Similar Datasets
Project description:Most 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.
| S-EPMC8352781 | biostudies-literature
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
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 | GSE168363 | GEO
Project description:In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels
| PRJNA707031 | ENA
Project description:In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [RNA-seq]
| PRJNA707032 | ENA
Project description:In vivo adenine base editing of PCSK9 in mice and macaques reduces LDL-cholesterol levels [amplicon-seq]
| PRJNA707033 | ENA
Project description:Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
| S-EPMC2999929 | biostudies-literature
Project description:DNA base editors use deaminases fused to a programmable DNA-binding protein for targeted nucleotide conversion. However, the most widely used TadA deaminases lack post-translational control in living cells. Here, we present a split adenine base editor (sABE) that utilizes chemically induced dimerization (CID) to control the catalytic activity of the deoxyadenosine deaminase TadA-8e. sABE shows high on-target editing activity comparable to the original ABE with TadA-8e (ABE8e) upon rapamycin induction while maintaining low background activity without induction. Importantly, sABE exhibits a narrower activity window on DNA and higher precision than ABE8e, with an improved single-to-double ratio of adenine editing and reduced genomic and transcriptomic off-target effects. sABE can achieve gene knockout through multiplex splice donor disruption in human cells. Furthermore, when delivered via dual adeno-associated virus vectors, sABE can efficiently convert a single A•T base pair to a G•C base pair on the PCSK9 gene in mouse liver, demonstrating in vivo CID-controlled DNA base editing. Thus, sABE enables precise control of base editing, which will have broad implications for basic research and in vivo therapeutic applications.
| S-EPMC10495389 | biostudies-literature
Project description:Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE-P1S (Adenine Base Editor-Plant version 1 Simplified) containing the ecTadA*7.10-nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE-P1 consisting of the ecTadA-ecTadA*7.10-nSpCas9 (D10A) fusion. We found that the protein expression level of ABE-P1S is higher than that of ABE-P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE-P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10-nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH-Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.
| S-EPMC7004905 | biostudies-literature
Project description:Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores -2.56 and -2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (-28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (-47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [(14)C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.
| S-EPMC4617395 | biostudies-literature