Base editing of ANGPTL3 and methods of using same for treatment of disease
Inventors
Chadwick, Alexandra • Rajeev, Kallanthottathil G. • Rohde, Ellen • Cheng, Christopher • Reiss, Caroline
Assignees
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Publication Number
US-12115230-B2
Publication Date
2024-10-15
Expiration Date
Abstract
Provided herein are compositions for gene modification or editing and methods of using same to treat or prevent certain conditions. Specific compositions and methods capable of safely and effectively editing gene targets expressed in the liver to durably lower LDL-C thereby treating a leading cause of cardiovascular disease are disclosed.
Core Innovation
The invention relates to compositions and in vivo therapeutic regimens for editing gene targets using lipid nanoparticles that deliver a base editor fusion protein, or an mRNA encoding the base editor fusion protein, together with a guide RNA. The base editor protein comprises a DNA binding domain and a deaminase, and the guide RNA comprises a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence corresponding to a protospacer on ANGPTL3.
The disclosed subject matter describes chemically modified gRNAs and sgRNAs and guide RNA/base-editor fusion protein complexes that increase cellular half-life and provide resistance to degradation. Gene editing is directed to ANGPTL3 and also references PCSK9, with additional discussion of APOC3 and LPA, and includes splice-site base editing and A·T to G·C alterations that disrupt splicing, aberrant transcripts, and functional protein expression.
The disclosure further describes nucleobase alteration outcomes, including frame shift, premature stop codon, insertion or deletion, and aberrant transcript effects, together with constraints on editing outside the protospacer and off-target control. It also describes lipid nanoparticle formulations containing amino or ionizable lipids, PEG-lipids, cholesterol, and phospholipids, and presents these compositions as intended for therapeutic lipid-related outcomes and methods of treatment.
Claims Coverage
The consolidated independent claims are directed to a gene-editing composition comprising an mRNA encoding a base editor protein and a guide RNA with a tracr scaffold and an ANGPTL3-targeting spacer. Across the claims, the principal inventive features are the sequence-identity constrained base editor mRNA and the tracr/scaffold plus ANGPTL3 protospacer guide RNA.
Sequence-identity constrained base editor mRNA for ANGPTL3 editing
An mRNA encoding a base editor protein comprising a DNA binding domain and a deaminase, wherein the mRNA comprises a sequence having at least 95% sequence identity to SEQ ID NO: 2192, or to the coding sequence of SEQ ID NO: 2192.
Tracr scaffold guide RNA with ANGPTL3 protospacer spacer
A guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein, and a spacer sequence that corresponds to a protospacer on ANGPTL3.
Tighter sequence identity to SEQ ID NO: 2192
The mRNA sequence has at least 99% sequence identity to SEQ ID NO: 2192.
Base editor protein sub-regions including TadA, Cas9 nickase, and NLS
A base editor protein that contains an adenine tRNA deaminase (TadA) region, a Cas9 nickase region, and a nuclear localization sequence (NLS) region.
Chemically modified guide RNA nucleotides
The guide RNA includes a chemical modification on one or more nucleotides.
ANGPTL3 nucleobase change directed by the guide RNA
The guide RNA directs a base editor protein to introduce a nucleobase change in the ANGPTL3 gene.
The claim coverage centers on an mRNA encoding a base editor protein with sequence identity requirements relative to SEQ ID NO: 2192, combined with a guide RNA that uses a tracr scaffold and an ANGPTL3 protospacer spacer. Dependent refinements further specify tighter identity constraints, base editor sub-regions, guide RNA chemical modification, and ANGPTL3 nucleobase change outcomes.
Stated Advantages
Chemically modified gRNAs can enhance binding affinity to Cas/base-editor components.
Chemically modified gRNAs can increase stability and half-life.
Chemically modified gRNAs can reduce off-target effects.
Minimal cytokine/complement activation that returns to baseline by approximately 336 hours.
High on-target liver editing efficiencies.
Lipid-lowering effect, including at least about 35% LDL-C reduction for PCSK9-targeting.
