RECOMBINANT PROTEINS WITH A SELECTIVE INACTIVATION ACTIVITY ON TARGET PROTEINS
The present invention refers to the field of genetic engineering and pharmacology. In particular, the present invention relates to recombinant proteins having a selective inactivation activity on target proteins, genetic constructs encoding said proteins, cell line comprising said genetic constructs and pharmaceutical compositions comprising said genetic constructs as the active ingredient. More in particular, the present invention relates to genetic constructs encoding recombinant proteins capable of specifically recognizing target proteins within the endoplasmic reticulum (ER) and inducing their degradation after retro- translocation to the cytosol. The present invention also relates to the medical uses of said recombinant proteins or genetic constructs, in cases where defined secretory or membrane bound target proteins are required to be inactivated, including proteins involved in diseases characterized by the accumulation of aberrantly folded proteins, such as neurodegenerative disorders. Other medical uses of the proteins or of the genetic constructs of the inventions refers to the following disease: tumours, genetic diseases, hypersensitivity disorders, bacterial, parasitic or viral infections. The present invention also provides the use of said protein in the field of biotechnology, i.e. the use of the above protein or the genetic construct for the selective inactivation of target proteins.
Background of the invention
Proteins synthesised within the secretory pathway initiate their folding while in the lumen of the endoplasmic reticulum (ER) (Wickner, W. and Schekman, R. (2005). "Protein translocation across biological membranes." Science 310: 1452-1456). A number of different ER-resident proteins, known as chaperones, as well as enzymes involved in co- and post-translational modifications were shown to assist protein folding (Ellgaard, L. and Helenius, A. (2003). "Quality control in the endoplasmic reticulum." Nat Rev Mol Cell Biol 4: 181 -191 ; Williams, D.B. (2006). "Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum." J Cell Sci 119: 615-623). To be properly transported, membrane and secretory proteins are subjected to the ER quality control mechanism: if protein folding is aberrant or delayed, proteins are subjected to additional folding cycles or selected for ER-associated degradation (ERAD) (Travers, K.J., Patil, C.K., Wodicka, L, Lockhart, D.J., Weissman, J.S. and Walter, P. (2000). "Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation." Cell 101 : 249-258; Ellgaard, L. and Helenius, A. (2001 ). "ER quality control: towards an understanding at the molecular level." Curr Opin Cell Biol 13: 431 -437; Vembar, S.S. and Brodsky, J.L. (2008). "One step at a time: endoplasmic reticulum-associated degradation." Nat Rev Mol Cell Biol 9: 944-957).
In this process, proteins that fail to reach their terminal folding or maturation state, and thus selected for ERAD, are retro-translocated to the cytosol, a process also known as dislocation, for degradation by the 26S proteasome complex (Pickart, CM. and Cohen, R.E. (2004). "Proteasomes and their kin: proteases in the machine age." Nat Rev Mol Cell Biol 5: 177-187; Brodsky, J.L. and Wojcikiewicz, R.J. (2009). "Substrate-specific mediators of ER associated degradation (ERAD)." Curr Opin Cell Biol 21 : 516-521 ). Different ER-resident proteins have been described to be involved in retro-translocation, such as Derlin 1 , whose expression is up-regulated under ER stress and is required for degradation of some substrates (Kamauchi, S., Nakatani, H., Nakano, C. and Urade, R. (2005). "Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana." Febs J 272: 3461 -3476), and Sec61 , a component of the translocon, which was found associated to various ERAD substrates (Schafer, A. and Wolf, D.H. (2009). "Sec61 p is part of the endoplasmic reticulum-associated degradation machinery." Embo J 28: 2874-2884). Following recognition, misfolded proteins are retro- translocated into the cytosol by the aid of ATPase p97 (or Cdc48 in yeast) for ubiquitinylation and subsequent degradation by the proteasome (Ye, Y., Shibata, Y., Kikkert, M., van Voorden, S., Wiertz, E. and Rapoport, T.A. (2005). "Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane." Proc Natl Acad Sci U S A 102: 14132-14138). For proteasomal degradation proteins need to be ubiquitinylated through the participation of different ER-associated ubiquitin ligases, whose catalytic domains are located on the cytosolic side. In yeast three different pathways of ERAD have been described, depending on the type of protein and location of the misfolded lesion: whether in the cytosolic part; in the lumenal part of soluble or membrane proteins; or in membrane spanning domains (Taxis, C, Hitt, R., Park, S.H., Deak, P.M., Kostova, Z. and Wolf, D.H. (2003). "Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD." J Biol Chem 278: 35903-35913; Vashist, S. and Ng, D.T. (2004). "Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control." J Cell Biol 165: 41 -52.). Depending on the localization of the misfolded moiety, different ubiquitin ligases are involved: misfolded cytosolic domains require Doa10p (Swanson, R., Locher, M. and Hochstrasser, M. (2001 ). "A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation." Genes Dev 15: 2660- 2674.), whereas misfolded lumenal domains require the ER-associated ubiquitin ligase Hrdl p (Bordallo, J., Plemper, R.K., Finger, A. and Wolf, D.H. (1998). "Der3p/Hrd1 p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins." Mol Biol Cell 9: 209-222; Bays, N.W., Gardner, R.G., Seelig, L.P., Joazeiro, C.A. and Hampton, R.Y. (2001 ). "Hrd1 p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation." Nat Cell Biol 3: 24-29).
The yeast ubiquitin ligase Hrdl p is an ER-associated protein that forms a complex with another ER-resident glycoprotein known as Hrd3p (Hampton, R.Y., Gardner, R.G. and Rine, J. (1996). "Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein." Mol Biol Cell 7: 2029-2044): this complex mediates substrate recruitment in the lumen and its degradation via ubiquitinylation and translocation to proteasomal machinery. Hrd3p seems to play a crucial role in the identification of misfolded proteins and their recognition as ERAD substrates, recruiting them to the site of dislocation. Truncation mutants of Hrd3p have shown that the NH2-terminal lumenal domain is involved in substrate recognition, while the second lumenal domain stabilizes its association with the ubiquitin ligase Hrdl p (Gardner, R.G., Swarbrick, G.M., Bays, N.W., Cronin, S.R., Wilhovsky, S., Seelig, L, Kim, C. and Hampton, R.Y. (2000). "Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrdl p by Hrd3p." J Cell Biol 151 : 69-82).
The lumenal domain of Hrd3p that mediates association with Hrdl p presents a high homology with the mammalian protein SEL1 L, a component of an ER multiprotein complex implicated in the recognition and dislocation of misfolded proteins (Mueller, B., Lilley, B.N. and Ploegh, H.L. (2006). "SEL1 L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER." J Cell Biol 175: 261 -270; Mueller, B., Klemm E.; Spooner E.; Claessen JH. and Ploegh, L. (2008). "SEL1 L nucleates a protein complex required for dislocation glycoproteins" PNAS. August 28. 2008. vol. 105. no. 34. 12325-2330). SEL1 L [ACCESSION NO. GENBANK AF052059] is an ER resident type-l transmembrane glycoprotein (773 residues) with a large lumenal region containing 5 N-linked glycans, a trans-membrane domain and a short proline rich cytoplasmic tail (34aa) (Biunno, I., Cattaneo, M., Orlandi, R., Canton, C, Biagiotti, L, Ferrero, S., Barberis, M., Pupa, S.M., Scarpa, A. and Menard, S. (2006). "SEL1 L a multifaceted protein playing a role in tumor progression." J Cell Physiol 208: 23- 38). SEL1 L belongs to the family of unfolded protein response genes which are induced during ER stress generated by the accumulation of misfolded proteins (Kaneko, M. and Nomura, Y. (2003). "ER signaling in unfolded protein response." Life Sci 74: 199-205).
