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WO2012070008A2 - Protéines recombinantes ayant une activité d'inactivation sélective sur des protéines cibles - Google Patents

Protéines recombinantes ayant une activité d'inactivation sélective sur des protéines cibles Download PDF

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WO2012070008A2
WO2012070008A2 PCT/IB2011/055274 IB2011055274W WO2012070008A2 WO 2012070008 A2 WO2012070008 A2 WO 2012070008A2 IB 2011055274 W IB2011055274 W IB 2011055274W WO 2012070008 A2 WO2012070008 A2 WO 2012070008A2
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protein
target
degradation
terminal portion
proteins
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WO2012070008A3 (fr
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Oscar Burrone
Marco Bestagno
Lara Vecchi
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International Centre for Genetic Engineering and Biotechnology
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International Centre for Genetic Engineering and Biotechnology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/80—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies
    • C07K2317/81—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies functional in the endoplasmatic reticulum [ER] or the Golgi apparatus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present invention refers to the field of genetic engineering and pharmacology.
  • 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.
  • 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.
  • ER endoplasmic reticulum
  • 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.
  • 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).
  • 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).
  • 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).
  • Hybrid1 p/Der3p 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 NH 2 -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).
  • Hrd3p 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.
  • SEL1 L 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).
  • 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).
  • E2s ubiquitin conjugating enzymes
  • 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).
  • a number of diseases are characterized by proteins in aberrant conformations that can generate toxic effects on cells.
  • these diseases are identified as “conformational diseases” or “protein misfolding diseases” or "ER stress-related diseases”.
  • conformational diseases or “protein misfolding diseases” or "ER stress-related diseases”.
  • 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.
  • 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.
  • mTOR mammalian target of rapamycin
  • 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 ⁇ , 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.
  • this misfolded form is prion protein PrP, which has been identified on various cancer cells.
  • 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.
  • 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
  • a technique of specific degradation of target molecule by means of fusion proteins 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;
  • 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;
  • 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.
  • 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.
  • introduction moiety antibody fragments like scFv
  • 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.
  • 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.
  • said C-terminal portion of mammalian SEL1 L is that portion comprising at least the degradation inducing domain.
  • 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).
  • 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.
  • said protein involved in ERAD is mammalian, more preferably human.
  • the Vpu protein is specifically excluded.
  • 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.
  • ERAD protein 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.
  • 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).
  • 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.
  • 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.
  • the above proteins further comprise a tag, preferably SV5.
  • 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.
  • the invention is 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.
  • said vectors are pcDNA-scFv 9E1 -degradin, pcDNA-scFv 9E1 -degradin- Hygro or pcDNA-Ligand FcE -degradin.
  • 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.
  • 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).
  • 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.
  • compositions 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.
  • 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 scFv 9E1 recognition domain and either a truncated SEL1 L (pcDNA-scFv 9E1 - SEL1 LA, containing residues 713-773 of SEL1 L) or the KDEL ER-retention signal (pcDNA-scFv 9E1 -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.
  • scFv 9E1 -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.
  • 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.
  • ER incomplete glycosylated
  • Golgi/secreted terminally glycosylated
  • 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 L FceRI -degradin both on sda (A) and mda (B).
  • FACS analysis of HEK-293 cells non-transfected or stably transfected to express mda alone or mda and scFv 9E1 -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 (scFv 9E1 -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.
  • the protein is a "recombinant protein” that is derived from recombinant DNA.
  • this protein is also named "fusion protein”.
  • rDNA Recombinant DNA
  • 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).
  • 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.
  • the 12 aa long SV5 tag (GKPIPNPLLGLD; SEQ ID. NO.1 ) was included in the recombinant degradins decribed in Fig. 1A.
  • the scFv when fused to the SEL1 L fragment, provides recognition specificity to the degradin within the ER lumenal side.
  • the recognition moiety of the protein of the invention is a ligand
  • the protein herein called by the inventor also as “ligand-degradin”
  • ligand-degradin can interact with specific target receptors or soluble proteins exposed in the ER lumen.
  • the degradin is specific for the FCERI D chain target.
  • the recognition domain is the scFv derived from the anti-FcsRIa monoclonal antibody 9E1 .
