Gene transfer of apolipoprotein A-V improves the hypertriglyceridemic phenotype of apoa5 (-/-) mice

Abstract

Objective: Apolipoprotein (apo) A-V is a low abundance protein with a profound influence on plasma triacylglycerol levels. In human populations, single nucleotide polymorphisms and mutations in APOA5 positively correlate with hypertriglyceridemia. As an approach to preventing the deleterious effects of chronic hypertriglyceridemia, apoA-V gene therapy has been pursued.

Methods and results: Recombinant adeno-associated virus (AAV) 2/8 harboring the coding sequence for human apoA-V or a control AAV2/8 was transduced into hypertriglyceridemic apoa5 (-/-) mice. After injection of 1×10(12) viral genome AAV2/8-apoA-V, maximal plasma levels of apoA-V protein were achieved at 3 to 4 weeks, after which the concentration slowly declined. Complementing the appearance of apoA-V was a decrease (50±6%) in plasma triacylglycerol content compared with apoa5 (-/-) mice treated with AAV2/8-β-galactosidase. After 8 weeks the mice were euthanized and plasma lipoproteins separated. AAV2/8-apoA-V-transduced mice displayed a dramatic reduction in very low-density lipoprotein triacylglycerol content. Vector generated apoA-V in plasma associated with both very low-density lipoprotein and high-density lipoprotein fractions.

Conclusions: Taken together, the data show that gene transfer of apoA-V improves the severe hypertriglyceridemia phenotype of apoa5 (-/-) mice. Given the prevalence of hypertriglyceridemia, apoA-V gene therapy offers a potential strategy for maintenance of plasma triacylglycerol homeostasis.

Figure 1

Characterization of adeno-associated virus (AAV) 2/8–transduced livers. Eight weeks after administrating AAV2/8-apolipoprotein (apo) A-V or AAV2/8-LacZ (1×10¹² vg) into apoa5 (−/−) mice, livers were harvested. A, Gene amplification of cytomegalovirus promoter DNA in liver extracts from each of 8 mice transduced with AAV2/8-LacZ or AAV2/8-apoA-V. B, Histogram depicting apoA-V mRNA expression level in liver tissue extracts from AAV2/8-LacZ, APOA5 transgenic (Tg) and each of 8 AAV2/8-apoA-V–transduced mice. APOA5 mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase expression. C, α-apoA-V immunoblot (left lanes) recombinant human apoA-V standard, AAV2/8-LacZ– injected apoa5 (−/−) mouse liver extract, and human apoA-V Tg mouse liver extract, respectively. Lanes labeled 1 to 8 refer to individual liver extracts from each of 8 AAV2/8-apoA-V–transduced mice.

Figure 2

Adeno-associated virus (AAV) 2/8 tissue distribution after injection. Eight weeks after gene transfer of 1×10¹² vg AAV2/8-apolipoprotein (apo) A-V into apoa5 (−/−) mice, indicated tissues were harvested and analyzed for (A) cytomegalovirus (CMV) promoter DNA. Inset shows polymerase chain reaction (PCR) amplification of CMV promoter region and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from genomic DNA from tissue extracts of mouse #4 (see Figure 1C). Lane assignments correspond to specific tissues, as indicated in the corresponding histogram. Histogram depicts quantitative PCR (qPCR) analysis of tissue extracts from 5 individual AAV2/8-apoA-V–transduced mice. Values were calculated as copies of CMV promoter DNA per diploid copy of mouse genomic DNA. B, Tissue distribution of apoA-V mRNA. Inset, PCR amplification of apoA-V cDNA and GAPDH cDNA from tissue extracts of mouse #4. Histogram depicts qPCR analysis of apoA-V mRNA relative to GAPDH in tissue extracts of 5 individual AAV2/8-apoA-V–transduced mice. Values are the mean±SEM (n=5).

Figure 3

Effect of adeno-associated virus (AAV) 2/8 gene transfer on plasma lipid and apolipoprotein (apo) A-V content. Apoa5 (−/−) mice (8 mice per group) were transduced with AAV2/8-apoA-V or AAV2/8-LacZ (1×10¹² vg each). A, α-apoA-V immunoblot of recombinant apoA-V standard (left) and pooled plasma samples from AAV2/8-apoA-V–transduced mice obtained each week for 8 weeks. B, Triacylglycerol content in plasma from 8 AAV2/8-LacZ–transduced animals (filled bars) and 8 AAV2/8-apoA-V–transduced animals (open bars). C, Cholesterol content in plasma from AAV2/8-LacZ–transduced animals (filled bars) and AAV2/8-apoA-V–transduced animals (open bars). Values reported as percent change vs week 0 values as mean±SEM (n=8).

Figure 4

Human apolipoprotein (apo) A-V immunoblot. Recombinant human apoA-V was separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with plasma collected from AAV2/8-LacZ mice (Lane 1) and AAV2/8-apoA-V (Lane 2) mice, 8 weeks after infection.

Figure 5

Effect of adeno-associated virus (AAV) 2/8 gene transfer on lipid and apolipoprotein distribution. Plasma samples collected 8 weeks after injection of AAV2/8- apolipoprotein (apo) A-V or AAV2/8-LacZ (1×10¹² vg) were pooled (from 8 animals) and subjected to fast protein liquid chromatography to separate lipoproteins. A, Triacylglycerol content in lipoprotein fractions from AAV2/8-LacZ mouse plasma (filled circles) and AAV2/8-apoA-V mouse plasma (open circles). B, Cholesterol content in lipoprotein fractions from AAV2/8-LacZ mouse plasma (filled circles) and AAV2/8-apoA-V mouse plasma (open circles). C, A 4% to 20% acrylamide gradient Bis–Tris gel was used for immunoblot analysis of lipoprotein fractions probed with antibodies directed against apoB, apoA-V, and apoA-I. HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and VLDL, very low–density lipoprotein.