Originally identified to function during ERAD, it has also been associated with cell fate determination, cell differentiation, cell transformation and cancer progression (Biunno, I., Cattaneo, M., Orlandi, R., Canton, C, Biagiotti, L, Ferrero, S., Barberis, M., Pupa, S.M., Scarpa, A. and Menard, S. (2006). "SEL1 L a multifaceted protein playing a role in tumor progression." J Cell Physiol 208: 23- 38). SEL1 L interacts with HRD1 (the mammalian homologue of yeast Hrdl p), Derlinl and Derlin 2 and with the ATPase p97, which has been shown to have a role in extracting proteins from the ER membrane during retrotranslocation (Baker, B.M. and Tortorella, D. (2007). "Dislocation of an endoplasmic reticulum membrane glycoprotein involves the formation of partially dislocated ubiquitinated polypeptides." J Biol Chem 282: 26845-26856). The SEL1 L-HRD1 complex has ubiquitin ligase activity and forms a complex with the ubiquitin conjugating enzymes (E2s) UBC6 and UBC7 (Kostova, Z., Tsai, Y.C. and Weissman, A.M. (2007). "Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation." Semin Cell Dev Biol 18: 770-779). In addition, SEL1 L-HRD1 complex interacts through the N-terminal lumenal portion of SEL1 L with the ER- resident lectins OS-9 and XTP3-B, which are also responsible of ERAD substrate targeting (Christianson, J.C., Shaler, T.A., Tyler, R.E. and Kopito, R.R. (2008). "OS-9 and GRP94 deliver mutant alphal -antitrypsin to the Hrd1 -SEL1 L ubiquitin ligase complex for ERAD." Nat Cell Biol 10: 272-282; Bernasconi, R., Galli, C, Calanca, V., Nakajima, T. and Molinari, M. (2010). "Stringent requirement for HRD1 , SEL1 L, and OS-9/XTP3-B for disposal of ERAD-LS substrates." J Cell Biol 188: 223-235). These lectins seem to be involved in coordinating substrate recognition in the ER lumen with ubiquitin conjugation in the cytoplasm.
A number of diseases are characterized by proteins in aberrant conformations that can generate toxic effects on cells. In the art, these diseases are identified as "conformational diseases" or "protein misfolding diseases" or "ER stress-related diseases". For a general discussion see, for example, WO02/04954, WO04/07546, WO2007/137237, WO2009/061906, US2007/0202544, US2008/01 18938 and the references cited therein.
Methods for treating this kind of diseases are disclosed. WO2006/1 16716 discloses materials and methods for enhanced degradation of mutant proteins associated with human diseases, providing administration of a compound that enhances autophagic protein degradation. Specifically, the compound inhibits the mammalian target of rapamycin (mTOR) or Ras homolog enriched in brain. This reference discloses rapamycin and its analogues and a farnesyl transferase inhibitor. US2007/015577 adopts a similar approach in treating protein conformational disorder also using rapamycin or a derivative thereof as an mTOR inhibitor.
US2007/0204352 discloses a method for treating a neurological disease depending on misfolded protein comprising altering the activity of a protein of the ubiquitin-proteasome degradation system or an autophagy protein by means of a vector expressing a second protein of the ubiquitin-proteasome degradation system or an autophagy protein. A similar method is also disclosed in US2009/1 1 1768, focused on proteins of the SURF family, SEC22 family and Acyl CoA oxidase enzymes. A method for treating Huntington's Disease is provided in US2008/045607. This method comprises inducing in neuronal cells the expression or activity of a protein that induces unfolded protein response. This protein is selected from the group consisting of IRE1 a, IRE1 β, ATF6, PERK or XBP-1 . This method prevents ER stress in neuronal stress by reducing or suppressing Htt-mediated toxicity. Disorders mediated by a misfolded form of superoxide dismutase (SOD) are treated by administering an antibody against said misfolded SOD, as disclosed in US2008/0206251 .
ANKRD16 protein and its use in the treatment of neurodegenerative disease or a proteopathy is disclosed in WO2008/127680. US2009/17588 aims at treating cancer with agents that bind to epitopes unique to misfolded proteins forms of surface proteins presented by cancer cells. In particular this misfolded form is prion protein PrP, which has been identified on various cancer cells.
A further approach to misfolded protein diseases is disclosed in US2008/0227700. Here, the aim is to stabilize and prevent aggregation of abnormally folded and compromised proteins. The solution provided by this reference is a series of peptides, called intellipeptides. Specific embodiments are provided in relation to αβ crystal I in. As a background art, Rodriguez-Gonzalez A.; Cyrus K.; Salcius M.; Kim K.; Crews CM.; Deshaies RJ. and Sakamoto KM.; Tergetin Steroid Hormone Receptors for Ubiquitination and Degradation in Breast and Prostate Cancer, Oncogene (2008) 27, 7201 -721 1 , provide proteolysis targeting chimeric molecules (Protacs) that target proteins for destruction by exploiting the ubiquitinin-dependent proteolytic system of eukaryotic cells. Protacs specifically inhibit the proliferation of hormone- dependent breast and prostate cancer cells through degradation of the estrogen receptor a and androgen receptor, respectively.
Filesi I.; Cardinale A.; Mattei S. and Biocca S.; Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrPSc formation; Journal of Neurochemistry, 2007, 101 , 1516-1526, generate the secreted version of 8H4 intrabody in order to compel PrPc outside the cells and induce proteasomal degradation of endogenous prion proteins.
It is felt the need of a molecule capable of specifically inactivate proteins. It also felt the need of a molecular tool, which is able to detect a protein into the ER, whether secretory or membrane-bound, and to induce its selective degradation. It is also felt the need of a molecular tool, which is specific and selective towards the target protein, but at the same time is flexible to be easily designed and constructed for a wide range of target proteins. The need for a molecule capable of inducing proteasome-mediated degradation of specific target proteins is still felt.
Summary of the invention
It has now been found and is an object of the present invention a protein comprising a C-terminal portion comprising at least the COOH-terminal 372 amino acids of the human SEL1 L and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein. It has surprisingly been found that said protein has a selective inactivation effect on the recognised target protein by inducing its proteasome-mediated degradation following retro- translocation to the cytosol. Other objects of the invention are: a protein comprising a mammalian SEL1 L C-terminal portion comprising the degradation-inducing domain that induces selective degradation of a target protein and a heterologous N-terminal portion consisting of a target-specific recognition domain for said target protein, wherein said recombinant protein has a selective inactivation activity on said target protein; a protein comprising a C-terminal portion comprising the degradation-inducing domain of a protein involved in ERAD and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein, said protein having a selective inactivation activity on the target protein, with the proviso that said C-terminal portion is of a non-viral protein; in particular said C-terminal portion is different from the one of the Vpu protein; a protein comprising a C-terminal portion comprising the degradation-inducing domain of a mammalian protein involved in ERAD and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein, said protein having a selective inactivation activity on the target protein.
These proteins are herein named by the present inventors "degradins".
Description of the invention
A technique of specific degradation of target molecule by means of fusion proteins is known and involves the fusion of a chemokine (a ligand) with the cytoplasmic portion of the HIV protein Vpu to obtain inactivation of the specific chemokine receptor (Coffield, V.M., Jiang, Q. and Su, L. (2003). "A genetic approach to inactivating chemokine receptors using a modified viral protein." Nat Biotechnol 21 : 1321 -1327). The protein of the invention is essentially different from the above fusion protein known in the art for the following reasons: 1 ) instead of a viral protein, the protein of the invention comprises a mammalian ER-resident protein known to be involved in recruiting misfolded proteins for ERAD, said protein being preferably human SEL1 L;
2) the protein of the invention comprises only a portion of the above protein, retaining the ability to efficiently interact with the proteins involved in retrotranslocation and degradation;
3) the use of a heterologous recognition moiety, such as scFvs or any other peptidic target-specific binding moiety (antibody-derived domains, binding peptides, fragments or domains from receptors [for ligands], or ligands [for receptors], etc.) allows to enormously extend the repertoire of proteins that can be targeted for degradation: whichever secretory or membrane-bound protein can theoretically be directed towards proteasome-mediated degradation, once a specific ligand is identified. The possibility to use different recognition moieties derived from many different sources (in particular from monoclonal antibodies, libraries of antibody fragments, binding peptides, ligands or receptors) provides a high degree of flexibility to the described system.
A clear advantage over other described methods of selective inactivation of target proteins, for example via siRNA interference, consists in the possibility to design degradins with the ability to discriminate among different sterical conformations of the same protein, for instance, by using as recognition moiety antibody fragments, like scFv, with such characteristic. In the sense of the present invention, it can be also referred to as "intrabody" (for a definition see for example Kvam E, Nannenga BL, Wang MS, Jia Z, Sierks MR, Messer A., Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity. PLoS One. 2009 May 28;4(5):e5727. and WO02062197).
Therefore, the protein of the invention represents also a solution to the problem of the specific treatment of diseases due to proteins in aberrant conformations that can generate toxic effects on cells and related diseases, without toxic and side effects to healthy proteins. Object of the invention is a protein comprising a C-terminal portion comprising at least the COOH-terminal 372 amino acids of the human SEL1 L and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein, and having a selective inactivation activity on said target protein.
