DK2650366T3 - Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet - Google Patents
Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet Download PDFInfo
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- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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Claims (14)
1
1. Fremgangsmåde tsi at spaite en dobbeitstrenget DNA~ måigenkendelsessekvens i en eukaryot celle, hvilken fremgangsmåde omfatter: at indføre i den nævnte eukaryote celle 5 en nucleinsyre, der koder en meganuciease; eller et meganucleaseprotein; hvilken nævnte meganuciease spalter den nævnte dobbeltstrengede DNA-målgenkendelsessekvens; og hvilken meganucleasen er en rekombinant meganuciease, der omfatter: 10 et polypeptid, der har mindst 85 % sekvenslighed med grupperne 2-153 af l-Crel-meganucleasen med SEQ ID NO: 1; og som har en specificitet for et genkendelsessekvens-halv-site, der afviger med mindst ét basepar fra et halv-sste inden en l-Grel· meganuciease genkendelsessekvens, som er valgt fra gruppen beståen-15 de af SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 og SEQ ID NO: 5, hvilken specificitet i position -1 er blevet ændret: (i) til et A ved en modifikation, der er valgt fra gruppen bestående af D75C, D75L, D75Y, K139Y, T46A, og T46C; (ii) til et G ved en modifikation, der er valgt fra gruppen bestående af 20 T46E, T46D, og D75E; eller (iii) til et T ved en modifikation, der er valgt fra gruppen bestående af D75H, D75Q, D75Y, K139H, T46H, og T46Q; (iv) til enhver base på en sensestreng ved en T46G modifikation; med det forbehold, at fremgangsmåden ikke er en fremgangsmåde til 25 behandling af en menneske- eller dyrekrop ved terapi; med det forbehold, at fremgangsmåden ikke er en fremgangsmåde til modificering af kønscellers genetiske identitet af mennesker og med det yderligere forbehold, at cellen ikke er en human embryonisk stamcelle eller human blastocyst-celle. 30
2. Fremgangsmåden ifølge krav 1, hvor den rekombinante meganuclease yderligere omfatter mindst en anden specificitet-ændrende aminosyresubstitution, der svarer til en substitution, som er valgt fra gruppen bestående af: I24C, I24K, I24R, Q28A, Q26E. Q26K, Q26S, K28A, K28C, K28H, K28Q, K28R, K28S, N30E, N30K, N30Q, N30R. S32A, S32C. S32D. S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E. Y33F, Y33H, Y33L Y33L, Y33R. Y33V, Q38C, Q38E. Q38H. Q38L Q38K, Q38L, Q38N. Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D. Q44E, Q44I, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, T46A, T46C, T46D. T46E, T46G, T46H, T46K, T46Q, T46R, Y66K, Y66M, R68C, R68E, R68F, R68H, R68K, R68L R68M, R68Q, R68Y, R70A, R70G, R70D, R70E, R70G, R70H, R70K, R7QL, R70Q, R70S. D75C, D75E, D75H. D75L D75G, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79G, S79S, S79V, K139H og K139Y.
3. Fremgangsmåde ifølge ethvert af de foregående krav, hvor den dobbeltstrengede DNA-målgenkendelsessekvens er omfattet i et kromosom i den eukaryote celle.
4. Fremgangsmåden ifølge krav 3, hvilken fremgangsmåde yderligere omfatter: at transfektere den nævnte eukaryote celle med en nucleinsyresekvens, der indbefatter en eksogen sekvens, hvilken nævnte exogene sekvens inserteres i kromosomet ved spaltningsstedet for derved at frembringe en genetisk modificeret eukaryot celle, der indbefatter den exogene sekvens, som inserteres i det nævnte kromosom af den nævnte eukaryote celle, eventuelt hvilken nævnte nucleinsyre yderligere omfatter sekvenser, der er homologe med sekvenser, som flankerer spaltningsstedet, og hvilken nævnte exogene sekvens inserteres ved det nævnte spaltningssted ved homolog rekombination, eller hvilken nucleinsyre mangler væsentlig homolog! med spaltningsstedet og den nævnte exogene sekvens inserteres i det nævnte kromosom ved ik-ke-homolog forbindelse af enderne.
