Inhibition of gene transcription by polyamide DNA-binding...

Details Inhibition of gene transcription by polyamide DNA-binding... Inhibition of gene transcription by polyamide DNA-binding...

2000-04-25

2003-12-09

Travers, Russell (Department: 1617)

Drug, bio-affecting and body treating compositions

Solid synthetic organic polymer as designated organic active...

Nitrogen heterocycle

Reexamination Certificate

active

06660255

ABSTRACT:

BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to polyamides that bind to predetermined sequences in the minor groove of double stranded DNA that are useful for diagnosis and treatment of diseases associated with gene transcription. This invention is related to modulation of cellular or viral gene expression required for maintenance and replication of pathogens in infectious disease, such as HIV-1 and CMV. This invention is also related to modulation of cellular gene expression in non-infectious disease conditions, such as cancers involving oncogenes, e.g., her-2
eu.
Gene Therapy Approaches for HIV:
Considerable effort has been expended over the past decade to devise methods to interfere with HIV-1 gene expression in living cells in the hope that therapeutic strategies will come from these studies (recently reviewed in Kohn, D. B. and N. Sarver, Gene therapy for HIV-1 infection, in
Antiviral Chemotherapy
, J. Mills, P. A. Volberding, and L. Corey, Editors. 1996, Plenum Press: New York. p. 421-427.). One approach includes interference with the translation of messenger RNA into protein by the introduction of antisense oligonucleotides into lymphoid cells, as discussed in Kohn, D. B. and N. Sarver, Gene therapy for HIV-1 infection, in
Antiviral Chemotherapy
, J. Mills, P. A. Volberding, and L. Corey, Editors. 1996, Plenum Press: New York. p. 421-427; Bordier, B., et al.,
Proc. Natl. Acad. Sci. U.S.A
., 92: 9383-9387 (1995) and Lisziewicz, J., et al.,
Proc. Natl. Acad. Sci. U.S.A
., 91: 7942-7946 (1994).
Another approach involves ribozyme-mediated destruction of specific regions of HIV-1 mRNA. See Sun, L. Q., et al.,
Proc. Natl. Acad. Sci. U.S.A
., 92: 7272-7276 (1995); Yamada, O., et al.,
J. Virol
., 70: 1596-1601 (1996) and Zhou, C., et al.,
Gene
, 149: 33-39 (1994). Decoy molecules, corresponding to HIV-1 RNA domains that bind regulatory proteins required for the HIV-1 life cycle (TAR RNA which binds Tat or the Rev-response element) have been used as inhibitors of HIV-1 replication (Sullenger, B. A., et al.,
Cell
, 63: 601-608 (1990). In addition, trans-dominant mutant versions of these regulatory proteins, introduced into cells with retroviral expression vectors, have been shown to inhibit HIV-1 replication (Bevec, D., et al.,
Proc. Natl. Acad. Sci. U.S.A
., 89: 9870-9874, 1992.).
Other approaches for direct inhibition of gene transcription, including designed or selected zinc finger peptides that recognize pre-determined DNA sequences, are described in Wu, H., et al.,
Proc. Natl. Acad. Sci. U.S.A
., 92: 344-348 (1995) and Thiesen, H.-J.,
Gene Expr
., 5: 229-243.(1996). DNA-cleaving ribozymes have also been tried (Raillard, S. A. and G. F. Joyce,
Biochemistry
, 35: 11693-11701(1996)). Triple helix-forming oligonucleotides have been used to block HIV-1 integration: Bouziane, M., et al.,
J. Biol. Chem
., 271: 10359-10364 (1996). Triple helix-forming oligonucleotides have also been used specifically cleave HIV-1 DNA with a metalloporphyrin group attached to the oligonucleotide, as described by Bigey, P., G. Pratviel, and B. Meunier,
Nucleic Acids Res
., 23: 3894-3900 (1995). Additionally, the DNA-binding calicheamicin oligosaccharides have the potential for use in anti-HIV-1 therapy but have not as yet been applied to this disease. See Ho, S. N., et al.,
Proc. Natl. Acad. Sci
., 91: 9203-9307 (1994) and Liu, C., et al.,
Biochemistry
, 93: 940-944 (1996).
For any gene therapy approach to be successful, several criteria must be met by the therapeutic agent: First, tile agent must not possess any general cell toxicity and should not elicit an immune response. Second, the agent must be cell-permeable or amenable to viral delivery and, in the case of the DNA-binding agents, the therapeutic agent must transit to the nucleus and bind the target sequence with high affinity and specificity in the context of cellular chromatin. Third, binding of the agent to its DNA or RNA target sequence must interfere with gene transcription or protein translation.