Preferably said C-terminal portion comprises the 372 COOH-terminal amino acids of the human SEL1 L.
Another object is a protein comprising a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein and a C-terminal portion of SEL1 L comprising the degradation-inducing domain that induces selective degradation of said target protein.
Within the scope of the present invention, it is intended that said C-terminal portion of mammalian SEL1 L is that portion comprising at least the degradation inducing domain. In particular, the above C-terminal portion comprising the degradation-inducing domain can comprise 167 COOH-terminal residues (starting from the third cluster of TPR repeats) or even the last 130 residues (including only the Hrd3p-like and the proline rich motifs) of human SEL1 L (Biunno, I., Cattaneo, M., Orlandi, R., Canton, C, Biagiotti, L, Ferrero, S., Barberis, M., Pupa, S.M., Scarpa, A. and Menard, S. (2006). "SEL1 L a multifaceted protein playing a role in tumor progression." J Cell Physiol 208: 23-38).
In particular, said degradation-inducing domain is capable of inducing proteasome- mediated degradation of the target protein following retro-translocation to the cytosol. A further object is therefore a protein comprising a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein and a C- terminal portion consisting of the degradation-inducing domain of a protein involved in ERAD and having a selective inactivation activity on the target protein. In particular said protein involved in ERAD is mammalian, more preferably human. The Vpu protein is specifically excluded.
Preferably, two proteins involved in ERAD which are ER proteins involved in the recognition of misfolded proteins, are OS-9 and XTP3-B. These two proteins seem to transiently interact either directly or indirectly with SEL1 L for the recruitment of substrates to the ubiquitin ligase HRD1 (Bernasconi, R., Galli, C, Calanca, V., Nakajima, T. and Molinari, M. (2010). "Stringent requirement for HRD1 , SEL1 L, and OS-9/XTP3-B for disposal of ERAD-LS substrates." J. Cell Biol. 188: 223- 235). Therefore, the fusion for a target recognition moiety to sequences of the C- terminal portion of OS-9 or XTP-3B results in a fusion-protein that recognizes proteins for degradation through ERAD, even if properly folded.
Another ERAD protein that can be exploited is the protein EDEM, that has been described to bind to misfolded proteins and to mark them for degradation (Cormier, J.H., Tamura, T., Sunryd, J.C. and Hebert, D.N. (2009) "EDEM1 recognition and delivery of misfolded proteins to the SEL1 L-containing ERAD complex." Mol. Cell. 34: 627-633). The use for a target recognition moiety fused to the C-terminal portion of EDEM can force also properly folded target proteins to enter the ERAD pathway.
In addition, the target recognition moiety could be fused to either Derlinl , a protein forming a putative retrotraslocation channel (Lilley, B.N. and Ploegh, H.L. (2004) "A membrane protein required for dislocation of misfolded proteins from the ER" Nature 429: 834-840), or directly to the ubiquitin ligase HRD1 (Kikkert, M., Doolman, R., Dai, M., Avner, R., Hassink, G., van Voorden, S., Thanedar, S., Roitelman, J., Chau, V. and Wiertz, E. (2004). "Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum" J. Biol Chem. 279: 3525-3534). In the case of a Derlinl -degradin, direct retro- translocation of the target protein could be achieved, while, in the case of a HRD1 - degradin, the target protein would be recognized for its polyubiquitinylation and subsequent degradation. Recombinant proteins according to the present invention with C-terminal of OS-9, XTP3-B, EDEM, Dehinl and HRD1 are other embodiments and the definitions of said C-terminal portion provided above for the embodiment of SEL1 L apply also here. It is intended that said C-terminal portion in the whole scope of the present invention is that portion comprising at least the degradation inducing domain. It is also intended that said C-terminal portion comprises also variants thereof, including mutations, deletions, substitutions which do not alter the function of the degradation inducing domain. In the sense of the present invention, any change in the length of the C-terminal portion is within the scope of the invention, provided that the recombinant protein maintains the inactivation activity.
In a preferred embodiment, the above proteins further comprise a tag, preferably SV5. In another preferred embodiment, the recognition domain of the above proteins is a scFv, preferably the scFv derived from the anti-FcsRIa monoclonal antibody 9E1 , or a ligand, preferably the FCE ligand consisting of domains £CH3-£CH4 from human IgE.
Other objects of the invention are a genetic construct encoding the protein of the invention, a cell line comprising said genetic construct and an expression vector comprising said construct. The genetic construct may take the form of DNA, RNA and mRNA.
Preferably said vectors are pcDNA-scFv9E1-degradin, pcDNA-scFv9E1-degradin- Hygro or pcDNA-LigandFcE-degradin. Alternatively, the genetic construct can be embedded in an expression system, such as for example a plasmid or a viral vector, and this is another object of the present invention. A further object is the protein of the invention or the genetic construct of the invention for use as a medicament, in particular for the treatment of any type of disease where degradation of a defined secretory or membrane bound protein is required in order to ameliorate or cure the disease, wherein the disease is a disease due to proteins in aberrant conformations that can generate toxic effects on cells, as in the case of neurodegenerative disorders, a tumour, a genetic disease, a hypersensitivity disorder, like allergies, a bacterial, parasitic or viral infection.
In the particular treatment of tumors the proteins or the genetic constructs of the invention can block their growth, for instance targeting growth factor receptors, or metastatic invasion (by targeting metalloproteinases, or cathepsins).
Other medical uses of the protein of the invention or the genetic construct of the invention can be related to the blocking of neoangiogenesis or in metabolic disorders, when blocking expression of a receptor or a metabolite can help in reducing or eliminating the pathological manifestations.
Other objects are a pharmaceutical composition for gene therapy comprising the genetic construct of the invention and the use of the protein or of the genetic construct of the invention for the selective inactivation of target proteins.
Object of the present invention is also the application of the degradin system for the treatment of the above defined diseases comprising the administration to a subject affected by such a disease an effective amount of the genetic construct of the invention. Therefore a further object is a method for the selective inactivation of target proteins comprising the administration of the genetic construct of the invention. The present invention will now be described in detail also by means of examples and figures.
In the figures: Fig. 1 . Schematic representation of the constructs used. A: maps of plasmids expressing degradins (scFv-degradin and ligand-degradin); B: maps of plasmids expressing targets (FCERI a-chain domains D1 -D2 either in secretory, sda, and membrane form, mda); C: maps of plasmids expressing control proteins containing the scFv9E1 recognition domain and either a truncated SEL1 L (pcDNA-scFv9E1- SEL1 LA, containing residues 713-773 of SEL1 L) or the KDEL ER-retention signal (pcDNA-scFv9E1-KDEL).
Fig. 2. Western blotting analysis of cell culture supernatants (A) or cellular extracts (B) from HEK-293 cells co-transfected with vectors expressing the model secretory target protein sda and the indicated degradins. Blots were developed with anti- FCERI antibodies to detect sda, with anti-SV5 antibodies to detect the degradins and with anti-actin antibodies to normalize the amount of extracts loaded. In A is shown that scFv9E1-degradin was able to completely block secretion of sda, while control irrelevant degradin (irr-degradin: a degradin containing a scFv of irrelevant specificity) did not. Analysis of the total cellular extracts (B) showed that only a very low amount of the target protein was intracellularly retained, in an incompletely glycosylated form indicating that the protein was retained in the ER and did not reach the Golgi compartment (arrows), while scFv9E1 fused to the ER retention sequence KDEL, which was able to block secretion only partially, produced instead large accumulation of the incompletely glycosylated protein within the cell. Interestingly, scFv9E1-KDEL was also found in part secreted in the supernatant, thus limiting its retention capability. We therefore concluded that degradin-mediated blocking of secretion was not only due to intracellular retention, but also to active degradation.
Arrows indicate the incomplete glycosylated (ER) and terminally glycosylated (Golgi/secreted) forms of the target protein sda.
