scholarly journals In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

2021 ◽  
Author(s):  
Tanja Rothgangl ◽  
Melissa K. Dennis ◽  
Paulo J. C. Lin ◽  
Rurika Oka ◽  
Dominik Witzigmann ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Point Mutations ◽  
Density Lipoprotein ◽  
Negative Regulator ◽  
Base Editing ◽  
Guide Rna ◽  
Humoral Immune ◽  
Cholesterol Levels ◽  
Adenine Base

AbstractMost known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

scholarly journals Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

2021 ◽  
Author(s):  
Han Zhang ◽  
Nathan Bamidele ◽  
Pengpeng Liu ◽  
Ogooluwa Ojelabi ◽  
Xin D. Gao ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Cell Types ◽  
Vector System ◽  
Adeno Associated Virus ◽  
Base Editing ◽  
Guide Rna ◽  
Gene Therapies ◽  
Compact Size ◽  
Adenine Base

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽  
2019 ◽  
pp. eaaw7166 ◽  
Author(s):  
Shuai Jin ◽  
Yuan Zong ◽  
Qiang Gao ◽  
Zixu Zhu ◽  
Yanpeng Wang ◽  
...  
Keyword(s):  
High Fidelity ◽  
Single Nucleotide Variants ◽  
Disease Treatment ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Wide ◽  
Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

scholarly journals LRP5 Regulates HIF-1α Stability via Interaction with PHD2 in Ischemic Myocardium

2021 ◽  
Vol 22 (12) ◽  
pp. 6581
Author(s):  
Sujin Ju ◽  
Leejin Lim ◽  
Kwanhwan Wi ◽  
Changwon Park ◽  
Young-Jae Ki ◽  
...  
Keyword(s):  
Target Genes ◽  
Apoptotic Cell ◽  
Low Density Lipoprotein ◽  
Hypoxia Inducible Factor ◽  
Density Lipoprotein ◽  
Ischemic Myocardium ◽  
Negative Regulator ◽  
Hypoxic Conditions ◽  
Density Lipoprotein Receptor

Low-density lipoprotein receptor-related protein 5 (LRP5) has been studied as a co-receptor for Wnt/β-catenin signaling. However, its role in the ischemic myocardium is largely unknown. Here, we show that LRP5 may act as a negative regulator of ischemic heart injury via its interaction with prolyl hydroxylase 2 (PHD2), resulting in hypoxia-inducible factor-1α (HIF-1α) degradation. Overexpression of LRP5 in cardiomyocytes promoted hypoxia-induced apoptotic cell death, whereas LRP5-silenced cardiomyocytes were protected from hypoxic insult. Gene expression analysis (mRNA-seq) demonstrated that overexpression of LRP5 limited the expression of HIF-1α target genes. LRP5 promoted HIF-1α degradation, as evidenced by the increased hydroxylation and shorter stability of HIF-1α under hypoxic conditions through the interaction between LRP5 and PHD2. Moreover, the specific phosphorylation of LRP5 at T1492 and S1503 is responsible for enhancing the hydroxylation activity of PHD2, resulting in HIF-1α degradation, which is independent of Wnt/β-catenin signaling. Importantly, direct myocardial delivery of adenoviral constructs, silencing LRP5 in vivo, significantly improved cardiac function in infarcted rat hearts, suggesting the potential value of LRP5 as a new target for ischemic injury treatment.

scholarly journals Apolipoprotein B gene polymorphisms in people in the East Mediterranean area of Turkey

2004 ◽  
Vol 10 (1-2) ◽  
pp. 125-130
Author(s):  
L. Tamer ◽  
K. Tanriverdi ◽  
B. Ercan ◽  
A. Unlu ◽  
N. Sucu ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Low Density Lipoprotein ◽  
Point Mutations ◽  
Density Lipoprotein ◽  
Mediterranean Area ◽  
Healthy Individuals ◽  
East Mediterranean ◽  
Apo B ◽  
Cholesterol Levels ◽  
East Mediterranean Region

Point mutations in the receptor binding domain of low density lipoprotein may increase cholesterol levels in blood. Three mutations of Apo B-100 protein result in defective binding [Arg 3500 —-> [corrected] Gln, Arg 3500 —-> [corrected] Trp and Arg 3531 —-> [corrected] Cys]. We estimated the frequency of Apo B point mutations [codon 3500] C9774T [Arg 3500 —-> [corrected] Trp] and G9775A [Arg 3500 —-> [corrected] Gln] in 179 atherosclerotic, 145 hyperlipidaemic individuals and 272 healthy individuals in the east Mediterranean region of Turkey. Lipid and lipoprotein levels were measured with routine biochemical analyser and Apo B mutation was detected using real-time PCR. Neither mutation was found. In this region, Apo B-100 protein mutations are rare and causes of hyperlipidaemia and atherosclerosis may therefore be unrelated to them

scholarly journals Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor–related protein 1 (LRP1) on megakaryocytes

