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In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [RNA-seq]

ABSTRACT: The majority of known pathogenic point mutations in the human genome are C•G to T•A substitutions. Adenine base editors (ABEs), comprised of nuclease-impaired Cas9 fused to adenine deaminases, enable direct repair of these mutations, making them promising tools for precision in vivo genome editing therapies. However, prior to application in patients, thorough safety and efficacy studies in relevant model organisms are needed. Here, we apply adenine base editing in vivo in the liver of mice and cynomolgus macaques to install a splice site mutation in PCSK9 and reduce blood low-density lipoprotein (LDL) levels, a well-known risk factor for cardiovascular disease. Intravenous delivery of ABE-encoding mRNA and a locus-specific single guide (sg)RNA utilizing lipid nanoparticle (LNP) technology induce up to 67% editing in the liver of mice and up to 34% editing in the liver of macaques, leading to a reduction of plasma PCSK9 and LDL levels. We observed rapid clearance of ABE mRNA after LNP-mediated delivery, and neither sgRNA-dependent nor sgRNA-independent off-target mutations are detected in genomic DNA. Together, our findings support safety and feasibility of adenine base editing to treat patients with monogenetic liver diseases.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE168363 | GEO | 2021/04/13

REPOSITORIES: GEO

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In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [amplicon-seq]

Project description:The majority of known pathogenic point mutations in the human genome are C•G to T•A substitutions. Adenine base editors (ABEs), comprised of nuclease-impaired Cas9 fused to adenine deaminases, enable direct repair of these mutations, making them promising tools for precision in vivo genome editing therapies. However, prior to application in patients, thorough safety and efficacy studies in relevant model organisms are needed. Here, we apply adenine base editing in vivo in the liver of mice and cynomolgus macaques to install a splice site mutation in PCSK9 and reduce blood low-density lipoprotein (LDL) levels, a well-known risk factor for cardiovascular disease. Intravenous delivery of ABE-encoding mRNA and a locus-specific single guide (sg)RNA utilizing lipid nanoparticle (LNP) technology induce up to 67% editing in the liver of mice and up to 34% editing in the liver of macaques, leading to a reduction of plasma PCSK9 and LDL levels. We observed rapid clearance of ABE mRNA after LNP-mediated delivery, and neither sgRNA-dependent nor sgRNA-independent off-target mutations are detected in genomic DNA. Together, our findings support safety and feasibility of adenine base editing to treat patients with monogenetic liver diseases.

2021-04-13 | GSE168364 | GEO

In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels

Project description:In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels

| PRJNA707031 | ENA

In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [RNA-seq]

Project description:In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [RNA-seq]

| PRJNA707032 | ENA

In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [amplicon-seq]

Project description:In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [amplicon-seq]

| PRJNA707033 | ENA

Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

Project description:Plant biotechnology needs new methods that accelerate design-build-test-learn cycles to develop new gene editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle –from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors and used it to develop an optimized LbCas12a-ABE system. Sequential improvement of five components –NLS, crRNA, LbCas12a nuclease, adenine deaminase and linker– led to a systematic, stepwise increase in ABE activity at extrachromosomal GFP reporter (from 0.5% to 40%) and endogenous target sites. We confirmed the activity of LbCas12a-ABE in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.

2022-11-21 | GSE200450 | GEO

Base Editing Correction of a Hypertrophic Cardiomyopathy-Causing MYH7 Mutation in Human Cardiomyocytes and Humanized Mice

Project description:The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by mutations in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia, and sudden cardiac death. The dominant-negative c.1208 G>A (p.R403Q) mutation in b-myosin (MYH7) is a common and well-studied mutation that leads to increased cardiac contractility and HCM onset. Here we identify an adenine base editor (ABE) and single-guide RNA system that can efficiently correct this human pathogenic mutation with minimal off-target and bystander editing. We show that delivery of base editing components rescues pathological manifestations of HCM in iPSC-cardiomyocytes derived from HCM patients and in a humanized mouse model of HCM. Our findings demonstrate the use of base editing to treat inherited cardiac diseases and prompt the further development of ABE-based therapies to correct a variety of monogenic mutations causing cardiac disease.

2022-10-25 | GSE201755 | GEO

Transcriptome-wide deamination analysis

Project description:Adenosine base editing to recreate the HPFH mutations is a promising approach to induce HbF. To measure Cas-independent off-target editing of mRNA, we electroporated CD34+ cells from three different donors with RNP consisting of ABE7.10 or ABE8e complexed with the -globin −175 A>G targeting sgRNA. Six days later, this was followed by high-depth RNA-seq. The rates of A-to-I conversion were similar in edited and unedited control cells, which is consistent with previous reports that off-target RNA editing was not detected after high-level adenine base editing of HUDEP2 cells or HSPCs.

2023-04-28 | GSE228821 | GEO

A-T to G-C base editing efficiency at targeted gene sites in HEK293T cell line

Project description:A-T to G-C base editing efficiency at targeted gene sites in HEK293T cells using the dCas12f-ABE design or the Cas12f-ABE design. Found that the total A-T to G-C conversion efficiency of Circular gRNAs exhibited about two-fold increase compared with U6 gRNAs. We further analyzed the pattern for A-T to G-C conversion on the target site, and observed that the most efficient base editing occurred in a narrow window A3 (3bp downstream of the PAM) similar to U6 gRNAs. In summary, Circular gRNAs with dCas12f-ABE design could enhance A-T to G-C base editing efficiency in a narrow window.

2024-03-11 | GSE260967 | GEO

Directed Evolution of an Adenine Base Editor withIncreased Activity and Context Compatibility [sgRNA-target library]

Project description:Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. ABEs function most effectively when the target A is in a TA context. Deficient ABE processing of RA (R = A or G) is most evident when the target A is outside the comfortable editing window or when delivery is suboptimal. In the current study, we report directed evolution of TadA8r, a new variant of the Escherichia coli tRNA-specific adenosine deaminase (TadA) with ultra-fast deoxyadenosine deamination and no context bias.

2023-09-14 | GSE242012 | GEO

Precise genomic editing of a pathogenic RBM20 mutation rescues dilated cardiomyopathy

Project description:Mutations in RNA binding motif protein 20 (RBM20) are a common cause of dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine rich (RS-rich) domain, resulting in mis-localization of RBM20 to ribonucleoprotein granules within the cytoplasm, abnormal splicing of cardiac genes, and cardiomyocyte dysfunction. We used adenine base editing (ABE) and prime editing to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell-derived cardiomyocytes. We also created humanized Rbm20R636Q mutant mice, which succumbed to severe cardiac dysfunction, heart failure and premature death. Systemic delivery of ABE components by adeno-associated virus in these mice restored cardiac function and extended life span. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.

2022-08-11 | GSE210783 | GEO

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