient transfection may lead to unphysiological high concentrations of the fusion proteins. We next transfected the constructs identified as active in Example 1 stably into the test cell line MEF5/N9, in short M5N9.
A. Plasmid Constructions
All plasmids and their purification are described in Example 1, except for the pPgk-hygro-pA plasmid (
B. Cell Culture and Stable Transfections
To generate stably transfected MEF5/N9 cells, 2·106 cells were coelectroporated with 32 μg ER(T2) fusion plasmid DNA linearised with ScaI and 8 μg pPgk-hygro-pA plasmid DNA linearised with XhoI and plated into two 96-well plates. The cells were grown in DMEM medium (Invitrogen) supplemented with 2 mM Glutamine and 10% fetal calf serum at 37° C., 5% CO2 in humid atmosphere, and passaged upon trypsinization. Two days after transfection the medium was supplemented with 250 U/ml of hygromycin (Calbiochem) for the selection of stable integrants. Upon the growth of resistant colonies these were individually expanded in the presence of hygromycin.
C. Immunoblot Detection of Er(T2) Fusion Proteins in Transfected Mef5/N9 Cells
To directly demonstrate expression of the transfected fusion protein vector, protein lysates of hygromycin resistant clones were prepared for western blot analysis.
One million cells were lysed in 500 μl of lysis buffer (50 mM Tris-HCl [pH 6.8], 50 mM dithiothreitol, 10% glycerol, 2% SDS) supplemented with protease inhibitor cocktail (Roche Diagnostics GmbH). Cell lysates were incubated for 7 min at 100° C. and sonificated for 10 s. Cell debris were pelleted by centrifugation and an aliquot of the supernatant was applied for protein concentration analysis by using the BCA protein assay kit (Pierce, Rockford, Ill.). 0.01% of bromphenolblue, diluted in lysis buffer was added to the remaining lysate. For western blot analysis, 40 μg protein was incubated for 3 min at 100° C. before separating the samples on a 10% SDS-polyacrylamide gel (NuPAGE® Novex Bis-Tris gels; Invitrogen). Electrophoresis was performed according to the manufacturer's protocol in MES running buffer (50 mM MES, 50 mM Tris-HCl, 1 mM EDTA, 0.1% SDS, [pH 7.2]). Following subsequent blotting to BioTrace PVDF transfer membrane (Pall Corporation) in blotting buffer (25 mM bicine, 25 mM bis-Tris, 1 mM EDTA, 0.005 mM chlorobutanol, 10% methanol), membranes were blocked in 5% skim milk (BD Biosciences) over night at 4° C. Western blot analysis was performed with the ECL kit (Amersham Biosciences) using either the rabbit anti-ERα antibody HC-20 (#SC543, Santa Cruz Biotechnology), diluted 1:1000 in 2.5% skim milk/TBST or mouse anti-β-actin antibody AC15 (BIOZOL, Eching, Germany), diluted 1:5000 in 2.5% skim milk/TBST. Between each incubation step and before signal detection, membranes were rinsed several times with TBST (25 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween-20). Membrane stripping occurred for 5 min in stripping buffer (10% acetic acid, 10% methanol) on a shaker.
Results
To generate mammalian cell clones with a stable genomic integration of active ER(T2) fusion genes, the murine fibroblast cell line MEF5/N9 was electroporated with linearised DNA of ER(T2) fusion constructs together with the plasmid pPgk-hygro (
As shown in
We have shown in transient transfection assays (example 1) that fusions between ER(T2) and Caspase 8, myristoylated Caspase 8, proCaspase 9, Caspase 9 without CARD, or proCaspase 3, induced cell death under control of 4-OH-tamoxifen. However, this assay system does not provide final evidence for the efficacy of the various constructs. To investigate differences in the efficacy of the various expression plasmids, we generated stably transfected MEF5/N9 clones expressing the ER(T2) fusion proteins in a low copy number. Transfected MEF5/N9 clones were incubated with 4-OH-tamoxifen and examined for cell viability.
