PAH-modulating compositions and methods

Patent metadata

Field	Value
Publication number	US-12544458-B2
Application number	US-202318495276-A
Country	US
Kind code	B2
Filing date	Oct 26, 2023
Priority date	Sep 8, 2021
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

Abstract

The disclosure provides, e.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject. Gene modifying systems for treating phenylketonuria (PKU) are described.

First claim

The invention claimed is:

1 . A template RNA comprising, from 5′ to 3′:
a) a gRNA spacer that is complementary to a first portion of a human PAH gene, wherein the gRNA spacer comprises an RNA sequence of nucleotides 1 to 20 of SEQ ID NO: 37,221;
b) a gRNA scaffold that binds a SpyCas9 domain;
c) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, wherein the heterologous object sequence comprises a nucleotide sequence of nucleotides 97 to 105 of SEQ ID NO: 37,221; and
d) a primer binding site (PBS) sequence comprising at least 3 bases with 100% identity to a third portion of the human PAH gene, wherein the PBS sequence comprises a nucleotide sequence of nucleotides 106 to 116 of SEQ ID NO: 37,221.

2 . The template RNA of claim 1 , wherein the mutation to be corrected in the human PAH gene is R408W.

3 . The template RNA of claim 1 , wherein the gRNA spacer has a length of 20 nucleotides.

4 . The template RNA of claim 1 , wherein the heterologous object sequence has a length of 9-16 nucleotides.

5 . The template RNA of claim 1 , wherein the heterologous object sequence comprises, from 5′ to 3′, a post-edit homology region, a mutation region, and a pre-edit homology region.

6 . The template RNA of claim 1 , wherein the heterologous object sequence consists of an RNA sequence of GGGCCGAGG.

7 . The template RNA of claim 1 , wherein the PBS sequence has a length of 11-12 nucleotides.

8 . The template RNA of claim 1 , wherein the PBS sequence consists of an RNA sequence of 106 to 116 of SEO ID NO: 37,221.

9 . The template RNA of claim 1 , wherein the gRNA scaffold comprises an RNA sequence having at least 90% identity to nucleotides 21 to 96 of SEQ ID NO: 37,221.

10 . The template RNA of claim 1 , wherein the gRNA scaffold comprises an RNA sequence of nucleotides 21 to 96 of SEQ ID NO: 37,221.

11 . The template RNA of claim 1 , which comprises an RNA sequence having at least 90% identity to SEQ ID NO: 37,221.

12 . The template RNA of claim 1 , which comprises an RNA sequence of SEQ ID NO: 37,221.

13 . The template RNA of claim 1 , which comprises one or more chemically modified nucleotides.

14 . The template RNA of claim 13 , which comprises the RNA sequence and chemical modifications of SEQ ID NO: 30,500.

15 . A gene modifying system comprising:
a template RNA of claim 1 , and a gene modifying polypeptide comprising a SpyCas9 nickase domain, or a nucleic acid encoding the gene modifying polypeptide.

16 . The gene modifying system of claim 15 , which comprises the nucleic acid encoding the gene modifying polypeptide, wherein the nucleic acid comprises RNA.

17 . The gene modifying system of claim 15 , wherein the gene modifying polypeptide comprises:
a reverse transcriptase (RT) domain; a SpyCas9 nickase domain; and a linker disposed between the RT domain and the SpyCas9 nickase domain.

18 . The gene modifying system of claim 17 , wherein the RT domain is an RT domain from a murine leukemia virus (MMLV), a porcine endogenous retrovirus (PERV), an Avian reticuloendotheliosis virus (AVIRE), a feline leukemia virus (FLV), a simian foamy virus (SFV), a bovine leukemia virus (BLV), a Mason-Pfizer monkey virus (MPMV), a human foamy virus (HFV), or a bovine foamy/syncytial virus (BFV/BSV).

19 . The gene modifying system of claim 18 , wherein the simian foamy virus is a SFV3L.

20 . The gene modifying system of claim 17 , which further comprises a second strand-targeting gRNA spacer that directs a second nick to a second strand of the human PAH gene.

21 . A pharmaceutical composition, comprising the gene modifying system of claim 15 and a pharmaceutically acceptable excipient or carrier.

22 . The pharmaceutical composition of claim 21 , wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.

23 . A method for modifying a target site in the human PAH gene in a cell, the method comprising contacting the cell with the gene modifying system of claim 15 , or DNA encoding the same, thereby modifying the target site in the human PAH gene in a cell.

24 . A method for treating a subject having phenylketonuria (PKU) or hyperphenylalaninemia, the method comprising administering to the subject the gene modifying system of claim 15 thereby treating the subject havingPKU or hyperphenylalaninemia.

25 . A method of making the template RNA of claim 1 , the method comprising synthesizing the template RNA by in vitro transcription, solid-phase synthesis, or by introducing a DNA encoding the template RNA into a host cell under conditions that allow for production of the template RNA.

26 . A template RNA comprising a sequence having at least 90% identity to SEQ ID NO: 37221.

27 . The template RNA of claim 26 , which comprises a sequence having at least 95% identity to SEQ ID NO: 37221.

28 . The template RNA of claim 26 , which comprises a sequence having at least 99% identity to SEQ ID NO: 37221.

Assignees

• Flagship Pioneering Innovations Vi Llc

Inventors

• Altshuler Robert Charles
• Bothmer Anne Helen
• Chee Daniel Raymond
• Cotta-Ramusino Cecilia Giovanna Silvia
• Kim Kyusik
• Kotlar Randi Michelle
• Mcallister Gregory David
• Ray Ananya
• Roquet Nathaniel
• Sanchez Carlos
• Steinberg Barrett Ethan
• Salomon William Edward
• Citorik Robert James
• Querbes William
• Apponi Luciano Henrique
• Wang Zhan

Classifications

• 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

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• C07K2319/09

  containing a nuclear localisation signal · CPC title

• C12N2310/20

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