  • scFv 9E1 -degradin-gene SEC secretion signal, intron, scFv 9E1 , SV5 tag, SEL1L 402-773; SEQ ID. NO 2):
  • the recognition domain is a ligand of the target, a truncated version of Fez, consisting of the domains ⁇ 3- ⁇ 4 from human IgE ⁇ heavy chain (Fig. 1A).
  • Ligand -degradin-gene SEC secretion signal, intron, SV5 tag. DCH3-CH4, SEL1L 402-773; SEQ ID. NO 4):
  • Ligand -degradin protein (SEC secretion signal, SV5 tag, DCH3-CH4, SEL1L 402-773; SEQ ID. NO 5):
  • 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.
  • vectors used are plasmids pcDNA and pcDNA-Hygro (Invitrogen).
  • 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).
  • the plasmids comprising the DNA constructs encoding the proteins of the invention are pcDNA-scFv 9E1 -degradin, pcDNA-scFv 9E1 -degradin-Hygro or pcDNA-Ligand FcD -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.
  • 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).
  • AAV vectors Adeno-associated viral vectors
  • AAV vectors 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).
  • 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).
  • pseudotyping spinal motor neurons, pancreatic islet cells, liver cells, fibroblasts, myocytes, epithelial cells or hematopoietic cells
  • HSV Herpes Simplex virus
  • 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.
  • 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.
  • silencing of specific receptors could help in reducing hypersensitivity-related diseases, like allergies.
  • 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.
  • cathepsins or metalloproteinases that cancer cells use for cell matrix degradation and tissue invasion.
  • degradins could act on chemokine receptor expression, in order to avoid metastasis formation.
  • 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.
  • the activity of degradins could be directed against tyrosine kinase receptors that bind to growth factors and survival factors.
  • 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 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.
  • TSE Transmissible Spongiform Encephalopaties
  • AD Alzheimer Disease
  • a p i - 2 amyloid ⁇ peptide 1 -42
  • a p i - 2 amyloid ⁇ peptide 1 -42
  • 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 ).
  • 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)
  • Bacterial pathogens such as Salmonella typhimurium and Chlamydia trachomatis
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • administration to individuals can be done through methods known in the art.
  • 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.
  • PCR reaction was performed with about 100 ng cDNA, 1 ⁇ of each primer, supplier provided buffer at 1 x concentration, 1 mM MgSO 4 , 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 scFv 9E1 degradin (pcDNA-scFv 9E1 -degradin).
  • scFv 9E1 - SELI LA A further truncated version of degradin (scFv 9E1 - SELI LA), completely lacking the SEL1 L lumenal portion, was obtained by amplifying the cDNA with primers SEL1 L ⁇ -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-scFv 9E1 -KDEL).
  • 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 scFv 9E1 -degradin was in addition subcloned into the pcDNA-Hygro vector (Invitrogen) for the generation of double transfected stable clones (pcDNA-scFv 9E1 -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).
  • HEK 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • 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 ⁇ 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 ⁇ g 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 were lysed with 100 ⁇ SDS buffer (100 mM Tris-HCI pH 6.8, 6% SDS, 10% glycerol) and subsequently sonicated to disrupt DNA or with 100 ⁇ of TNN lysis buffer (100 mM Tris-HCI pH 8.0, 250 mM NaCI, 0.5% NP40) supplemented with protease inhibitors cocktail (Sigma).
  • HEK 293 clones either transfected only with mda or double transfected with mda and scFv 9E1 -degradin were analyzed by FACS: about 10 6 cells were incubated with the anti-FcsRI mAb 9E1 and fluorescein coniugated anti-mouse IgG (KPL) and analyzed in a FACSCalibur (Becton Dickinson).
  • 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).
  • FcsRIa the extracellular domains of the a-chain of the human high affinity receptor for IgE
  • sda secretory
  • mda membrane-bound
  • the present inventors used either the scFv derived from the anti-FcsRIa monoclonal antibody 9E1 (mAb-9E1 ) (scFv 9E1 - degradin), or the Fes ligand (L Fc£ -degradin), represented by domains sCH3-sCH4 from human IgE (Fig.
  • 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).
  • scFv 9E1 -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.
  • Figure 4 shows a strong degradation activity of L Fc£ -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).
  • scFv 9E1 -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.