A. Viability of Transfected Mef5/N9 Cells Upon Treatment with 4-OH-Tamoxifen
To examine 4-OH-tamoxifen-dependent cell death, 1·105 MEF5/N9 cells expressing ER(T2) fusion proteins were plated into each well of a six-well plate. The following day, for each sample the medium in two of four individual wells were replaced with medium supplemented with 10−8 M 4-hydroxy-tamoxifen. 24 hours after induction with 4-OH-tamoxifen, cells were photographed through a Leica DMI6000B fluorescence microscope with phase contrast either at 10-fold or 20-fold magnification.
B. MTT Cytotoxicity Assay
To quantify 4-OH-tamoxifen-inducible cell death, we applied the colorimetric MTT assay (Mossman, J. Immunol. Methods, 65, 55 (1983)) allowing the quantification of cell survival. Cells (4·103) were plated on 96-well plates. The assay was carried out in three replicas for each sample treated with and without 4-OH-tamoxifen. After 12 h, medium was replaced either with fresh medium or with medium supplemented with 10−8 M 4-OH-tamoxifen. Following 48 h of 4-OH-tamoxifen treatment, sterile filtered MTT reagent in PBS (Sigma) was added at the concentration of 0.5 mg/ml. Incubation occurred for 4 hours at 37° C. With ongoing reaction time, dark violet MTT formazan crystals appeared on the bottom of the wells containing living cells. The crystals were dissolved in 150 μl 0.04 N HCl/isopropanol to give a homogeneous blue solution suitable for absorbance measurement at a wavelength of 570 nm on the Synergy™ HT Multi-Detection Microplate Reader (BioTek).
Results
To examine 4-OH-tamoxifen-inducible apoptosis in stably transfected MEF5/N9 clones, we first visualized cell viability after 24 hours of incubation with 4-OH-tamoxifen at a concentration of 10−8 M.
The results obtained from the transient transfection assays could partially be confirmed. Interestingly, only the fusion proteins of ER(T2) and proCaspase 9, myristoylated Caspase 8, Caspase 8 and Caspase 9 with deleted CARD were able to induce 4-OH-tamoxifen-dependent cell death in stably transfected MEF5/N9 cells. All clones tested were growing well, like the parental cell line, in absence of 4-OH-tamoxifen (
We further quantified cell viabilities by the MTT cytotoxicity assay following 48 h of incubation with 10−8 M 4-OH-tamoxifen. Representative results from two independently performed experiments are shown in
Taken together, we could identify three highly efficient ER(T2) fusion constructs, namely Casp8-ER(T2), myrCasp8-ER(T2) and Casp9full-ER(T2) inducing 4-OH-tamoxifen-dependent apoptosis in the test cell line MEF5/N9 within a short time. However, the reported activity of the Fas-ER(T2) fusion protein (Kodaira et al., Jpn. J. Cancer Res., 89, 741-747 (1998)) originally planned as a positive control could not be reproduced in the cell line MEF5/N9. This finding demonstrates that Fas-ER(T2) possibly shows activity only in specific cell types, namely in those expressing endogeneous fas receptor and in which apoptosis can be induced in a fas-dependent manner. In contrast, we have demonstrated for the first time that fusion constructs of ER(T2) and Caspases provide a highly efficient system to conditionally ablate mammalian cells. Moreover, since Caspase 8 and Caspase 9 are ubiquitously expressed in mammalian tissues and are both involved in different pathways of apoptosis, the potential universal application of ER(T2) fusions to Caspase 8 or 9 for inducible cell ablation is of commercial relevance in biotechnology.
We could demonstrate in example 3 that 4-OH-tamoxifen induced cell death of MEF5 clones expressing Caspase fusion proteins. In order to provide direct evidence that the cell death occurs via induced apoptosis the TUNEL DNA fragmentation assay was used. This method detects DNA fragmentation, one of the late events that result from apoptotic signaling cascades. As shown in
In Situ Cell Death Detection Assay
TUNEL, or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, is acknowledged as a method of choice in the rapid detection of DNA fragmentation that results from apoptotic signaling cascades (Gavrieli et al., J. Cell Biol., 119(3), 493-501 (1992)). Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments as well as single strand breaks (“nicks”) in high molecular weight DNA. Those DNA strand breaks can be identified by terminal deoxynucleotidyl transferase (TdT), which catalyzes polymerization of labeled nucleotides to free 3′-OH DNA ends in a template-independent manner. To detect 4-OH-tamoxifen induced cell death in MEF5/N9 cells, we applied the In Situ Cell Death Detection Kit, POD by Roche according to the manufacturer's instructions. Incorporated fluorescein is detected by anti-fluorescein antibody, conjugated with horse-radish peroxidase (POD). After substrate reaction, stained cells can be analyzed under light microscopy.
