Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1999-09-23
2002-02-26
LeGuyader, John L. (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C536S023100, C536S024300, C536S024500, C424S450000
Reexamination Certificate
active
06350853
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to compositions comprising a peptide nucleic acid (PNA) which is conjugated to a lipophilic group and incorporated into liposomes. The PNA is composed of naturally-occurring nucleobases or non-naturally-occurring nucleobases which are covalently bound to a polyamide backbone. The PNA compositions of the present invention may further comprise a PNA modified by an amino acid side chain. The PNA compositions of the present invention exhibit enhanced cellular uptake and distribution. PNA compositions which were incorporated into liposomes demonstrated increased cellular uptake and more diffuse distnbution than PNA compositions without liposomes.
BACKGROUND OF THE INVENTION
The function of a gene starts by transcription of its information to a messenger RNA (mRNA). By interacting with the ribosomal complex, mRNA directs synthesis of proteins. This protein synthesis process is known as translation. Translation requires the presence of various cofactors, building blocks, amino acids and transfer RNAs (tRNAs), all of which are present in normal cells.
Most conventional drugs exert their effect by interacting with and modulating one or more targeted endogenous proteins, e.g., enzymes. Typically, however, such drugs are not specific for targeted proteins but interact with other proteins as well. Thus, use of a relatively large dose of drug is necessary to effectively modulate the action of a particular protein. If the modulation of a protein activity could be achieved by interaction with or inactivation of mRNA, a dramatic reduction in the amount of drug necessary and in the side-effects of the drug could be achieved. Further reductions in the amount of drug necessary and the side-effects could be obtained if such interaction is site-specific. Since a functioning gene continually produces mRNA, it would be even more advantageous if gene transcription could be arrested in its entirety. Oligonucleotides and their analogs have been developed and used as diagnostics, therapeutics and research reagents. One example of a modification to oligonucleotides is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and 2′-O-methyl ribose sugar moieties. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for the treatment of various disease states. Although some improvements in diagnostic and therapeutic uses have been realized with these oligonucleotide modifications, there exists an ongoing demand for improved oligonucleotide analogs.
There are several known nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences. Among these, the peptide nucleic acids (PNAs), as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
PNAs are useful surrogates for oligonucleotides in binding to DNA and RNA. Egholm et al.,
Nature,
1993, 365, 566, and references cited therein. The current literature reflects the various applications of PNAs. Hyrup et al.,
Bioorganic & Med. Chem.,
1996, 4, 5; and Nielsen,
Perspectives Drug Disc. Des.,
1996, 4, 76.
PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone. Each subunit of the peptide backbone is attached to a naturally-occurring or non-naturally-occurring nucleobase. One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. The synthesis of PNAs via preformed monomers was previously described in WO 92/20702 and WO 92/20703, the contents of which are herein incorporated by reference. More recent advances in the structure and synthesis of PNAs are illustrated in WO 93/12129 and U.S. Pat. No. 5,539,082, issued Jul. 23, 1996, the contents of both being herein incorporated by reference. Further, the literature is replete with publications describing synthetic procedures, biological properties and uses of PNAs. For example, PNAs possess the ability to effect strand displacement of double-stranded DNA. Patel,
Nature,
1993, 365, 490. Improved synthetic procedures for PNAs have also been described. Nielsen et al.,
Science,
1991, 254, 1497; and Egholm,
J. Am. Chem. Soc.,
1992, 114, 1895. PNAs form duplexes and triplexes with complementary DNA or RNA. Knudson et al.,
Nucleic Acids Research,
1996, 24, 494; Nielsen et al.,
J. Am. Chem. Soc.,
1996, 118, 2287; Egholm et al.,
Science,
1991, 254, 1497; Egholm et al.,
J. Am. Chem. Soc.,
1992, 114, 1895; and Egholm et al.,
J Am. Chem. Soc.,
1992, 114, 9677.
PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (T
m
). This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA) duplexes is that T
m
is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
Triplex formation by oligonucleotides has been an area of intense investigation since sequence-specific cleavage of double-stranded deoxyribonucleic acid (DNA) was demonstrated. Moser et al.,
Science,
1987, 238, 645. The potential use of triplex-forming oligonucleotides in gene therapy, diagnostic probing, and other biomedical applications has generated considerable interest. Uhlmann et al.,
Chemical Reviews,
1990, 90, 543. Pyrimidine oligonucleotides have been shown to form triple helix structures through binding to homopurine targets in double-stranded DNA. In these structures the new pyrimidine strand is oriented parallel to the purine Watson-Crick strand in the major groove of the DNA and binds through sequence-specific Hoogsteen hydrogen bonding. The sequence specificity is derived from thymine recognzig adenine (T:A-T) and protonated cytosine recognizing guanine (C
+
:G-C). Best et al.,
J. Am. Chem. Soc.,
1995, 117, 1187. In a less well-studied triplex motif, purine-rich oligonucleotides bind to purine targets of double-stranded DNA. The orientation of the third strand in this motif is anti-parallel to the purine Watson-Crick strand, and the specificity is derived from guanine recognizing guanine (G:G-C) and thymine or adenine recognizing adenine (A:A-T or T:A-T). Greenberg et al.,
J. Am. Chem. Soc.,
1995, 117, 5016.
Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)
2
/DNA(RNA) triplexes of high thermal stability. Egholm et al.,
Science,
1991, 254, 1497; Eghohm et al.,
J. Am. Chem. Soc.,
1992, 114, 1895; Egholm et al.,
J Am. Chem. Soc.,
1992, 114, 9677. The formation of triplexes involving two PNA strands and one nucleotide strand has been reported in U.S. patent application Ser. No. 08/088,661, filed Jul. 2, 1993, the contents of which are incorporated herein by reference. The formation of triplexes in which the Hoogsteen strand is parallel to the DNA purine target strand is preferred to formation of anti-parallel complexes. This allows for the use of bis-PNAs to obtain triple helix structures with increased pH-independent thermal stability
Knudsen Helle
Nielsen Peter E.
Larson Thomas G
LeGuyader John L.
Nielsen Peter E.
Woodcock & Washburn LLP
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