Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-22
2001-02-06
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06183966
ABSTRACT:
TECHNICAL FIELD
The present invention generally relates to an apparatus and method for ranking nucleic acid sequences based on stability of nucleic acid oligomer sequence binding interactions to select sequence zones for antisense targeting.
BACKGROUND OF THE INVENTION
Bodily states in mammals, including disease states, are at least in part directly affected by proteins. Those proteins, acting directly or through enzymatic functions, contribute in large proportion to many diseases in animals and humans. In the past, therapeutic treatment has focused on interactions with those proteins in an effort to moderate the disease. Attempts have also been made to moderate the actual production of such proteins by interaction with molecules that directly affect their synthesis, i.e., the DNA sequence that underlies the gene product. It is well known that by interfering with the production of the proteins, the effect of the therapeutic results can be maximized. Likewise, therapeutic approaches have been developed that interfere with gene expression, leading to undesired formations which need to be patrolled. There are numerous methods that have been formulated for inhibiting specific gene expression which have been adopted to some degree and have been defined as antisense nucleic acids. The basic approach is that an oligonucleotide analog complimentary to a specific targeted messenger RNA or mRNA sequence is used. Pertinent references include those of Stein and Cohen (1988); Walder (1988); Marcus-Sekura (1988); Zon (1988); Van der Krol (1988) and Matteucci and Wagner (1996). Each of the foregoing concern general antisense theory and prior techniques.
Background of Antisense Technology
The term “antisense” generally connotes an approach to chemotherapy which is based upon the complementary pairing of an antisense oligonucleotide, or ASO, with a target nucleic acid. The use of an ASO compound requires a complementarity of the antisense base sequence to a target zone of an mRNA, so that the ASO will bind to that mRNA target sequence and will bring about selective inhibition of gene expression (D. A. Melton, 1988; Stein and Cohen, 1988; and Toulmé and HélIIne, 1988). A more thorough understanding of such technology can be found in a book edited by Cohen (Oligonucleotides—
Antisense Inhibitors of Gene Expression
(1989)), and a recent review by Mercola and Cohen (1995).
Referring to
FIG. 1
, the schematic representation of the process of transfer of information from the DNA genome to a protein product can be seen. If the gene product (protein) is one whose controlled synthesis is essential for the well-being of the organism or cell, then a defective synthesis or an unwanted protein can lead to illness or death.
FIG. 1
also illustrates that an antisense oligonucleotide (or oligo) can be used to inhibit or terminate protein synthesis.
A summary of the relevant portion of the disclosure from the review by Cohen (Id., 1989, pp. 1-6) is set forth below. There are five basic assumptions included in this approach:
(1) Cellular uptake: It is assumed that the oligo will cross the cell membrane and will be able to reach its target sequence within the cell.
(2) Stability: The oligo will be stable under in vivo conditions, and will reach the target sequence in significant quantity.
(3) Hybridization: The oligo will hybridize with the target sequence so that a DNA:RNA hybrid will be produced.
(4) Inhibition of expression: The formation of this hybrid will prevent the expression of the gene(s) coded for by the hybridized mRNA.
(5) Selectivity of binding: The oligo will not be bound non-selectively to many other sites, particularly protein sites so as to have its effective concentration, or potency reduced.
The results of early studies that showed selective inhibition of gene expression in Rous sarcoma virus with a synthetic 13-mer (Zamenik and Stephenson, 1978) indicated that this oligo was indeed penetrating into cells. However, the general resistance to this possibility, and the difficulty in synthesizing oligos of such length until the mid-1980's, slowed progress in this area.
Once automated oligo synthesis became possible, further attempts were made to test this antisense approach. It has now been established that antisense oligonucleotides can be actively taken up by cells and can accumulate in the cellular nucleus (G. D. Gray et al., 1997).
Initial attempts to inhibit gene expression naturally concentrated on normal oligos with phosphodiester linkages. However, it has been believed that since these compounds are known to be subject to enzymatic hydrolysis by nucleases in vivo and in vitro, they could not form the basis of an effective antisense strategy. This problem of low oligo stability could explain the high concentrations of oligos that were found to be required to bring about inhibition of expression in some of the early work. Further, the breakdown of an informational molecule in which the integrity of the base sequence is integral to its mode of action would obviously be a devastating restriction on the strategy of the antisense approach.
Any chemical modification in an oligo can lead to a change in hybridization with its target mRNA sequence at physiological temperature. Since the object of this strategy is to ensure that hybridization occurs, it is mandatory that the hydrogen bonding capability of the bases not be impaired. It is for this reason that the modifications that are made in the oligo are usually in the backbone, and not in the bases or the sugars. Nevertheless, there are many possible modifications of the three portions of the nucleotide unit, only a few of which have so far been investigated. However, in order not to disrupt the formation of Watson-Crick base pairing, it is preferable that any modification that is made should be rather conservative. For example, the substitution of one sulfur atom for any oxygen or phosphate is perhaps the most conservative substitution that can be envisaged that accomplishes the aim of nuclease stability (Eckstein, 1985) without significantly impairing the hybridization of the oligo. The prospect for hybridization of an oligo with a mRNA must also take account of the fact that RNAs can have complex folded tertiary structures and, thus, the target sequence should be contained within a single-stranded accessible region.
The mechanics of inhibition of gene expression were originally presumed to arise from interference of the hybrid DNA:RNA duplex with ribosomal processing. This mechanism has been termed translation arrest or hybridization arrest. However, subsequent work has shown that ribonucleases that hydrolyze such hybrids, namely RNase-H, are actively involved in the mechanism of action (Walder and Walder, 1988). This was shown very clearly by comparison of normal oligos which have the beta configuration of the base-sugar linkage, and non-natural alpha-oligos, which form hybrids with RNA that are not susceptible to RNase-H, and which were concomitantly found not to produce translational arrest (Cavenave et al., 1989). The most potent antisense effects are obtained when RNA translation is blocked by RNase-H cleavage (Matteucci and Wagner, 1996).
It is important for the effectiveness of the antisense oligo approach that the oligo bind selectively to the target complementary sequence. If the oligo is bound non-selectively to the other sites, particularly protein sites that are present in the cell, the potency of any oligo as a putative drug would be severely limited. Eckstein has shown in a series of elegant studies (Eckstein, 1985) that phosphorothioate oligos have a tendency to bind to protein sites and to inhibit nucleases. This “problem” can, in fact, be turned into an advantage if the oligos interact specifically with a protein site, and thus inhibit, for example, a process that is required for vital proliferation. This has been found to be the case with the phosphorothioate analogues which inhibit certain polymerases in a sequence non-specific manner. Such an inhibition cannot be described as an antisense effect. However, the inhib
Clark Christopher L.
Gray Donald M.
Board of Regents University of Texas System
Houtteman Scott W.
Locke Liddell & Sapp LLP
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