Title:
METHOD FOR SELECTING IMPROVED VECTORS
Document Type and Number:
WIPO Patent Application WO/2000/071693
Kind Code:
A2
Abstract:
A method is provided for selecting an improved retroviral genome having an improved packaging efficiency which method comprises: a) introducing one or more random mutations into a retroviral genome comprising a packaging signal; b) introducing the mutagenised retroviral genome into a host cell expressing viral polypeptides required for packaging the retroviral genome; c) determining whether retroviral packaging efficiency in the cell is improved as compared with a retroviral genome comprising a non-mutated packaging signal; d) selecting a viral genome which has improved packaging efficiency.
Inventors:
KINGSMAN ALAN JOHN (GB)
SLINGSBY JASON (GB)
YAP MELVYN (GB)
SLINGSBY JASON (GB)
YAP MELVYN (GB)
Application Number:
PCT/GB2000/001964
Publication Date:
November 30, 2000
Filing Date:
May 22, 2000
Export Citation:
Assignee:
OXFORD BIOMEDICA LTD (GB)
KINGSMAN ALAN JOHN (GB)
SLINGSBY JASON (GB)
YAP MELVYN (GB)
KINGSMAN ALAN JOHN (GB)
SLINGSBY JASON (GB)
YAP MELVYN (GB)
International Classes:
A61K35/76; C12N15/09; A61K48/00; A61P31/00; A61P35/00; A61P37/00; C07K14/15; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N15/867; C12Q1/68; (IPC1-7): C12N15/00
Domestic Patent References:
| WO1993020220A1 | 1993-10-14 | |||
| WO2000040741A2 | 2000-07-13 |
Foreign References:
| US5747323A | 1998-05-05 | |||
| US5278056A | 1994-01-11 |
Other References:
ARYA S K ET AL: "HUMAN IMMUNODEFICIENCY VIRUS TYPE 2 LENTIVIRUS VECTORS FOR GENE TRANSFER: EXPRESSION AND POTENTIAL FOR HELPER VIRUS-FREE PACKAGING" HUMAN GENE THERAPY,XX,XX, vol. 9, no. 9, 10 June 1998 (1998-06-10), pages 1371-1380, XP000906951 ISSN: 1043-0342
SONEOKA YUKO ET AL: "A transient three-plasmid expression system for the production of high titer retroviral vectors." NUCLEIC ACIDS RESEARCH, vol. 23, no. 4, 1995, pages 628-633, XP000569533 ISSN: 0305-1048 cited in the application
DORIA-ROSE NICOLE A ET AL: "In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal." JOURNAL OF VIROLOGY, vol. 72, no. 10, October 1998 (1998-10), pages 8073-8082, XP002155091 ISSN: 0022-538X
MCCANN E M ET AL: "LOCATION OF CIS-ACTING SIGNALS IMPORTANT FOR RNA ENCAPSIDATION IN THE LEADER SEQUENCE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 2" JOURNAL OF VIROLOGY,US,THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 71, no. 5, 1997, pages 4133-4137, XP000909426 ISSN: 0022-538X
HARRISON G P ET AL: "THE HUMAN IMNUNODEFICIENCY VIRUS TYPE 1 PACKAGING SIGNAL AND MAJOR SPLICE DONOR REGION HAVE A CONSERVED STABLE SECONDARY STRUCTURE" JOURNAL OF VIROLOGY,US,NEW YORK, US, vol. 66, no. 7, 1992, pages 4144-4153, XP000909419 ISSN: 0022-538X
BORNSCHEUER U T ET AL: "Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones" BIOTECHNOLOGY AND BIOENGINEERING - COMBINATORIAL CHEMISTRY,US,WILEY, NEW YORK, NY, vol. 58, no. 5, 5 June 1998 (1998-06-05), pages 554-559, XP002144278 ISSN: 0006-3592
BUCHSCHACHER G L ET AL: "Development of lentiviral vectors for gene therapy for human diseases" BLOOD,US,W.B.SAUNDERS COMPAGNY, ORLANDO, FL, vol. 95, no. 8, 15 April 2000 (2000-04-15), pages 2499-2504, XP000916373 ISSN: 0006-4971
