DK2931898T3 - Konstruktion og optimering af systemer, fremgangsmåder og sammensætninger til sekvensmanipulation med funktionelle domæner - Google Patents
Konstruktion og optimering af systemer, fremgangsmåder og sammensætninger til sekvensmanipulation med funktionelle domæner Download PDFInfo
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Claims (49)
1. Ikke-naturligt forekommende eller konstruerede sammensætninger, der omfatter: et fremføringssystem, der er operabelt konfigureret til at fremføre CRISPR-Cas-kompleksbestanddele eller polynukleotidsekvenser, der omfatter eller koder for bestanddelene, ind i en eukaryot celle, hvor CRISPR-Cas-komplekset er operabelt i den eukaryote celle; 1. en eller flere CRISPR-Cas-komplekspolynukleotidsekvenser, der omfatter eller koder til ekspression i den eukaryote celle: (a) en styresekvens, der er i stand til at hybridisere til en målsekvens i en eukaryot celle, (b) en tracr-parringssekvens og (c) en tracr-sekvens, og II. et CRISPR-enzym, der omfatter et eller flere heterologe, funktionelle domæner eller et polynukleotid, der koder for et CRISPR-enzym, der omfatter et eller flere heterologe, funktionelle domæner til ekspression i den eukaryote celle; hvor: tracr-parringssekvensen hybridiserer til tracr-sekvensen, styresekvensen leder sekvensspecifik binding af et CRISPR-kompleks til målsekvensen, CRISPR-komplekset omfatter CRISPR-enzymet, der er kompleksbundet med (1) styresekvensen, der er hybridiseret til målsekvensen, og (2) tracr-parringssekvensen, der er hybridiseret til tracr-sekvensen, CRISPR-enzymet omfatter en eller flere mutationer, således at enzymet har ændret nuklease-aktivitet sammenlignet med vildtypeenzymet, hvor den ene eller de flere heterologe, funktionelle domæner omfatter mindst et eller flere cellekernelokaliseringssignal(er) (NLS - nuclear localization signal(s) ) .
2. Sammensætning ifølge krav 1, hvor fremføringssystemet omfatter et vektorsystem, der omfatter en eller flere vektorer, og hvor bestanddel (I) omfatter et første regulatorelement, der er operabelt koblet til en polynukleotidsekvens, der omfatter styresekvensen, tracr-parringssekvensen og tracr-sekvensen, og hvor bestanddel (II) omfatter et andet regulatorelement, der er operabelt koblet til en polynukleotidsekvens, der koder for CRISPR-enzymet.
3. Sammensætning ifølge krav 1, hvor fremføringssystemet omfatter et vektorsystem, der omfatter en eller flere vektorer, og hvor bestanddel (I) omfatter et første regulatorelement, der er operabelt koblet til styresekvensen og tracr-parringssekvensen, og et tredje regulatorelement, der er operabelt koblet til tracr-sekvensen, og hvor bestanddel (II) omfatter et andet regulatorelement, der er operabelt koblet til en polynukleotidsekvens, der koder for CRISPR-enzymet .
4. Sammensætning ifølge et hvilket som helst af kravene 2 eller 3, hvor bestanddel I og II er placeret på den samme eller forskellige vektorer i systemet.
5. Sammensætning ifølge krav 4, hvor bestanddel I og II er placeret på den samme vektor.
6. Sammensætning ifølge et hvilket som helst af ovennævnte krav, som er en multipleks sammensætning, der omfatter flere styresekvenser, der er i stand til at hybridisere til flere målsekvenser.
7. Sammensætning ifølge et hvilket som helst af kravene 2 til 6, hvor den ene eller de flere vektorer omfatter en eller flere virale vektorer.
8. Sammensætning ifølge krav 7, hvor den ene eller de flere virale vektorer omfatter en eller flere retrovirus-, lentivirus-, adenovirus-, adenoassocieret virus- eller herpes simplex-virusvektorer.
9. Sammensætning ifølge et hvilket som helst af kravene 1 til 6, hvor fremføringssystemet omfatter et gærsystem, et lipofektionssystem, et mikroinjektionssystem, et biolistisk system, virosomer, liposomer, immunliposomer, polykationer, lipid:nukleinsyre-konjugater eller kunstige virions.
10. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet omfatter en eller flere mutationer i to eller flere katalytisk aktive domæner.
11. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet har reduceret eller elimineret nukleaseaktivitet sammenlignet med vildtypeenzymet.
12. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den ene eller de flere mutationer omfatter en mutation af D10-SpCas9, H840-SpCas9, N854-SpCas9 eller N863-SpCas9 eller tilsvarende rester i andre CRISPR-enzymer.
13. Sammensætning ifølge krav 12, hvor den ene eller de flere mutationer omfatter D10A, H840A, N854A eller N863A.
14. Sammensætning ifølge et hvilket som helst af krav 12 eller 13, hvor den ene eller de flere mutationer omfatter to mutationer.
