Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-11-03
2004-06-08
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S458000
Reexamination Certificate
active
06746868
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to peptide nucleic acid (PNA) conjugates and a method for binding these conjugates to DNA for diagnostic and therapeutic applications. More particularly, the invention relates to PNA linked to fluorophores or peptides which hybridize to DNA and can be used to monitor the intracellular location of exogenous transfected DNA and to promote various intracellular processes.
BACKGROUND OF THE INVENTION
Plasmid based, non-viral gene delivery systems have been shown to be promising for the treatment of major inherited and acquired diseases, and for the development of a new approach to vaccination (Wolff et al.,
Science
247:1465-1468, 1990; Ulmer et al.,
Science
259:1745-1749,1993; Donnelly et al.,
Life Sci
. 60:163-172, 1997; Gao et al.,
Gene Ther
. 2:710-722, 1995; Felgner,
Ann. N.Y. Acad. Sci
.772:126-139, 1995). Although the numbers of human gene therapy trials with these technologies are increasing, their efficiencies and clinical potencies are currently limited due to low levels of in vivo gene product expression (Felgner,
Hum. Gene Ther
.7:1791-1793, 1996). Commonly used approaches for increasing in vivo expression include improving the DNA delivery system (Gao et al.,
Gene Therapy
2:710-722, 1995; Felgner, supra.; Behr,
Bioconj. Chem
. 5:382-389, 1994) or optimizing the DNA sequence at the level of the promoter, enhancer, intron or terminator (Hartikka et al.,
Hum. Gene Ther
. 7:1205-1217, 1996; Liang et al.,
Gene Therapy
3:350-356, 1996).
In conventional small molecule drug development, it is common to make systematic chemical modifications of the biologically active agent itself in order to improve its bioavailability and efficacy. This research and development activity is referred to as medicinal chemistry. The ability to carry out a medicinal chemistry approach to improve the bioavailability of DNA is presently lacking because the methods that have been employed to directly modify DNA either reduce or destroy its ability to be transcribed. In addition, the available approaches to chemically modify plasmids which involve photolysis, nick translation, or the use of chemically active nucleotide analogs, randomly attack the DNA so that the final product is chemically heterogeneous and poorly defined.
Several methodologies, including electron microscopy, fluorescence in situ hybridization (FISH), in situ polymerase chain reaction (PCR), DNA intercalating dyes and radio-, biotin-, gold-, or fluorescent-labeled DNA, have been used to follow the delivery of DNA in cells (Loyter et al.,
Proc. Natl. Acad. Sci. U.S.A
. 79:422-426, 1982; Tsuchiya et al.
J. Bacteriol
. 170:547-551, 1988; Chowdhury, 1993; Zabner et al.,
J. Biol. Chem
. 270:18997-19007, 1995; Dowty et al.,
Proc. Natl. Acad. Sci. U.S.A
. 92:4572-4576, 1995; Bordignon et al.,
Science
270:470-475, 1995; Dean,
Exp. Cell Res
. 230:293-302, 1997). However, these methods have practical and technical limitations, which can lead to difficulties in interpretation. Electron microscopy, FISH and in situ PCR require cell to fixation, lysis, and lengthy manipulations, and these procedures have been shown to influence the detection sensitivity and pattern of DNA distribution in cells. DNA intercalating fluorescent dyes, bind weakly to plasmid and exchange with endogenous nucleic acid raising questions about the intracellular source of the fluorescent signal. Other covalent fluorescent labeling methods which utilize nick translation or photoaffinity labeling result in chemical breakdown of the starting material, and thus any observations made with these materials may not be representative of the behavior of the original intact plasmid. None of the technologies presented above allow direct detection of structurally and functionally intact plasmid in a real-time fashion in viable cells.
