JPWO2021113536A5

JPWO2021113536A5 -

Info

Publication number: JPWO2021113536A5
Application number: JP2022533105A
Authority: JP
Publication date: 2023-12-12

Claims

A lipid nanoparticle or microparticle for delivering a DNA targeting system to muscle cells, the DNA targeting system comprising:
at least one gRNA molecule that targets a fragment of a mutant dystrophin gene or a polynucleotide encoding said at least one gRNA molecule ; and/or
A lipid nanoparticle or microparticle comprising a Cas9 nuclease or a polynucleotide encoding said Cas9 nuclease.

The lipid nanoparticle or microparticle according to claim 1 , wherein the polynucleotide encoding the Cas9 nuclease is mRNA.

the at least one gRNA molecule comprises a first gRNA molecule and a second gRNA molecule;
the first gRNA molecule and the second gRNA molecule each include a targeting domain;
The first gRNA molecule is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, encoded by a polynucleotide comprising a nucleotide sequence selected from SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 83, or SEQ ID NO: 110, or a fragment or complementary strand thereof, or selected from SEQ ID NO: 112 to 124 nucleotide sequence or a fragment or complementary strand thereof;
The second gRNA molecule is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 84, or a fragment or complementary strand thereof;
the first gRNA molecule and the second gRNA molecule include different targeting domains;
Lipid nanoparticles or microparticles according to claim 1 .

the at least one gRNA or the polynucleotide encoding the at least one gRNA molecule and the Cas9 nuclease or the polynucleotide encoding the Cas9 nuclease are encapsulated within the same lipid nanoparticle or microparticle; or
The at least one gRNA or the polynucleotide encoding the at least one gRNA molecule and the Cas9 nuclease or the polynucleotide encoding the Cas9 nuclease are each encapsulated in separate lipid nanoparticles.
Lipid nanoparticles or microparticles according to claim 1 .

The lipid nanoparticles or microparticles include solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), lipid-drug conjugate (LDC) nanoparticles, lipid nanocapsules (LNCs), and polymer-lipid hybrid nanoparticles (PLNs). , and solid lipid microparticles ( SLMs ).

The at least one gRNA molecule is an exon selected from exons 1-8, 10, 11, 12, 14, 16-22, 43-59, and 61-66 of the mutant dystrophin gene, or The lipid nanoparticles of claim 1 or Microparticles.

The DNA targeting system further comprises a donor sequence comprising exons of the wild-type dystrophin gene or a functional equivalent thereof, wherein the exons include exons 1-8, 10, 11, 12, 14, Lipid nanoparticles or microparticles according to claim 1 , selected from 16-22, 43-59, and 61-66.

The DNA targeting system generates a first double-strand break in a first intron adjacent to exon 51 of the human dystrophin gene and a second double-strand break in a second intron adjacent to exon 51 of the human dystrophin gene. The lipid nanoparticle or microparticle according to claim 1 , which induces the following.

Lipid nanoparticles or microparticles according to claim 2 , wherein the mRNA is a modified mRNA comprising one or more modifications selected from: N-terminal NLS, C-terminal NLS, HA tag, and uridine substitution .

Lipid nanoparticles or microparticles according to claim 1 , wherein the muscle cells are selected from skeletal muscle cells, cardiomyocytes, and smooth muscle cells.

A composition comprising a lipid nanoparticle or microparticle according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

A composition for use in the treatment of Duchenne muscular dystrophy in a subject , comprising a lipid nanoparticle or microparticle according to any one of claims 1 to 10 .

13. The composition of claim 12 , wherein the subject experiences no or a limited humoral response cross-reactive with the Cas9 nuclease after administration of the composition .

A composition for use in genome editing of a mutant dystrophin gene in a subject , comprising the lipid nanoparticle or microparticle according to any one of claims 1 to 10 .

Genome editing of the mutant dystrophin gene includes removing a premature stop codon, correcting a disrupted reading frame, regulating splicing by destroying a splice acceptor site, regulating splicing by destroying a splice donor sequence, removing exon 51, or a combination thereof.

Dystrophin expression in the subject increases by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% after editing. A composition according to claim 14 .

15. The composition of claim 14 , wherein the lipid nanoparticles or microparticles are administered to the subject before or within 1-2 days after birth.

15. The composition of claim 14 , wherein the lipid nanoparticles or microparticles are administered to the subject intramuscularly, intravenously, or a combination thereof.

15. The composition of claim 14 , wherein administration of the composition results in expression of a functional or partially functional dystrophin protein in the subject.

A kit comprising a lipid nanoparticle or microparticle according to any one of claims 1 to 10 .