YAP MELVYN W ET AL: "Effects of stoichiometry of retroviral components on virus production." JOURNAL OF GENERAL VIROLOGY, vol. 81, no. 9, September 2000 (2000-09), pages 2195-2202, XP002155092 ISSN: 0022-1317
SONEOKA YUKO ET AL: "A transient three-plasmid expression system for the production of high titer retroviral vectors." NUCLEIC ACIDS RESEARCH, vol. 23, no. 4, 1995, pages 628-633, XP000569533 ISSN: 0305-1048 cited in the application
DORIA-ROSE NICOLE A ET AL: "In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal." JOURNAL OF VIROLOGY, vol. 72, no. 10, October 1998 (1998-10), pages 8073-8082, XP002155091 ISSN: 0022-538X
MCCANN E M ET AL: "LOCATION OF CIS-ACTING SIGNALS IMPORTANT FOR RNA ENCAPSIDATION IN THE LEADER SEQUENCE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 2" JOURNAL OF VIROLOGY,US,THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 71, no. 5, 1997, pages 4133-4137, XP000909426 ISSN: 0022-538X
HARRISON G P ET AL: "THE HUMAN IMNUNODEFICIENCY VIRUS TYPE 1 PACKAGING SIGNAL AND MAJOR SPLICE DONOR REGION HAVE A CONSERVED STABLE SECONDARY STRUCTURE" JOURNAL OF VIROLOGY,US,NEW YORK, US, vol. 66, no. 7, 1992, pages 4144-4153, XP000909419 ISSN: 0022-538X
BORNSCHEUER U T ET AL: "Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones" BIOTECHNOLOGY AND BIOENGINEERING - COMBINATORIAL CHEMISTRY,US,WILEY, NEW YORK, NY, vol. 58, no. 5, 5 June 1998 (1998-06-05), pages 554-559, XP002144278 ISSN: 0006-3592
BUCHSCHACHER G L ET AL: "Development of lentiviral vectors for gene therapy for human diseases" BLOOD,US,W.B.SAUNDERS COMPAGNY, ORLANDO, FL, vol. 95, no. 8, 15 April 2000 (2000-04-15), pages 2499-2504, XP000916373 ISSN: 0006-4971
YAP MELVYN W ET AL: "Effects of stoichiometry of retroviral components on virus production." JOURNAL OF GENERAL VIROLOGY, vol. 81, no. 9, September 2000 (2000-09), pages 2195-2202, XP002155092 ISSN: 0022-1317
Attorney, Agent or Firm:
Harding, Charles Thomas (D. Young & Co. 21 New Fetter Lane London
EC4A 1DA, GB)
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Claims:
CLAIMS
| 1. | A method for selecting an improved retroviral genome having an improved packaging efficiency which method comprises: (a) introducing one or more random mutations into a retroviral genome comprising a packaging signal ; (b) introducing the mutagenised retroviral genome into a host cell expressing viral polypeptides required for packaging the retroviral genome; (c) determining whether retroviral packaging efficiency in the cell is improved as compared with a retroviral genome comprising a nonmutated packaging signal; (d) selecting a viral genome which has improved packaging efficiency. |
| 2. | A method according to claim 1 wherein an additional step (e) of determining the sequence of all or part of the viral genome to identify the sequence of the packaging signal. |
| 3. | A method according to claim 1 or 2 wherein step (a) is carried out in a bacterial strain which introduces random mutations into the retroviral genome. |
| 4. | A method according to claim 3 wherein the bacterial strain is a mutagenic strain of Epicurian coli. |
| 5. | A method according to any one of the preceding claims wherein the host cell is a mammalian cell. |