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Abstract

La présente invention concerne une protéine recombinante comprenant un domaine de reconnaissance spécifique d'une cible et un domaine d'induction de dégradation, ladite protéine ayant une activité d'inactivation sélective sur des protéines cibles, une construction génétique codant pour la protéine mentionnée ci-dessus, un vecteur et une lignée cellulaire comprenant ladite construction génétique. L'invention concerne également la protéine ou la construction génétique mentionnée ci-dessus pour l'utilisation en tant que médicament, en particulier pour le traitement de tout type de maladie, où la dégradation d'une protéine liée à la membrane ou sécrétoire définie est nécessaire afin d'améliorer ou guérir la maladie, ladite maladie étant plus particulièrement un trouble neurodégénératif, une tumeur, une maladie génétique, un trouble d'hypersensibilité, une infection bactérienne, parasitaire ou virale. L'invention concerne en outre une composition pharmaceutique comprenant ladite construction génétique et l'utilisation de la protéine ou de la construction génétique mentionnée ci-dessus pour l'inactivation sélective de protéines cibles.
PCT/IB2011/055274 2010-11-25 2011-11-24 Protéines recombinantes ayant une activité d'inactivation sélective sur des protéines cibles Ceased WO2012070008A2 (fr)

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ES2548784A1 (es) * 2014-09-22 2015-10-20 Universidade De Santiago De Compostela Proteína muNS capaz de formar inclusiones en el retículo endoplasmático, métodos de uso y usos de la misma
WO2022161502A1 (fr) * 2021-02-01 2022-08-04 羿尊生物医药（浙江）有限公司 Système de dégradation de protéine ciblée et son utilisation

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004954A2 (fr) 2000-07-07 2002-01-17 Applied Research Systems Ars Holding N.V. Diagnostic precoce de maladies conformationelles
WO2002062197A2 (fr) 2000-12-19 2002-08-15 Hospital For Special Surgery Marqueurs de predisposition pour des maladies et cibles pour therapie
WO2004007546A1 (fr) 2002-07-11 2004-01-22 Eidgenoessische Technische Hochschule Zuerich Proteines mutantes et leur utilisation pour la fabrication de medicaments et le traitement d'humains ou d'animaux souffrant de maladies post-transcriptionnelles
WO2006116716A2 (fr) 2005-04-27 2006-11-02 University Of Florida Materiaux et methodes permettant d'ameliorer la degradation de proteines mutantes associees avec une maladie humaine
US20070015577A1 (en) 2005-07-18 2007-01-18 Weistech Technology Co., Ltd. Joypad with a display unit
US20070202544A1 (en) 2003-10-09 2007-08-30 Fumihiko Urano Methods For Diagnosing And Treating Endoplasmic Reticulum (er) Stress Diseases
US20070204352A1 (en) 2005-02-25 2007-08-30 Caldwell Guy A Regulators of protein misfolding and aggregation and methods of using the same
WO2007137237A2 (fr) 2006-05-19 2007-11-29 The Scripps Research Institute Traitement du mauvais repliement de protéines
US20080045607A1 (en) 2004-05-05 2008-02-21 Lindquist Susan L Compositions and Methods for Treatment of Protein Misfolding Diseases
US20080118938A1 (en) 2006-09-06 2008-05-22 Lisbell Estrada Methods and Compositions for the Detection of Protein Folding Disorders
US20080206251A1 (en) 2005-12-02 2008-08-28 Cashman Neil R Methods and Compositions to Treat and Detect Misfolded-SOD1 Mediated Diseases
US20080227700A1 (en) 2004-11-04 2008-09-18 Ghosh Joy G Compositions and Methods for Treatment of Protein Misfolding and Protein Aggregation Diseases
