Methods and compositions for modulating a genome

Patent metadata

Field	Value
Publication number	US-12565666-B2
Application number	US-202217929455-A
Country	US
Kind code	B2
Filing date	Sep 2, 2022
Priority date	Mar 4, 2020
Publication date	Mar 3, 2026
Grant date	Mar 3, 2026

Abstract

Methods and compositions for modulating a target genome are disclosed.

First claim

What is claimed is:

1 . A system for modifying DNA comprising: (a) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) an endonuclease domain, wherein the polypeptide comprises the amino acid sequence of SEO ID NO: 2044 or a sequence having at least 90% identity thereto; and (b) a template RNA or DNA encoding the template RNA comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence.

2 . The system of claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEO ID NO: 2044.

3 . The system of claim 1 , wherein the polypeptide comprises a nuclear localization signal (NLS).

4 . The system of claim 1 , wherein the nucleic acid encoding the polypeptide and the template RNA are two separate nucleic acids.

5 . The system of claim 1 , wherein (a) comprises RNA encoding the polypeptide and wherein (b) comprises template RNA.

6 . The system of claim 1 , wherein the nucleic encoding the polypeptide comprises a coding sequence that is codon-optimized for expression in human cells.

7 . The system of claim 1 , wherein the heterologous object sequence encodes a human polypeptide, or a fragment or variant thereof.

8 . The system of claim 1 , wherein the template RNA comprises the nucleic acid sequence of SEQ ID NO: 2042 or 2043, or a sequence having at least 80% identity thereto.

9 . The system of claim 1 , wherein the template RNA comprises a 5′ UTR sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 2042.

10 . The system of claim 1 , wherein the template RNA comprises a 3′ UTR sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 2043.

11 . The system of claim 1 , which is capable of inducing an insertion, deletion, or alteration of a protein coding sequence to a genome of a mammalian cell.

12 . The system of claim 11 , wherein the insertion is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.

13 . The system of claim 1 , which is capable of inducing an insertion, deletion, or alteration of a non-coding sequence to a genome of a mammalian cell.

14 . A method of modifying a target DNA strand in a cell, tissue, or subject, the method comprising administering the system of claim 1 to the cell, tissue, or subject, wherein the system reverse transcribes the template RNA sequence into the target DNA strand, thereby modifying the target DNA strand.

15 . The method of claim 14 , wherein the cell is:
(a) a human cell; (b) a primary cell; and/or (c) a T cell.

16 . A lipid nanoparticle (LNP) comprising the system of claim 1 .

17 . The system of claim 1 , wherein the heterologous object sequence comprises a regulatory sequence.

18 . The system of claim 17 , wherein the regulatory sequence:
(a) is a promoter; (b) is an enhancer; (c) is a binding site for an endogenous regulatory component; (d) is a miRNA binding site; or (e) alters the expression of an endogenous gene or non-coding RNA.

19 . The system of claim 1 , wherein the template RNA comprises:
(a) a polyA site; (b) a regulatory element of Woodchuck Hepatitis Virus (WPRE); and/or (c) a Kozak sequence.

Assignees

• Flagship Pioneering Innovations Vi Llc

Inventors

• Cleaver Stephen
• Steinberg Barrett Ethan
• Rubens Jacob Rosenblum
• Citorik Robert James
• Salomon William Edward
• Wang Zi Jun
• Cotta-Ramusino Cecilia Giovanna Silvia
• Li Donghui

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/1276

  RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title