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Title:
METHOD TO IMPROVE TRANSLATION OF POLYPEPTIDES BY USING UNTRANSLATED REGIONS FROM HEAT-SHOCK PROTEINS
Document Type and Number:
WIPO Patent Application WO/2000/053785
Kind Code:
A2
Abstract:
Untranslated regions associated with the heat shock response can be used to obtain increased efficiency of translation of polypeptides that are not necessarily normally associated with the heat shock response. This allows the development of greatly improved expression systems. The invention is also useful, for example, in the treatment of a patient suffering from a deficiency in the expression of a polypeptide and in the provision of vaccines.

Inventors:
COSTE HERVE JEAN-CLEMENT (FR)
ELLIS JONATHAN HENRY (GB)
Application Number:
PCT/EP2000/002031
Publication Date:
September 14, 2000
Filing Date:
March 09, 2000
Export Citation:
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Assignee:
GLAXO GROUP LTD (GB)
COSTE HERVE JEAN CLEMENT (FR)
ELLIS JONATHAN HENRY (GB)
International Classes:
A61K48/00; A61P31/00; C07H21/04; C07K14/47; C12N15/85; C12Q1/68; (IPC1-7): C12N15/85; C12N5/10; A61K48/00
Domestic Patent References:
WO1987000861A11987-02-12
WO1994011521A11994-05-26
Foreign References:
US5659122A1997-08-19
Other References:
HUNT C R ET AL: "Inducible expression of cDNAs in a vector based upon the mouse HSP70 heat-shock promoter;" J.CELL.BIOCHEM.; SUPPL.12D, 260, 1988, XP000933846
DATABASE GENEMBL [Online] 16 July 1988 (1988-07-16) HUNT,C. AND MORIMOTO, R.I.: "Human heat shock protein (hsp 70) gene, complete cds" XP002144203 cited in the application -& HUNT, C. AND MORIMOTO, R.I.: "Conserved features of eukaryotic hsp-70 genes revealeed by comparison with the nucleotide sequence of human hsp-70" PROC. NATL.ACAD. SCI. USA, vol. 82, no. 19, 1985, pages 6455-6459, XP000929693
MOSELEY POPE L ET AL: "Heat stress regulates the human 70-kDa heat-shock gene through the 3'-untranslated region." AMERICAN JOURNAL OF PHYSIOLOGY, vol. 264, no. 6 PART 1, 1993, pages L533-L537, XP000925808 ISSN: 0002-9513
PITTO LETIZIO ET AL: "Role of the leader sequence during thermal repression of translation in maize, tobacco, and carrot protoplasts." PLANT PHYSIOLOGY (ROCKVILLE), vol. 100, no. 4, 1992, pages 1827-1833, XP000929495 ISSN: 0032-0889
HESS MARK A ET AL: "Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'-UTR secondary structure specifically inhibits heat shock protein mRNA translation." NUCLEIC ACIDS RESEARCH, vol. 24, no. 12, 1996, pages 2441-2449, XP002144202 ISSN: 0305-1048 cited in the application
LIARAKOS CHARLES D ET AL: "The translation efficiency of ovalbumin mRNA is determined in part by a 5'-end hairpin structure." ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 315, no. 1, 1994, pages 54-59, XP000925807 ISSN: 0003-9861
JOSHI CHANDRASHEKHAR P ET AL: "5' untranslated leader sequences of eukaryotic mRNAs encoding heat shock induced proteins." NUCLEIC ACIDS RESEARCH, vol. 23, no. 4, 1995, pages 541-549, XP000929506 ISSN: 0305-1048
Attorney, Agent or Firm:
Rees, Marion L. (UB6 0NN, GB)
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Claims:
Claims
1. A DNA molecule that can be transcribed to provide an RNA molecule having an untranslated region that provides an increased efficiency of translation of a polypeptide when operably linked to a region encoding said polypeptide; wherein said DNA molecule does not encode a mammalian Hsp70.
2. A DNA molecule according to claim 1 that does not comprise the bovine hsp70 promoter.
3. A DNA molecule according to any preceding claim; wherein said untranslated region is at least 175 nucleotides long.
4. A DNA molecule according to any preceding claim; wherein said untranslated region is at least 200 nucleotides long.
5. A DNA molecule according to any preceding claim; wherein said untranslated region is about 215 nucleotides long.
6. A DNA molecule according to any preceding claim; wherein said untranslated region is a 5'untranslated region.
7. A DNA molecule according to any preceding claim, comprising: a) the sequence: <BR> <BR> <BR> 5'ataacggctagcctgaggagctgctgcgacagtccactacctttttcgagagtgactcccgttgtcccaaggcttccc<BR> <BR> <BR> <BR> <BR> <BR> agagcgaacctgtgcggctgcaggcaccggcgcgtcgagtttccggcgtccggaaggaccgagctcttctcgcgg<BR> <BR> <BR> <BR> <BR> atccagtgttccgtttccagcccccaatctcagagccgagccgacagagagcagggaaccgc3', b) the complement of the sequence given in a), or c) a sequence having substantial sequence identity with a sequence as defined in a) or b) above.
