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APO-skip, a treatment for familial hypercholesterolemia, reduces plasma LDL-cholesterol in human APOB transgenic mice
Genetic mutations in low-density lipoprotein (LDL)-receptors cause familial hypercholesterolaemia (FH), which is characterized by extremely high levels of plasma LDL and premature cardiovascular disease. Current hypocholesterolaemic drugs, such as statins, are inadequate and alternative therapies to reduce LDL are needed. The structural apolipoprotein in LDL particles is APOB100. We have developed APO-skip antisense oligonucleotides (ASOs) that induce the skipping of APOB100 exon 27 to generate a truncated form, APOB87. This mimics the truncated APOB isoforms found in families with hypobetalipoproteinaemia who have very low LDL-cholesterol and show no atherosclerosis. In initial studies we achieved a mean skipping efficiency of 58.7% (sk/(fl+sk)*100; n=3) in vitro and up to 29.1% in vivo with a 50-mer APO-skip, 5’ and 3’ end-protected with phosphorothioate (PTO) residues, which targeted the donor and acceptor as well as a branch point splice site. Weekly i.v. injection, hepatic targeting, of this 50-mer APO-skip (25mg/Kg) complexed to the cationic lipid Invivofectamine 2.0 resulted in a 51% relative reduction in LDL-cholesterol in transgenic mice expressing human APOB. Here, we report efforts to increase therapeutic potential by reducing ASO length and optimizing backbone chemistry. As we found that APO-skip ASOs targeting exon sequences of APOB caused only low levels of exon 27 skipping, we focused on intronic target sequences. After extensive sequence optimization we identified 34- and 39-mer APO-skip ASOs that still target all three splice sites, but in a discontinuous sequence. Interestingly, the invariant 3’ AG and 5’ GT nucleotides of the 3’ and 5’ splice sites, respectively, proved non-essential in the target sequence. Disruption of the strong 3D structure as modelled with the RNA structure and mfold programs seemed more significant. Importantly, these shorter APO-skip ASOs give comparable exon skipping efficiencies in vitro (HepG2 cells) compared to our original 50-mer. Moreover, preliminary in vivo data suggest that these short APO-skip ASOs are able to reduce LDL-cholesterol without requiring a delivery reagent. Our findings re-affirm that ASO-mediated exon skipping of hepatic APOB100 is a promising therapeutic approach to treat FH.
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