Using polyamide nucleic acid oligomers to engender a...

Details Using polyamide nucleic acid oligomers to engender a... Using polyamide nucleic acid oligomers to engender a...

1998-10-08

2004-04-20

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S325000, C435S377000, C514S04400A, C536S018700, C536S024100, C536S024500

Reexamination Certificate

active

06723560

ABSTRACT:

BACKGROUND
1. Technical Field
The invention relates to administering polyamide nucleic acid oligomers to living cells such that the polyamide nucleic acid oligomers engender a sequence specific biological response.
2. Background Information
Polyamide nucleic acids (PNAs) are DNA analogs containing neutral amide backbone linkages. Unlike DNA oligomers, PNA oligomers can bind DNA by displacing one strand of the duplex to form a stable D-loop structure (Peffer et al.,
Proc. Natl. Acad. Sci. USA
90:10648-10652 (1993) and Møllegaard et al.,
Proc. Natl. Acad. Sci. USA
91:3892-3895 (1994)). Interestingly, binding of PNA oligomers to DNA is independent of DNA strand polarity, allowing PNA oligomers to bind in both parallel and anti-parallel fashion (Egholm et al.,
Nature
365:566-568 (1993) and Peffer et al.,
Proc. Natl. Acad. Sci. USA
90:10648-10652 (1993)). In addition, PNA oligomers are less susceptible to enzymatic degradation (Demidov et al.,
Biochem. Pharmacol.
48:1310-1313 (1994)) and bind RNA with higher affinity than analogous DNA oligomers. Taken together, these properties suggest that PNA oligomers have great potential in both antigene and antisense approaches for regulating gene expression.
The success of an oligonucleotide analog as an antigene or antisense agent requires that the oligonucleotide be taken up by cells in reasonable quantities such that the oligonucleotide reaches its target at a sufficient concentration. PNA oligomers, however, have low phospholipid membrane permeability (Wittung et al.,
FEBS Letters
365:27-29 (1995)) and have been reported to be taken up by cells very poorly (Hanvey et al.,
Science
258:1481-1485 (1992); Nielsen et al.,
Bioconjugate Chem.
5:3-7 (1994); Bonham et al.,
Nucleic Acids Res.
23:1197-1203 (1995); Gray et al.,
Biochem. Pharmacol.
48:1465-1476 (1997)), which would appear to limit their potential uses in antigene and antisense approaches.
Recent strategies devised to improve cellular uptake of PNA oligomers involve conjugating other molecules to PNA sequences. Specifically, conjugating a small peptide sequence that binds the insulin-like growth factor 1 receptor (IGF1R) to a PNA oligomer increases cellular uptake of labeled PNA sequences by IGF1R-expressing cells, whereas conditions using unconjugated PNA sequences or cells lacking IGF1R show negligible cellular uptake (Basu S. and Wickstrom E.,
Bioconjugate Chem.
8:481-488 (1997)). These results suggest that conjugating receptor ligand molecules to PNA oligomers can increase cellular uptake; however, the ability of these receptor ligand-conjugated PNA oligomers to influence biological activity once inside the target cells remains unknown. Further, PNA oligomers will gain entrance only into cells expressing that particular targeted receptor. Thus, an appropriate ligand molecule would have to be designed and coupled to PNA oligomers for each cell type of interest. In addition, the level of receptor expression can influence the permeability of ligand-conjugated PNA oligomers.
The use of PNA oligomers to manipulate brain protein expression, an approach that would greatly aid the understanding of brain function as well as neurological disease, has an additional problem. The endothelial wall of capillaries in both brain and spinal cord creates a barrier (blood-brain barrier; BBB) that excludes the uptake of molecules into these organs. Although specialized transport systems operate within the BBB to allow certain circulating molecules to cross, many pharmaceutical agents are not recognized and thus have poor BBB permeability. This appears true for PNA molecules since the transport of PNAs across the BBB is reported to be negligible (Pardridge et al.,
Proc. Natl. Acad. Sci. USA
92:5592-5596 (1995)). Therefore, PNA oligomers targeting brain proteins administered outside the central nervous system need to cross two barriers, the BBB and the plasma membrane of individual cells within brain, whereas PNA oligomers administered directly into brain need to cross one barrier, the plasma membrane of individual cells.
Various drug delivery strategies can circumvent the BBB permeability problem (Pardridge,
Pharmacol. Toxicol.
71:3-10 (1992); Pardridge,
Trends Biotechnol.
12:239-245 (1994)). For example, PNA molecules can undergo transport through the BBB when the amino terminus is biotinylated and linked to a streptavidin conjugated monoclonal antibody specific for transferrin receptor (OX26-SA; Pardridge et al.,
Proc. Natl. Acad. Sci. USA
92:5592-5596 (1995)). The OX26-SA antibody delivers linked molecules to the brain presumably by receptor-mediated endocytosis, given the high transferrin receptor concentrations located on the BBB. These studies suggest that antibody-conjugation strategies provide a mechanism for PNA oligomers to cross the BBB. No data, however, exist as to whether the biotinylated PNA linked to OX26-SA actually enters cells or not. In addition, the utility of PNA delivery methods that rely on conjugating other molecules to PNA oligomers remains unclear since these other molecules may influence the desired functionality of particular PNAs.
SUMMARY
The present invention relates to PNA oligomers that influence biological activity in a sequence specific manner. Specifically, this invention relates to the discovery that PNA oligomers administered extracellularly cross biological barriers and elicit a sequence specific biological response in living cells. This discovery is in direct opposition to the current understanding of the physical properties of PNA oligomers and has far-reaching implications for both gene therapy and research purposes. Further, extracellularly administering PNA oligomers to living cells circumvents the need to micro-inject PNA oligomers directly into cells as well as the need to permeabilize cells. In addition, this invention provides for the treatment of cells in vivo such that a behavioral response is observed in an organism. Thus, this invention describes methods and materials that allow any polypeptide to be manipulated and studied in living cells. For example, the expression of a specific polypeptide can be knocked-out in adult organisms for the duration of PNA oligomer treatment. In addition to greatly aiding the advancement of basic scientific research, this ability to manipulate polypeptide expression and thus function in a sequence specific manner is clearly beneficial to gene therapy approaches involving the treatment of cancer, aging, behavioral diseases, infections, and auto-immune diseases.
In addition, this invention relates to the following two discoveries: 1) antisense PNA oligomers administered to an animal cross biological barriers and modulate translation in a sequence specific manner, and 2) sense PNA oligomers administered to an animal cross biological barriers and modulate transcription in a sequence specific manner. Thus, this invention provides two powerful in vivo methods for manipulating polypeptide expression in a sequence specific manner. Clearly, having the ability to modulate two distinct steps involved in polypeptide synthesis will further the advancement of basic scientific research and provide useful methods for treating disease. The term “antisense PNA oligomer” refers to any PNA oligomer having sequence specificity for an RNA molecule found within a cell. The term “sense PNA oligomer” refers to any PNA oligomer having sequence specificity for a region of nucleic acid that can be used as the template strand during transcription, including reverse transcription.
This invention also relates to the discovery that a mismatch PNA oligomer crosses biological barriers and engenders a biological response to a degree less than that engendered by a PNA oligomer targeting the same sequence but having, for example, no base pair mismatches. In other words, this invention relates to the discovery that the degree to which a biological response is engendered can be modulated by changing the sequence of a particular PNA oligomer to introduce base pair mismatches with a target sequence. Having the ability to alter th

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