Title:
GENETIC MODIFICATION OF SOMATIC CELLS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2000/051424
Kind Code:
A2
Abstract:
The production of genetically modified animals, in which the genetic modifications are engineered in somatic cells cultured $i(in vitro) by gene targeting, is described. Genetically modified cells may then be used as nuclear donors loci in animal chromosomes which are suitable sites for transgene addition to cells.
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Inventors:
COLMAN ALAN (GB)
SCHNIEKE ANGELIKA ELISABETH (GB)
KIND ALEXANDER JARVIS (GB)
AYARES DAVID LEE (US)
DAI YIFAN (US)
SCHNIEKE ANGELIKA ELISABETH (GB)
KIND ALEXANDER JARVIS (GB)
AYARES DAVID LEE (US)
DAI YIFAN (US)
Application Number:
PCT/GB2000/000778
Publication Date:
September 08, 2000
Filing Date:
March 03, 2000
Export Citation:
Assignee:
PPL THERAPEUTICS SCOTLAND LTD (GB)
COLMAN ALAN (GB)
SCHNIEKE ANGELIKA ELISABETH (GB)
KIND ALEXANDER JARVIS (GB)
AYARES DAVID LEE (US)
DAI YIFAN (US)
COLMAN ALAN (GB)
SCHNIEKE ANGELIKA ELISABETH (GB)
KIND ALEXANDER JARVIS (GB)
AYARES DAVID LEE (US)
DAI YIFAN (US)
International Classes:
A01K67/027; C12N15/10; C12N15/90; H04L12/24; A61K48/00; (IPC1-7): A01K67/027
Domestic Patent References:
| WO1998037183A1 | 1998-08-27 | |||
| WO1998030683A2 | 1998-07-16 | |||
| WO1997007668A1 | 1997-03-06 | |||
| WO1997007669A1 | 1997-03-06 | |||
| WO1998056903A1 | 1998-12-17 |
Other References:
POLEJAEVA, I.A. AND CAMPBELL, K.H.S.: "New advances in somatic cell nuclear transfer: Application in transgenesis" THERIOGENOLOGY, vol. 53, no. 1, 1 January 2000 (2000-01-01), pages 117-126, XP000940942
SCHNIEKE, A.E. ET AL.: "Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts" SCIENCE, vol. 278, 19 December 1997 (1997-12-19), pages 2130-2133, XP002067036 cited in the application
CIBELLI, J.B. ET AL.: "Embryonic stem cells: Bovine chimeric offspring produced by transgenic embryonic stem cells generated from somatic cell nuclear transfer embryos" ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, vol. 49, no. 1, 18 January 1998 (1998-01-18), page 236 XP000764040
ROBL. J.M. ET AL.: "Somatic cell nuclear transplantation in cattle" ANNUAL MEETING SOC. STUDY REPROD, vol. 58, 8 August 1998 (1998-08-08), page 25 XP000791151
CAMPBELL, K.H.S. AND WILMUT, I.: "Totipotency or multipotentiality of cultured cells: Applications and progress" THERIOGENOLOGY, vol. 47, 1997, pages 63-72, XP000940776
WAKAYAMA, T. ET AL.: "Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei" NATURE, vol. 394, 23 July 1998 (1998-07-23), pages 369-374, XP002900702 cited in the application
MCCREATH, K.J. ET AL.: "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells" NATURE, vol. 405, 29 July 2000 (2000-07-29), pages 1066-1069, XP000919025
SCHNIEKE, A.E. ET AL.: "Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts" SCIENCE, vol. 278, 19 December 1997 (1997-12-19), pages 2130-2133, XP002067036 cited in the application
CIBELLI, J.B. ET AL.: "Embryonic stem cells: Bovine chimeric offspring produced by transgenic embryonic stem cells generated from somatic cell nuclear transfer embryos" ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, vol. 49, no. 1, 18 January 1998 (1998-01-18), page 236 XP000764040
ROBL. J.M. ET AL.: "Somatic cell nuclear transplantation in cattle" ANNUAL MEETING SOC. STUDY REPROD, vol. 58, 8 August 1998 (1998-08-08), page 25 XP000791151
CAMPBELL, K.H.S. AND WILMUT, I.: "Totipotency or multipotentiality of cultured cells: Applications and progress" THERIOGENOLOGY, vol. 47, 1997, pages 63-72, XP000940776
WAKAYAMA, T. ET AL.: "Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei" NATURE, vol. 394, 23 July 1998 (1998-07-23), pages 369-374, XP002900702 cited in the application
MCCREATH, K.J. ET AL.: "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells" NATURE, vol. 405, 29 July 2000 (2000-07-29), pages 1066-1069, XP000919025
