Familial hypercholesterolemia (FH) is characterized by elevated levels of low-density lipoprotein cholesterol (LDL-C) and premature cardiovascular disease (CVD). FH is an autosomal dominant genetic disease (with a gene dosage effect) that is caused by mutations in genes encoding low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB) or subtilisin converting enzyme 9 (PCSK9). Around 90% of FH is caused by LDLR mutations. Mutations in LDLR lead to a lower expression level of functional LDLR, resulting in reduced hepatic LDL receptor-mediated clearance of LDL, which causes excessive LDL-C in circulating blood, ultimately resulting in the occurrence and accumulation of atherosclerotic plaques. The severity of atherosclerosis is closely related to the level and activity of low-density lipoprotein receptors (LDLRs) in liver tissue.
The incidence of homozygous familial hypercholesterolemia (HoFH) in most countries has been previously reported to be as few as one in a million. HoFH rapidly develops into severe atherosclerosis and cardiovascular disease (CVD) in infants and young children, causing an early death. As heterozygous FH (HeFH) patients have functional LDLR protein, current FH drug treatments (such as MG-CoA reductase inhibitors [statins]) are usually only effective for HeFH. Notable, high-intensity statins and PCSK9 inhibitors have limited therapeutic effect on HoFH. These differences indicate that even low levels of LDLR expression and activity can significantly influence the development and severity of FH.
The CRISPR/Cas9 system is an effective gene editing tool that can correct pathological mutations at the DNA level and is a promising treatment for human diseases caused by genetic errors. Adeno-associated virus (AAV) is regarded as the most promising vector for gene therapy applications due to its high safety and stable long-term expression. At the time of writing, gene therapy using AAV has been used in clinical practice for several years, with AAV-mediated treatments approved for more than 80 diseases.
The research groups of Dr. Bin Zhou (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) and Dr. Hefeng Huang (International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University) co-published an article titled“In Vivo AAV-CRISPR/Cas9-mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia” in the journal Circulation.
In this study, researchers find that adeno-associated virus (AAV) delivers CRISPR/Cas9 to achieve Ldlr gene correction that can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutant mice generated by Cyagen. The nonsense point mutation mouse line, LdlrE208X, is based on a gene mutation relevant to familial hypercholesterolemia - providing a potential therapeutic approach for the treatment of patients with the rare disease (Zhao H, Li Y, et al., 2020, Circulation).
Figure 1. The Research Map
In this paper, the author first screened for unknown new mutations of low-density lipoprotein receptor (LDLR), such as E207X, from clinical homozygous patients with familial hypercholesterolemia. The author verified through in vitro cell experiments that the expression of a mutant LDLR gene is related to these functional changes. So, how can this be used to confirm the causal relationship between this newly discovered point mutation and familial hypercholesterolemia?
The author realized that it was difficult to be convincing only with results from in vitro experiments, so they replicated the human LDLR-E207X point mutation in mice to study the relationship between this new mutation and disease occurrence. When the authors reached out to Cyagen for their help generating the LDLR-E207X point mutation mouse model, we knew we would be able to work wonders for their project. By comparing the sequence of human LDLR and mouse Ldlr gene, we found that the human LDLR E207X point mutation site was located in E208X of the mouse Ldlr gene. We designed the corresponding gRNA and donor DNA by using CRISPR/Cas9 gene editing technology and successfully constructed the Ldlr-E208X point mutation mouse model via micro-pronuclear injection. The results showed that the loss of LDLR protein expression function was caused by the introduction of a stop codon in this point mutation and the mouse model showed obvious hypercholesterolemia and atherosclerosis phenotype when provided with a high-fat diet. The Ldlr-E208X point mutation mouse model not only demonstrated the causal relationship between Ldlr-E208X point mutation and hypercholesterolemia and atherosclerosis in clinical patients, but also established a reliable mouse model of Familial Hypercholesterolemia.
Figure 2. Generation and characterization of Ldlr-E208X (LdlrE208X) knock-in point mutation mouse line.
Figure 3. Atherosclerosis in Ldlr-E208X mice after a high-fat diet regimen.
Using the described mouse model, the author explored the possibility of using gene modification therapy on the disease. To implement the CRISPR/Cas9 gene therapy technology mediated by liver specific Adeno-Associated Virus Vector Serotype 8 (AAV8), the liver specific expression vectors of AAV8-Cas9, AAV8 gRNA, and normal Ldlr donors were constructed, respectively. The newly born Ldlr-E208X point mutation mice were treated by a subcutaneous injection of the vectors. The results showed that after the AAV8-CRISPR/Cas9 gene therapy, the Ldlr protein expression in the mice appeared to have made a moderate recovery and the hypercholesterolemia and atherosclerotic phenotype of the mice were also somewhat improved - indicating that AAV8-CRISPR/Cas9 has a certain effect on in vivo Ldlr repair therapy.
From the discovery of a possible new pathogenic gene in clinical patients to the construction of the point mutation mouse model, the causal relationship between the point mutation and the disease was verified. The LDLR E207X point mutation can damage the function of the gene and increase the content of the low-density lipoproteins (LDL) in blood, which leads to acute hypercholesterolemia and atherosclerotic phenotype. It was further demonstrated that AAV8-CRISPR/Cas9 gene therapy can alleviate the disease phenotype. Given that AAV8-CRISPR/Cas9 expression vectors can modify the function of LDLR gene in liver and thus lower LDL content in blood, this could play a therapeutic role in alleviating hypercholesterolemia and atherosclerosis. Collectively, these results have helped put forward hypotheses for the pathogenesis and potential treatment options for Familial Hypercholesterolemia.
Figure 3: Partial recovery of low-density lipoprotein receptor (LDLR) protein and amelioration of atherosclerosis (AS) phenotypes in LdlrE208X after AAV–CRISPR/Cas9 treatment.
From research ideas and strategies to technical methods - this article is a good reference for future applications of genetically modified mouse models across gene function studies, the establishment of disease models, and improved preclinical testing of potential treatments to evaluate efficacy.
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Gene Therapy Related Resource:
>> Rare Disease Model Collaboration Program
>> Case Studies in Gene Therapy - Applications of Accurate Mouse Models
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References：
In Vivo AAV-CRISPR/Cas9-mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia. DOI:10.1161/CIRURCHA1191: 042476.
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