Peptide nucleic acids (PNA) have been developed to hybridize to single and double stranded nucleic acids. PNA are nucleic acid analogs in which the entire deoxyribose-phosphate backbone has been exchanged with a chemically completely different, but structurally homologous, polyamide (peptide) backbone containing 2-aminoethyl glycine units. Unlike DNA, which is highly negatively charged, the PNA backbone is neutral. Therefore, there is much less repulsive energy between complementary strands in a PNA-DNA hybrid than in the comparable DNA-DNA hybrid, and consequently they are much more stable. PNA can hybridize to DNA in either a Watson/Crick or Hoogsteen fashion (Demidov et al.
Proc. Natl. Acad. Sci. U.S.A
. 92:2637-2641, 1995; Egholm,
Nature
365:566-568, 1993; Nielsen et al.,
Science
254:1497-1500, 1991; Dueholm et al.,
New J. Chem
. 21:19-31, 1997). Molecules called PNA “clamps” have been synthesized which have two identical PNA sequences joined by a flexible hairpin linker containing three 8-amino-3,6-dioxaoctanoic acid units. When a PNA clamp is mixed with a complementary homopurine or homopyrimidine DNA target sequence, a PNA-DNA-PNA triplex hybrid can form which has been shown to be extremely stable (Bentin et al.,
Biochemistry
35:8863-8869, 1996; Egholm et al.,
Nucleic Acids Res
. 23:217-222, 1995; Griffith et al.,
J. Am. Chem. Soc
. 117:831-832, 1995).
The sequence-specific and high affinity duplex and triplex binding of PNA have been extensively described (Nielsen et al.,
Science
254.1497-1500, 1991; Eghohm et al.,
J. Am. Chem. Soc
. 114:9677-9678, 1992; Egholm et al.,
Nature
365:566-568, 1993; Almarsson et al.,
Proc. Natl. Acad. Sci. U.S.A
. 90:9542-9546, 1993; Demidov et al.,
Proc. Natl. Acad. Sci. U.S.A
. 92:2637-2641, 1995). They have also been shown to be resistant to nuclease and protease digestion (Demidov et al.,
Biochem. Pharm
. 48:1010-1313, 1994). PNA has been used to inhibit gene expression (Hanvey et al.,
Science
258:1481-1485,1992; Nielsen et al.,
Nucl. Acids. Res
., 21:197-200, 1993; Nielsen et al.,
Gene
149:139-145, 1994), to block restriction enzyme activity (Nielsen et al., supra., 1993), to act as an artificial transcription promoter (Mollegaard,
Proc. Natl. Acad. Sci. U.S.A
. 91:3892-3895, 1994) and as a pseudo restriction endonuclease (Demidov et al.,
Nucl. Acids. Res
. 21:2103-2107, 1993). Recently, PNA has also been shown to have antiviral and antitumoral activity mediated through an antisense mechanism (Norton,
Nature Biotechnol
., 14:615-619, 1996; Hirschman et al.,
J. Investig. Med
. 44:347-351, 1996).
The ideal probe for irreversible chemical modification of plasmid will not dam the plasmid, and thus will not interfere with its transcription or intracellular trafficking. The plasmid structure, biological activity and stability would be the same with or without probe. The probe should be sequence-specific in order to differentiate delivered plasmid from endogenous nucleic acid and the probe itself should not have any influence on plasmid function. All of the technologies discussed above for chemically modifying plasmid DNA result in DNA damage and interfere with its transcriptional activity. Further, none of the technologies mentioned above allow direct detection of structurally and functionally intact plasmid in a real-time fashion on viable cells. The present invention provides a straightforward and versatile approach to permanently introduce new physical and biological properties into DNA by irreversible plasmid modification.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a composition comprising a nucleic acid molecule and a conjugated peptide nucleic acid (PNA) molecule associated with said DNA molecule, wherein the PNA molecule contains a region complementary to the DNA molecule. In one aspect of this preferred embodiment, the nucleic acid molecule is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA molecule is a plasmid. In another aspect of this preferred embodiment, the plasmid encodes a reporter gene. Alternatively, the plasmid encodes a therapeutic gene. Advantageous
Bennett C. Frank
Felgner Philip L.
Zelphati Olivier
ISIS Pharmaceuticals Inc.
McKelvey Terry
Vogel Nancy
Woodcock & Washburn LLP
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