| 6. | A method according to any one of the preceding claims wherein the host cell comprises at least: (i) a first nucleotide sequence encoding a retroviral gagpol polypeptide; (ii) a second nucleotide sequence encoding a retroviral envelope polypeptide; and (iii) a third nucleotide encoding a retroviral genome comprising a nonmutated packaging signal, wherein the third nucleotide is present as part of a nucleic acid vector which lacks a selectable marker; the mutagenised retroviral genome is present as part of a nucleic acid vector which contains the selectable marker; and the ratio of the vector comprising the third nucleotide to the vector comprising the mutagenised retroviral genome is greater than 2 : 1, preferably greater than 5: 1. |
| 7. | A method according to any one of the preceding claims wherein the packaging efficiency, measured as the proportion of the number of infectious retroviral particles to the total number of retroviral particles produced is greater than 25%. |
| 8. | A retroviral genome obtained by the method of any one of the preceding claims. |
| 9. | A retroviral genome according to claim 8 having a packaging efficiency, measured as the proportion of the number of infectious retroviral particles to the total number of retroviral particles produced is greater than 25%. |
| 10. | A retroviral packaging signal obtainable from a retroviral genome according to claim 8 or 9. |
| 11. | A nucleic acid comprising a retroviral packing signal according to claim 10. |
| 12. | A retroviral vector comprising a retroviral packing signal according to claim 10. |
| 13. | A retroviral vector according to claim 12 for use in producing infectious retroviral particles. |
| 14. | A retroviral vector according to claim 12 or 13, which is a lentiviral vector. |
| 15. | A producer cell comprising a retroviral genome according to claim 8 or 9. a retroviral packaging signal according to claim 10, or a retroviral vector according to claim 12,13 or 14. |
| 16. | A method for enhancing the efficiency of retroviral packaging which method comprises expressing in a producer cell at least a first nucleotide sequence encoding a retroviral gagpol polypeptide, a second nucleotide sequence encoding a retroviral envelope polypeptide and a third nucleotide sequence encoding a retroviral genome wherein the ratio of the first nucleotide sequence to the second nucleotide sequence and the ratio of the first nucleotide sequence to the third nucleotide sequence is x: y and x: z. respectively wherein x is less than 1 and y and z are 1. |
| 17. | A method according to claim 16 wherein x is less than 0.5. |
| 18. | A method according to claim 16 or 17 wherein the packaging efficiency, measured as the proportion of the number of infectious retroviral particles to the total number of retroviral particles produced is greater than 25%. |
| 19. | A composition comprising infectious retroviral particles produced according to the method of any one of claims 16 to 18. |
| 20. | A composition according to claim 19 for use in therapy. |
| 21. | A producer cell which expresses at least a first nucleotide sequence encoding a retroviral gagpol polypeptide, a second nucleotide sequence encoding a retroviral envelope polypeptide and a third nucleotide sequence encoding a retroviral genome wherein the ratio of the first nucleotide sequence to the second nucleotide sequence and the ratio of the first nucleotide sequence to the third nucleotide sequence is x: y and x: z, respectively wherein x is less than 1 and y and z are 1. |