15. Sammensætning ifølge krav 14, hvor mutationerne omfatter D10A-SpCas9 og H840A-SpCas9 eller tilsvarende rester i andre CRISPR-enzymer.
16. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet er et DNA-bindingsprotein, som ikke styrer spaltning af den ene eller den anden streng i placeringen af målsekvensen.
17. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den ene eller de flere mutationer er i et katalytisk aktivt domæne af CRISPR-enzymet, der omfatter RuvCI, RuvCII eller RuvCIII.
18. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet omfatter to eller flere heterologe, funktionelle domæner.
19. Sammensætning ifølge et hvilket som helst af ovennævnte krav, der omfatter mindst to eller flere NLS'er.
20. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den mindst ene eller de flere eller mindst to eller flere NLS'er er i eller tæt på enzymets aminoterminal og/eller i eller tæt på enzymets carboxyterminal.
21. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor et funktionelt domæne omfatter et transkriptionsaktivatordomæne.
22. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor et funktionelt domæne omfatterVP64.
23. Sammensætning ifølge et hvilket som helst af kravene 1 til 20, hvor et funktionelt domæne er et transkriptionsrepressordomæne.
24. Sammensætning ifølge krav 23, hvor det funktionelle domæne omfatter et KRAB-domæne eller et SID-domæne.
25. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den enzymkodende sekvens koder for to eller flere heterologe, funktionelle domæner.
26. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den enzymkodende sekvens koder for et eller flere heterologe, funktionelle domæner, der er fusioneret til CRISPR-enzymet.
27. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den enzymkodende sekvens yderligere omfatter en linkersekvens mellem hvilke som helst to domæner.
28. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor et eller flere funktionelle domæner har en eller flere af følgende aktiviteter: methylaseaktivitet, demethylaseaktivitet, transkriptionsaktiveringsaktivitet, transkriptionsrepressionsaktivitet, transkriptionsfrigøre1sesfaktoraktivitet, histonmodificeringsaktivitet, RNA-spaltningsaktivitet og nukleinsyrebindingaktivitet.
29. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor et funktionelt domæne binder DNA.
30. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor et funktionelt domæne påvirker transkriptionen af målnukleinsyren.
31. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor den eukaryote celle omfatter en mammaliacelle.
32. Sammensætning ifølge krav 31, hvor mammaliacellen omfatter en human celle.
33. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet er kodonoptimeret til ekspression i en eukaryot celle.
34. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet er et type II-Cas9-CRISPR-enzym.
35. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor Cas9-enzymet er fra en organisme, der er udvalgt fra gruppen, der omfatter af slægterne Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium og Corynebacter.
36. Sammensætning ifølge et hvilket som helst af kravene 1-34, hvor Cas9 er fra Staphylococcus aureus.
37. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor CRISPR-enzymet er et Cas9-enzym, som er et kimært Cas9-protein, der omfatter fragmenter fra forskellige Cas9-homologer.
38. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor målsekvensen grænser op til et Protospacer Adjacent Motif (PAM), der genkendes af CRISPR-enzymet.
39. Sammensætning ifølge et hvilket som helst af kravene 2 til 38, hvor det første og andet regulatorelement hvert omfatter en vævsspecifik promotor.
40. Sammensætning ifølge krav 39, hvor den vævsspecifikke promotor leder ekspression af CRISPR-transkripter til blod.
41. Sammensætning ifølge et hvilket som helst af ovennævnte krav, hvor tracr-sekvensen er 30 eller flere nukleotider lang.
42. Fremgangsmåde til modulering af genekspression i et genomisk locus af interesse i en celle i en eukaryot organisme ved etablering af kontakt mellem cellen og sammensætningen ifølge et hvilket som helst af ovennævnte krav, forudsat at organismen ikke er et menneske eller et dyr.
43. Ex vivo- eller in vi tro-fremgangsmåde til modulering af genekspression i et genomisk locus af interesse i en celle i en eukaryot organisme ved etablering af kontakt mellem cellen og sammensætningen ifølge et hvilket som helst af ovennævnte krav, forudsat at fremgangsmåden ikke omfatter en proces til modificering af den genetiske identitet af germlinjen hos et menneske.
44. Anvendelse af en sammensætning ifølge et hvilket som helst af kravene 1 til 41 til ex vivo- eller in vitro-qen-eller genomredigering, forudsat at redigeringen ikke omfatter en proces til modificering af den genetiske identitet af germlinjen hos et menneske.
45. Sammensætning ifølge et hvilket som helst af kravene 1 til 41 til anvendelse som et terapeutisk middel.
46. Sammensætning til anvendelse ifølge krav 45, hvor det terapeutiske middel er til gen- eller genomredigering eller til genterapi.
47. In vitro- eller ex vivo-eukaryot værtscelle, der omfatter en sammensætning ifølge et hvilket som helst af kravene 1 til 41, forudsat at værtscellen ikke er en human germlinjecelle.
48. In vitro- eller ex vivo-eukaryot værtscelle, der omfatter CRISPR-Cas-komplekset som defineret ifølge et hvilket som helst af kravene 1 til 41, forudsat at værtscellen ikke er en human germlinjecelle.
