Design and validation of a specific scavenger receptor class AI binding peptide for targeting the inflammatory atherosclerotic plaque.

OBJECTIVE: Scavenger receptor A (SR-A) is abundantly expressed by macrophage and plays a critical role in foam cell formation and atherogenesis. In search of selective SR-AI antagonists, we have used affinity selection of a phage displayed peptide library on the synthetic extracellular domain of SR-AI. METHODS AND RESULTS: Phage selection led to an almost 1,000-fold enrichment of SR-AI binding phage, which bound avidly to human THP-1 cells. A 15-mer corresponding to the peptide insert of the major SR-AI binding phage (PP1) displaced phage binding to SR-AI. Peptides, docked to a streptavidin scaffold, were effectively internalized by macrophages in an SR-AI-dependent manner. The enriched phage pool and streptavidin bound PP1 exhibited marked uptake by hepatic macrophages in mice. Importantly, PP1 significantly increased streptavidin as well as particulate accumulation in advanced aortic plaques, and in particular intraplaque macrophage, of apolipoprotein E(-/-) mice. CONCLUSIONS: We have identified a novel peptide antagonist selective for SR-AI; this antagonist could be a valuable tool in SR-AI targeted imaging of atherosclerotic lesions.

A targeted peptide nucleic acid to down-regulate mouse microsomal triglyceride transfer protein expression in hepatocytes.

Peptide nucleic acids (PNA's) have shown to hold potential as antisense drugs. In this study we have designed PNA drugs for the microsomal triglyceride transfer protein (MTP), which is known to play a critical role in the assembly of atherogenic lipoproteins, and have converted the most potent drug into a liver-targeted prodrug. First, we have synthesized three PNA sequences targeting domains on the mouse MTP mRNA, which were not involved in intrastrand base-pairing interactions as jugded from its secondary structure. Only one of the PNA's, PNA569, showed dose-dependent inhibition of MTP expression in a cell-free system for coupled transcription/translation of MTP. Second, to improve the cellular uptake of this PNA drug, we have conjugated PNA569 to a high affinity ligand for the asialoglycoprotein receptor, K(GalNAc)(2). As compared to the parent PNA, the prodrug PNA-K(GalNAc)(2) was found to display to a markedly improved capacity to inhibit MTP mRNA expression in parenchymal liver cells. A glycoconjugated nonsense control appeared to be ineffective. In conclusion, the design of a targeted PNA is described to reduce MTP expression in parenchymal liver cells by 70%. The presented approach for targeted tissue-specific down-regulation of genes by PNA's may be valid for other genes as well.

Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes.

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.