First-in-human autologous implantation of genetically modified adipocytes expressing LCAT for the treatment of familial LCAT deficiency.

Background: Familial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a severe inherited disease without effective treatment. Patients with FLD develop severe low HDL, corneal opacity, hemolytic anemia, and renal injury. Objective: We developed genetically modified adipocytes (GMAC) secreting LCAT (LCAT-GMAC) for ex vivo gene therapy. GMACs were prepared from the patient's adipocytes to express LCAT by retroviral gene transduction to secrete functional enzymes. This study aimed to evaluate the safety and efficacy of LCAT-GMAC implantation in an FLD patient. Methods: Proliferative preadipocytes were obtained from a patient using a ceiling culture and retrovirally transduced with LCAT. After obtaining enough cells by expansion culture of the transduced cells, the resulting LCAT-GMACs were implanted into a patient with FLD. To evaluate the safety and efficacy, we analyzed the outcome of the autologous implantation for 24 weeks of observation and subsequent 240 weeks of the follow-up periods. Results: This first-in-human autologous implantation of LCAT-GMACs was shown to be safe by evaluating adverse events. The LCAT-GMAC implantation increased serum LCAT activity by approximately 50% of the baseline and sustained over three years. Consistent with increased LCAT activity, intermediate-density lipoprotein (IDL) and free cholesterol levels of the small and very small HDL fractions decreased. We found the hemoglobin/haptoglobin complex in the hemolyzed pre-implantation sera of the patient. After one week of the implantation, the hemoglobin/haptoglobin complex almost disappeared. Immediately after the implantation, the patient's proteinuria decreased temporarily to mild levels and gradually increased to the baseline. At 48 weeks after implantation, the patient's proteinuria deteriorated with the development of mild hypertension. By the treatment with antihypertensives, the patient's blood pressure normalized. With the normalization of blood pressure, the proteinuria rapidly decreased to mild proteinuria levels. Conclusions: LCAT-GMAC implantation in a patient with FLD is shown to be safe and appears to be effective, in part, for treating anemia and proteinuria in FLD.

A novel homozygous frameshift mutation in the APOA1 gene associated with marked high-density lipoprotein deficiency.

The proband was a 53-year-old Japanese woman. Despite having no atherosclerotic vascular lesions on a physiological examination, markedly decreased levels of high-density lipoprotein (HDL) were always noted at her annual medical checkup. She also had corneal opacities but neither xanthoma nor tonsillar hypertrophy. A biochemical examination showed decreased levels of both apolipoprotein A-I (apoA-I) (<5 mg/dL) and lecithin-cholesterol acyltransferase (LCAT) activity. Her brother and son also had low concentrations of HDL-cholesterol, suggesting the presence of a genetic abnormality. Therefore, a sequence analysis of the genes for ABCA1, LCAT and apoA-I proteins was performed in the proband. The analysis of the APOA1 gene revealed a novel homozygous two-nucleotide deletion in exon 4 (c.614_615delTC), which causes a frameshift after residue 205 of the apoA-I protein (p.Leu205fs). Since no mutation has been found in the ABCA1 or LCAT gene, functional abnormalities of the carboxyl-terminal region of the apoA-I protein in lipid binding might have caused the low HDL-cholesterol levels and decreased LCAT activity, possibly associated with corneal opacities but not premature CAD, in the patient.

Generation and characterization of monoclonal antibodies against mature hepcidin and its application to neutralization and quantitative alteration assay.

Hepcidin regulates the quantity of ferroportin (FPN) on cellular membrane. In our cell assay expressing ferroportin labeled with green fluorescence, FPN was internalized and degraded only after treatment with hepcidin-25, not hepcidin-22 or hepcidin-20, leading to accumulation of cellular iron. Thus we generated murine monoclonal antibodies (mAbs) against hepcidin-25, and then characterized and validated their functions. Among them, several mAbs showed a neutralizing activity that may prevent ferroportin internalization induced by hepcidin-25. To measure hepcidin level in various fluids, mAbs specific for human and rat hepcidin-25 were selected. As for rat, a sandwich ELISA developed using clone rHN1 as capture antibody and biotinylated clone mHW1 as a detection reagent had high sensitivity, allowing for the detection of 1-100 ng/mL of hepcidin-25. Rat hepcidin-25 level in plasma was measured at an average concentration of 63.0 ng/mL in healthy condition, and at 218.2 ng/mL after stimulation of lipopolysaccharide.

Mitochondrial reactive oxygen species generation by the SDHC V69E mutation causes low birth weight and neonatal growth retardation.

We have previously demonstrated that excessive mitochondrial reactive oxygen species caused by mutations in the SDHC subunit of Complex II resulted in premature death in C. elegans and Drosophila, tumors in mouse cells and infertility in transgenic mice. We now report the generation and initial characterization of conditional transgenic mice (Tet-mev-1) using our uniquely developed Tet-On/Off system, which equilibrates transgene expression to endogenous levels. The mice experienced mitochondrial respiratory chain dysfunction that induced reactive oxygen species overproduction. The mitochondrial oxidative stress resulted in excessive apoptosis leading to low birth weight and growth retardation in the neonatal developmental phase in Tet-mev-1 mice.

Cell growth of the mouse SDHC mutant cells was suppressed by apoptosis throughout mitochondrial pathway.

SDHC E69 cells, which overproduce superoxide anions in their mitochondria, were previously established that had a mutation in the SDHC gene of complex II of the respiratory chain. We now demonstrate that tumors formed by NIH 3T3 and SDHC E69 cells showed significant histological differences. Cytoplasmic cytochrome c release from mitochondria was significantly elevated in SDHC E69 cells and was likely caused by superoxide anion overproduction from mitochondria. In addition, the p53 and Ras signal transduction pathways were activated by oxidative stress and may play a key role in the supernumerary apoptosis in SDHC E69 cells. Our results suggest that the development and growth characteristics of hereditary paragangliomas, which are defective in the same complex of electron transport as mouse SDHC E69 cells, may be caused by apoptosis induction by mitochondrial oxidative stress.