WO2008127680A2 (fr) 2007-04-11 2008-10-23 The Jackson Laboratory Diagnostic et traitement de maladies causées par des protéines mal pliées
US20090017588A1 (en) 2005-02-02 2009-01-15 Texas Instruments Incorporated Systems and methods that selectively modify liner induced stress
US20090111768A1 (en) 2007-08-08 2009-04-30 Guy Caldwell Regulators of protein misfolding and neuroprotection and methods of use
WO2009061906A2 (fr) 2007-11-06 2009-05-14 Mayo Foundation For Medical Education And Research Traitement des pathologies de mauvais repliement des protéines

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004954A2 (fr) 2000-07-07 2002-01-17 Applied Research Systems Ars Holding N.V. Diagnostic precoce de maladies conformationelles
WO2002062197A2 (fr) 2000-12-19 2002-08-15 Hospital For Special Surgery Marqueurs de predisposition pour des maladies et cibles pour therapie
WO2004007546A1 (fr) 2002-07-11 2004-01-22 Eidgenoessische Technische Hochschule Zuerich Proteines mutantes et leur utilisation pour la fabrication de medicaments et le traitement d'humains ou d'animaux souffrant de maladies post-transcriptionnelles
US20070202544A1 (en) 2003-10-09 2007-08-30 Fumihiko Urano Methods For Diagnosing And Treating Endoplasmic Reticulum (er) Stress Diseases
US20080045607A1 (en) 2004-05-05 2008-02-21 Lindquist Susan L Compositions and Methods for Treatment of Protein Misfolding Diseases
US20080227700A1 (en) 2004-11-04 2008-09-18 Ghosh Joy G Compositions and Methods for Treatment of Protein Misfolding and Protein Aggregation Diseases
US20090017588A1 (en) 2005-02-02 2009-01-15 Texas Instruments Incorporated Systems and methods that selectively modify liner induced stress
US20070204352A1 (en) 2005-02-25 2007-08-30 Caldwell Guy A Regulators of protein misfolding and aggregation and methods of using the same
WO2006116716A2 (fr) 2005-04-27 2006-11-02 University Of Florida Materiaux et methodes permettant d'ameliorer la degradation de proteines mutantes associees avec une maladie humaine
US20070015577A1 (en) 2005-07-18 2007-01-18 Weistech Technology Co., Ltd. Joypad with a display unit
US20080206251A1 (en) 2005-12-02 2008-08-28 Cashman Neil R Methods and Compositions to Treat and Detect Misfolded-SOD1 Mediated Diseases
WO2007137237A2 (fr) 2006-05-19 2007-11-29 The Scripps Research Institute Traitement du mauvais repliement de protéines
US20080118938A1 (en) 2006-09-06 2008-05-22 Lisbell Estrada Methods and Compositions for the Detection of Protein Folding Disorders
WO2008127680A2 (fr) 2007-04-11 2008-10-23 The Jackson Laboratory Diagnostic et traitement de maladies causées par des protéines mal pliées
US20090111768A1 (en) 2007-08-08 2009-04-30 Guy Caldwell Regulators of protein misfolding and neuroprotection and methods of use
WO2009061906A2 (fr) 2007-11-06 2009-05-14 Mayo Foundation For Medical Education And Research Traitement des pathologies de mauvais repliement des protéines

Non-Patent Citations (61)

* Cited by examiner, † Cited by third party
Title
"Remington's or Delivery Technologies for Biopharmaceuticals", 2009, WILEY
"Remington's Pharmaceutical Sciences", MACK PUB.
BAKER, B.M.; TORTORELLA, D.: "Dislocation of an endoplasmic reticulum membrane glycoprotein involves the formation of partially dislocated ubiquitinated polypeptides", J BIOL CHEM, vol. 282, 2007, pages 26845 - 26856
BAYS, N.W.; GARDNER, R.G.; SEELIG, L.P.; JOAZEIRO, C.A.; HAMPTON, R.Y.: "Hrd1 p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation", NAT CELL BIOL, vol. 3, 2001, pages 24 - 29, XP002275252, DOI: doi:10.1038/35050524