Attorney, Agent or Firm:
Howard, Paul Nicholas (Carpmeals & Ransford 43 Bloomsbury Square
London WC1A 2RA, GB)
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Claims:
CLAIMS
| 1. | A method of preparing a somatic cell for nuclear transfer comprising modifying the genetic material of the somatic cell by a genetic targeting event. |
| 2. | A method as claimed in claim 1, wherein the genetic targeting event is mediated by homologous recombination. |
| 3. | A method, as claimed in claim 1 or claim 2, wherein the modification is inactivation, removal or modification of a gene; upregulation of a gene, gene replacement or transgene placement. |
| 4. | A method as claimed in any one of claims 1 to 3, wherein the genetic targeting event results in a gene targeted cell clone: randomly targeted cell clone ratio of equal to or greater than 1: 100. |
| 5. | A method as claimed in any one of claims 1 to 4 wherein the gene targeting event is carried out at a locus abundantly expressed in the host somatic cell. |
| 6. | A method as claimed in any one of claims 1 to 5 wherein a structural gene is placed adjacent to an endogenous promoter. |
| 7. | A method as claimed in claim 6 wherein the endogenous promoter is that of a collagen gene. |
| 8. | A method as claimed in claim 6 wherein the endogenous promoter is that of a milk protein gene. |
| 9. | A method as claimed in claim 6 wherein the endogenous promoter directs abundant expression in fibroblast cells. |
| 10. | A method as claimed in claim 6 wherein the endogenous promoter directs abundant expression in endothelial cells. |
| 11. | A method as claimed in any one of claims 1 to 10 wherein the genetic targeting event is mediated by lipofection. |
| 12. | A method as claimed in any one of claims 1 to 11 wherein the genetic targeting event involves the use of a gene targeting vector which vector comprises a long region of homology to the target locus. |
| 13. | A method as claimed in any one of claims 1 to 12 wherein the genetic targeting event involves the use of a gene targeting vector which is in a circular form. |
| 14. | A method as claimed in any one of claims 1 to 13 wherein the genetic targeting event includes the artificial induction of gene expression or the induction of chromatin changes in the cell. |
| 15. | A method as claimed in any one of claims 1 to 14 wherein the genetic targeting event is facilitated by an agent which inhibits histone deacetylation or by expression in the cell of a factor which stimulates transcription at the target locus. |
| 16. | A method as claimed in any one of claims 1 to 15, wherein the somatic cell is a primary somatic cell. |
| 17. | A method as claimed in any one of claims 1 to 16, wherein the somatic cell is an epithelial cell, or a fibroblast cell, or an endothelial cell, or a muscle cell. |
| 18. | A method of nuclear transfer comprising a method as claimed in any one of claims 1 to 17 and a method comprising transfer of the genetic material from the somatic cell to a recipient cell. |
| 19. | A method, as claimed in claim 18, wherein the transfer of the genetic material from the somatic cell, to a recipient cell, provides an animal embryo. |
| 20. | A method, as claimed in claim 18 or claim 19 further comprising the production of a totipotent or pluripotent cloned cell population. |
| 21. | A transgenic cell, suitable for nuclear transfer obtainable by a method as claimed in any one of claims 1 to 17. |
| 22. | A transgenic embryo or a transgenic fetus obtainable by a method as claimed in claim 19. |
| 23. | A method for preparing a transgenic animal, comprising causing an animal to develop to term from the embryo as claimed in claim 22 and optionally breeding from the animal. |
| 24. | A transgenic animal obtainable by the method as claimed in claim 23. |
