In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model

Abstract

BackgroundPlasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles.ResultsTo address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified.ConclusionsHere, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0624-2) contains supplementary material, which is available to authorized users.

ObLiGaRe doxycycline Inducible (ODIn) Cas9 system driving pre-clinical drug discovery, from design to cancer treatment

“…To determine whether targeted gene editing in the liver could be achieved in the ODInCas9 mouse, the ODInCas9 mouse was crossed with the human PCSK9 knock-in hypercholesterolemic mouse [24]. The liver was chosen as the target organ model as lipid nanoparticle (LNP) coated sgRNAs readily accumulate in the organ.…”

“…Male and female ODinCas9 were bred to generate homozygous mice to understand the inheritance pattern of the cassette and to generate an experimental cohort. OdinCas9 mice were crossed to the described human knock in PCSK9 mice [24, 44] to generate mice heterozygous for both constructs. In all experiments mice were randomised to groups to ensure that each group had similar body weight.…”

“…Induction of Cas9 required doxycycline hyclate (2mg/ml; Sigma Aldrich, MI, USA) supplemented with 1% sucrose added to the drinking water for 3 nights. To test the efficacy of guide delivery to the liver, OdinCas9 mice crossed with the human PCSK9 mice were injected intravenously with LNP containing guide for PCSK9 [24, 44], To generate models of NSCLC ODInCas9 mice (12-16 weeks), after 2 nights on Dox, mice were dosed by oropharyngeal aspiration with 1 × 10 10 GC ( KrasG12D;Stk11 -/- ), or 1 × 10 11 GC ( KrasG12D;Trp53 -/- , KrasG12C;Trp53 -/- ) genome copies of adeno-associated (AAV9) diluted to a final volume of 50μl in PBS. 24h post dosing the mice were returned to normal drinking water.…”

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

“…240 Now, we have a thousand dollar genome and a million dollar gene therapy. Given the explosion of research and development into CRISPRbased gene therapy and the expansion of its application from rare genetic disease to large-market ubiquitous conditions such as chronic pain, 241 cardiovascular disease, 242,243 and Alzheimer's, 7 it is not entirely implausible nor unprecedented in the decades to come to hope for a few thousand dollar gene therapy.…”

PCSK9 Variants in Familial Hypercholesterolemia: A Comprehensive Synopsis

“…Subsequently, severely diminished levels of blood PCSK9 are seen in mice with humanized livers with almost 50% of highly specific human hepatocyte variants, thus demonstrating the safety and effectiveness of CRISPR-Cas9 in reducing human PCSK9 levels ( Wang et al, 2016 ). In addition, CRISPR/Cas9-mediated genome editing decreases PCSK9 levels in both human and murine hypercholesterolemic models, which could be a valuable tool in the search for novel therapeutic approaches against hypercholesterolemia ( Carreras et al, 2019 ).…”