49. Afkom af værtscellen ifølge krav 47 eller 48, forudsat at afkommet ikke omfatter en menneskekrop.
Applications Claiming Priority (13)
Application Number | Priority Date | Filing Date | Title |
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US201261736527P | 2012-12-12 | 2012-12-12 | |
US201361748427P | 2013-01-02 | 2013-01-02 | |
US201361758468P | 2013-01-30 | 2013-01-30 | |
US201361769046P | 2013-02-25 | 2013-02-25 | |
US201361802174P | 2013-03-15 | 2013-03-15 | |
US201361791409P | 2013-03-15 | 2013-03-15 | |
US201361806375P | 2013-03-28 | 2013-03-28 | |
US201361814263P | 2013-04-20 | 2013-04-20 | |
US201361819803P | 2013-05-06 | 2013-05-06 | |
US201361828130P | 2013-05-28 | 2013-05-28 | |
US201361835936P | 2013-06-17 | 2013-06-17 | |
US201361835931P | 2013-06-17 | 2013-06-17 | |
PCT/US2013/074736 WO2014093655A2 (en) | 2012-12-12 | 2013-12-12 | Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains |
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DK2931898T3 true DK2931898T3 (da) | 2016-06-20 |
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US (3) | US8993233B2 (da) |
EP (1) | EP2931898B1 (da) |
DK (1) | DK2931898T3 (da) |
ES (1) | ES2576128T3 (da) |
PL (1) | PL2931898T3 (da) |
PT (1) | PT2931898E (da) |
WO (1) | WO2014093655A2 (da) |
Families Citing this family (518)
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WO2011002988A1 (en) | 2009-07-01 | 2011-01-06 | Transposagen Biopharmaceuticals, Inc. | Genetically modified rat models for severe combined immunodeficiency (scid) |
CA2853829C (en) | 2011-07-22 | 2023-09-26 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
GB201117313D0 (en) | 2011-10-07 | 2011-11-16 | Gt Biolog Ltd | Bacterium for use in medicine |
US11021737B2 (en) | 2011-12-22 | 2021-06-01 | President And Fellows Of Harvard College | Compositions and methods for analyte detection |
GB201122458D0 (en) | 2011-12-30 | 2012-02-08 | Univ Wageningen | Modified cascade ribonucleoproteins and uses thereof |
US9737480B2 (en) | 2012-02-06 | 2017-08-22 | President And Fellows Of Harvard College | ARRDC1-mediated microvesicles (ARMMs) and uses thereof |
IN2014DN09261A (da) | 2012-04-25 | 2015-07-10 | Regeneron Pharma | |
PE20190844A1 (es) | 2012-05-25 | 2019-06-17 | Emmanuelle Charpentier | Modulacion de transcripcion con arn de direccion a adn generico |
DK3444342T3 (da) | 2012-07-11 | 2020-08-24 | Sangamo Therapeutics Inc | Fremgangsmåder og sammensætninger til behandlingen af lysosomale aflejringssygdomme |
US10648001B2 (en) | 2012-07-11 | 2020-05-12 | Sangamo Therapeutics, Inc. | Method of treating mucopolysaccharidosis type I or II |
JP2015527889A (ja) * | 2012-07-25 | 2015-09-24 | ザ ブロード インスティテュート, インコーポレイテッド | 誘導可能なdna結合タンパク質およびゲノム撹乱ツール、ならびにそれらの適用 |
KR101656237B1 (ko) | 2012-10-23 | 2016-09-12 | 주식회사 툴젠 | 표적 DNA에 특이적인 가이드 RNA 및 Cas 단백질을 암호화하는 핵산 또는 Cas 단백질을 포함하는, 표적 DNA를 절단하기 위한 조성물 및 이의 용도 |
JP6502259B2 (ja) | 2012-11-16 | 2019-04-17 | トランスポサジェン バイオファーマシューティカルズ, インコーポレイテッド | 部位特異的酵素および使用方法 |
SG10202107423UA (en) * | 2012-12-06 | 2021-08-30 | Sigma Aldrich Co Llc | Crispr-based genome modification and regulation |
SG10201801969TA (en) | 2012-12-12 | 2018-04-27 | Broad Inst Inc | Engineering and Optimization of Improved Systems, Methods and Enzyme Compositions for Sequence Manipulation |
US8697359B1 (en) | 2012-12-12 | 2014-04-15 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
EP3252160B1 (en) | 2012-12-12 | 2020-10-28 | The Broad Institute, Inc. | Crispr-cas component systems, methods and compositions for sequence manipulation |
ES2883590T3 (es) | 2012-12-12 | 2021-12-09 | Broad Inst Inc | Suministro, modificación y optimización de sistemas, métodos y composiciones para la manipulación de secuencias y aplicaciones terapéuticas |
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WO2014093655A3 (en) | 2014-07-31 |
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EP2931898A2 (en) | 2015-10-21 |
US20140186958A1 (en) | 2014-07-03 |
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