Compositions, systems, and methods for epigenetic regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression
Inventors
Kwon, Jennifer • CONGDON, Kendra
Assignees
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Publication Number
US-12098399-B2
Publication Date
2024-09-24
Expiration Date
Abstract
Provided in some aspects are epigenetic-modifying DNA-targeting systems, such as CRISPR-Cas/guide RNA (gRNA) systems for the transcriptional repression of genes to promote a cellular phenotype that leads to reduction of low-density lipoprotein (LDL). In some embodiments, the epigenetic-modifying DNA-targeting systems bind to or target a target site of at least one gene or regulatory element thereof that regulate LDL. In some embodiments, the systems are multiplexed systems that bind to or target a target site in at least two genes or regulatory elements thereof. Also provided herein are methods and uses related to the provided epigenetic-modifying DNA targeting systems in connection with treatments for cardiovascular disease and familial hypercholesterolemia.
Core Innovation
The invention relates to an epigenetic-modifying DNA-targeting system that uses CRISPR/dCas9 to perform transcriptional repression without DNA breakage. The system is a DNA-targeting platform in which a guide RNA directs a dead SpCas9 fusion protein to DNA target sites in gene or regulatory elements, and it includes a fusion protein architecture combining a DNMT3A/L domain with dSpCas9 and a KRAB domain.
The fusion protein includes one or more nuclear localization signals, with nuclear localization signals positioned between the DNMT3A/L domain and dSpCas9 and between dSpCas9 and the KRAB domain. The disclosure also describes sequence-defined components, including gRNA spacers/scaffolds, dCas variants, and transcriptional repressor or effector domain options.
The described effector arrangement supports epigenetic modification and transcriptional repression of target genes including LDL-regulating genes such as PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB. The specification further describes multiplexed targeting, liver targeting, in-cell and in vivo testing, and sustained epigenetic effects in cellular settings and in vivo liver testing.
Claims Coverage
The independent claims cover sequence-specific epigenetic-modifying DNA-targeting systems. Across the claims, there are two main inventive features: a DNMT3A/L-dSpCas9-KRAB fusion protein with specified nuclear localization signal placement, and a gRNA with specified sequence identity.
Nuclear localization signal-positioned DNMT3A/L-dSpCas9-KRAB fusion with specified gRNA
An epigenetic-modifying DNA-targeting system comprising a fusion protein in which a DNMT3A/L domain is followed by dSpCas9 followed by KRAB, with one or more nuclear localization signals positioned between DNMT3A/L and dSpCas9 and between dSpCas9 and KRAB, and a gRNA comprising the sequence set forth in SEQ ID NO: 66.
Sequence-defined DNMT3A/L-dSpCas9-KRAB fusion with specified gRNA
An epigenetic-modifying DNA-targeting system comprising a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 278 and a gRNA comprising the sequence set forth in SEQ ID NO: 66.
Overall, the claim coverage is sequence-specific and defines particular fusion-protein amino acid sequences together with particular sequence-defined gRNAs for epigenetic-modifying DNA targeting.
Stated Advantages
Reduces LDL/LDL-C by transcriptional repression of LDL-regulating genes in liver cells.
Provides sustained PCSK9 repression and LDL lowering as supported by referenced figures.
Enables multiplexed targeting to lower LDL.
Targets a treatment context for cardiovascular disease and familial hypercholesterolemia.
Documented Applications
Use for treatment of cardiovascular disease and familial hypercholesterolemia by reducing LDL through transcriptional repression of LDL-regulating genes in liver cells.
Exemplary target application includes transcriptional repression of PCSK9 and other LDL-regulating genes in liver cells.
In-cell and in vivo testing of epigenetic-modifying DNA-targeting systems using the dSpCas9-KRAB-DNMT3A/L fusion and promoter-targeting gRNAs, including repression of PCSK9 mRNA and associated CpG methylation at or near the PCSK9 promoter target site.
Multiplexed gRNA combinations to enhance LDL-relevant cellular phenotypes, including increased LDL uptake and LDL receptor surface expression.
Liver-humanized mouse testing using lipid nanoparticles (LNPs), with readouts including circulating LDL and ELISA, knockdown, and reported durability and correlations between targeted methylation and PCSK9 protein reduction.
Non-human primate testing (cynomolgus macaque) using PCSK9-targeting gRNAs delivered as LNPs, reporting reductions in PCSK9 protein and LDL-C with targeted CpG methylation and durability after dosing/redosing.
Identification and testing of gRNAs via FlowFISH in Huh7 cells, including use of a lentiviral gRNA together with dSpCas9-KRAB-DNMT3A/L mRNA.
Sustained PCSK9 mRNA repression in primary human hepatocytes after transfection, including time-course qRT-PCR measurement.
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