Fig. 3. Western blotting analysis of cellular extracts from HEK-293 cells co- transfected with vectors expressing the model membrane-bound target protein mda and the indicated degradins. Blots were developed with anti-FcsRI antibodies to detect mda, with anti-SV5 antibodies to detect the degradins and with anti-actin antibodies to normalize the amount of extracts loaded. Arrows indicate the incomplete glycosylated (ER) and terminally glycosylated (Golgi/secreted) forms of the target protein mda. Similarly to the secretory form, expression of the membrane-bound mdD was also heavily impaired by scFv9E1-degradin, but not by the control irr-degradin, which, instead, allowed a large proportion of target protein to reach the terminal glycosylated state. Also in this case, scFv9E1-KDEL induced mainly intracellular retention, with only modest degradation
Fig. 4. Western blotting analysis of cell culture supernatants and cellular extracts from HEK-293 cells co-transfected with vectors expressing the model target proteins sda (A) and mda (B) and the indicated scFv- and ligand-degradins showed a strong degradation activity of LFceRI-degradin both on sda (A) and mda (B). Analysis of cell culture supernatants from a cell line co-transfected for expression of an irrelevant SV5-tagged secretion protein and the corresponding degradins (C) showed that both degradins were specific for the target, since secretion of the irrelevant molecule, which is not recognised by either of the two degradins, was unaffected. Fig. 5. FACS analysis of HEK-293 cells non-transfected or stably transfected to express mda alone or mda and scFv9E1-degradin showed a strong reduction in cell surface expression of md D (A), which corresponded to a similar impairment of the total amount of protein detectable in Western blotting of cellular extracts from the same clones (B); mda and sda expressions were detected by anti-FcsRI antibodies.
Fig. 6. Western blotting analysis of cell culture supernatants and cellular extracts from HEK-293 cells co-transfected with vectors expressing the model target proteins sdaD (A) and mda (B) and a degradin construct where the complete lumenal domain of SEL1 L (up to residue 712) was deleted (scFv9E1-SEL1 LA, Fig. 1 C), showed that, while the truncated degradin was still expressed in the ER where it produced a large retention of both target proteins, the degradation activity was highly reduced.
Fig.7. Western blotting analysis of cellular extracts from HEK-293 cells co- transfected with vectors expressing the model target protein mda and the indicated degradins, after treatment with the proteasome inhibitor MG132 or the same amount of MG132 solvent DMSO, showed that a reduced activity of the degradins, visible as an increase in the amount of the intracellularly retained mda, was observed in cells treated with the proteasome inhibitor, thus indicating that degradin-mediated degradation involves proteasome activity. Modes to carry out the invention
According to the present invention, the protein is a "recombinant protein" that is derived from recombinant DNA. In the context of the present invention, this protein is also named "fusion protein".
Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together.
According to the present invention, with the expression "selective inactivation activity" it is intended the constitutive knocking down of specific target proteins to a quantity not significantly detectable or a quantity such as, at therapeutic doses of the protein of the invention, there are no negative effects due to the target proteins. Said selective inactivation is preferably due to specific degradation.
The present degradation-inducing fusion protein, herein also termed "degradin", is an ER-resident transmembrane protein with its binding moiety within the lumenal side; this allows recognition of ER localized target proteins (proteins in transit through the ER or ER resident). In the scope of the present invention, the definitions "binding moiety", "target- specific recognition domain", "recognition moiety", "recognition specific domain" and "recognition domain" have the same meaning.
The activity of degradins has been demonstrated equally efficient, independently whether the target protein was secretory or membrane-bound. The target proteins can therefore be secretory or membrane-bound proteins and the inactivation is due to specific degradation. The recognition moiety can be represented by any protein domain able to recognize a specific target, preferably a scFv from an antibody directed against the defined target or a ligand for a target receptor or viceversa. The recognition moiety specifically recognises the target within the ER and, thanks to the construction of the invention with the SEL1 L portion, induces its degradation after retro- translocation to the cytosol.
ScFv molecules are engineered antibody fragments that retain the specificity of the original antibody, and can be easily modified to direct their localisation to different intracellular compartments, A ligand is intended as any protein sequence with ability to bind the defined target.
The protein of the invention can also comprise a tag.
A tag is whichever peptide sequence that can be added to a defined protein for different purposes such as polyhistidine tag for purification, an epitope tag for detection or immunopreciptation by antibodies, or a tag for in vivo biotinylation. In particular, to facilitate detection, in a preferred embodiment the 12 aa long SV5 tag (GKPIPNPLLGLD; SEQ ID. NO.1 ) was included in the recombinant degradins decribed in Fig. 1A.
According to an embodiment of the present invention, when fused to the SEL1 L fragment, the scFv provides recognition specificity to the degradin within the ER lumenal side. Similarly, when the recognition moiety of the protein of the invention is a ligand, the protein, herein called by the inventor also as "ligand-degradin", can interact with specific target receptors or soluble proteins exposed in the ER lumen. When a target molecule within the ER is engaged by the recognition-specific domain of a degradin, it is retro-translocated to the cytosol by means of the activity of SEL1 L domain and degraded.
In an embodiment of the invention, the degradin is specific for the FCERI D chain target. Particularly, in one embodiment the recognition domain is the scFv derived from the anti-FcsRIa monoclonal antibody 9E1 . scFv9E1-degradin-gene (SEC secretion signal, intron, scFv 9E1 , SV5 tag, SEL1L 402-773; SEQ ID. NO 2):
ATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGCTACAGgfaagggg ctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacagGTG TGCACTCTCAAATTGTGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCC AGGGGAGAGGGTCACCATAACCTGCAGTGTCAGTTCAACTGTAAGCTACAT GCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAAGTCTTGATTCATGA CACATCCAAGTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGTTCT GGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCC ACTTATTACTG C C AG C AAAG G AGTTATTTC C C G CTC AC GTTC G GTATTG G G A CCAAGCTGGAGCTGAAAggcagcactagtggtagcggcaaaccaggttccggcgaaggctc gagcaaaggcCAGGTGCAGCTGAAGGAGTCGGGACCTGGCCTGGTGGCGCCC TCACAGAGTCTGTCCATCACTTGCACTGTCTCTGGATTTTCATTAACCAGTTA TGGTATACACTGGATTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGG GAGTAATATGGGCTGGTGGAAACACAAGTTATAATTCGGCTCTCATGTCCA G ACTG AG CATC AG C AAAG AC AACTC C AG GAG C C AAGTTATCTTAC AAATG A AC AGTCTG C AAACTG ATG AC AC AG C C ATATACTACTGTG C C AG AG AG G AC C GGATGTATTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCT CCTCCqqaqctaqtGGCAAACCAATCCCAAACCCACTGCTGGGCCTGGATqctaqc GGAAGTGACATTGTACCTCAGAGTAATGAGACAGCTCTCCACTACTTTAAG AAAGCTGCTGACATGGGCAACCCAGTTGGACAGAGTGGGCTTGGAATGGC CTACCTCTATGGGAGAGGAGTTCAAGTTAATTATGATCTAGCCCTTAAGTAT TTCCAGAAAGCTGCTGAACAAGGCTGGGTGGATGGGCAGCTACAGCTTGGT TCCATGTACTATAATGGCATTGGAGTCAAGAGAGATTATAAACAGGCCTTG AAGTATTTTAATTTAGCTTCTCAGGGAGGCCATATCTTGGCTTTCTATAACCT AGCTCAGATGCATGCCAGTGGCACCGGCGTGATGCGATCATGTCACACTGC AGTGGAGTTGTTTAAGAATGTATGTGAACGAGGCCGTTGGTCTGAAAGGCT TATGACTGCCTATAACAGCTATAAAGATGGCGATTACAATGCTGCAGTGATC CAGTACCTCCTCCTGGCTGAACAGGGCTATGAAGTGGCACAAAGCAATGCA GCCTTTATTCTTGATCAGAGAGAAGCAAGCATTGTAGGTGAGAATGAAACTT ATCCCAGAGCTTTGCTACATTGGAACAGGGCCGCCTCTCAAGGCTATACTG TGGCTAGAATTAAGCTCGGAGACTACCATTTCTATGGGTTTGGCACCGATGT AGATTATGAAACTGCATTTATTCATTACCGTCTGGCTTCTGAGCAGCAACAC AGTGCACAAGCTATGTTTAATCTGGGATATATGCATGAGAAAGGACTGGGC ATTAAACAGGATATTCACCTTGCGAAACGTTTTTATGACATGGCAGCTGAAG CCAGCCCAGATGCACAAGTTCCAGTCTTCCTAGCCCTCTGCAAATTGGGCG TCGTCTATTTCTTGCAGTACATACGGGAAACAAACATTCGAGATATGTTCAC CCAACTTGATATGGACCAGCTTTTGGGACCTGAGTGGGACCTTTACCTCATG ACCATCATTGCGCTGCTGTTGGGAACAGTCATAGCTTACAGGCAAAGGCAG CACCAAGACATGCCTGCACCCAGGCCTCCAGGGCCACGGCCAGCTCCACC CCAGCAGGAGGGGCCACCAGAGCAGCAGCCACCACAGTAA scFv9E1-degradin protein (SEC secretion signal, scFv 9E1 , SV5 tag, SEL1L 402- 773; SEQ ID. NO 3):
