The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

BACKGROUND: In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA(6679 )(RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. METHODS: We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. RESULTS: The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. CONCLUSION: This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme

Modulation of Muscle Regeneration, Myogenesis, and Adipogenesis by the Rho Family Guanine Nucleotide Exchange Factor GEFT

Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions

Metabolic and physicochemical bases of hyperapobetalipoproteinemia

The goal of this thesis was to elucidate the physicochemical and metabolic bases of Hyperapobetalipoproteinemia (HyperapoB). This disorder, which is likely the commonest metabolic abnormality associated with premature coronary artery disease, was defined as a combination of a normal, or near-normal, LDL cholesterol in the face of an elevated LDL apoB.LDL, even in normals, is heterogeneous. The experimental findings herein confirm this. They also extend this concept to indicate that familial hypercholesterolemia (FH) and HyperapoB each imprint LDL in different and characteristic ways, each an exaggeration of the typical relations between LDL composition and size in normals. At one extreme is HyperapoB, which is characterized by most of the LDL particles being smaller and denser than normal because they contain less cholesteryl ester but the same amount of apoB as normal. At the other is FH, which is characterized by larger, cholesteryl ester-enriched particles. There is, as well, a predictable relation between LDL particle size and the immunoreactivity of certain apoB epitopes.Turnover studies of hepatic apoB using traditional analytic models showed that hepatic apoB is overproduced in HyperapoB, a finding which stands in marked contrast to the impaired catabolism of apoB in FH. A new multi-compartmental model of LDL metabolism has been developed which appears to elucidate several of the basic mechanisms involved in the pathogenesis of HyperapoB. All the data to date indicate that the characteristic abnormalities of LDL in HyperapoB are all consequences of the overproduction of hepatic apoB. Obviously, the goal for future research must be to understand the basis for this overproduction. A preliminary study with adipose tissue suggested that the overproduction of hepatic apoB might be secondary to a defect in peripheral tissue triglyceride biosynthesis

PCSK9 deficiency reduces atherosclerosis, apolipoprotein B secretion, and endothelial dysfunction.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interacts directly with cytoplasmic apoB and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis. We backcrossed Pcsk9-/- mice with atherosclerosis-prone Ldlr-/-Apobec1-/- (LDb) mice to generate Ldlr-/-Apobec1-/-Pcsk9-/- (LTp) mice. Deletion of PCSK9 resulted in decreased hepatic apoB secretion, increased autophagic flux, and decreased plasma levels of IDL and LDL particles. The LDLs from LTp mice (LTp-LDLs) were less atherogenic and contained less cholesteryl ester and phospholipids than LDb-LDLs. Moreover LTp-LDLs induced lower endothelial expression of the genes encoding TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62, and TRAF6 Collectively, these effects were associated with substantially less atherosclerosis development (>4-fold) in LTp mice. The absence of PCSK9 in LDb mice results in decreased lipid and apoB levels, fewer atherogenic LDLs, and marked reduction of atherosclerosis. The effect on atherogenesis may be mediated in part by the effects of modified LDLs on endothelial cell receptors and proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDL receptor activity

PCSK9 deficiency reduces atherosclerosis, apolipoprotein B secretion, and endothelial dysfunction.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interacts directly with cytoplasmic apoB and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis. We backcrossed Pcsk9-/- mice with atherosclerosis-prone Ldlr-/-Apobec1-/- (LDb) mice to generate Ldlr-/-Apobec1-/-Pcsk9-/- (LTp) mice. Deletion of PCSK9 resulted in decreased hepatic apoB secretion, increased autophagic flux, and decreased plasma levels of IDL and LDL particles. The LDLs from LTp mice (LTp-LDLs) were less atherogenic and contained less cholesteryl ester and phospholipids than LDb-LDLs. Moreover LTp-LDLs induced lower endothelial expression of the genes encoding TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62, and TRAF6 Collectively, these effects were associated with substantially less atherosclerosis development (>4-fold) in LTp mice. The absence of PCSK9 in LDb mice results in decreased lipid and apoB levels, fewer atherogenic LDLs, and marked reduction of atherosclerosis. The effect on atherogenesis may be mediated in part by the effects of modified LDLs on endothelial cell receptors and proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDL receptor activity

Long-term Expression of Apolipoprotein B mRNA-specific Hammerhead Ribozyme via scAAV8.2 Vector Inhibits Atherosclerosis in Mice

Target substrate-specific hammerhead ribozyme cleaves the specific mRNA efficiently and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. The goal of this study is to demonstrate that long-term reduction of apoB gene expression using hammerhead ribozyme would result in inhibition of atherosclerosis development. We designed two hammerhead ribozymes targeted at the nucleotides of apoB mRNA GUC2326 (designated RB1) and GUA6679 (designated RB15), and we used self-complementary adeno-associated virus 8.2 (scAAV8.2) vector to deliver these active ribozymes of RB1, RB15, combination of RB1/RB15, and an inactive hammerhead ribozyme RB15 mutant to atherosclerosis-prone LDb mice (Ldlr−/−Apobec1−/−). LDb mice lack both low density lipoproteins (LDL) receptor (Ldlr−/−) and apoB mRNA editing enzyme (Apobec1−/−) genes and develop atherosclerosis spontaneously. After the RB1, RB15, or combination of RB1/RB15 ribozymes treatment, the LDb mice had significantly decreased plasma triglyceride and apoB levels, resulting in markedly decreased of atherosclerotic lesions, Furthermore, the active ribozymes treatment decreased the levels of diacylglycerol acyltransferase 1 (Dgat1) mRNA and the levels of multiple diacylglycerol (DAG) molecular species. These results provide the first evidence that decreased apoB levels results to reduction of Dgat1 expression and triglyceride levels (TAG), which had a significant impact on the development of atherosclerosis

Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases plasma cholesterol levels by promoting LDL receptor degradation. Current antibody inhibitors block the interaction between PCSK9 and LDL receptors, significantly decrease plasma cholesterol levels, and provide beneficial clinical outcomes. To reduce the action of PCSK9 in plasma, a novel strategy that will produce a panel of non-native, conformationally-altered isomers of PCSK9 (X-PCSK9) to develop active immunotherapy targeting of native PCSK9 and inhibiting/blocking the interaction of PCSK9 with LDL receptor, thus decreasing plasma cholesterol levels is proposed. The authors used the scrambled disulfide bond technique to generate conformationally-altered isomers of the catalytic domain of mouse PCSK9. The focus was on the immune response of four X-isomers and their effects on plasma cholesterol and triglyceride levels in both C57BL/6J and Apoe−/− mice. The authors showed that the four immunogens produced significant immunogenicity against native PCSK9 to day 120 after immunization of C57BL/6J and Apoe−/− mice. This resulted in significantly decreased plasma cholesterol levels in C57BL/6J mice, and to a lesser degree in Apoe−/− mice. The X-PCSK9-B1 treated mice had increased LDL receptor mRNA and protein levels at day 120 after treatment. Thus, this study provides a new, potentially promising approach that uses long-term immunotherapy for a treatment of hypercholesterolemia