Optimized mRNA encoding CAS9 for use in LNPs
Inventors
Cheng, Christopher J. • Scharenberg, Andrew • Wang, Kui • SANE, Shailendra
Assignees
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Publication Number
US-12263227-B2
Publication Date
2025-04-01
Expiration Date
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.
Core Innovation
The invention relates to a lipid-based nanoparticle (LNP) composition that includes a nucleic acid molecule encoding a synthetic RNA-guided endonuclease. The nucleic acid molecule is 3.8 kb or less in length, and the synthetic RNA-guided endonuclease comprises amino acid sequences of SEQ ID NO: 1 through SEQ ID NO: 5 or sequences having at least 95% sequence identity to SEQ ID NO: 1 through SEQ ID NO: 5.
The LNP composition includes one or more lipid moieties selected from amino lipids, ionizable lipids, neutral lipids, PEG lipids, helper lipids, and cholesterol or cholesterol derivatives. The composition can further include guide RNA or a nucleic acid molecule that encodes the guide RNA, and the nucleic acid molecule can include UTR, Kozak signal, NLS, codon optimization, sequence optimization, and base modifications.
The document describes base modifications including pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5-methyl-cytidine. The invention is described as using short nucleic acids encoding synthetic RNA-guided endonucleases in LNPs to achieve improved LNP physicochemical and functional performance characteristics.
The document also describes comparative effects versus reference LNPs carrying longer (>4 kb) SpCas9 mRNA, including slower physicochemical and stability degradation, slower functional performance decline, larger average particle diameter, improved genome editing efficiency, and improved stability after storage at about 2–8°C for about 7–9 days with reduced editing-efficiency drop.
Claims Coverage
The consolidated claim coverage identifies one main inventive feature set centered on an LNP composition comprising a nucleic acid molecule encoding a synthetic RNA-guided endonuclease, with a 3.8 kb or less length constraint and selected lipid moieties. Additional dependent features include guide RNA inclusion, stability and particle-size limitations, and comparative performance versus a reference SpCas9 LNP.
Short nucleotide sequence encoding a synthetic RNA-guided endonuclease
A nucleic acid molecule comprising a nucleotide sequence encoding a synthetic RNA-guided endonuclease, wherein the nucleic acid molecule is 3.8 kb or less in length.
Specified synthetic endonuclease amino acid sequences or high-identity variants
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.
Lipid moieties selected from an LNP lipid set
One or more lipid moieties selected from amino lipids, ionizable lipids, neutral lipids, PEG lipids, helper lipids, and cholesterol or cholesterol derivatives.
Guide RNA inclusion
An LNP composition further provided with a guide RNA or a nucleic acid molecule that encodes the guide RNA.
Stability and functional-performance comparison versus a reference SpCas9 LNP
An LNP composition having improved stability characteristics, reduced decline in functional performance, and/or a larger average particle diameter versus a reference LNP composition that includes an SpCas9-encoding nucleic acid.
Specific named lipid components in the LNP
The lipid nanoparticle composition includes C12-200, cholesterol, DOPE, and/or PEG-DMPE.
The claim coverage centers on an LNP composition defined by a 3.8 kb or less nucleic acid encoding a synthetic RNA-guided endonuclease with SEQ ID NOs 1–5 or at least 95% identity variants, together with selected lipid moieties. The merged claim features also include guide RNA inclusion, specified lipid components, and comparative stability, functional performance, and particle-size limitations versus a reference SpCas9 LNP.
Stated Advantages
Improved LNP physicochemical and stability characteristics versus reference LNP compositions carrying longer (>4 kb) SpCas9 mRNA.
Slower functional performance decline versus reference LNP compositions carrying longer (>4 kb) SpCas9 mRNA.
Larger average particle diameter versus a reference LNP composition carrying an SpCas9-encoding nucleic acid.
Improved genome editing efficiency versus reference LNPs carrying longer SpCas9 mRNA.
Improved stability after storage at about 2–8°C for about 7–9 days with reduced editing-efficiency drop.
Documented Applications
In vivo genome editing targeting specific loci, including the albumin locus.
Treatment of diseases described in the document, including hemophilia A (FVIII) in the context of targeting at an albumin locus.
Treatment of cardiovascular disease described in the document, including targeting at an albumin locus for LPA/apo(a) and discussion of ApoE targeting.
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