Corresponding lipid changes for ANGPTL3 targeting, including smaller LDL-C and triglyceride reductions.
ANGPTL3 protein reduction and triglyceride reduction are described following liver-targeted editing.
Editing is described as primarily limited to liver, with low off-target editing outside the protospacer.
Increases cellular half-life of gRNAs/sgRNAs.
Provides resistance to degradation of the guide RNA.
Increased stability, including ex vivo/in vitro half-life increase of the base-editor guide RNA/base-editor fusion protein complexes.
Disrupts splicing via splice site A·T to G·C alterations.
Reduces or abolishes functional protein expression.
Anticipated or reported reductions in LDL cholesterol, triglycerides, blood PCSK9 protein level, and blood ANGPTL3 protein level.
Enables treatment or prevention of atherosclerotic vascular and metabolic conditions, including atherosclerotic cardiovascular disease, atherosclerotic vascular disease, coronary heart disease, stroke, and diabetes.
Documented Applications
Liver-targeted gene editing of ANGPTL3 in mammalian subjects, including cynomolgus monkeys, using lipid nanoparticles for mRNA and guide RNA delivery.
Evaluation of downstream lipid phenotypes including reduction of ANGPTL3 protein and triglyceride reduction, with assessment of liver markers and cytokines.
In vivo and in vitro evaluation of SpCas9- and adenine base editor (ABE8.8/SpCas9) editing delivered via LNP-delivered mRNA and gRNAs in non-human primates and primary hepatocytes targeting ANGPTL3 or PCSK9.
Assessment of edit durability/maintenance in primary hepatocytes across multiple tracr/spacer guide modifications.
Evaluation of ABE mRNA GC-content variation with reported functional impact via mouse day-5 editing differences.
Treating diseases via LNP-administered, chemically modified gRNAs targeting PCSK9 or ANGPTL3, including atherosclerotic vascular disease, hypertriglyceridemia, and diabetes [procedural detail omitted for safety].
Splice-site editing outcomes at PCSK9 and ANGPTL3, including splice donor/acceptor disruption leading to aberrant/unstable transcripts [procedural detail omitted for safety].
PCSK9 base editing in mice and non-human primates with reductions in PCSK9 protein and LDL-C following LNP delivery.
ANGPTL3 base editing in non-human primates with reductions in ANGPTL3 protein and triglycerides following LNP delivery.
Dual PCSK9/ANGPTL3 editing using a single test article, including repeat dosing outcomes, tissue distribution, and low off-target editing at selected sites.
Assessment of ABE8.8 adenine base editors for gRNA-independent RNA editing in primary human and cynomolgus hepatocytes, including on-target editing and gRNA specificity after spacer/tracr modifications.
Off-target evaluation of adenine base editor activity using candidate selection with ONE-seq and NGS validation, including indel frequency measurements at selected sites.
In vivo therapeutic regimens for splice-site base editing targeting ANGPTL3, including single-dose and multiple-dose schedules using LNPs delivering a base editor fusion protein or encoding nucleic acid and a guide RNA.
Therapeutic use in cardiovascular and metabolic diseases, including atherosclerotic cardiovascular disease, atherosclerotic vascular disease, coronary heart disease, stroke, and diabetes.
Therapeutic gene modification targeting ANGPTL3 and/or PCSK9 in liver cells.
Gene editing of lipid-cholesterol pathway genes, including ANGPTL3 and PCSK9, by base editing that disrupts splicing to reduce functional protein expression.
Evaluation of base-editing outcomes using next generation sequencing, Sanger sequencing, ONE-seq candidate off-target site identification, targeted NGS validation, RT-PCR, and lipid-panel assays.
Assessment of lipid endpoints using enzymatic lipid-panel assays and an indirect Friedewald-type calculation of LDL-C from total cholesterol, HDL-C, and triglycerides.
Use of base-editor guide RNA/base-editor fusion protein complexes as gene-editing compositions within therapeutics and methods of treatment for conditions associated with LPA, including atherosclerosis, coronary heart disease, and stroke.
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