8. A DNA molecule according to any preceding claim wherein said untranslated region has a AG of below10 kCal/mol.
9. A DNA molecule according to any preceding claim; wherein said sequence has a AG that is below30 kCal/Mol.
10. A DNA molecule according to any preceding claim; wherein said sequence has a AG that is below40 kCal/Mol.
11. A DNA molecule according to any preceding claim; wherein said untranslated region has a AG of below50 kCal/Mol.
12. A DNA molecule according to any preceding claim; wherein expression of said polypeptide is heat shock responsive.
13. An RNA molecule obtainable by transcribing a DNA molecule according to any of claims 1 to 12.
14. A vector comprising a DNA molecule according to any of claims 1 to 12.
15. An expression system comprising a DNA molecule according to any of claims 1 to 12 or a vector according to claim 14.
16. An expression system according to claim 15 which comprises one or more cells.
17. An expression system according to claim 16 comprising one or more eukaryotic cells.
18. An expression system according to claim 16 comprising one or more mammalian cells.
19. An expression system according to claim 16 comprising one or more human cells.
20. An expression system according to claim 15 which is a cell free expression system.
21. A method of obtaining a polypeptide comprising expressing the polypeptide using an expression system according to any of claims 15 to 20 and, optionally, purifying the polypeptide.
22. A method according to claim 21 comprising the step of providing the expression system with a heat shock.
23. A polypeptide when obtained via a method according to claim 21 or claim 22.
24. A method of treating a deficiency in the expression of a polypeptide, comprising providing a patient with a DNA molecule as described in any of claims 1 to 12 which encodes said polypeptide, with a vector comprising said DNA molecule, or with a cell comprising said DNA molecule or vector.
25. A method of treating a deficiency in the expression of a polypeptide, comprising providing a patient with a DNA molecule that can be transcribed to provide the untranslated region defined in any of claims 1 to 12; wherein said molecule is provided in a manner to allow it to become operably liked with a sequence already present in the patient which encodes said polypeptide.
26. A method of treating a disorder (e. g. an infection) treatable by providing an increased immune response, comprising providing a patient with a vaccine comprising a DNA molecule as described in any of claims 1 to 12 or comprising a vector including said DNA molecule.
27. A method according to claim 24 or 25; wherein a DNA molecule or vector is provided under conditions allowing it to integrate within the patient's genome.
28. A method according to claim 24; wherein a cell is provided under conditions allowing it to be maintained within the patient.
29. A method according to claim 28 wherein said cell is a cell that has been removed from the patient and has been modified prior to being reintroduced to the patient.
30. A method of treating adeficiency in the expression of a polypeptide, comprising providing the patient with an RNA molecule according to claim 13 or with a polypeptide according to claim 23.
31. A pharmaceutically acceptable composition comprising a DNA molecule according to any of claims 1 to 12, an RNA molecule according to claim 13, a polypeptide according to claim 23 or a cell as described in any of claims 16 to 19.
32. A vaccine comprising a DNA molecule according to any of claims 1 to 10, or a vector including said DNA molecule.
33. The use of a DNA molecule according to any of claims 1 to 12, of an RNA molecule according to claim 13, of a vector according to claim 14, or of an expression system according to any of claims 15 to 20, in achieving increased expression of a polypeptide.
34. The invention as substantially herein before described with reference to the accompanying drawings and examples.
35. A DNA molecule according to any one of claims 1 to 12 for use in therapy.
36. A DNA molecule according to claim 35 for use in therapeutic or prophylactic vaccination.
37. A DNA molecule according to claim 35 or 36 when administered by particle bombardment.
38. A DNA molecule according to claim 35,36 or 37 for use in achieving an increased immune response.
39. Method of therapeutic or prophylactic vaccination comprising administering an effective amount of a DNA molecule as claimed in any one of claims 1 to 12.