Description:
INTERNATIONALSEARCHREPORT Inter; nal ApplicationNo PCT/GB00/01964 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT Category Citationofdocument,withindication,whereappropriate,oftherele
vantpassagesRelevanttoclaimNo. ASONEOKAYUKOETAL:"Atransient1,7-13, three-plasmidexpressionsystemforthe15 productionofhightiterretroviral vectors." NUCLEICACIDSRESEARCH, vol.23,no.4,1995,pages628-633, XP000569533 ISSN:0305-1048 citedintheapplication thewholedocument AWO9320220A(WHITEHEADBIOMEDICALINST)1-3, 14October1993(1993-10-14)5-12,14, 15,20 page3,line1-page4,line25 page7,line10-page12,line18 AUS5747323A(DARLIXJEAN-LUCETAL)1,2,6, 5May1998(1998-05-05)8-13,16, 20 column4,line3-28 column5,line28-column6,line48 column7,line34-column8,line7 AUS5278056A(BANKARTHURETAL)1-3,5,6, 11January1994(1994-01-11)8-12,15, 20 column2,line34-column3,line6 ADORIA-ROSENICOLEAETAL:"Invivo1 selectionofRoussarcomavirusmutants withrandomizedsequencesinthepackaging signal." JOURNALOFVIROLOGY, vol.72,no.10,October1998(1998-10), pages8073-8082,XP002155091 ISSN:0022-538X thewholedocument AMCCANNEMETAL:"LOCATIONOFCIS-ACTING1 SIGNALSIMPORTANTFORRNAENCAPSIDATIONIN THELEADERSEQUENCEOFHUMAN IMMUNODEFICIENCYVIRUSTYPE2" JOURNALOFVIROLOGY,US,THEAMERICAN SOCIETYFORMICROBIOLOGY, vol.71,no.5,1997,pages4133-4137, XP000909426 ISSN:0022-538X thewholedocument 2 2 INTERNATIONALSEARCHREPORT inter na) ApplicationApplication PCT/GB00/01964 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT CategoryCitationofdocument,withindication,whereappropriate,o
ftherelevantpassagesRelevanttoclaimNo. AHARRISONGPETAL:"THEHUMAN1 IMNUNODEFICIENCYVIRUSTYPE1PACKAGING SIGNALANDMAJORSPLICEDONORREGIONHAVE ACONSERVESTABLESECONDARYSTRUCTURE" JOURNALOFVIROLOGY,US,NEWYORK,US, vol.66,no.7,1992,pages4144-4153, XP000909419 ISSN:0022-538X thewholedocument ABORNSCHEUERUTETAL :"Directed4 evolutionofanesteraseforthe stereoselectiveresolutionofakey intermediateinthesynthesisof epothilones" BIOTECHNOLOGYANDBIOENGINEERING- COMBINATORIALCHEMISTRY,US,WILEY,NEW YORK,NY, vol.58,no.5,5June1998(1998-06-05), pages554-559,XP002144278 ISSN:0006-3592 page556,left-handcolumn,last paragraph;figure2 P,XBUCHSCHACHERGLETAL:"Developmentof14,20 lentiviralvectorsforgenetherapyfor humandiseases" BLOOD,US,W.B.SAUNDERSCOMPAGNY,ORLANDO, FL, vol.95,no.8, 15April2000(2000-04-15),pages 2499-2504,XP000916373 ISSN:0006-4971 thewholedocument TYAPMELVYNWETAL:"Effectsof1-21 stoichiometryofretroviralcomponentson virusproduction." JOURNALOFGENERALVIROLOGY, vol.81,no.9,September2000(2000-09), pages2195-2202,XP002155092 ISSN:0022-1317 thewholedocument EWO0040741A(ARYASURESHK;USHEALTH1-3,5, (US))13July2000(2000-07-13)8-15, 18-20 page3,line1-page5,line17 page19,line26-page31,line5; examples1-21 2 INTERNATIONALSEARCHREPORT InternalApplication No informationonpatentfamily members PCT/GB00/01964 PatentdocumentPublicationPatentfamily Publication citedinsearchreportdatemember(s)date WO9320220A14-10-1993AU678152B22-05-1997 AU3799493A08-11-1993 CA2117884A14-10-1993 EP0633942A18-01-1995 JP8500005T09-01-1996 US5861282A19-01-1999 US5919458A06-07-1999 US5747323A05-05-1998NONE US5278056A11-01-1994AU3069189A25-08-1989 CA1315719A06-04-1993 WO8907150A10-08-1989 WO0040741A13-07-2000AU2495300A24-07-2000