| 25. | A transgenic animal as claimed in claim 24 which is a sheep, cow, bull, goat, pig, horse, camel, rabbit or rodent. |
| 26. | A transgenic animal which is bred from an animal as claimed in claim 24 or claim 25. |
| 27. | A method for obtaining a clonal pluripotent or totipotent cell population comprising culturing a cell line from a transgenic embryo or a transgenic fetus as claimed in claim 22. |
| 28. | A clonal pluripotent or totipotent cell population obtainable according to a method as claimed in claim 27. |
| 29. | A method for modifying the genetic material of a somatic cell while maintaining the totipotency of the cell, the method comprising a genetic targeting event. |
| 30. | A method as claimed in claim 29, wherein the genetic targeting event is mediated by homologous recombination. |
| 31. | A method as claimed in claim 29 or claim 30 wherein the modification is inactivation, removal or modification of a gene, upregulation of a gene, gene replacement or transgene placement. |
| 32. | A method as claimed in any one of claims 29 to 31, wherein the genetic targeting event results in a gene targeted cell clone: randomly targeted cell clone ratio of equal to or greater than 1: 100. |
| 33. | A method as claimed in any one of claims 29 to 32 wherein the gene targeting event is carried out at a locus abundantly expressed in the host somatic cell. |
| 34. | A method as claimed in any one of claims 29 to 33 wherein a structural gene is placed adjacent to an endogenous promoter. |
| 35. | A method as claimed in claim 34 wherein the endogenous promoter is that of a collagen gene. |
| 36. | A method as claimed in claim 34 wherein the endogenous promoter is that of a milk protein gene. |
| 37. | A method as claimed in claim 34 wherein the endogenous promoter directs abundant expression in fibroblast cells. |
| 38. | A method as claimed in claim 34 wherein the endogenous promoter directs abundant expression in endothelial cells. |
| 39. | A method as claimed in any one of claims 29 to 38 wherein the genetic targeting event is mediated by lipofection. |
| 40. | A method as claimed in any one of claims 29 to 39 wherein the genetic targeting event involves the use of a gene targeting vector which vector comprises a long region of homology to the target locus. |
| 41. | A method as claimed in any one of claims 29 to 40 wherein the genetic targeting event involves the use of a gene targeting vectory which is in a circular form. |
| 42. | A method as claimed in any one of claims 29 to 41 wherein the somatic cell is a primary somatic cell. |
| 43. | A method as claimed in any one of claims 29 to 42, wherein the genetic targeting event includes the artificial induction of gene expression or the induction of chromatin changes in the cell. |
| 44. | A method as claimed in any one of claims 29 to 43, wherein the genetic targeting event is facilitated by an agent which inhibits histone deacetylation or by expression in the cell of a factor which stimulates transcription at the target locus. |
| 45. | A method as claimed in any one of claims 29 to 44, in combination with a method comprising the transfer of the genetic material from the somatic cell to a recipient cell. |
| 46. | The use of artificial induction of gene expression or induction of chromatin changes in the genetic targeting of a cell. |
| 47. | The use of a gene targeting vector which is in a circular form in the modification of the genetic material of a cell by a gene targeting event. |
| 48. | The use, as claimed in claim 46 or claim 47, wherein the cell is somatic or non somatic. |
| 49. | The use, as claimed in any one of claims 46 to 48, wherein the genetic targeting is facilitated by an agent which inhibits histone deacetylation or by expression in the cell of a factor which stimulates transcription at the target locus. |
| 50. | The use, as claimed in any one of claims 46 to 49, in combination with the nuclear transfer of the genetic material of the cell into a suitable recipient cell. |