Each of the potential approaches listed above has its own unique advantages and limitations. For example, while nucleic acid-based approaches (antisense, decoy and triple helix-forming oligonucleotides and ribozymes) have the potential for sequence selectivity and can effectively inhibit transcription or translation in vitro, these molecules suffer from poor cell permeability and other delivery systems, such as retroviral vectors in the case of the ribozymes (Zhou, C., et al., 1994) or liposomes or other delivery strategies in the case of antisense or triple helix oligonucleotides, must be used for effective gene inhibition (reviewed in Kohn & Sarver, 1996). Similarly, zinc finger peptides must be introduced via a gene therapy approach with an appropriate viral expression vector since these peptides cannot directly enter cells. See Choo, Y., et al.,
Nature
, 372: 642-645 (1994).
One additional problem with gene therapy approaches is that they must be performed on lymphoid cells ex vivo and, once an “HIV-protected” cell population is established, these cells must then be introduced into the patient.
In contrast to gene therapy approaches, HIV protease inhibitors taken in combination with standard anti-retroviral agents (AZT) have recently shown success in clinical trials. Wei, X. et al.,
Nature
, 373: 117-122 (1995); Ho, D. D. et al.,
Nature
, 373: 123-126 (1995).
The key to the anti-HIV properties of these drugs is that they strike at two separate phases of the virus life cycle, limiting the ability of spontaneous mutations to result in inhibitor-resistant strains of the virus. Small molecule inhibitors of HIV-1 RNA transcription which would target a third phase of the virus life cycle would be highly desirable. Cell-permeable sequence-specific DNA-binding ligands would circumvent the problems associated with other forms of gene therapy and could compliment the protease inhibitor-anti-retroviral agent cocktail approach mentioned above. The calicheamicin oligosaccharides satisfy some of the requirements for a therapeutic agent; these molecules are sufficiently hydrophobic to pass through cell membranes but these molecules possess severely limited sequence specificity (4 base pairs) and bind DNA with very low affinities (100 &mgr;M or higher required for inhibition of protein-DNA interactions . See Ho, S. N., et al.,
Proc. Natl. Acad. Sci
., 91: 9203-9307 (1994) and Liu, C., et al.,
Biochemistry
, 93: 940-944 (1996)).
Thus, new classes of cell-permeable molecules that possess higher degrees of DNA sequence specificity and affinity are needed for the treatment of AIDS and other infectious diseases. We describe below the successful development of a new class of highly specific designed small molecule ligands with great potential for inhibition of HIV-1 gene transcription.
The HIV-1 Enhancer and Promoter:
A recent review has summarized our current knowledge of the protein factors required for the control of RNA initiation and elongation by RNA polymerase II at the HIV-1 promoter (Jones, K. A. and B. M. Peterlin. 1994. Control of RNA initiation and elongation at the HIV-1 promoter.
Annu. Rev. Biochem
., 63: 717-743). Thus only those aspects of HIV-1 transcription that relate to transcription inhibition are discussed herein. For HIV, the template for synthesis of both new viral RNA and messenger RNA (for viral protein synthesis) is the integrated provirus, the product of reverse transcription of the viral RNA in the infected cell. HIV-1 utilizes the transcription machinery of the host cell but encodes its own trans-activators, Tat and Rev, that are responsible for RNA elongation and utilization. The HIV-1 promoter is located in the U3 region of the leftward (5′) long terminal repeat (see FIG. 11 below, taken from Jones and Peterlin, 1994). The core promoter and enhancer elements span a region of approximately 250 base pairs and include TATA and initiator elements and the binding sites for the following cellular transcription factors: Sp1, NF-&kgr;B, LEF-1, Ets-1 and USF. Sequences upstream of t

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