RSYFPLTFGIGTKLELKGSTSGSGKPGSGEGSSKGQVQLKESGPGLVAPSQSL SITCTVSGFSLTSYGIHWIRQPPGKGLEWLGVIWAGGNTSYNSALMSRLSISKD NSRSQVILQMNSLQTDDTAIYYCAREDRMYWYFDVWGAGTTVTVSSqasGKPIP NPLLGLDasGSPiVPQSNETAtHYFKKAAPMGNPVGQSGLGMAYtYGRGVQVN YDLALKYFQKAAEQGWVDGQLQLGS YYNGIGVKRDYKQALKYFNLASQGG HILAFYNLAQ HASGTGV RSCHTAVELFKNVCERGRWSERL TAYNSYKDG DYNAAVIQYLLtAEQGYEVAQSNAAFILDQREASIVGENETYPRALLHWNRAAS QGYTVARI LGDYHFYGFGTDVDYETAFIHYRLASEQQHSAQAMFNLGYMHEK GLGIKQDIHLAKRFYD AAEASPDAQVPVFLALCKLGWYFLQYiRETNIRDMFT QLDMDQLLGPEWDLYL TIIALLLGTVIAYRQRQHQDMPAPRPPGPRPAPPQQ EGPPEQQPPQ
In a second embodiment, the recognition domain is a ligand of the target, a truncated version of Fez, consisting of the domains εΟΗ3-εΟΗ4 from human IgE ε heavy chain (Fig. 1A). Ligand -degradin-gene (SEC secretion signal, intron, SV5 tag. DCH3-CH4, SEL1L 402-773; SEQ ID. NO 4):
ATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGCTACAGgfaagggg ctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacagGTG TGCACTCGaaaGGCAAACCAATCCCAAACCCACTGCTGGGCCTGGATaqttqtqc agatTCGAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCCAGCCCGTT CGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCT GGCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGA AGCCTGTGAACCACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACG TTAACCGTCACGTCCACCCTGCCGGTGGGCACCCGAGACTGGATCGAGGG GGAGACCTACCAGTGCAGGGTGACCCACCCCCACCTGCCCAGGGCCCTCA TGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCGGAAGTCTAT GCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCG CCTGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGC ACAACGAGGTGCAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCG CAAGACCAAGGGCTCCGGCTTCTTCGTCTTCAGCCGCCTGGAGGTGACCAG G G C C G AATG G GAG C AG AAAG ATG AGTTC ATCTG C C GTG C AGTC C ATG AG G CAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTAAATCCCGGG
Ligand -degradin protein (SEC secretion signal, SV5 tag, DCH3-CH4, SEL1L 402-773; SEQ ID. NO 5):
MGWSLILLFLVAVATGVHSeGKPIPNPLLGLDscadSNPRGVSAYLSRPSPFDLFIR KSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPV GTRDWIEGHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQN
The above degradins specific for the FCERI D chain target find application in the field of allergy prevention, because of their effectiveness in inactivating the FCERI .
The invention relates to the genetic construct encoding the recombinant protein. Said genetic construct can be a DNA or RNA construct.
The invention further relates to an expression system or expression construct that allows the expression of the protein of the invention in mammalian cells.
Any expression vector suitable for protein expression in mammalian cells such as plasmids or viral vectors known in the art may be attached to a DNA sequence encoding the proteins of the invention to enable production in an appropriate host. In a particular embodiment of the invention vectors used are plasmids pcDNA and pcDNA-Hygro (Invitrogen).
The person skilled in the art can easily prepare a DNA as described above by using conventional techniques of genetic engineering. More specifically, DNAs of the present invention can be inserted in any expression vector able to sustain expression in mammalian cells (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning. A laboratory manual. New York, Cold Spring Harbor Laboratory Press). In a particular embodiment of the invention, the plasmids comprising the DNA constructs encoding the proteins of the invention are pcDNA-scFv9E1-degradin, pcDNA-scFv9E1-degradin-Hygro or pcDNA-LigandFcD-degradin.
Said genetic constructs are introduced into host cells by transfection with any procedure useful for entry into a particular cell, for example biological or physical methods, able to produce a cell having the recombinant DNA stably integrated into its genome or existing as episomal element, so that DNA molecules used in the present invention are expressed by the cell host.
Physical methods to introduce a double-stranded DNA or RNA in a host cell include, but are not limited to, precipitation with calcium phosphate, lipofection, DEAE-dextran, particle bombardment, microinjection, electroporation, immunoliposomes, lipids, cationic lipids, phospholipids or liposomes or similar.
The skilled person will understand that any method can be used to deliver to cells the genetic information in the form of DNA or RNA.
Viral vectors such as adenoviruses, lentiviruses, retroviruses, Adeno-associated Virus (AAV), Herpes Simplex Virus (HSV) or Sendai Virus and non-viral methods such as liposome-mediated delivery, electroporation or biolistic delivery (Gene Gun) can be used to administer the genetic construct of the invention (DNA or RNA) encoding the degradin proteins of the invention in cells or living subjects to be treated. The administration includes for example, in-vivo and ex-vivo methods. Said subjects to be treated are preferably human, but are not particularly limited to, and they can also be animals.
Adenoviral vectors are a useful tool in gene therapy because of their ability to accommodate large foreign DNAs and because of the possibility to transduce both proliferating and quiescent cells. Adenoviral vectors have already been used for cancer gene therapy trials and, through the improvement of adenoviral tropism they have been used for gene transfer into muscle cells and hepatocytes (Breyer, B., Jiang, W., Cheng, H., Zhou, L, Paul, R., Feng, T. and He T.C. (2001 ) "Adenoviral vector-mediated gene transfer for human gene therapy." Curr. Gene Ther. 1 : 149-162).
Especially important are Adeno-associated viral vectors (AAV vectors), that are non-pathogenic and non-immunogenic vectors with a broad range of target cells and able to transduce both dividing and non-dividing cells. AAV vectors have been used in clinical trials for several diseases including neurological diseases, heart failure, genetic disorders and several types of cancer (dos Santos Coura, R. and Nardi, N.B. (2007) "The state of the art of adeno-associated virus-based vectors in gene therapy" Virol. J. 4: 99). In addition, the existence of several natural AAV variants have allowed the exploitation of different receptor to enter the host cell, thus extending the repertoire of transduceable cell types (liver, heart, CNS, kidney, etc.) (Zentilin, L. and Giacca M. (2008) "Adeno-associated virus vectors: versatile tools for in vivo gene transfer" Contrib. Nephrol. 159: 63-77).
Retroviruses, like MLV, or Lentiviruses, like HIV-1 , have also been successfully used to transduce different cell types after pseudotyping (spinal motor neurons, pancreatic islet cells, liver cells, fibroblasts, myocytes, epithelial cells or hematopoietic cells) (Escors, D. and Breckpot, K. (2010) "Lentiviral vectors in gene therapy: their current status and future potential" Arch. Immunol. Ther. Exp. 58: 107-1 19).
Another viral vector that can be used is the Herpes Simplex virus (HSV): it can infect a large number of either dividing and non-diving cells and offers the advantage of incorporating large size of foreign DNA; in addition, it is able to persist as an episome in neuronal cells, thus allowing long lasting foreign gene expression without the need of integration (Manservigi, R., Argnani, R. and Marconi, P. (2010) "HSV recombinant vectors for gene therapy" Open Virol. J. 4: 123-156). The recombinant protein is expressed in eukaryotic cells, for example in the mammalian cell lines CHO, Sp2/0 or BHK21 , or in the human cell lines HEK 293 or HeLa. The recombinant protein can also be expressed living organisms and even in patients. The usage of tissue-specific promoters to direct expression of degradins in specific tissues could allow degradation of targets only in selected tissues. For instance promoters of keratin 14 for keratinocytes, of L-plastin for bladder and ovarian cells, of PSA for prostate cells, of myosin heavy chain for cardiac tissue and skeletal muscle, of albumin for hepatocytes, of CD1 1 c for denditric cells, of CD4 for T cells, of Ig Heavy chain for B lymphocytes, etc.
An object of the invention is the protein of the invention or the genetic construct of the invention for use as a medicament.
Degradins are of wide and still unpredicted usage in several different diseases.
In a possible application, silencing of specific receptors could help in reducing hypersensitivity-related diseases, like allergies.
