APOB is the principal structural apolipoprotein in LDL, very (V)LDL, intermediate-density lipoprotein (IDL), lipoprotein(a) (Lp(a)) and chylomicron particles [48
]. The APOB pre-mRNA is composed of 29 exons, all of which are constitutively spliced into the mature APOB mRNA. There are two natural protein isoforms, the full-length APOB100 and the C-terminal truncated APOB48 forms. APOB100 is synthesized in the liver and is required for the assembly of VLDL, IDL, LDL and Lp(a) particles. Along with APOE, APOB100 is the ligand for the LDL receptor. Excess levels of the APOB100-containing particles LDL and Lp(a) have been implicated in atherogenesis [49
]. The APOB48 isoform is synthesized in the intestine, is identical to the N-terminal 48% of the APOB100 protein, and is required for chylomicron assembly and intestinal fat transport. These chylomicrons are cleared from the circulation by interaction of APOE with the chylomicron remnant receptor; APOB48 cannot bind to the LDL receptor as it lacks the C-terminal domain necessary for binding. The APOB48 isoform is generated by tissue-specific RNA editing of the CAA codon to a premature UAA termination codon in the intestine. The APOB48 mRNA is edited by a protein complex known as the editosome, which consists of the catalytic subunit APOBEC-1 (APOB mRNA-editing enzyme, catalytic polypeptide-1) and accessory factors. The RNA editing site and mooring sequence necessary for editosome binding and function are within exon 26 of the APOB mRNA [50
]. Because of the central role of APOB100 in atherosclerosis, this isoform has become a major therapeutic target. Downregulation of APOB100 expression is expected to decrease total cholesterol, LDL and Lp(a) levels, and therefore prevent the development of atherosclerosis.
Modifying APOB splicing was hypothesized to cause the expression of an alternative isoform of APOB. Exon 27 was selected as the target for three reasons. First, translation of the APOB mRNA lacking exon 27 (skip 27 mRNA) would generate a C-terminally truncated isoform of APOB100, APOB87SKIP27
, which is similar to the C-terminal truncations observed in some patients with mutations in APOB
causing hypobetalipoproteinemia [51
]. Heterozygotes for these mutant alleles of APOB have low cholesterol and LDL levels, have lowered risks of heart attacks, and live significantly longer than normal, although these individuals may be susceptible to fat accumulation in the liver [52
]. Second, the 5' splice site of exon 27, when scored for its similarity to the splice-site consensus sequence using the Shapiro and Senapathy position weight matrix, was the weakest of all the 5' splice sites of the internal exons of APOB [53
]. As constitutive 5' splice sites in general demonstrate better scores than the alternatively spliced 5' splice sites [54
], APOB exon 27 would be the most amenable to alternative splicing. Third, as the sequences necessary for RNA editing are present in exon 26, the skip 27 mRNA should be edited as usual in the intestine and APOB48 expression should be unaffected ().
Skipping APOB exon 27 causes the expression of APOB87SKIP27, a C-terminally truncated isoform of APOB
-methyl ASOs targeting the splice sites flanking APOB exon 27 and to the BPS of intron 26–27 were designed and transfected into HepG2 cells, which naturally express APOB100 [55
]. Combination ASOs targeting two elements simultaneously in a single ASO induced exon 27 skipping, and this was most effective when both the 3' splice site and BPS were targeted. In contrast, targeting predicted ESE motifs within exon 27 to interfere with SR protein binding did not have a significant effect on exon skipping, suggesting that the constitutive incorporation of exon 27 is more dependent on the splice sites than on exonic elements, or that there is functional redundancy between the exonic elements [55
Chabot and colleagues successfully utilized A1-tailed bifunctional oligonucleotides to induce alternative splicing in Bcl-x pre-mRNA [56
]. A bifunctional oligonucleotide consisting of a combination ASO targeting two splice sites plus an RNA tail was designed to bind the hnRNP A1 protein to encourage skipping of exon 27; however, this A1 tail did not augment exon-skipping in APOB pre-mRNA, but paradoxically partially reversed the skipping of exon 27 [55
]. Therefore, the splice site and BPS ASOs may not function through a simple interference with splicing-factor binding. Instead, hybridization of the combination ASOs is likely to induce a secondary structure that is unfavorable to exon 27 inclusion. It is possible that recruitment of hnRNP A1 is counterproductive, as this may cause unwinding of the secondary structure [55
Lastly, we demonstrated that combination ASO-transfected HepG2 cells were able to translate the skip 27 mRNA to a shortened isoform of APOB100, APOB87SKIP27
, which included the N-terminal 87% (3929 amino acids) of APOB100, along with a divergent 37-amino acid peptide at the C-terminus and ending in a premature termination codon within the exon 28 sequence [55
]. Therefore, the induction of APOB87SKIP27
expression in vivo
should lead to decreased LDL and cholesterol levels, such as occurs in patients with hypobetalipoproteinemia.
As mentioned previously, because intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression. This is in contrast to methods that rely on a generalized downregulation of APOB mRNA levels, such as chimeric ASO-mediated RNase H degradation [57
], and RNAi [58
]. These latter methods have been demonstrated to reduce APOB100 levels and therefore circulating levels of cholesterol and LDL, but they also reduce the levels of APOB48 and circulating levels of chylomicrons, an unwanted side effect expected to interfere with the transport of fat from the intestine.