Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein SB-715992 and a significant lowering of serum LDL in mice. ApoB targeting is specific and dose-dependent and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70% the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality. hemizygous mice have serum lipid levels very similar to those of healthy humans which make them suitable for investigation of lipid changes in response to different treatment regimens and are very similar in lipid composition to ApoE3-Leiden/CETP transgenic mice (10 11 We have used mice to explore the effect of targeting ApoB mRNA in the liver using chemically modified siRNAs. ApoB is the main lipoprotein required for synthesis and secretion of VLDL particles from the liver (12). Levels of ApoB protein LDL and total cholesterol (TC) are highly correlated with increased risk for atherosclerosis. Patients with FH who show reduced uptake of apoB-bound LDL from the circulation are at high risk for development of coronary heart disease and atherosclerosis (13 14 Contrarily humans with very SB-715992 low levels of plasma apoB reported in particular cases of familial hypobetalipoproteinemia (FHBL) are at a reduced risk for coronary atherosclerosis (15). Because targeting of ApoB has proven difficult with conventional small molecule approaches it presents an attractive target for development of a putative RNAi-based therapeutic. RNA interference (RNAi) is a regulatory sequence-dependent RNA silencing mechanism that uses small double-stranded RNA (dsRNA) molecules to direct gene silencing in a homology-based manner (16). These molecules also known as short-interfering RNAs (siRNA) recruit a RNA-induced silencing complex (RISC) to the target mRNA and eventually lead to site-specific cleavage of the target mRNA and its subsequent degradation (17). RNAi-mediated gene silencing has been extensively used for SB-715992 target validation as it enables fast and relatively inexpensive screens without the need to generate knockout (KO) animals. We used chemically modified siRNAs in a mouse model with a human-like lipid profile Mouse monoclonal to CD32.4AI3 reacts with an low affinity receptor for aggregated IgG (FcgRII), 40 kD. CD32 molecule is expressed on B cells, monocytes, granulocytes and platelets. This clone also cross-reacts with monocytes, granulocytes and subset of peripheral blood lymphocytes of non-human primates.The reactivity on leukocyte populations is similar to that Obs. to interrogate ApoB pathways. We demonstrated that LNP-formulated siRNAs can be successfully used in hemizygous mice to achieve hepatic ApoB mRNA knockdown and that this reduction in ApoB mRNA levels results in significant reductions in serum ApoB protein changes in genes in the lipid and fatty acid pathways significant and prolonged reductions in serum total cholesterol triglycerides and LDL levels as well as correlative hepatic steatosis. MATERIALS AND METHODS siRNA synthesis and characterization Chemically modified siRNAs used in these studies were synthesized and characterized as previously described SB-715992 (18–20). ApoB lead siRNAs used in these experiments are listed in Table 1 (all in the 5′-3′ direction). siRNA sequences contained the following chemical modifications added to the 2′ position of the ribose sugar when indicated: deoxy (d) 2 fluoro (flu) or 2′ O-methyl (ome). Modification abbreviations are given preceding SB-715992 the base to which they were applied immediately. Passenger strands are blocked with an inverted abasic nucleotide on the 5′ and 3′ ends (iB). Nontargeting control siRNAs (nt controls) used in the experiments are listed in Table 2 (all in the 5′-3′ direction). Nontargeting siRNA sequences contained the same chemical modifications as ApoB siRNAs described above. TABLE 1. List of lead ApoB siRNAs used for in vitro and in vivo screens TABLE 2. List of nt control siRNAs used for in vitro and in vivo screens Encapsulation of siRNA siRNAs were encapsulated into two different types of liposomes. Liposome 201 (LNP201) with a lipid composition of CLinDMA (2-{4-[.