Another important field of application is in the treatment of tumors: degradins can be used with the aim of blocking metastatic phenotype of tumors or in order to avoid tumor growth by inducing their regression.
The metastatic phenotype could be blocked for instance by the down-regulation of cathepsins or metalloproteinases that cancer cells use for cell matrix degradation and tissue invasion. In addition, there are mounting evidences that development of organ-specific metastasis is governed by the interaction between chemokine receptors expressed by tumor cells and chemokines expressed by the target organ. Therefore degradins could act on chemokine receptor expression, in order to avoid metastasis formation.
A reduction in tumor growth could also be achieved by acting on the angiogenetic pathway, for example by inhibiting VEGF receptor expression or the WNT signalling pathway: WNT is a soluble ligand that has been described to be involved in angiogenesis as well as in cell fate determination, differentiation, proliferation, motility and apoptosis (Lustig, B. and Behrens, J. (2003). "The Wnt signalling pathway and its role in tumor development." J. Cancer Res. Clin. One. 129: 199-221 ); WNT pathway could therefore be blocked by inhibiting with specific degradins the expression of the WNT receptor frizzled or of the co-receptors LRP 5 and 6.
Another way to block tumor growth could be obtained by promoting degradation of either growth factors that tumor cells use as in the case of autocrine growth stimulation or their corresponding receptors. For example the activity of degradins could be directed against tyrosine kinase receptors that bind to growth factors and survival factors.
In the case of viral infections, degradins can be used to block intracellularly the formation of infective viruses, by inducing the degradation of proteins essential for the formation of complete and infective viral particles. Preliminary experiments performed with degradins obtained from antibodies specific for viral envelope proteins have already shown a dramatic reduction of viral titres in cells expressing the degradin. Alternativaly they could be used to inactivate the expression of specific virus receptors, like CCR5 receptor for HIV, thus blocking virus entry into the cell.
Degradins could also be exploited to study particular phenotypes arising from the knock-out of specific proteins; in addition, they could be used to target proteins involved in metastatic behaviour of cancer cells, such as metalloproteinases or cathepsins, with the aim of blocking the metastatic transformation of tumors. Also, silencing of specific receptors could help in reducing hypersensitivity-related diseases, like allergies.
An important concept is that they can target non-misfolded proteins and induce their degradation. A clear advantage over other described methods of selective inactivation of target proteins (for example via siRNA interference) consists in fact in the ability of antibody to discriminate among different sterical conformations of the same protein: this possibility could turn out to be particularly useful when the aim is to eliminate proteins in aberrant conformations, that can generate toxic effects on cells. An object of the invention is the recombinant protein or the genetic construct of the invention for treatment of diseases due to proteins in aberrant conformations, or protein aggregate-related diseases or ER stress-related diseases. Examples of said diseases can be found in neurodegenerative diseases, bacterial and viral infections.
Neurodegenerative disorders are good candidates for applications of this technique: Transmissible Spongiform Encephalopaties (TSE) class of diseases is an example of how conformational changes can subvert a normal phenotype to a pathological phenotype. TSEs are caused by very peculiar infective agents, the prions, autocatalitically replicating proteins (Bieschke, J., Weber, P., Sarafoff, N., Beekes, M., Giese, A. and Kretzschmar, H. (2004). "Autocatalytic self-propagation of misfolded prion protein." Proc Natl Acad Sci U S A 101 : 12207-1221 1 ) responsible of BSE, Creutzfeld-Jacob disease and Scrapie ( Prusiner, S.B. (1998). "Prions." Proc Natl Acad Sci U S A 95: 13363-13383). In prion caused diseases, the cellular protein Prpc undergoes conformational modifications, giving rise to an insoluble version of the protein named Prpsc. This leads to the intracellular accumulation of Prpsc aggregates, with consequent cell destruction and pathological manifestations. It has been suggested that dysfunctions in the ubiquitin-proteasome may be involved in the accumulation of Prpsc (Deriziotis, P. and Tabrizi, S.J. (2008). "Prions and the proteasome." Biochim Biophvs Acta 1782: 713-722; Paul, S. (2008). "Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches." Bioessavs 30: 1 172- 1 184); therefore, degradin-mediated re-directing to the proteasome of the aberrantly folded Prpsc may help in contrasting aggregates formation. Another neurodegenerative disorder, Alzheimer Disease (AD), is characterized by pathological accumulation of a protein metabolite, the amyloid β peptide 1 -42 (A p i- 2), either extracellular or in the neuronal ER (Cuello, A.C. (2005). "Intracellular and extracellular Abeta, a tale of two neuropathologies." Brain Pathol 15: 66-71 ), probably caused by the conversion of natural monomers to toxic oligomers (Walsh, D.M. and Selkoe, D.J. (2007). "A beta oligomers - a decade of discovery." J Neurochem 101 : 1 172-1 184); also in this case, a degradin specific for the toxic form of A p i- 2 could help in reduce the disease symptomatology. It has been shown that in some neurodegenerative disorders the proteasome can actively participate in clearing aggregated proteins, when the production of abnormal protein is stopped (Martin-Aparicio, E., Yamamoto, A., Hernandez, F., Hen, R., Avila, J. and Lucas, J.J. (2001 ). "Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington's disease." J Neurosci 21 : 8772-8781 ); therefore, degradin technology can be applied not only to block the synthesis of new aberrantly folded proteins, but also to favour proteasome-mediated clearing of preexisting aggregates.
In the context of the present invention, examples of neurodegenerative diseases and their possible target proteins are: tauopathies, like Alzheimer's disease (tau protein, Amyloid beta); Parkinson's disease (Alpha-synuclein, tau); Creutzfeld- Jakob disease (Amyloid protein); Kuru (Amyloid protein); GSS disease (Amyloid protein); various systemic amyloidosis (Fragments of serum amyloid-A, beta 2- microglobulin, Transthyretin); Huntington's disease (Huntingtin); Polyglutamine diseases, like spinal & bulbar muscular atrophy (Atrophin-1 , ataxins); Prion diseases, like Bovine Spongiform Encephalopathy (BSE) (Prion protein); Amyotrophic Lateral Sclerosis (Superoxide dismutase); Alexander's disease (Glial fibrillary acidic protein); Charcot-Marie Tooth disease (PMP-22); ocular diseases, like Retinitis Pigmentosa (Rhodopsin) and Macular Degeneration (Amyloid-beta, crystal I ins) (see, for example, Sanders and Myers, Annu. Rev. Biophys. Biomol. Struct, 33: 25-51 , 2004).
Other applications of the degradin system include the inactivation of bacterial antigens in transit through the ER. Bacterial pathogens, such as Salmonella typhimurium and Chlamydia trachomatis, are able to persist within the cell in intracellular vacuoles that protect bacteria from the innate immune response; within these elements bacterial replication can occur (Rathman, M., Barker, L.P. and Falkow, S. (1997). "The unique trafficking pattern of Salmonella typhimurium- containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry." Infect Immun 65: 1475-1485; Beatty, W.L. (2006). "Trafficking from CD63-positive late endocytic multivesicular bodies is essential for intracellular development of Chlamydia trachomatis." J Cell Sci 119: 350-359). Although vacuoles isolate bacteria from cellular structures, some metabolites released by pathogens can enter the secretory pathway: examples are S. typhimurium Slrp protein, possibly involved in interference with MHC I peptide presentation, due to the induction of ER stress (Bernal-Bayard, J., Cardenal-Munoz, E. and Ramos- Morales, F. (2010). "The Salmonella type III secretion effector, salmonella leucine- rich repeat protein (SlrP), targets the human chaperone ERdj3." J Biol Chem 285: 16360-16368) and C. trachomatis proteins and lipopolysaccarides (MOMP, IncA and LPS), whose trafficking through the ER could as well induce ER stress (Giles, D.K. and Wyrick, P.B. (2008). "Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells." Microbes Infect 10: 1494-1503). In these cases the siRNA approach would not be useful, because the bacterial RNA does not enter the cell, while degradins act at the protein level and can therefore be efficient in contrasting the effects of bacterial infections.
A further application of present invention is the selective redirecting of antigens to the proteasome in dendritic cells, to activate antigen-specific cytotoxic CD8+ T- lymphocytes. Cytotoxic T-lymphocytes (CD8+) are in fact activated by the interaction with peptide-loaded class I MHC molecules on the surface of specialized antigen-presenting cells, like dendritic cells. MHC loading requires antigen to be previously processed by the proteasome, that executes the initial proteolytic degradation to produce peptides that are in turn transported to the ER and loaded to class I MHC molecules.
