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1.  Exon Skipping of Hepatic APOB pre-mRNA with Splice-switching Oligonucleotides Reduces LDL Cholesterol In Vivo 
Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-Apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting VLDL assembly and increasing LDL clearance. We demonstrate that these “APO-skip” SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for more than 6 d. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to Pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.
PMCID: PMC3589156  PMID: 23319054
apolipoprotein B; exon skipping; familial hypercholesterolemia; oligoribonucleotides; quantitative reverse-transcription polymerase chain reaction; splice-switching oligonucleotides
2.  Splicing therapeutics in SMN2 and APOB 
Splicing therapeutics are defined as the deliberate modification of RNA splicing to achieve therapeutic goals. Various techniques for splicing therapeutics have been described, and most of these involve the use of antisense oligonucleotide-based compounds that target key elements in the pre-mRNA to control splicing in the nucleus. In this review, recent developments in splicing therapeutics for the treatment of two specific diseases are described: correcting the alternative splicing of survival of motor neuron (SMN)2 pre-mRNA to compensate for the defective SMN1 gene in spinal muscular atrophy, and re-engineering the splicing of apolipoprotein B pre-mRNA to lower circulating cholesterol levels.
PMCID: PMC3140428  PMID: 19330716
Antisense oligonucleotide; APOB; apolipoprotein B; cholesterol; RNA splicing; SMN2; spinal muscular atrophy
3.  Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB 
Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels.
We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26–27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27.
The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.
PMCID: PMC1784105  PMID: 17233885
4.  Characterization of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Mutations in Familial Isolated Pituitary Adenoma Families 
Human Mutation  2010;31(8):950-960.
Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation-negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc.
PMCID: PMC3065644  PMID: 20506337
pituitary adenoma; FIPA; acromegaly; AIP; tumor suppressor

Results 1-4 (4)