Targeted Replacement of Mouse Apolipoprotein A-I with Human ApoA-I or the Mutant ApoA-IMilano

Quality control in the apoA-I secretory pathway

“…Recently, investigators have shown that primary mouse hepatocytes from mice homozygous for a targeted substitution of apoA-I Milano or R173C apoA-I for wild-type mouse apoA-I had a 42% reduction in hepatic secretion of the mutant protein (27). This dominant-negative mutation has been extensively studied in humans and in animal models.…”

“…In one study, transgenic mice were created expressing ⌬ 6 apoA-I, a mutant form of apoA-I lacking the entire proline-punctuated 22 amino acid helix 6 (residues 143-164) (24,25). In other studies, an adenoviral construct expressing the dominant-negative helix 6 point mutation, L159R apoA-I, was expressed in apoA-I knockout mice (26), and in yet another study, a targeted replacement for the dominant-negative mutant apoA-I Milano (R173C apoA-I), a helix 7 point mutation, was examined (27). In each of these studies, the data suggest that mutations within apoA-I helices 6 and/or 7 cause decreased hepatic production and contribute to the dominant-negative phenotype observed in individuals heterozygous for these mutations (23,25).…”

Gain-of-Function Mutations and Therapeutic Implications

“…ApoA-I Milano has been studied extensively, including transgenic and somatic gene transfer studies of atherosclerosis in animals 21 and even in a clinical trial of repeated intravenous infusion of an apoA-I Milano /phospholipids complex in patients with CHD. 22,23 However, there have been remarkably few studies comparing apoA-I Milano directly to wild-type apoA-I in vivo 24 and none with regard to atherosclerosis. AAV1-, AAV2-, and AAV5-based vectors have been used to express apoA-I Milano at very low concentrations after intramuscular or intravenous injection, 25 but no comparison with wild-type apoA-I was made.…”

Apolipoprotein A-I Mutations and Clinical Evaluation

“…Koukos and colleagues [88] performed adenovirus-mediated gene transfer of mutants into apoA-I-knockout mice, and revealed that apoA-I L141R Pisa and L159R Fin inhibit an early step in the biogenesis of HDL through inefficient esterification of the cholesterol in pre-β1-HDL particles by endogenous LCAT, and that both defects can be corrected by treatment with LCAT [88]. They also showed that the apoA-I levels in apoA-I -knockout mice expressing the apoA-I R160L Oslo and R151C Paris mutants were reduced by 68% and 55%, respectively, and that apoA-I R151C Paris generated subpopulations of different sizes that migrated between pre-β-and α-HDL particles and formed mostly spherical and a few discoidal particles [87]. An artificial point mutation (pR160V/H162A) and deletion (L144-P165del) in helix 6 were also associated with abolished LCAT activity and reduced plasma spherical HDL [89].…”

“…For example, transgenic mice expressing a mutant form of apoA-I that lacked the entire proline-punctuated 22-amino acid helix 6 (residues 143-165) were created [84,85]. In other studies, an adenoviral construct expressing a dominant-negative helix 6 point mutation, L159R Fin apoA-I, was expressed in apoA-I-knockout mice, and a targeted replacement for the dominantnegative mutant apoA-I R173C Milano , a helix 7 point mutation, was examined [86,87]. Both of these mutants decreased the plasma HDL-C and apoA-I levels.…”