Abstract 15669: Generation and LDLR Functional Correction of Familial Hypercholesterolemia iPSC With Physiologically Responsive Non-Integrating BAC Vector

Abstract

Familial Hypercholesterolemia (FH) is a monogenic disorder of low-density lipoprotein receptor (LDLR) dysfunction, resulting in pathological LDL cholesterol levels and accelerated cardiovascular disease. Many FH patients are refractory to pharmacologic therapies and require cholesterol apheresis or liver transplant. Induced pluripotent stem cells (iPSC) are a promising cell-based option for ameliorating such disorders, though clinically relevant approaches for their functional correction remain to be explored. Homozygous FH fibroblasts were used to generate iPSC (FH-iPSC) using non-integrating synthetic modified mRNAs. Pluripotence was confirmed via immunocytochemistry (OCT4, SOX2, SSEA4, TRA 1-60 & TRA 1-81), in vitro spontaneous differentiation, and in vivo teratoma formation. To mitigate LDLR dysfunction, FH-iPSC were non-virally transfected with a physiologically sensitive episomal LDLR BAC-based plasmid, circumventing cytotoxic LDLR over-expression, insertional mutagenesis, and transgene activation. Transfected colonies (FH-iPSC-LDLR) were selected via Hygromycin-B resistance. After six months of continuous culture, the plasmid was rescued by alkaline lysis, isolated, and introduced into CHO-a7 (LDLR-/-) cells. AgeI restriction digest analysis was performed and DiI-LDL uptake qualitatively demonstrated plasmid functionality. FH-iPSC were also differentiated into hepatocyte-like cells (FH-HLC) and tested for definitive endoderm (SOX17, HNF4α, GATA4) and hepatocyte lineage (AFP, Albumin) markers via PCR and immunocytochemistry. FH-HLC stored lipids and cleared Indocyanine Green. Corrected FH-HLC (FH-HLC-LDLR) qualitatively exhibited markedly increased DiI-LDL internalization compared to dysfunctional FH-HLC (Figure 1). These experiments support a proof-of-principle that patient-specific iPSC can be generated and functionally rescued with clinically relevant vectors to produce therapeutic cell lines.