Methods for correcting alpha-antitrypsin point mutations

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 nucleic acid encoding a mutant α-antitrypsin protein to correct a point mutation associated with a disease or disorder, e.g., with chronic obstructive pulmonary disease (COPD) disease. The methods provided are useful for correcting an α-antitrypsin point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Description

RELATED APPLICATION[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. provisional patent application, U.S. Ser. No. 61 / 915,386 filed Dec. 12, 2013, and U.S. provisional patent application, U.S. Ser. No. 61 / 980,333 filed Apr. 16, 2014, the entire contents of each of which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Targeted editing of nucleic acid sequences, for example, the introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases.1 An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency of installing the desired modification; (2) minimal off-target activity; and (3) the ability to be programmed to edit precisely any site in a given nucleic acid, e.g., any site within the human genome.2 Current genome engineering tools, including engineered zinc finger nucleases ...

AI Technical Summary

Benefits of technology

This patent describes a system for editing nucleic acids using a fusion protein of Cas9 and a nucleic acid editing enzyme. The system can be used to target specific sites in the genome and has been shown to have high efficiency and no stochasticity. The fusion protein can be designed to install a specific type of base modification at a precise location in genomic DNA with enzyme-like efficiency. The patent also provides strategies, systems, reagents, methods, and kits for targeted nucleic acid editing. Overall, this system offers a powerful new approach for gene editing-based research tools and human therapeutics.

Problems solved by technology

Significantly, 80-90% of protein mutations responsible for human disease arise from the substitution, deletion, or insertion of only a single nucleotide.6 No genome engineering tools, however, have yet been developed that enable the manipulation of a single nucleotide in a general and direct manner.

The resulting mismatch is recognized by the cell's endogenous repair system and fixed, leading to a change in the sequence of either the chimera or the genome.

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