Two days prior to the TUNEL staining, round glass cover slips (12 min Ø) were sterilized with 70% EtOH, dried under laminar flow and coated with Poly-L-lysine at a concentration of 50 μg/ml over night at 37° C., which allows a better attachment of cells on the cover slips. The following day, the cover slips were rinsed 3 times with PBS and dried for 1 hour under laminar flow. 2.5·105 cells were seeded onto cover slips in a 6-well plate. On the test day, medium was replaced either by fresh medium or medium supplemented with 10−8 M 4-OH-tamoxifen. Cells were fixated in 4% PFA (diluted in PBS, pH 7.4) for 1 hour at room temperature following 2, 3 and 7 hours of incubation. After fixation, cells were rinsed three times in PBS, covered with 100% methanol and incubated for 10 min or over night in a freezer and rinsed in PBS. Endogenous peroxidase activity was blocked by immersing cover slips for 10 min in 3% H2O2 in methanol prior to cell permeabilization. Cells were subsequently rinsed with PBS and incubated in permeabilization solution (0.1% Triton X-100 in freshly prepared 0.1% sodium citrate) for 2 min on ice. To generate a positive control, DNA strand breaks were induced by incubating permeabilized cells with 2 U/ml DNase I in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mg/ml BSA for 10 min at room temperature, prior to labeling procedure. Cover slips were rinsed twice with PBS before incubating each sample with 25 μl TUNEL reaction mixture for 60 min at 37° C. in a humidified atmosphere in the dark. To avoid evaporative loss, cover slips were covered with parafilm. For a negative control 25 μl of Label solution without enzyme solution were added. Following labeling, cover slips were rinsed 3 times with PBS for 10 min each. To convert the fluorescence signal to a chromogenic signal, 25 μl of the POD solution were added on each sample which was covered with parafilm and incubated in a humidified chamber for 30 min at 37° C. Subsequently, cover slips were rinsed 3 times with PBS and incubated in freshly prepared 0.05% DAB in 0.1 M Tris-HCl (pH 7.4) supplemented with 0.02% H2O2 for 10-20 min at room temperature. The conversion procedure was controlled under a light microscope. The reaction was stopped with PBS until brown precipitates were well visible inside the cells. To remove DAB, cover slips were rinsed several times in PBS. Optionally, nuclear staining was performed by incubating cover slips for 15 min in 2.5 μg/ml DAPI diluted in PBS. Cover slips were mounted with a water-soluble, non-fluorescing mounting medium (Aqua-Poly/Mount; Polysciences Inc., Eppelheim, Germany) under SuperFrost slides (VWR International). Prior to the analysis of the samples under a light microscope, mounting medium was dried for 24 hours at room temperature.
Results
To provide direct evidence for 4-OH-tamoxifen-induced apoptosis in MEF5/N9 clones expressing Caspase-8 or -9 fusion proteins, an in situ application of the TUNEL assay was performed. This method detects DNA fragmentation, one of the late events that result from apoptotic signaling cascades. Cells were seeded onto cover slips and incubated with 4-OH-tamoxifen at a concentration of 10−8 M. Since general data of apoptotic kinetics do not exist so far, cells were fixated at different time points. Thus, cells were incubated with 4-OH-tamoxifen for 2, 3, 5 and 7 hours before fixation in 4% PFA and permeabilized in Methanol at −20° C. Fragmented DNA was labeled by incorporated fluorescein which was recognized by an anti-fluorescein antibody, conjugated with horse-radish-peroxidase (POD). POD converts the substrate DAB into insoluble brown precipitates which can be visualized under a light microscope. Samples were performed in duplicates and documented with a Zeiss Axioplan 2 fluorescence microscope. Nuclei were additionally stained with DAPI (A2-E2; A4-F4). As a positive control served wildtype MEF5/N9 cells that were incubated with DNase I to initiate DNA strand breaks. These cells were stained typically dark brown (see
Transient transfections into M5/N9 and HeLa cells were performed with FuGene transfection reagent (Roche) following the manufacturer's instructions. Briefly, semiconfluent cultures in a 24-well plate were transfected in triplicate with 112 ng of the β-galactosidase expression vector CMVβ(Invitrogen) and the same amount of pCAG-Cre-ER(T2), pCAG-Casp8-ER(T2) or pCAG-Casp9-ER(T2) plasmid DNA. After 12 h 4-OH-tamoxifen was added at a concentration of 10−8 M and the cultures were lysed at 48 h. Cell lysis and the measurement of β-galactosidase activity by chemiluminescence were performed with the β-galactosidase reporter gene assay (Roche) using a Centro LB960 plate luminometer (Berthold).