BEATTY, W.L.: "Trafficking from CD63-positive late endocytic multivesicular bodies is essential for intracellular development of Chlamydia trachomatis", J CELL SCI, vol. 119, 2006, pages 350 - 359
BERNAL-BAYARD, J.; CARDENAL-MUNOZ, E.; RAMOS-MORALES, F.: "The Salmonella type III secretion effector, salmonella leucine- rich repeat protein (SIrP), targets the human chaperone ERdj3", J BIOL CHEM, vol. 285, 2010, pages 16360 - 16368
BERNASCONI, R.; GALLI, C.; CALANCA, V.; NAKAJIMA, T.; MOLINARI, M.: "Stringent requirement for HRD1, SEL1 L, and OS-9/XTP3-B for disposal of ERAD-LS substrates", J CELL BIOL, vol. 188, 2010, pages 223 - 235, XP055001474, DOI: doi:10.1083/jcb.200910042
BERNASCONI, R.; GALLI, C.; CALANCA, V.; NAKAJIMA, T.; MOLINARI, M.: "Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates", J. CELL BIOL., vol. 188, 2010, pages 223 - 235, XP055001474, DOI: doi:10.1083/jcb.200910042
BIESCHKE, J.; WEBER, P.; SARAFOFF, N.; BEEKES, M.; GIESE, A.; KRETZSCHMAR, H.: "Autocatalytic self-propagation of misfolded prion protein", PROC NATL ACAD SCI U S A, vol. 101, 2004, pages 12207 - 12211, XP002399092, DOI: doi:10.1073/pnas.0404650101
BIUNNO, I.; CATTANEO, M.; ORLANDI, R.; CANTON, C.; BIAGIOTTI, L.; FERRERO, S.; BARBERIS, M.; PUPA, S.M.; SCARPA, A.; MENARD, S.: "SEL 1 L a multifaceted protein playing a role in tumor progression", J CELL PHYSIOL, vol. 208, 2006, pages 23 - 38
BIUNNO, I.; CATTANEO, M.; ORLANDI, R.; CANTON, C.; BIAGIOTTI, L.; FERRERO, S.; BARBERIS, M.; PUPA, S.M.; SCARPA, A.; MENARD, S.: "SEL1L a multifaceted protein playing a role in tumor progression", J CELL PHYSIOL, vol. 208, 2006, pages 23 - 38
BORDALLO, J.; PLEMPER, R.K.; FINGER, A.; WOLF, D.H.: "Der3p/Hrd1 p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins", MOL BIOL CELL, vol. 9, 1998, pages 209 - 222
BREYER, B.; JIANG, W.; CHENG, H.; ZHOU, L.; PAUL, R.; FENG, T.; HE T.C.: "Adenoviral vector-mediated gene transfer for human gene therapy", CURR. GENE THER., vol. 1, 2001, pages 149 - 162
BRODSKY, J.L.; WOJCIKIEWICZ, R.J.: "Substrate-specific mediators of ER associated degradation (ERAD", CURR OPIN CELL BIOL, vol. 21, 2009, pages 516 - 521, XP026434717
CHRISTIANSON, J.C.; SHALER, T.A.; TYLER, R.E.; KOPITO, R.R.: "OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD", NAT CELL BIOL, vol. 10, 2008, pages 272 - 282, XP055001475, DOI: doi:10.1038/ncb1689
COFFIELD, V.M.; JIANG, Q.; SU, L.: "A genetic approach to inactivating chemokine receptors using a modified viral protein", NAT BIOTECHNOL, vol. 21, 2003, pages 1321 - 1327, XP002471890, DOI: doi:10.1038/nbt889
CORMIER, J.H.; TAMURA, T.; SUNRYD, J.C.; HEBERT, D.N.: "EDEM1 recognition and delivery of misfolded proteins to the SEL 1 L-containing ERAD complex", MOL. CELL, vol. 34, 2009, pages 627 - 633
CUELLO, A.C.: "Intracellular and extracellular Abeta, a tale of two neuropathologies", BRAIN PATHOL, vol. 15, 2005, pages 66 - 71
DERIZIOTIS, P.; TABRIZI, S.J.: "Prions and the proteasome", BIOCHIM BIOPHVS ACTA, vol. 1782, 2008, pages 713 - 722, XP025691381, DOI: doi:10.1016/j.bbadis.2008.06.011
DOS SANTOS COURA, R.; NARDI, N.B.: "The state of the art of adeno-associated virus-based vectors in gene therapy", VIROL. J., vol. 4, 2007, pages 99
ELLGAARD, L.; HELENIUS, A.: "ER quality control: towards an understanding at the molecular level", CURR OPIN CELL BIOL, vol. 13, 2001, pages 431 - 437
ELLGAARD, L.; HELENIUS, A.: "Quality control in the endoplasmic reticulum", NAT REV MOL CELL BIOL, vol. 4, 2003, pages 181 - 191, XP009139072
