Nucleobase editors comprising nucleic acid programmable DNA binding proteins

A technology for binding proteins and fusion proteins, used in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc.

Pending Publication Date: 2020-03-24
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] A disadvantage of current techniques is that both NHEJ and HDR are stochastic processes, which often result in modest gene editing efficiencies and unwanted genetic changes that can compete with desired changes 8

Method used

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  • Nucleobase editors comprising nucleic acid programmable DNA binding proteins
  • Nucleobase editors comprising nucleic acid programmable DNA binding proteins
  • Nucleobase editors comprising nucleic acid programmable DNA binding proteins

Examples

Experimental program
Comparison scheme
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preparation example Construction

[0650] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or later developed in the art of pharmacology. Generally, such preparation methods comprise the steps of bringing into association one or more active ingredients with excipients and / or one or more other auxiliary ingredients, then, if necessary and / or desired, shaping the product and / or Or packaged as desired single-dose or multi-dose units.

[0651] The pharmaceutical formulation may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, Thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21 st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimo...

Embodiment 1

[0659] Embodiment 1: Cas9 deaminase fusion protein

[0660] A number of Cas9:deaminase fusion proteins were generated and the resulting fusions were characterized for their deaminase activity. The following deaminases were tested:

[0661] Human AID (hAID):

[0662] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSLSRAFQLLRILLLR(

[0663] Human AID-DC (hAID-DC, a truncated form of hAID with 7-fold increased activity):

[0664] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLR:(0) IDSLLM

[0665] Rat APOBEC1 (rAPOBEC1):

[0666] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQL...

Embodiment 2

[0699] Example 2: Deamination of DNA Target Sequences

[0700] Exemplary deamination targets. The dCas9:deaminase fusion proteins described herein can be delivered to a cell in vitro or ex vivo or to a subject in vivo, and when the target nucleotide is in position 3-11 with respect to the PAM, can be used to achieve C to T or G to A transition. Exemplary deamination targets include, but are not limited to, the following: CCR5 truncation: Any codon encoding Q93, Q102, Q186, R225, W86, or Q261 of CCR5 can be deaminated to generate a stop codon, which results in CCR5 The non-functional truncation of , is used in HIV treatment. APOE4 Mutations: The mutant codons encoding the C11R and C57R mutant APOE4 proteins can be deaminated to revert to wild-type amino acids, which find application in the treatment of Alzheimer's disease. eGFP truncation: Any codon encoding Q158, Q184, Q185 can be deaminated to generate a stop codon, or the codon encoding M1 can be deaminated to encode I,...

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PUM

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Abstract

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome ofa cell or subject, e. g., within the human genome. In some embodiments, fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e. g., Cpf 1 or variants thereof, and nucleic acid editing proteins or protein domains, e. g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e. g., fusion proteins of a napDNAbp (e. g., CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute) and nucleic acid editing proteins or domains, are provided.

Description

[0001] Background of the invention [0002] Targeted editing of nucleic acid sequences, such as targeted cleavage of genomic DNA or targeted introduction of specific modifications to genomic DNA is a very promising approach to study gene function and also has the potential to provide new treatments for human genetic diseases 1 . An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency of installing desired modifications; (2) minimal off-target activity; and (3) can be programmed to precisely edit any site in a given nucleic acid, such as the human genome capability at any site within the 2 . Current genome engineering tools, including engineered zinc finger nucleases (ZFNs) 3 , Transcription activator-like effector nuclease (TALEN) 4 , and more recently the RNA-guided DNA endonuclease Cas9 5 Achieve sequence-specific DNA cleavage in the genome. This programmable cleavage can result in DNA mutation at the cleavage site by non-homologous ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
CPCC12N15/102C12N15/111C12N15/62C12N2310/20A61K38/00C12N9/22C12N9/78Y02A50/30C12N15/11C12N2800/80
Inventor D.R.刘A.C.科摩L.陈H.A.里斯
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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