Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood–brain barrier and specifically reduce gene expression

Abstract

Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i.p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.Peptide nucleic acids (PNAs), a new type of DNA analog ( Fig. 1), hold great promise as antisense or antigene drugs, because they are electrically neutral oligomers that are stable against nucleases and proteases, bind independently of salt concentration to their complementary nucleic acids, and have higher affinity for nucleic acids than do DNA͞DNA duplexes (1, 2). Additionally, PNA͞DNA duplexes are much more gene specific, because they are less tolerant of mismatches than are DNA͞DNA duplexes (3). Initial enthusiasm for their use as antisense or antigene drugs was dampened by the fact that these molecules pass poorly into cells (4, 5). Our laboratory reported that unmodified (carrier-free) PNAs, on their direct injection into rat brain, enter neuronal cells and inhibit protein synthesis in a gene-specific manner (6).To determine both the mechanism of action of PNAs and whether PNAs could pass the blood-brain barrier (BBB), brain neurotensin (NT) receptors (NTR1) again were targeted. After a single i.p. injection of antisense PNA to NTR1 (targeted to mRNA) behavioral and physiological responses to NT (antinociception and hypothermia) were specifically and almost completely lost. These results were accompanied by specific reductions in receptor sites as determined by radioligand binding assays. However, there were no changes in mRNA levels. A sensitive assay developed to detect the amount of PNAs in tissue (gel shift assay) confirmed the presence of PNA in brain after i.p. injection. Therefore, these results provided evidence that any antisense strategy targeted to brain proteins can work by i.p. delivery and by crossing the normal (i.e., not compromised by malignancy) BBB. Also, of ...

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

“…[38][39][40] Moreover, although in vivo, there is little evidence for antigene activity of PNAs, several groups have reported that PNAs can induce downregulation of target mRNA and have suggested either an antigene mechanism by interaction with target DNA, which inhibits the transcription machinery, or that PNA interaction with mRNA targets triggers its degradation through a hypothetical mechanism. [11][12][13][14] However, the PNAs used in these studies do not target homo-purine motifs, and would not be able to form a stable complex with DNA in order to block RNA polymerase. 10,38,39 Likewise, the antisense PNA that targets the first codons of the open reading frame of cyclin B1 gene should not be able to act as an antigene molecule.…”

“…The inefficiency of PNAs in vivo is in part related to their poor propensity to cross the cell membrane and/or to their inappropriate cellular localization. 10 Recent work has demonstrated that cellular uptake of free PNAs can occur, although this requires very high concentrations of PNAs, except in neuronal cells, 11,12 and yields limited biological response. [13][14][15] In order to improve delivery of PNAs into cells and to reduce the doses required for detectable effects, several chemical modifications based on covalently linked cell-penetrating peptides have been successfully used.…”

Isolation of Peptide Transport System-6 from Brain Endothelial Cells: Therapeutic Effects with Antisense Inhibition in Alzheimer and Stroke Models

“…Phosphorothioate oligonucleotides are enzymatically resistant (Jaeger and Banks, 2004) and many are taken up by and transported (Banks et al, , 2006) across brain endothelial cells. Peptide nucleic acids, another class of antisense molecules, also effectively cross the BBB (Tyler et al, 1999). The antisenses developed here against PTS-6 were taken up by the capillaries and by brain tissue after the i.v.…”

Artificial (Pseudo)peptides for Molecular Recognition and Catalysis

“…Finally, in a very exciting and controversial study Richelson and coworkers have claimed that antigene and antisense PNA can pass the blood-brain barrier and can bind in-vivo to the neurotensin receptor (NTR1) in rats [52,53]. Specifically, intraperitoneal injection of PNA inhibited the hypothermic and antinociceptive activities of neurotensin microinjected in the brain.…”