ESCORS, D.; BRECKPOT, K.: "Lentiviral vectors in gene therapy: their current status and future potential", ARCH. IMMUNOL. THER. EXP., vol. 58, 2010, pages 107 - 119, XP055179407, DOI: doi:10.1007/s00005-010-0063-4
FILESI I.; CARDINALE A.; MATTEI S.; BIOCCA S.: "Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent Prpsc formation", JOURNAL OF NEUROCHEMISTRY, vol. 101, 2007, pages 1516 - 1526
GARDNER, R.G.; SWARBRICK, G.M.; BAYS, N.W.; CRONIN, S.R.; WILHOVSKY, S.; SEELIG, L.; KIM, C.; HAMPTON, R.Y.: "Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1 p by Hrd3p", J CELL BIOL, vol. 151, 2000, pages 69 - 82, XP002998209, DOI: doi:10.1083/jcb.151.1.69
GILES, D.K.; WYRICK, P.B.: "Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells", MICROBES INFECT, vol. 10, 2008, pages 1494 - 1503, XP025744641, DOI: doi:10.1016/j.micinf.2008.09.001
HAMPTON, R.Y.; GARDNER, R.G.; RINE, J.: "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, vol. 7, 1996, pages 2029 - 2044, XP000653013
KAMAUCHI, S.; NAKATANI, H.; NAKANO, C.; URADE, R.: "Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana", FEBS J, vol. 272, 2005, pages 3461 - 3476
KANEKO, M.; NOMURA, Y.: "ER signaling in unfolded protein response", LIFE SCI, vol. 74, 2003, pages 199 - 205, XP002990812, DOI: doi:10.1016/j.lfs.2003.09.007
KIKKERT, M.; DOOLMAN, R.; DAI, M.; AVNER, R.; HASSINK, G.; VAN VOORDEN, S.; THANEDAR, S.; ROITELMAN, J.; CHAU, V.; WIERTZ, E.: "Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum", J. BIOL CHEM., vol. 279, 2004, pages 3525 - 3534, XP002990810, DOI: doi:10.1074/jbc.M307453200
KOSTOVA, Z.; TSAI, Y.C.; WEISSMAN, A.M.: "Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation", SEMIN CELL DEV BIOL, vol. 18, 2007, pages 770 - 779, XP022372969, DOI: doi:10.1016/j.semcdb.2007.09.002
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, vol. 4, no. 5, 28 May 2009 (2009-05-28), pages E5727, XP002726336, DOI: doi:10.1371/journal.pone.0005727
LILLEY, B.N.; PLOEGH, H.L.: "A membrane protein required for dislocation of misfolded proteins from the ER", NATURE, vol. 429, 2004, pages 834 - 840
LUSTIG, B.; BEHRENS, J.: "The Wnt signalling pathway and its role in tumor development", J. CANCER RES. CLIN. ONC., vol. 129, 2003, pages 199 - 221, XP002983435
MANSERVIGI, R.; ARGNANI, R.; MARCONI, P.: "HSV recombinant vectors for gene therapy", OPEN VIROL. J., vol. 4, 2010, pages 123 - 156
MARTIN-APARICIO, E.; YAMAMOTO, A.; HERNANDEZ, F.; HEN, R.; AVILA, J.; LUCAS, J.J.: "Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington's disease", J NEUROSCI, vol. 21, 2001, pages 8772 - 8781
MUELLER, B.; KLEMM E.; SPOONER E.; CLAESSEN JH.; PLOEGH, L.: "SEL1L nucleates a protein complex required for dislocation glycoproteins", PNAS, vol. 105, no. 34, 28 August 2008 (2008-08-28), pages 12325 - 2330, XP055002329, DOI: doi:10.1073/pnas.0805371105
MUELLER, B.; LILLEY, B.N.; PLOEGH, H.L: "SEL 1 L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER", J CELL BIOL, vol. 175, 2006, pages 261 - 270
PAUL, S.: "Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches", BIOESSAVS, vol. 30, 2008, pages 1172 - 1184, XP055001501, DOI: doi:10.1002/bies.20852
PICKART, C.M.; COHEN, R.E.: "Proteasomes and their kin: proteases in the machine age", NAT REV MOL CELL BIOL, vol. 5, 2004, pages 177 - 187
PRUSINER, S.B.: "Prions", PROC NATL ACAD SCI U S A, vol. 95, 1998, pages 13363 - 13383
RATHMAN, M.; BARKER, L.P.; FALKOW, S.: "The unique trafficking pattern of Salmonella typhimurium- containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry", INFECT IMMUN, vol. 65, 1997, pages 1475 - 1485
