Compositions and methods for treating hemoglobinopathies

Abstract

The present invention features compositions and methods for editing deleterious mutations associated with hemoglobinopathies, such as sickle cell disease (SCD). In particular embodiments, the invention provides methods for correcting mutations in a beta globin polynucleotide using modified adenosine base editors termed “ABE8” having unprecedented levels (e.g., >60-70%) of efficiency.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is the U.S. national phase application, pursuant to 35 U.S.C. § 371 of PCT International Application No. PCT / US2020 / 018193, filed Feb. 13, 2020, which claims priority to and benefit of U.S. Provisional Application Nos. 62 / 805,271 filed Feb. 13, 2019; 62 / 805,277, filed Feb. 13, 2019; 62 / 852,224, filed May 23, 2019; 62 / 852,228, filed May 23, 2019; 62 / 931,722, filed Nov. 6, 2019; 62 / 931,747, filed Nov. 6, 2019; 62 / 941,569, filed Nov. 27, 2019; and 62 / 966,526, filed Jan. 27, 2020, the contents of all of which are incorporated by reference herein in their entireties.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 29, 2020, is named 180802-042103PCTSequenceListing.txt and is 838,681 bytes in size.INCORPORATION BY REFERENCE[0003]All publications, patents, and ...

AI Technical Summary

Benefits of technology

[0006]As described below, the present invention features compositions and methods for editing deleterious mutations associated with sickle cell disease (SCD). In particular embodiments, the invention provides for the correction of SCD mutations using a modified adenosine deaminase base editor termed “ABE8” having unprecedented levels (e.g., >60-70%) of efficiency.

[0064]By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of a corresponding naturally-occurring polynucleotide or polypeptide while having certain modifications that enhance the analog's function relative to a naturally occurring polynucleotide or polypeptide. Such modifications could increase the analog's affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life, without altering, for example, ligand binding. An analog may include an unnatural nucleotide or amino acid.

[0130]The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). In some embodiments, the presently disclosed base editors can efficiently generate an “intended mutation,” such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor (e.g., an adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), specifically designed to generate the intended mutation.

[0132]The term “non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the wild-type protein.

Problems solved by technology

Vaso-occlusive events are associated with ischemia / reperfusion damage to tissues resulting in pain and acute or chronic injury affecting any organ system.

Consequently, Hb G-Makassar and HbS have been incorrectly identified and mistaken for each other by those skilled in the art, thus leading to misdiagnosis of Sickle Cell Disease (SCD).

Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyl adenine glycosylase (AAG)) can bind inosine but cannot create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage / repair mechanisms.

Until recently, all DNA editing platforms have functioned by inducing a DNA double strand break (DSB) at a specified genomic site and relying on endogenous DNA repair pathways to determine the product outcome in a semi-stochastic manner, resulting in complex populations of genetic products.

Though precise, user-defined repair outcomes can be achieved through the homology directed repair (HDR) pathway, a number of challenges have prevented high efficiency repair using HDR in therapeutically-relevant cell types.

In practice, this pathway is inefficient relative to the competing, error-prone non-homologous end joining pathway.

Further, HDR is tightly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells.

As a result, it has proven difficult or impossible to alter genomic sequences in a user-defined, programmable manner with high efficiencies in these populations.

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