PAC2 zebrafish fibroblasts were grown in Leibowitz's L15—with L-Glutamine medium supplemented with 1% Non-essential Amino Acids, 1% Penicillin/Streptomycin and 10% fetal calf serum (all Gibco) at 28° C. 3×106 Cells were transiently transfected with the plasmids CMVβ together with CAG-CreER(T2), CAG-myrCasp8ER(T2) or CAG-Casp9ER(T2) (500 ng each) using nanofectin transfection reagent (PAA). 24 hrs after transfection the medium was replaced and conditioned with 10−8 M 4-OH-tamoxifen. Before lysis, the cells were incubated for another 24 hrs at 28°.
Results
We examined the effects of various concentrations of 4-OHT and 17-β-estradiol (E2), the natural ligand of the estrogen receptor, on the induction of cell death in M5/N9 cells expressing myrCasp8-ERT2 as compared to control cells expressing Cre-ER(T2). Cell viability was determined following 48 hours of incubation with the respective ligand at concentrations ranging from 10−6 M to 10−12 M by using the MTT assay. 4-OHT at a concentration of 10−9 M reduced the viability of myrCasp8-ER(T2) expressing cells by 30% and led to complete cell death at 10−8 M or higher concentration (
To define whether fusion proteins of murine Caspases and the human ER(T2) domain induce apoptosis in cells of other vertebrates than mice we transiently transfected Caspase-ER(T2) or Cre-ER(T2) expression vectors with a β-galactosidase reporter plasmid into human HeLa cells and zebrafish Pac2 cells (Chen et al., J Virol 76:2192-2198 (2002)). The activity of β-Galactosidase cotransfected with Cre-ER(T2) was taken as positive control of cell viability and its decline as indication of the death of cotransfected cells. The levels of β-Galactosidase activity in HeLa and Pac2 cells were reduced by 95-99% for myrCasp8-ER(T2) and 91-93% for Casp9-ER(T2) in the presence of 4-OHT and reached the same values as obtained from murine M5/N9 fibroblasts (
Taking advantage of a mutant estrogen receptor ligand binding domain (ER(T2)) we developed novel Caspase fusion proteins for inducible apoptosis. We show that Caspase-ER(T2) fusion proteins become specifically activated by the synthetic ligand 4-OH-tamoxifen and rapidly induce apoptotic cell death in human, murine and zebrafish cells. This novel tool for targeted cell ablation greatly facilitates the generation of disease models as well as developmental and regeneration studies in model organisms.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07012138 | Jun 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/005013 | 6/20/2008 | WO | 00 | 9/27/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/155133 | 12/24/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4736866 | Leder et al. | Apr 1988 | A |
| 5583278 | Alt et al. | Dec 1996 | A |
| 5625122 | Mak | Apr 1997 | A |
| 5698765 | Mak | Dec 1997 | A |
| 5750825 | Yazaki et al. | May 1998 | A |
| 6399327 | Wallach et al. | Jun 2002 | B1 |
| 6699686 | Brocard et al. | Mar 2004 | B1 |
| 7754209 | Ledbetter et al. | Jul 2010 | B2 |
| 20090099826 | Shi | Apr 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 196 25 049 | Jan 1998 | DE |
| WO 9836052 | Aug 1998 | WO |
| WO 0132855 | May 2001 | WO |
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| 20110023137 A1 | Jan 2011 | US |