RODRIGUEZ-GONZALEZ A.; CYRUS K.; SALCIUS M.; KIM K.; CREWS CM.; DESHAIES RJ.; SAKAMOTO KM.: "Tergetin Steroid Hormone Receptors for Ubiquitination and Degradation in Breast and Prostate Cancer", ONCOGENE, vol. 27, 2008, pages 7201 - 7211
SAMBROOK, J.; FRITSCH, E.F.; MANIATIS, T.: "Molecular Cloning. A laboratory manual.", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK, J.; FRITSCH, E.F.; MANIATIS, T.: "Molecular Cloninq. A laboratory manual. New York", 1989, COLD SPRING HARBOR LABORATORY PRESS
SANDERS; MYERS, ANNU. REV. BIOPHYS. BIOMOL. STRUCT., vol. 33, 2004, pages 25 - 51
SCHAFER, A.; WOLF, D.H.: "Sec61 p is part of the endoplasmic reticulum-associated degradation machinery", EMBO J, vol. 28, 2009, pages 2874 - 2884
SWANSON, R.; LOCHER, M.; HOCHSTRASSER, M.: "A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation", GENES DEV, vol. 15, 2001, pages 2660 - 2674
TAXIS, C.; HITT, R.; PARK, S.H.; DEAK, P.M.; KOSTOVA, Z.; WOLF, D.H.: "Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD", J BIOL CHEM, vol. 278, 2003, pages 35903 - 35913
TRAVERS, K.J.; PATIL, C.K.; WODICKA, L.; LOCKHART, D.J.; WEISSMAN, J.S.; WALTER, P.: "Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation", CELL, vol. 101, 2000, pages 249 - 258, XP002235358, DOI: doi:10.1016/S0092-8674(00)80835-1
VANGELISTA, L.; CESCO-GASPERE, M.; LAMBA, D.; BURRONE, O.: "Efficient folding of the FcepsilonRl alpha-chain membrane- proximal domain D2 depends on the presence of the N-terminal domain D1", J MOL BIOL, vol. 322, 2002, pages 815 - 825, XP055150957, DOI: doi:10.1016/S0022-2836(02)00853-7
VANGELISTA, L.; CESCO-GASPERE, M.; LORENZI, R.; BURRONE, O.: "A minimal receptor-Ig chimera of human FcepsilonRl alpha-chain efficiently binds secretory and membrane IgE", PROTEIN ENG, vol. 15, 2002, pages 51 - 57, XP002484236, DOI: doi:10.1093/protein/15.1.51
VANGELISTA, L.; LAFFER, S.; TUREK, R.; GRONLUND, H.; SPERR, W.R.; VALENT, P.; PASTORE, A.; VALENTA, R.: "The immunoglobulin-like modules Cepsilon3 and alpha2 are the minimal units necessary for human gE-FcepsionR interaction", J CLIN INVEST, vol. 103, 1999, pages 1571 - 1578, XP001055643
VANGELISTA, L.; SOPRANA, E.; CESCO-GASPERE, M.; MANDIOLA, P.; DI LULLO, G.; FUCCI, R.N.; CODAZZI, F.; PALINI, A.; PAGANELLI, G.; B: "Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner", J IMMUNOL, vol. 174, 2005, pages 5602 - 5611
VASHIST, S.; NG, D.T.: "Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control", J CELL BIOL, vol. 165, 2004, pages 41 - 52
VEMBAR, S.S.; BRODSKY, J.L.: "One step at a time: endoplasmic reticulum-associated degradation", NAT REV MOL CELL BIOL, vol. 9, 2008, pages 944 - 957, XP008129175, DOI: doi:10.1038/nrm2546
WALSH, D.M.; SELKOE, D.J.: "A beta oligomers - a decade of discovery", J NEUROCHEM, vol. 101, 2007, pages 1172 - 1184
WICKNER, W.; SCHEKMAN, R.: "Protein translocation across biological membranes", SCIENCE, vol. 310, 2005, pages 1452 - 1456
WILLIAMS, D.B.: "Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum", J CELL SCI, vol. 119, 2006, pages 615 - 623
YE, Y.; SHIBATA, Y.; KIKKERT, M.; VAN VOORDEN, S.; WIERTZ, E.; RAPOPORT, T.A.: "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, vol. 102, 2005, pages 14132 - 14138
ZENTILIN, L.; GIACCA M.: "Adeno-associated virus vectors: versatile tools for in vivo gene transfer", CONTRIB. NEPHROL., vol. 159, 2008, pages 63 - 77

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