Blood ◽  
2009 ◽  
Vol 114 (11) ◽  
pp. 2290-2298 ◽  
Author(s):  
Michele P. Lambert ◽  
Yuhuan Wang ◽  
Khalil H. Bdeir ◽  
Yvonne Nguyen ◽  
M. Anna Kowalska ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Colony Growth ◽  
Platelet Factor 4 ◽  
Negative Regulator ◽  
Low Density ◽  
Related Protein ◽  
Platelet Factor

Abstract Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis, but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor–related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells, is maximally present on large, polyploidy megakaryocytes, and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP), an antagonist of LDL family member receptors, or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition, using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4High). Further, shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally, infusion of RAP into hPF4High mice was able to increase baseline platelet counts without affecting other lineages, suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1, which is transiently expressed during megakaryopoiesis.

Strategies to prevent cleavage of the linker region between ligand-binding repeats 4 and 5 of the LDL receptor

2019 ◽  
Vol 28 (22) ◽  
pp. 3734-3741 ◽  
Author(s):  
Thea Bismo Strøm ◽  
Katrine Bjune ◽  
Luís Teixeira da Costa ◽  
Trond P Leren
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Lipid Lowering ◽  
Ldl Receptors ◽  
Linker Region ◽  
Cholesterol Levels

Abstract A main strategy for lowering plasma low-density lipoprotein (LDL) cholesterol levels is to increase the number of cell-surface LDL receptors (LDLRs). This can be achieved by increasing the synthesis or preventing the degradation of the LDLR. One mechanism by which an LDLR becomes non-functional is enzymatic cleavage within the 10 residue linker region between ligand-binding repeats 4 and 5. The cleaved LDLR has only three ligand-binding repeats and is unable to bind LDL. In this study, we have performed cell culture experiments to identify strategies to prevent this cleavage. As a part of these studies, we found that Asp193 within the linker region is critical for cleavage to occur. Moreover, both 14-mer synthetic peptides and antibodies directed against the linker region prevented cleavage. As a consequence, more functional LDLRs were observed on the cell surface. The observation that the cleaved LDLR was present in extracts from the human adrenal gland indicates that cleavage of the linker region takes place in vivo. Thus, preventing cleavage of the LDLR by pharmacological measures could represent a novel lipid-lowering strategy.

scholarly journals Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates

2021 ◽  
Author(s):  
Lisa N Kasiewicz ◽  
Souvik Biswas ◽  
Aaron Beach ◽  
Huilan Ren ◽  
Chaitali Dutta ◽  
...  
Keyword(s):  
Gene Editing ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipid Nanoparticles ◽  
Atherosclerotic Cardiovascular Disease ◽  
Low Density ◽  
Wild Type ◽  
Guide Rna ◽  
Delivery Technology

Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand - N-acetylgalactosamine (GalNAc) - which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.

scholarly journals GOLIATH regulates LDLR availability and plasma LDL cholesterol levels

2020 ◽  
Author(s):  
Bethan L. Clifford ◽  
Kelsey E. Jarrett ◽  
Joan Cheng ◽  
Angela Cheng ◽  
Marcus Seldin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Lipoprotein Receptors ◽  
Cholesterol Levels ◽  
Genome Wide

AbstractIncreasing the availability of hepatic low-density lipoprotein receptors (LDLR) remains a major clinical target for reducing circulating plasma LDL cholesterol (LDL-C) levels. Here, we identify the molecular mechanism underlying genome-wide significant associations in the GOLIATH locus with plasma LDL-C levels. We demonstrate that GOLIATH is an E3 ubiquitin ligase that ubiquitinates the LDL Receptor resulting in redistribution away from the plasma membrane. Overexpression of GOLIATH decreases hepatic LDLR and increases plasma LDL-C levels. Silencing of Goliath using antisense oligonucleotides, germline deletion, or AAV-CRISPR in vivo strategies increases hepatic LDLR abundance and availability, thus decreasing plasma LDL-C. In vitro ubiquitination assays demonstrate RING-dependent regulation of LDLR abundance at the plasma membrane. Our studies identify GOLIATH as a novel post-translational regulator of LDL-C levels via modulation of LDLR availability, which is likely important for understanding the complex regulation of hepatic LDLR.

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