40. Method according to claim 39 wherein the DNA molecule is administered by particle bombardment.
41. Method according to claim 39 or 40 for use in achieving an increased immune response.
Description:
INTERNATIONALSEARCHREPORTintt cional Application No PCT/EP00/02031 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT Category Citation of document,withindication,whereappropriate,oftherelevantpassag esRelevanttoclaimNo. XWO9411521A(BIOSTARINC)1,3,4,6, 26May1994(1994-05-26)12-23, citedintheapplication26,31-41 page14,line14-page15,line22 page16,line30-page17,line23 page23,line1-16 page23,line24-page24,line19 XDATABASEGENEMBL'Online!1-23,31, 16July1988(1988-07-16)32,34 HUNT,C.ANDMORIMOTO,R.I.:"Humanheat shockprotein(hsp70)gene,completecds" XP002144203 citedintheapplication AccessionM11717 -&HUNT,C.ANDMORIMOTO,R.I.: "Conservedfeaturesofeukaryotichsp-70 genesrevealeedbycomparisonwiththe nucleotidesequenceofhumanhsp-70" PROC.NATL.ACAD.SCI.USA, vol.82,no.19,1985,pages6455-6459, XP000929693 figure2 XMOSELEYPOPELETAL:"Heatstress1-5, regulatesthehuman70-kDaheat-shockgene12-23, throughthe3'-untranslatedregion."31-34 AMERICANJOURNALOFPHYSIOLOGY, vol.264,no.6PART1,1993,pages L533-L537,XP000925808 ISSN:0002-9513 figure4 APITTOLETIZIOETAL:"Roleoftheleader1-41 sequenceduringthermalrepressionof translationinmaize,tobacco,andcarrot protoplasts." PLANTPHYSIOLOGY(ROCKVILLE), vol.100,no.4,1992,pages1827-1833, XP000929495 ISSN:0032-0889 figures1,3 page1829,right-handcolumn-page1830, left-handcolumn table1 AUS5659122A(AUSTINGLENNDOUGLAS)1-41 19August1997(1997-08-19) seeEXAMPLE ___ 4 INTERNATIONALSEARCHREPORTm,.tionaiApplication No PCT/EP00/02031 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT CategoryCitationofdocument,withindication,whereappropriate,o ftherelevantpassagesRelevanttoclaimNo. AHESSMARKAETAL:"Sequenceand1-41 structuredeterminantsofDrosophilaHsp70 mRNAtranslation:5'-UTRsecondary structurespecificallyinhibitsheatshock proteinmRNAtranslation." NUCLEICACIDSRESEARCH, vol.24,no.12,1996,pages2441-2449, XP002144202 ISSN:0305-1048 citedintheapplication thewholedocument ALIARAKOSCHARLESDETAL:"The1-41 translationefficiencyofovalbuminmRNA isdeterminedinpartbya5'-endhairpin structure." ARCHIVESOFBIOCHEMISTRYANDBIOPHYSICS, vol.315,no.1,1994,pages54-59, XP000925807 ISSN:0003-9861 page55,right-handcolumn,paragraph4 -page56,right-handcolumn,paragraph2; figure2 AJOSHICHANDRASHEKHARPETAL:"5'1-41 untranslatedleadersequencesof eukaryoticmRNAsencodingheatshock inducedproteins." NUCLEICACIDSRESEARCH, vol.23,no.4,1995,pages541-549, XP000929506 ISSN:0305-1048 figures1,2;tables2,3 4 FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 Continuation of Box 1.2 Present claims 1-41 relate to a product by reference to a desirable characteristic or property, namely, to a DNA molecule that can be transcribed to provide an RNA having an untranslated region that provides an increased efficency of translation of a polypeptide when operably linked to a region encoding said polypeptide. The claims cover all products having this characteristic or property, whereas the application provides support within the meaning of Article 6 PCT and/or disclosure within the meaning of Article 5 PCT for only a very limited number of such products. In the present case, the claims so lack support, and the application so lacks disclosure, that a meaningful search over the whole of the claimed scope is impossible. Independent of the above reasoning, the claims also lack clarity (Article 6 PCT). An attempt is made to define the product by reference to a result to be achieved. Again, this lack of clarity in the present case is such as to render a meaningful search over the whole of the claimed scope impossible. Consequently, the search has been carried out for those parts of the claims which appear to be clear, supported and disclosed, namely those parts relating to the sequence of the human hsp70 gene encoding for the 5'-untranslated region of the human hsp70 mRNA.

The applicant's attention is drawn to the fact that claims, or parts of claims, relating to inventions in respect of which no international search report has been established need not be the subject of an international preliminary examination (Rule 66.1 (e) PCT). The applicant is advised that the EPO policy when acting as an International Preliminary Examining Authority is normally not to carry out a preliminary examination on matter which has not been searched. This is the case irrespective of whether or not the claims are amended following receipt of the search report or during any Chapter II procedure. INTERNATIONALSEARCHREPORT Intt. tional ApplicationNo informationonpatent{4 {9 memben PatentdocumentPublicationPatentfamily Publication citedinsearchreportdate member(s)date WO8700861A12-02-1987AU 604214 B 13-12-1990 AU 6286086 A 05-03-1987 EP 0231368 A 12-08-1987 ES 2003085 A 16-10-1988 NO 871328 A 30-03-1987 ZA 8605702 A 25-03-1987 WO9411521A26-05-1994US 5521084 A 28-05-1996 CA 2148492 A 26-05-1994 EP 0672156 A 20-09-1995 JP 8505283 T 11-06-1996 US 5981224 A 09-11-1999 US 5733745 A 31-03-1998 US5659122A19-08-1997US 5362865 A 08-11-1994 AU 7833494 A 22-03-1995 CA 2169854 A 09-03-1995 EP 0716709 A 19-06-1996 WO 9506742 A 09-03-1995