Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Daniel J. Rader
Familial lecithin cholesterol acyltransferase (LCAT) deficiency (FLD), is a rare monogenic disease, characterized by abnormal lipid profile, corneal opacities, anemia and renal disease which progresses to renal failure. There is currently no approved treatment for patients with this disease. FLD is a good candidate for AAV mediated gene therapy but this therapeutic avenue has not been pursued until now. Here, we completed dose response studies in LCAT KO and LCAT KO/ human ApoA-I transgenic mice using AAV8 expressing human LCAT. AAV8-TBG-hLCAT induces significant increases in plasma % cholesterol esterified at a dose as low as 3e9 GC per mouse, which is approximately equivalent to 1.5e11 GC per kg. We also characterize the first gain-of-function variant of LCAT ever to be identified, hLCAT-V114M. We expressed this variant in 293 cells via transient transfection and found that it has 150% the activity of WT human LCAT. We also expressed this variant in mice using our AAV8 vector. In vivo, AAV8-TBG-hLCAT-V114M induced greater increases in plasma lipids than AA8-TBG-hLCAT at the same dose, and significant increase in plasma % cholesterol esterified was measured at 1e9 GC per mouse, approximately equivalent 5e10 GC per kg. Additionally, we expressed four codon optimized variants of human LCAT in vivo using AAV8. Codon optimization had little effect on the efficacy of hLCAT in mice, with only one variant, AAV-TBG-hLCAT-v1, inducing greater increases in HDL-C than WT hLCAT. Lastly, we were able to successfully induce renal disease in LCAT KO/SREBP1a transgenic mice using high-protein diet. When we treated these mice with 1e10 GC of AAV8-TBG-hLCAT, we found that it neither prevented nor reversed the progression of renal disease. Next, we will combine our gain-of-function LCAT, V114M, with the leading codon optimized variant and evaluate this new ‘optimally effective vector’ as a clinical candidate vector for use in a clinical trial.
Christopher, Devin Mae, "Pre-Clinical Development Of Aav Mediated Gene Therapy For Familial Lecithin Cholesterol Acyltransferase Deficiency" (2017). Publicly Accessible Penn Dissertations. 2228.