Peptide nucleic acid conjugates

Details Peptide nucleic acid conjugates Peptide nucleic acid conjugates

1997-01-07

2001-01-30

Riley, Jezia (Department: 1656)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S023100, C536S025300, C536S025310, C536S025320, C536S025330, C536S025340

Reexamination Certificate

active

06180767

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the inhibition or regulation of gene expression with oligonucleotides. More particularly, the invention relates to conjugates of peptide nucleic acids and molecules which enhance cellular uptake, and the use of such conjugates to selectively target specific cell types.
BACKGROUND OF THE INVENTION
DNA therapeutics show great potential for gene-specific, nontoxic therapy of a wide variety of disease. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to improve nuclease stability and cell membrane permeability (Knorre et al. (1994)
Design and Targeted reactions of Oligonucleotide Derivatives
, CRC Press, Boc a Raton, Fla.). Recently, a new class of compound, peptide nucleic acids (PNA) has shown potential as an antisense agent (Nielsen et al
Science,
254, 1497-1500, 1991). PNAs comprise nucleic acid mimics in which the sugar-phosphate backbone is replaced with a backbone based on amino acids. PNAs generally exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They are resistant to nuclease and protease attack. Melting temperatures of their duplexes with DNA or RNA are much higher than any of the known DNA compounds, both modified and unmodified. Recently, the solution structure of PNAs has also been determined by nuclear magnetic resonance (Brown et al.,
Science
265, 777-780, 1994).
The PNAs may be synthesized inexpensively on a large scale. PNAs may be synthesized by either solution phase or solid phase methods adapted from peptide synthesis. For example, PNAs can be synthesized from four protected monomers containing thymidine, cytosine, adenine and guanine via solid-phase peptide synthesis, by a modification of the Merrifield method (Merrifield,
J. Am. Chem. Soc.
85, 2149-2154, 1963; Merrifield,
Science
232, 341-347, 1986) employing, for example, BOC-Z protected monomers (Christensen et al.,
J. Peptide Science
3, 175-183, 1995).
PNAs recognize DNA and RNA in a sequence specific manner and form complexes which can be characterized by biophysical methods. The binding motif is context dependent; homopyrimidine PNAs combine with complementary polypurine targets to form stoichiometric 2:1 complexes, whereas PNAs containing both purine and pyrimidine bases afford a 1:1 heteroduplex with mis-match sensitivity comparable to that found in double-stranded (ds)DNA. The 2:1 complexes are formed when a second strand of the PNA binds the major groove of a PNA-DNA duplex through Hoogsteen base pairing. Thus, the triplex is comprised of a PNA/DNA duplex (formed by Watson-Crick hydrogen bonds) with a second PNA strand lying in the major grove of the duplex (held by Hoogsteen hydrogen bonds). These triplex complexes are so stable that “strand invasion” of dsDNA is possible. Binding of the PNA results in formation of a D-loop in the dsDNA. This characteristic is believed useful to manipulate gene expression at the transcriptional level. These 2:1 and 1:1 complexes mediate the antigene and antisense effects of PNAs via the steric blockade of enzyme complexes responsible for DNA transcription, cDNA synthesis, and RNA translation (Noble et al.,
Drug Development Research
34:184-195, 1995). PNAs may be used as antisense or antigene drugs, exploiting the sequence-dependent binding of the PNA portion to single stranded nucleic acids, particularly mRNA, or double-stranded dsDNA, respectively.
Although the biophysical data are very much in favor of the PNAs becoming very successful as nucleic acid binding agents, they suffer from a vital limitation in that they are taken up by cells very poorly.
Abbreviations
The following abbreviations may be used herein: A, adenine; aeg, 2-aminoethylglycine; AFP, alpha-fetoprotein; Bzl, benzyl; BOC, 1,-1-dimethylethoxycarbonyl; BHOC, benzhydryloxycarbonyl; C, cytosine; DECA, diethylcyclohexylamine; DIEA, diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF, N-N-dimethylformamide; EDCHA, ethyldicyclohexylamine; EGF, epidermal growth factor; G, guanine; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FMOC, (9H-fluoren-9-ylmethoxy); HATU,
O
-(7-azabenzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HDPU, O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-bis(tetramethylene)uronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HGR, heregulin; IGF1, insulin growth factor-1; IGF1R, insulin growth factor receptor; MDCHA, methyldicyclohexylamine; MOB, 4-methoxybenzyl; NGF, nerve growth factor; NMP, N-methylpyrrolidine; PBS, phosphate buffered saline; PNA, peptide nucleic acid; PyBOP, benzotriazolyl-tris-pyrrolidino-phosphonium hexafluorophosphate; Rapoport's reagent, benzyloxycarbonyl-N′-methylimidazole triflate; ST,
E. coli
heat-stable enterotoxin; T, thymine; TBTU,
O
-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TGF, transforming growth factor; TLC, thin layer chromatography; Z, phenylmethoxycarbonyl.
SUMMARY OF THE INVENTION
According to the present invention, a conjugate is provided comprising a peptide nucleic acid (PNA) oligomer conjugated to a ligand which is capable of binding to a cell surface receptor. The oligomers bind complementary DNA or RNA strands through the bases which are linked to a peptide backbone. The sequence of the bases specifies the target nucleic acid to which the oligomer binds.
According to one preferred embodiment of the invention, the PNA oligomer has a subunit sequence such that the oligomer is capable of forming (i) a triplex with a dsDNA segment or (ii) a duplex with a ssDNA segment or mRNA segment, to inhibit expression of a gene. According to another preferred embodiment, the peptide nucleic acid oligomer has a subunit sequence capable of (i) or (ii), to inhibit expression of a gene which encodes a cell receptor for the ligand. The invention further provides a method for inhibiting expression of a gene in an organism comprising administering such a conjugate to an organism.
The invention is also a method for killing a pathogenic organism, such as a virus, a bacteria or a eukaryotic parasite, comprising contacting said organism with a conjugate as described above, which conjugate binds to a target polynucleotide sequence of said pathogenic organism.


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