Optimized mRNA encoding CAS9 for use in LNPs

Patent metadata

Field	Value
Publication number	US-12263227-B2
Application number	US-201917298114-A
Country	US
Kind code	B2
Filing date	Nov 26, 2019
Priority date	Nov 28, 2018
Publication date	Apr 1, 2025
Grant date	Apr 1, 2025

Abstract

The present disclosure relates generally to novel lipid nanoparticle (LNP)-based compositions useful for, e.g., the delivery of a site-specific endonuclease or a nucleic acid molecule encoding same, into a target cell. Some embodiments of the disclosure relate to compositions and methods for editing the genome of a cell, which involve contacting the cell with an LNP composition as described herein.

First claim

What is claimed is:

1. A lipid-based nanoparticle (LNP) composition comprising: a nucleic acid molecule comprising a nucleotide sequence encoding a synthetic RNA-guided endonuclease; and one or more lipid moieties selected from the group consisting of amino lipids, ionizable lipids, neutral lipids, PEG lipids, helper lipids, and cholesterol or cholesterol derivatives, wherein the nucleic acid molecule is 3.8 kb or less in length, and wherein the synthetic RNA-guided endonuclease comprises amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO 5, or a sequence having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO 5.

2. The LNP composition of claim 1 , wherein the nucleic acid molecule is 3.7 kb or less in length.

3. The LNP composition of claim 1 , wherein the nucleic acid molecule is 3.5 kb or less in length.

4. The LNP composition of claim 1 , wherein the nucleic acid molecule is a messenger RNA (mRNA).

5. The LNP composition of claim 1 , wherein the nucleotide sequence encoding the synthetic RNA-guided endonuclease is operably linked to at least one additional nucleotide sequence.

6. The LNP composition of claim 5 , wherein the at least one additional nucleotide sequence comprises an untranslated terminal region (UTR), a consensus Kozak signal, a nucleotide sequence encoding a nuclear localization signal (NLS) chosen from a nucleoplasm NLS or a SV40 NLS, a nucleotide sequence encoding a linker peptide, a nucleotide sequence encoding a tag peptide, or a combination thereof.

7. The LNP composition of claim 1 , wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

8. The LNP composition of claim 1 , wherein the nucleotide sequence is codon-optimized for expression in a host cell.

9. The LNP composition of claim 8 , wherein the host cell is a mammalian cell.

10. The LNP composition of claim 9 , wherein the mammalian cell is a human cell, a murine cell, or a non-human primate (NHP) cell.

11. The LNP composition of claim 1 , further comprising a guide RNA (gRNA) or a nucleic acid molecule encoding the gRNA.

12. The LNP composition of claim 1 , wherein the LNP composition comprises C12-200, cholesterol, DOPE, PEG-DMPE, or a combination thereof.

13. The LNP composition of claim 1 , wherein
(a) the LNP composition has a lower rate of change in stability as compared to a reference LNP composition comprising a reference nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided endonuclease, wherein the reference nucleic acid molecule is SpCas9; or
(b) the LNP composition has a lower rate of decrease in functional performance as compared to that of a reference LNP composition comprising a reference nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided endonuclease, wherein the reference nucleic acid molecule is SpCas9; or
(c) the LNP composition has an average particle diameter larger than that of a reference LNP composition comprising a reference nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided endonuclease, wherein the reference nucleic acid molecule is SpCas9; or
(d) a combination of (a), (b), and (c).

14. The LNP composition of claim 13 , wherein
(e) the LNP composition has a rate of change in stability that is at least 5% less than the corresponding rate of the reference LNP composition; or
(f) the LNP composition has a rate of decrease in functional performance that is at least 5% less than the corresponding rate of the reference LNP composition; or
(g) the LNP composition has an average particle diameter that is at least 10% larger than the average particle diameter of the reference LNP composition; or
(h) a combination of (a), (b), and (c).

15. A method for delivering a nucleic acid molecule into a cell in vitro, comprising contacting the cell in vitro with an LNP composition according to claim 1 .

16. A method for editing a genome of a cell in vitro, comprising providing to the cell in vitro, an LNP composition according to claim 1 .

17. The LNP composition of claim 1 , wherein the synthetic RNA guided endonuclease comprises fragments from RNA-guided endonucleases obtained from at least two bacterial species.

18. The LNP composition of claim 17 , wherein the bacterial species are selected from the group consisting of Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus microti and Staphylococcus hyicus.

Assignees

• Crispr Therapeutics Ag
• Bayer Healthcare Llc

Inventors

• Cheng Christopher J
• Scharenberg Andrew
• Wang Kui
• Sane Shailendra

Classifications

• C12N2320/32

  Special delivery means, e.g. tissue-specific · CPC title

• C12N15/88

  using microencapsulation, e.g. using {amphiphile} liposome vesicle · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N9/22 Primary

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

• C12N2310/20

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