The degradin model can be exploited to efficiently generate a cytotoxic CD8+ response against an antigen when a specific monoclonal antibody is available: an antigen-specific degradin would efficiently direct the antigen towards proteasome degradation and generate peptides that can be loaded to MHC I molecules to activate a CD8+ response.
An antigen can also be artificially generated, even if a specific antibody is not available, by fusing the antigen to a different protein, against which is possible to create a specific degradin. Co-expression of the two constructs (recombinant antigen target and degradin) in the same antigen presenting cell (APC), such as DCs, will lead to target degradation and efficient peptide presentation in association with MHC I molecules, thus inducing lymphocyte activation. The genetic constructs encoding the target and degradin could be administered to a patient, for example by delivering the plasmid DNA by biolistic gene transfer, which efficiently transfects dermal APCs; a further level of control of specific expression in APCs can be achieved by using dendritic cell-specific promoter, such as CD1 1 c.
The present invention provides a pharmaceutical composition comprising as active ingredient a genetic construct encoding the protein of the invention (generally, but not exclusively in the form of a plasmid DNA) in an amount sufficient to inactivate the target protein in the relevant tissue. In connection with the pharmaceutical composition of the invention conventional vehicles such as gold particles or viral vectors, can be used appropriately and the composition can be formulated as a DNA-coated gold particles.
The preparation of pharmaceutical compositions of the present invention falls within the general knowledge of the expert in the field and do not require particular description. Examples of general knowledge are contained in Remington's Pharmaceutical Sciences, Mack Pub., latest edition.
In general, administration to individuals can be done through methods known in the art.
Any formulation suitable for the administration of genetic material is useful for carrying out the present invention. This technology is within the general knowledge of the skilled person, see for example the above cited Remington's or Delivery Technologies for Biopharmaceuticals, Lene Jorgensen and Hanne M0rck Nielsen eds., Wiley 2009,
In a particular embodiment, the present invention is carried out by means of gene delivery. Methods and means for gene therapy are well-known in the art. Examples of this kind of therapy are disclosed in US2009/01 1 1768, WO2008/127680 and the references cited therein. The pharmaceutical agent or pharmaceutical composition doses carrying out the present invention, in particular the method of treatment aspect in the present invention can be determined properly by a physician considering the type of dosage form, method of administration, the age of the patient, weight, the symptoms etc. Even this activity falls within the normal activities of person skilled in the art, for example following the guidelines of the regulatory authorities for drug development, such as the FDA or the EMEA.
Another embodiment of the present invention provides the use of the proteins herein disclosed as "laboratory tools" for the selective inactivation of target proteins. It is intended herein that "laboratory tool" means a laboratory reagent to be used for experiments in vitro or in vivo without any therapeutic purpose, but only for scientific investigation.
The following Example will further illustrate the invention.
Example Materials and Methods
Total RNA was extracted from the human cell line HEK 293 using the RNeasy Mini Kit (Qiagen). After cDNA synthesis with oligo-dT and MuMLV Reverse Transcriptase (Invitrogen) using 2 g of total RNA as substrate, a fragment of 1 138 bp, containing codons 423-794 of human SEL1 L (corresponding to positions 402- 773 of the mature protein) was amplified by PCR using primers SEL1 L-Nhel (AGTAGCTAGCGGAAGTGACATTGTACCTCA; SEQ ID. NO 6) and SEL1 L- EcoRI (TCAGAATTCTTACTGTGGTGGCTGCTGCTCT; SEQ ID. NO 7) and KOD High-Fidelity DNA polymerase (Novagen). PCR reaction was performed with about 100 ng cDNA, 1 μΜ of each primer, supplier provided buffer at 1 x concentration, 1 mM MgSO4, 0.2 mM of each dATP, dGTP, dCTP, dTTP and 1 U of KOD DNA polymerase, by incubating for 2' at 95°C followed by 30 cycles of 30" at 95°C, 30" at 55°C and 1 ' at 72°C. The amplified fragment was digested Nhel/EcoRI and inserted in a pcDNA vector (Invitrogen) expressing the anti-FcsRI scFv 9E1 (Vangelista et al., 2002b; Predonzani et al., 2008), modified by insertion in the Nhel site of a linker (gctagtGGCAAACCAATCCCAAACCCACTGCTGGGCCTGGATgctagc; SEQ ID. NO 8) encoding the SV5 tag (GKPIPNPLLGLD; SEQ ID. NO 1 ).
The final construct therefore encodes the construct scFv9E1 -SV5-SEL1 L that has been defined scFv9E1 degradin (pcDNA-scFv9E1-degradin).
A further truncated version of degradin (scFv9E1- SELI LA), completely lacking the SEL1 L lumenal portion, was obtained by amplifying the cDNA with primers SEL1 L Δ-Nhel (AGTAGCTAGCGATATGGACCAGCTTTTGGGA; SEQ ID. NO 9) and SELI L-EcoRI.
The construct used to compare ER retention operated by the KDEL retention sequence (Pelham, 1990) with the active degradation operated by degradins was obtained by inserting in the BspEI/Xhol sites of the said pcDNA-scFv-9E1 a linker encoding the SV5 tag followed by the KDEL sequence (tccgg a G GCAAACCAATCCCAAACCCACTGCTG G GCCTG G ATAGTACTAAAG A TGAGCTGtagctcgag; SEQ ID. NO 10) (pcDNA-scFv9E1-KDEL).
The control constructs containing irrelevant scFvs were obtained by substituting the Hindlll/BspEI cassette encoding the 9E1 scFv with the corresponding cassettes encoding different scFvs.
The cassette encoding the anti-FcsRI scFv9E1-degradin was in addition subcloned into the pcDNA-Hygro vector (Invitrogen) for the generation of double transfected stable clones (pcDNA-scFv9E1-degradin-Hygro).
The ligand-degradin expressing vector has been obtained from the previously described plasmid pCDNA-SEC-SV5-CH3/CH4, encoding a secretion signal (SEC), the SV5 tag and human IgE domains CH3-CH4 (Vangelista, L, Soprana, E., Cesco-Gaspere, M., Mandiola, P., Di Lullo, G., Fucci, R.N., Codazzi, F., Palini, A., Paganelli, G., Burrone, O.R. and Siccardi, A.G. (2005). "Membrane IgE binds and activates Fc epsilon Rl in an antigen-independent manner." J Immunol 174: 5602-561 1 ). This vector was modified by the addition of a linker to introduce a Nhel site after the CH4 domain gene, then a Hindlll/Nhel fragment was excised and transferred to the plasmid pcDNA-scFv9E1-degradin, yielding the construct pcDNA-LigandFcD-degradin. A truncated form of this construct was generated, lacking the sequence encoding the CH3 domain, which is the domain responsible for interaction with FCERI ( Vangelista, L, Laffer, S., Turek, R., Gronlund, H., Sperr, W.R., Valent, P., Pastore, A. and Valenta, R. (1999). "The immunoglobulin-like modules Cepsilon3 and alpha2 are the minimal units necessary for human IgE-FcepsilonRI interaction." J Clin Invest 103: 1571 -1578), to be used as a non-binding degradin control.
Cell culture and transfection
HEK 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS).
Transient transfections were performed by standard calcium phosphate technique (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning. A laboratory manual. New York, Cold Spring Harbor Laboratory Press) using 2.5 g of each plasmid in 6-well plates. 18 hours after transfection, medium was discarded and serum free medium was added for further 6 hours. When required, at the time of medium change, the proteasome inhibitor MG132 (Sigma) was added at a concentration of 20 μΜ and treatment was carried on for 6 hours. As a negative control, the same amount of MG132 solvent, DMSO, was added for the same time.
Stable transfection with pcDNA-mda was achieved by adding 400 μg ml G-418 (Invitrogen) when changing medium 24 h after transfection. HEK 293-mda were afterwards stably transfected with pCDNA-SEC-9E1 -SV5-SEL1 L-Hygro and selected with hygromycine (Invitrogen) 400 g/ml.
Cell extract preparation and Western blotting HEK 293 transfected cells (about 5x105 cells) were lysed with 100 μΙ SDS buffer (100 mM Tris-HCI pH 6.8, 6% SDS, 10% glycerol) and subsequently sonicated to disrupt DNA or with 100 μΙ of TNN lysis buffer (100 mM Tris-HCI pH 8.0, 250 mM NaCI, 0.5% NP40) supplemented with protease inhibitors cocktail (Sigma). 10 μΙ of cell extract or 20 μΙ of the corresponding supernatants were separated on 10% SDS-PAGE and transferred to PVDF membrane for immunodetection with anti-SV5 (Invitrogen) or 9E1 anti-Fc£RI antibodies, followed by incubation with HRP-labeled anti-mouse IgG (Jackson) and ECL reaction.