5. Fremgangsmåden ifølge krav 3, hvor den nævnte målsekvens afbrydes ved ikke-homolog forbindelse af enderne på spaltningsstedet for derved at frembringe en genetisk modificeret eukaryot celle ved at afbryde målsekvensen i det nævnte kromosom af den nævnte eukaryote celle. β. Fremgangsmåde ifølge ethvert foregående krav, hvor den nævnte ceile er en plantecelle.
7. Fremgangsmåden ifølge krav 8, hvor den nævnte nucieinsyre indføres I den nævnte celle ved en Agrobacteriurn-vektor, ballistisk injektion, mikroprojektil-bombardement eller elektroporering,
8. Fremgangsmåden ifølge krav 6, hvor den nævnte celle er en protoplast og den nævnte nucieinsyre indføres i den nævnte celle ved PEG-medieret transformation.
9. Fremgangsmåde ifølge et hvilket som helst af kravene 1-5, hvor den nævnte celle er en pattedyrcelle.
10. Fremgangsmåden ifølge krav 9, hvor den nævnte nucieinsyre indføres i den nævnte celle ved en adenovirus, en adeno-associeret virusvektor, ballistisk injektion, elektroporation, mikroinjektion eiler liposomtransfektion.
11. Fremgangsmåde ifølge et hvilket som helst af kravene 1-5 eller 9-10, hvor cellen er valgt fra gruppen bestående af en gamet, en zygot, en blastocyst celle, en embryonisk stamcelle, og en protoplast-celle, med det forbehold, at fremgangsmåden ikke er en fremgangsmåde til behandling af en menneske- eller dyrekrop ved terapi; med det forbehold, at fremgangsmåden ikke er en fremgangsmåde til modificering af kønscellers genetiske identitet af mennesker og med det yderligere forbehold, at cellen ikke er en human embryonisk stamcelle eller human blastocyst celle.
12. Rekombinant meganuclease, der omfatter: et polypeptid, som har mindst 85 % sekvenslighed med grupperne 2-153 af l-Grel-meganucieasen med SEG ID NO: 1; og som har specificitet for et genkendelsessekvens-haiv-site, der afviger med mindst ét basepar fra et halv-site inden en l-Crel-meganudease-genkendelsessekvens, som er valgt fra gruppen bestående af SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 og SEQ ID NO: 5, hvilken specificitet i position -1 er blevet ændret: (i) til et A ved en modifikation, der er valgt fra gruppen bestående af D75C, D75L, D75Y, K139Y, T46A, og T46C; (ii) til et G ved en modifikation, der er valgt fra gruppen bestående af T46E, T46D, og D75E; eller (iii) til et T ved en modifikation, der er valgt fra gruppen bestående af D75H, D75Q, D75Y, K139H, T48H, og T46Q; (iv) til enhver base på en sense-streng ved en T46G modifikation.
13. Rekombinant meganudease ifølge krav 12 til anvendelse i genterapi eller til behandling af patogene infektioner.
14. Anvendelse af den rekombinante meganudease ifølge krav 12 til fremstilling af et medikament til anvendelse i genterapi eller til behandling af patogene infektioner.
