In situ binary synthesis of biologically effective molecules

Details In situ binary synthesis of biologically effective molecules In situ binary synthesis of biologically effective molecules

1998-11-25

2002-12-10

Fredman, Jeffrey (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S325000, C435S091520, C536S024300

Reexamination Certificate

active

06492111

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to the synthesis, generation or release of molecules within cells and tissues, which molecules have significant, biological effects. Such molecules are synthesized which permit the identification of the cells or tissues, which destroy the cells or tissues, which moderate, modulate, or enhance cellular or tissue function, or which otherwise have a diagnostic, therapeutic, nutritional or other biological effect.
The in situ generation of such molecules is specific to particular cells and tissues such that the molecules thus formed or released are delivered specifically to the cells or tissues in question. Such high specificity of delivery overcomes many therapeutic impediments and can provide high “leverage” with concomitant low side effects.
BACKGROUND OF THE INVENTION
It has long been known to employ nucleic acid structures present in cells to serve as “targets” for diagnostic and therapeutic regimes. A classical example of such employment is evidenced by the field of antisense therapeutics. According to the antisense paradigm, a nucleic acid sequence or structure is identified as being associated with the production of a gene product—usually a peptide—which has deleterious effects upon cells or tissues. The target nucleic acid may be an oncogene, an mRNA associated with the genesis or development of a disease state or a hyperproliferative gene structures which proliferation is to be reduced or eliminated.
Under the antisense paradigm, oligonucleotides are designed which are specifically bindable to a nucleic acids sequence or structure, usually mRNA, the down regulation of which is desired. The oligonucleotide, usually in the form of a chemically modified analog or construct, is administered to the cells containing the targeted nucleic acid. The specific binding of the oligonucleotide interferes with expression of the mRNA, thus interfering with its function and expression. Protein normally expressed by the mRNA is either not expressed at all or is expressed in much lower quantity with a concomitant, beneficial therapeutic effect. The paradigm may also be used for diagnosis and research in ways which are well known to persons of ordinary skill in the art.
The antisense paradigm is well understood per se to persons of ordinary skill in the art and there are numerous review articles which describe in detail varying approaches to this practice.
Nucleic acids which can be used to target in accordance with some embodiments of this invention include mRNA molecules, which preferably have secondary structures such as stem-loop structures, or unique secondary structural sites, a such as the molecular interaction sites taught in U.S. application Ser. Nos. 09/076,440, 09/076,447, and 09/076,404, each of which is incorporated herein by reference in its entirety.
Potential target nucleic acids include, but are not limited to, mammalian, bacterial, fungal and viral mRNA. Oligonucleotides have been shown to bind to numerous targets including, for example, Candida (U.S. Pat. No. 5,691,461), protein kinase C (U.S. Pat. Nos. 5,703,054, 5,681,944, and 5,620,963), papillomavirus (U.S. Pat. Nos. 5,681,944 and 5,756,282), herpesvirus (U.S. Pat. No. 5,658,891), cytomegalovirus (U.S. Pat. Nos. 5,607,923 and 5,595,978), human immunodeficiency virus (U.S. Pat. No. 5,523,389), ras (U.S. Pat. Nos. 5,661,134, 5,582,986, and 5,576,208), Epstein-Barr virus (U.S. Pat. No. 5,242,906), cell adhesion molecules (U.S. Pat. Nos. 5,599,797 and 5,514,788), hepatitis virus (U.S. Pat. No. 5,576,302), Raf kinase (U.S. Pat. Nos. 5,654,284, 5,563,255, 5,656,612, and 5,744,361), p120 (U.S. Pat. No. 5,814,629), cell growth (U.S. Pat. No. 5,656,743), and multi-drug resistance associated protein (U.S. Pat. No. 5,510,239). Each of the above-identified patents is incorporated herein by reference in its entirety.
In addition to traditional antisense mechanisms, oligonucleotides have also been shown to act through mechanisms, for example, involving pseudo-half-knot formulations (U.S. Pat. No. 5,512,438), 5′-cap inhibition (U.S. Pat. No. 5,643,780), and triple helix formation (U.S. Pat. No. 5,834,185), each of which is incorporated herein by reference in its entirety. All such targets and many more may be used herein.
It has also been proposed to employ a pair of oligonucleotides to bind specifically with a mRNA in an antisense fashion. (see Viassov et al., “
Binary Systems of Oligonucleotide Conjugates for Sequence Specific Energy—Transfer Sensitized Photomodification of Nucleic Acids” NATO ASI Ser., Ser. C,
479 (DNA and RNA Cleavers and Chemotherapy of Cancer and Viral Diseases) 195-207, (1996). Viassov discloses that, along with one oligonucleotide there is a flourescent sensitizer, while the other oligonucleotide carries a structure which, when irradiated with ultraviolet radiation, is known to react with nucleic acids. Viassov demonstrates a rapid modification of the DNA to which the oligonucleotides were targeted when the two oligonucleotides are allowed to bind to adjacent sites on the DNA and be subsequently irradiated. The proximity of the photosensitizer to the group which transfers energy to the DNA was viewed to be important to the reaction with the DNA.
The technique of fluorescence resonance energy transfer (FRET) has been used, inter alia to identify point mutations in nucleic acids. A pair of oligonucleotides, each of which carries a fluorophores are caused to bind to nucleic acids. Through study of their fluorescence behavior, determination of the proximity of the fluorophores and, hence, the presence or absence of intervening base units, can give rise to the desired mutational information. FRET can also monitor ribozime interactions and seems to be useful generally in nucleic acid research.
Viassov et al in, “
Sequence Specific Cleavage of Yeast tRNA
-
Phe with Oligonucelotides Conjugated to Diimidazole Construction
”, Antisense Nucleic Drug Development, 7(1): 39-42, (1997), prepares oligonucleotides conjugated to a chemical construction having two histidine residues. Yurchenko et al, “
Cleavage of Leishmania Mini—Exon Sequence by Oligonucleotides Conjugated to a Diimidazole Construction
”, Nucleosides and Nucleosides, 16 (7-9): 1721-1725, (1996), is directed to similar subject matter. While it has been known to conjugate a plurality of oligonucleotides to a single nucleic acid in a sequence specific fashion, all such techniques are believe to have been directed to the incapacitation or destruction of the nucleic acid target. While this can certainly have some useful applications, it is limited in its applicability.
It has been greatly desired to provide for the targeting of nucleic acids in a way different from the traditional, antisense approach. Thus, improvements over antisense methodology and over the methodologies for nucleic acid cleavage of, e.g. Viassov and Yurchenko are greatly desired. In particular, a methodology that does not necessarily involve the incapacitation of the target nucleic acid but which has a vastly more significant therapeutic potential have long been desired. The present invention is directed to this new paradigm of therapeutics and diagnostics.
SUMMARY OF THE INVENTION
The present invention provides a dramatic divergence in oligonucleotide therapeutics, diagnostics and related paradigms. In accordance with the invention, a nucleic acid target which has been identified as being present in the cell or tissue of interest is caused to be the object of specific binding by a plurality of oligonucleotides. By virtue of the specific binding of the two or more oligonucleotides, which binding is caused to take place at adjacent sites on the target nucleic acid, a molecular species is either synthesized, formed or released. The molecular species has great biological effect within the cell or tissue which, of course, carries the target nucleic acid. By virtue of the formation, synthesis or release of such biologically effective molecules, cells having the target nucleic acid may either be identified, imaged, killed, benefitted or

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