| 51. | The use of an animal, which has been obtained from a cell following a genetic targeting event, to test for genetic changes due to the location of the genetic targeting. |
| 52. | The use, as claimed in claim 51, wherein the cell is a somatic cell and the production of the animal includes nuclear transfer. |
| 53. | The use, as claimed in claim 51 or claim 52, wherein the somatic cell is a primary somatic cell. |
| 54. | The use, as claimed in claim 52 or claim 53, wherein the cell is a fibroblast. |
| 55. | The use, as claimed in any one of claims 51 to 54, wherein the gene targeting event is as described in any one of claims 2 to 13. |
| 56. | A method for validating a locus for targeted gene therapy comprising: obtaining cells of a chosen type; introducing a desired genetic change at a selected locus; growing a clonal population of the targeted cells; and demonstrating through the production of an animal that the genetic changes are acceptable. |
| 57. | A method, as claimed in claim 56, wherein the production of the animal involves nuclear transfer. |
| 58. | A method, as claimed in claim 57, wherein the cell is a fibroblast. |
| 59. | A method as claimed in claim 56, wherein the cell is an embryonic stem (ES) cell or an embryonic germ (EG) cell. |
| 60. | A method as claimed in claim 59, wherein the animal is a chimeric animal. |
| 61. | A validated locus, obtainable by the method of any one of claims 56 to 60. |
Description:
INTERNATIONALSEARCH REPORT Intern... nal Application No PCT/GB00/00778 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT CategoryCitationofdocument,withindication,whereappropriate,o
ftherelevantpassagesRelevanttoclaimNo. XWO9830683A(UNIVERSITYOF1,3-6,9, MASSACHUSETTS)16July1998(1998-07-16)10,13, cited intheapplication14, 16-29, 31-34, 37,38, 41-43,45 page10,line5-page16,line15 page18,line17-page24,line22; examples1,2 XWO9707668A(ROSLININSTITUTE1,3-6, (EDINBURGH))6March1997(1997-03-06)9-11,13, citedintheapplication14, 16-29, 31-34, 37-39, 41-43,45 page5,line1-page7,line25 41-43, 45 page8,line24-line27 page15,line15-page16,line7 page18,line18-line22 XWO9707669A(ROSLININSTITUTE1,3-6, (EDINBURGH))6March1997(1997-03-06)9-11,13, cited intheapplication14, 16-29, 31-34, 37-39, 41-43,45 page3,line29-page7,line33 page15,line7-page17,line16 page19,line29-page20,line26 XSCHNIEKE,A.E.ET AL. :"HumanfactorIX1,3-6,8, transgenicsheepproducedbytransferof11,13, nucleifromtransfectedfetalfibroblasts"14, SCIENCE,16-26, vol.278,19December1997(1997-12-19),29, pages2130-2133,XP00206703631-34, citedintheapplication36,39, 41-43,45 thewholedocument XCIBELLI,J.B.ET AL. :"Embryonicstem1,3-5, cells:Bovinechimericoffspringproduced14, bytransgenicembryonicstemcells16-29, generatedfromsomaticcellnuclear31-33, transferembryos"42,43,45 ANNUALCONFERENCEOFTHEINTERNATIONAL EMBRYOTRANSFERSOCIETY, vol.49,no.1, 18January1998(1998-01-18),page236 XP000764040 thewholedocument 4 INTERNATIONAL SEARCHREPORT r--------------j PCT/GB00/00778 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT __ Category ° Citationofdocument,withindication,whereappropriate,oftherele
vantpassagesRelevanttoclaimNo. YROBL.J.M.ET AL. :"Somaticcellnuclear1-45 transplantationincattle" ANNUALMEETINGSOC.STUDYREPROD, vol.58,8August1998(1998-08-08),page 25XP000791151 thewholedocument YWO9856903A(PRESIDENTANDFELLOWSOF1-45 HARVARDCOLLEGE) 17December1998(1998-12-17) thewholedocument ACAMPBELL,K.H.S.AND WILMUT, I.:1-45 "Totipotencyormultipotentialityof culturedcells:Applicationsandprogress" THERIOGENOLOGY, vol.47,1997,pages63-72,XP000940776 thewholedocument AWAKAYAMA,T.ET AL. :"Full-term1-45 developmentofmicefromenucleated oocytesinjectedwithcumuluscellnuclei" NATURE, vol.394,23July1998(1998-07-23),pages 369-374,XP002900702 citedintheapplication thewholedocument TMCCREATH,K.J.ET AL. :"Productionof1-45 gene-targetedsheepbynucleartransfer fromculturedsomaticcells" NATURE, vol.405,29July2000(2000-07-29),pages 1066-1069,XP000919025 thewholedocument 4 InternationalapplicationNo. PCT/GB00/00778 INTERNATIONALSEARCHREPORTT/GB 00/00778 Box IObservationswherecertainclaimswerefoundunsearchable(Continu