15. Anvendelse af den rekombinante meganudease ifølge krav 12 i in vitro applikationer i diagnostik og i forskning.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US72751205P | 2005-10-18 | 2005-10-18 | |
| EP06826293.0A EP1945762B1 (en) | 2005-10-18 | 2006-10-18 | Rationally-designed meganucleases with altered sequence specificity and dna-binding affinity |
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| Publication Number | Publication Date |
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| DK2650366T3 true DK2650366T3 (da) | 2017-07-10 |
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| Application Number | Title | Priority Date | Filing Date |
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| DK12162075.1T DK2484758T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK13164623.4T DK2662442T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt designet meganuklease med ændret dimerdannelsesaffinitet |
| DK06826293.0T DK1945762T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK10191904T DK2360244T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganukleaser med ændret specificitet og DNA-bindingsaffinitet |
| DK13164585.5T DK2650366T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK17161899.4T DK3246403T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt designede meganucleaser med ændret sekvens-specificitet og dna-bindende affinitet |
| DK10191888.6T DK2357226T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt udformede meganukleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK13164564.0T DK2650365T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganukleaser med ændret sekvensspecificitet og dna-bindingsaffinitet |
| DK13164628.3T DK2628794T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganukleaser med ændret sekvensspecificitet og dna-bindingsaffinitet |
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| DK12162075.1T DK2484758T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK13164623.4T DK2662442T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt designet meganuklease med ændret dimerdannelsesaffinitet |
| DK06826293.0T DK1945762T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganucleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK10191904T DK2360244T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganukleaser med ændret specificitet og DNA-bindingsaffinitet |
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| DK17161899.4T DK3246403T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt designede meganucleaser med ændret sekvens-specificitet og dna-bindende affinitet |
| DK10191888.6T DK2357226T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt udformede meganukleaser med ændret sekvensspecificitet og DNA-bindingsaffinitet |
| DK13164564.0T DK2650365T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganukleaser med ændret sekvensspecificitet og dna-bindingsaffinitet |
| DK13164628.3T DK2628794T3 (da) | 2005-10-18 | 2006-10-18 | Rationelt konstruerede meganukleaser med ændret sekvensspecificitet og dna-bindingsaffinitet |
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| US (19) | US8021867B2 (da) |
| EP (13) | EP2357226B1 (da) |
| JP (9) | JP5937292B2 (da) |
| AT (1) | ATE549401T1 (da) |
| AU (1) | AU2006304668B2 (da) |
| CA (3) | CA2626262C (da) |
| DK (9) | DK2484758T3 (da) |
| ES (9) | ES2533370T3 (da) |
| WO (1) | WO2007047859A2 (da) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE60316124T3 (de) † | 2002-03-15 | 2018-03-22 | Cellectis | Hybride and einzelkettige meganukleasen und deren anwendungen |
| WO2009095742A1 (en) | 2008-01-31 | 2009-08-06 | Cellectis | New i-crei derived single-chain meganuclease and uses thereof |
| AU2004208031B2 (en) * | 2003-01-28 | 2009-10-08 | Cellectis | Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof. |
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| WO2007060495A1 (en) † | 2005-10-25 | 2007-05-31 | Cellectis | I-crei homing endonuclease variants having novel cleavage specificity and use thereof |
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| WO2009013559A1 (en) * | 2007-07-23 | 2009-01-29 | Cellectis | Meganuclease variants cleaving a dna target sequence from the human hemoglobin beta gene and uses thereof |
| WO2009019528A1 (en) * | 2007-08-03 | 2009-02-12 | Cellectis | Meganuclease variants cleaving a dna target sequence from the human interleukin-2 receptor gamma chain gene and uses thereof |
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| DK2215223T3 (da) | 2007-10-31 | 2013-07-22 | Prec Biosciences Inc | Rationelt konstruerede enkeltkæde-meganucleaser med ikke-palindrome genkendelsessekvenser |
| US20110041194A1 (en) * | 2007-11-28 | 2011-02-17 | Cellectis | I-msoi homing endonuclease variants having novel substrate specificity and use thereof |
| JP2011505809A (ja) * | 2007-12-07 | 2011-03-03 | プレシジョン バイオサイエンシズ，インク． | ヒトゲノムのＤＮａｓｅ高感受性領域に見出される認識配列を有する合理的に設計されたメガヌクレアーゼ |
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| WO2009114321A2 (en) | 2008-03-11 | 2009-09-17 | Precision Biosciencs, Inc. | Rationally-designed meganucleases for maize genome engineering |
| US20100071083A1 (en) * | 2008-03-12 | 2010-03-18 | Smith James J | Temperature-dependent meganuclease activity |
| WO2009131632A1 (en) | 2008-04-14 | 2009-10-29 | Sangamo Biosciences, Inc. | Linear donor constructs for targeted integration |
| CA2722797A1 (en) * | 2008-04-28 | 2009-11-05 | Precision Biosciences, Inc. | Fusion molecules of rationally-designed dna-binding proteins and effector domains |
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