Tweaking lentiviral vector design balances efficacy and safety in liver-directed gene therapy for familial hypercholesterolemia.

Elevated low-density lipoprotein (LDL) cholesterol is the hallmark of familial hypercholesterolemia (FH), an inherited disease mostly due to mutations in the LDL receptor (LDLR)-encoding gene. Here, we enforced overexpression of LDLR in hepatocytes by in vivo lentiviral vector (LV) gene therapy and normalized circulating LDL cholesterol in a mouse model of FH. One year after gene therapy, we detected a high incidence of hepatic tumors in treated mice. Analysis of LV genomic integration sites in the liver tumors revealed expanded clones bearing LV insertions within the Fyn-related kinase (Frk) gene, a known hepatic oncogene, inducing the formation of aberrant Ldlr-Frk transcripts. When we purposely overexpressed LDLR-FRK by LV gene transfer, we observed hepatocellular hyperplasia and hypertrophy, supporting a role of this chimeric protein in oncogenesis. Since high expression of this chimeric oncogene and the LDLR likely cooperated to cause liver toxicity, we reduced the strength of LDLR expression within LV. We achieved normalization of circulating LDL cholesterol in two FH mouse models and prevention of atherosclerosis, even under high-fat diet challenge, without any long-term liver tumorigenicity. Overall, our in-depth assessment of the efficacy and safety of in vivo gene therapy for FH provides new insights into mechanisms of action and potential vulnerabilities, with implications for future developments of lipid-lowering gene therapies.