To normalize the amount of extract loaded to the gels, blots were in addition incubated with anti-actin antibodies (Sigma) and HRP-labeled anti-rabbit IgG (Pierce).
HEK 293 clones either transfected only with mda or double transfected with mda and scFv9E1-degradin were analyzed by FACS: about 106 cells were incubated with the anti-FcsRI mAb 9E1 and fluorescein coniugated anti-mouse IgG (KPL) and analyzed in a FACSCalibur (Becton Dickinson).
To test the system the present inventors chose as a target for degradation the extracellular domains of the a-chain of the human high affinity receptor for IgE (FcsRIa), both in a secretory (sda) and in a membrane-bound (mda) form (Fig. 1 B). As recognition-specific moieties the present inventors used either the scFv derived from the anti-FcsRIa monoclonal antibody 9E1 (mAb-9E1 ) (scFv9E1- degradin), or the Fes ligand (LFc£-degradin), represented by domains sCH3-sCH4 from human IgE (Fig. 1A), that are known to be sufficient to bind the FcsRI (Vangelista, L, Laffer, S., Turek, R., Gronlund, H., Sperr, W.R., Valent, P., Pastore, A. and Valenta, R. (1999). "The immunoglobulin-like modules Cepsilon3 and alpha2 are the minimal units necessary for human IgE-FcepsilonRI interaction." J Clin Invest 103: 1571 -1578). The two versions of the target FcsRIa contain the two extracellular domains D1 and D2 of FcsRIa chain, which are sufficient to bind human IgE with high affinity, fused to the yCH3 dimerising domain of IgG heavy-chain, either in the secretory form (to yield the secretory FcsRIa construct, sda) (Vangelista, L, Cesco-Gaspere, M., Lamba, D. and Burrone, O. (2002a). "Efficient folding of the FcepsilonRI alpha-chain membrane- proximal domain D2 depends on the presence of the N-terminal domain D1 ." J Mol Biol 322: 815-825; Vangelista, L, Cesco-Gaspere, M., Lorenzi, R. and Burrone, O. (2002b). "A minimal receptor-lg chimera of human FcepsilonRI alpha-chain efficiently binds secretory and membrane IgE." Protein Eng 15: 51 -57), or in the membrane bound form (to yield the membrane FcsRIa construct, mda).
In order to test the activity of degradins the present inventors co-transfected HEK 293 cells with sda (secretory version of FcsRIa) and scFv9E1-degradin and analysed both the supernatant and total cellular extract by Western blotting. As shown in figure 2A, scFv9E1-degradin was able to completely block secretion of sda, while the control irrelevant degradin (irr-degradin: a degradin containing a scFv of irrelevant specificity) did not. Analysis of the total cellular extracts showed that only a very low amount of the target protein was intracellular^ retained, in an incompletely glycosylated form indicating that the protein was retained in the ER and did not reach the Golgi compartment (arrows), while scFv9E1 fused to the ER retention sequence KDEL, which was able to block secretion only partially, produced instead large accumulation of the incompletely glycosylated protein within the cell (Fig. 2B). Interestingly, scFv9E1-KDEL was also found in part secreted in the supernatant, thus limiting its retention capability. The inventors therefore concluded that degradin-mediated blocking of secretion was not only due to intracellular retention, but mainly to active degradation.
Similarly to the secretory form, expression of the membrane-bound mda was also heavily impaired by scFv9E1-degradin, but not by the control irr-degradin, which, instead, allowed a large proportion of target protein to reach the terminal glycosylated state. Also in this case, scFv9E1-KDEL induced mainly intracellular retention, with only modest degradation (Fig. 3). Further evidence of the broad applications of the SEL1 L based degradins emerged from experiments with the ligand-degradin (LFc£-degradin). Figure 4 shows a strong degradation activity of LFc£-degradin both on sda (A) and mda (B), although with an efficiency somehow lower than with the scFv-degradin. The activities of both degradins were specific for the target, since secretion of a control secretory molecule, which was not recognised by either of the two degradins, was unaffected (Fig. 4C).
To further demonstrate the ability of degradins to constitutively knock down the specific protein target, the scFv9E1-degradin was stably transfected into a HEK293- derived cell-line already expressing mda. Cytofluorimetric analysis showed a strong reduction in cell surface expression of mda (Fig. 5A), which corresponded to a similar impairment of the total amount of protein detectable in Western blotting, consistent with the expression of the degradin in such cells, as shown in Fig. 5B. Co-expression experiments of either sda or mda targets with a degradin construct where the complete lumenal domain of SEL1 L (up to residue 712) was deleted (scFv9E1-SEL1 LA, Fig. 1 C), showed that, while the truncated degradin was still expressed in the ER where it produced a large retention of both target proteins, the degradation activity was highly reduced (figure 6). This result demonstrates that the SEL1 L lumenal portion between amino acid residues 402 and 712 is essential for the degradation-inducing activity of the degradin. In addition, a reduced activity of the degradins, visible as an increase in the amount of the intracellular^ retained mda, was observed in cells treated with the proteasome inhibitor MG132 (Fig. 7), thus indicating that degradin-mediated degradation involves proteasome activity.
1 . Protein comprising a C-terminal portion comprising at least the COOH- terminal 372 amino acids of the human SEL1 L and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein, said protein having a selective inactivation activity on said target protein.
2. Protein comprising a mammalian SEL1 L C-terminal portion comprising the degradation-inducing domain that induces selective degradation of a target protein and a heterologous N-terminal portion consisting of a target-specific recognition domain for said target protein, wherein said protein has a selective inactivation activity on said target protein.
3. Protein comprising a C-terminal portion comprising the degradation-inducing domain of a protein involved in ERAD and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein, said protein having a selective inactivation activity on the target protein, with the proviso that said C-terminal portion is of a non-viral protein.
4. Protein comprising a C-terminal portion comprising the degradation-inducing domain of a protein involved in ERAD and a heterologous N-terminal portion consisting of a target-specific recognition domain for a target protein, said protein having a selective inactivation activity on the target protein, wherein said C-terminal portion is different from the C-terminal of Vpu protein.
5. Protein comprising a C-terminal portion comprising the degradation-inducing domain of a mammalian protein involved in ERAD and a heterologous N- terminal portion consisting of a target-specific recognition domain for a target protein, said protein having a selective inactivation activity on the target protein.
6. The protein according to any one of claims 3-5, wherein said C-terminal portion is the C-terminal portion of a protein selected from the group consisting of OS-9, XTP3-B, EDEM, Derlinl and HRD1 .
7. The protein according to any one of claims 1 -6 further comprising a tag.
8. The protein according to any one of claims 1 -7, wherein the recognition domain is a scFv or a ligand.
9. The protein according to claim 8, wherein the scFv is derived from the anti- FcsRIa monoclonal antibody 9E1 .
10. The protein according to claim 8, wherein the ligand is the FCE ligand consisting of domains εΟΗ3-εΟΗ4 from human IgE.
1 1 . A genetic construct encoding the protein of any one of claims 1 -10.
12. An expression system comprising the construct of claim 1 1 .
13. An expression vector comprising the construct of claim 1 1 .
14. The vector according to claim 13, selected from the group consisting of pcDNA-scFv9E1-degradin, pcDNA-scFv9E1-degradin-Hygro and pcDNA- LigandFcD-degradin.
15. A cell line comprising the genetic construct of claim 1 1 .
16. The protein of any one of claims 1 -10 or the genetic construct of claim 1 1 or the expression system of claim 12 or the expression vector of any one of claims 13-14 for use as a medicament.
17. The protein or the genetic construct or the expression system or the expression vector according to claim 16 for the treatment of a disease where degradation of a defined secretory or membrane bound protein is required in order to ameliorate or cure said disease, wherein said disease is selected from a neurodegenerative disorder, a tumour, a genetic disease, a hypersensitivity disorder, a bacterial, parasitic or viral infection, and a metabolic disorder.
18. Pharmaceutical composition for gene therapy comprising the genetic construct of claim 1 1 .
19. The protein of anyone of claims 1 -10 or the genetic construct of claim 1 1 for use for the selective inactivation of target proteins.
20. The protein of anyone of claims 1 -10 or the genetic construct of claim 1 1 for use as a laboratory tool for the selective inactivation of target proteins.