ationofitem1offirstsheet) ThisInternationalSearchReporthasnotbeenestablishedinrespecto
fcertainclaimsunderArticle17(2)(a)forthefollowingreasons: 1.0 ClaimsNos.: becausetheyrelatetosubjectmatternotrequiredtobesearchedbythi
sAuthority,namely: 2.ClaimsNos.: becausetheyrelatetopartsoftheInternationalApplicationthatdon
otcomplywiththeprescribedrequirementstosuch anextentthatnomeaningfulInternationalSearchcanbecarriedout,s
pecifically: 3.ClaimsNos.: becausetheyaredependentclaimsandarenotdraftedinaccordancewit
hthesecondandthirdsentencesofRule6.4(a). Box IfObservationswhereunityofinventionislacking(Continuationofi
tem2offirstsheet) ThisInternationalSearchingAuthorityfoundmultipleinventionsin
thisinternationalapplication,asfollows: 1.As allrequiredadditionalsearchfeesweretimelypaidbytheapplicant,
thisInternationalSearchReportcoversall searchableclaims. 2.As allsearchableclaimscouldbesearchedwithouteffortjustifyingana
dditionalfee,thisAuthoritydidnotinvitepayment ofanyadditionalfee. 3.As onlysomeoftherequiredadditionalsearchfeesweretimelypaidbythe
applicant,thisInternationalSearchReport coversonlythoseclaimsforwhichfeeswerepaid,specificallyclaims
Nos.: 4.Norequiredadditionalsearchfeesweretimelypaidbytheapplicant
.Consequently,thisInternationalSearchReportis restrictedtotheinventionfirstmentionedintheclaims;itiscovere
dbyclaimsNos.: 1-45 RemarkonProtestThe additional search feeswereaccompaniedbytheapplicant's protest. No protestaccompaniedaccompaniedpaymentpaymentadditionalsearchf
ees. u International Application No. PCT/GB 00/00778 FURTHERINFORMATIONCONTINUEDFROMPCT/ISA/21Q 1.Claims:1-45 Claims1-45refertoamethodofpreparingasomaticcell fornucleartransfercomprisingmodifyingthegenetic materialofthesomaticcellbyagenetictargetingevent, toamethodofnucleartransfercomprisingthetransferof thegeneticmaterialfromsaidmodifiedsomaticcelltoa recipientcell,toatransgenicembryoobtainedbysaid methodofnucleartransferandtoatransgenicanimal developedtotermfromsaidembryo. 2.Claims:46-50 Claims46-50refertothegeneraluseofartificial inductionofgeneexpressionorinductionofchromatin changesinthegenetictargetingofacell. 3.Claims:51-55 Claims51-55refertotheuseofananimal,whichhasbeen obtainedingeneralfromacellfollowingagenetic targetingevent. 4.Claims:56-61 Claims56-61refertoageneralmethodforvalidatinga locusfortargetedgenetherapyandtothevalidatedlocus obtainedbysaidmethod. INTERNATIONALSEARCHREPORT r-.,,.,------- Interno al Application izio nformation or patent family members pCT/GB 00/00778 PatentdocumentPublication Patent family Publication citedinsearchreportdate member (s) date WO9837183A27-08-1998AU 6300198 A 09-09-1998 EP 1009816 A 21-06-2000 WO9830683A16-07-1998US 5945577 A 31-08-1999 AU 6014598 A 03-08-1998 BR 9806872 A 18-04-2000 CN 1248288 T 22-03-2000 EP 1015572 A 05-07-2000 WO9707668A06-03-1997AU 6830996 A 19-03-1997 BR 9610013 A 21-12-1999 CA 2229657 A 06-03-1997 CN 1202085 A 16-12-1998 CZ 9800604 A 15-07-1998 EP 0847237 A 17-06-1998 GB 2318792 A, B 06-05-1998 GB 2340493A,B 23-02-2000 HU 9802485 A O1-02-1999 JP 2000506721 T 06-06-2000 NO 980846 A 29-04-1998 PL 325336 A 20-07-1998 WO9707669A06-03-1997AU 716956 B 09-03-2000 AU 6831096 A 19-03-1997 BR 9610034 A 21-12-1999 CA 2229568 A 06-03-1997 CN 1202084 A 16-12-1998 CZ 9800608 A 15-07-1998 EP Q84999Q A Q1-Q7-1998 EP 0930009 A 21-07-1999 EP 1005789 A 07-06-2000 GB 2318578A,B 29-04-1998 GB 2331751A,B 02-06-1999 HU 9900234 A 28-05-1999 JP 2000506722 T 06-06-2000 NO 980845 A 29-04-1998 NZ 316149 A 28-10-1999 PL 325331 A 20-07-1998 US 6147276 A 14-11-2000 WO9856903A17-12-1998AU 7729498 A 30-12-1998