Antisense oligonucleotides targeted against human...

Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives

Reexamination Certificate

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C435S006120, C435S091100, C435S325000, C435S375000

Reexamination Certificate

active

06420551

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to oligonucleotide (ODN) based therapeutics, particularly the treatment of infections of the human immunodeficiency virus (HIV).
BACKGROUND OF THE INVENTION
The present invention relates to ODNs suitable for use in treatment of HIV infected individuals by inhibition of replication of HIV in infected cells.
HIV is responsible for the disease that has come to be known as acquired immune deficiency syndrome (AIDS). Although initially recognized in 1981, no cure has yet been found for this inevitably fatal disease. HIV is spread by a variety of means such as sexual contact, infected blood or blood products and perinatally. Because of the complexity of HIV infection and the paucity of effective therapies, a great deal of effort has been expended in developing methods for detecting, treating and preventing infection. Diagnostic procedures have been developed for identifying infected persons, blood and other biological products.
The HIV genome has been well characterized. Its approximately 10 kb encode sequences containing regulatory segments for HIV replication as well as the gag, pol and env genes coding for the core proteins, the reverse transcriptase-protease-endonuclease, and the internal and external envelope glycoproteins, respectively. HIV tends to mutate at a high rate causing great genetic variation between strains of the viruses and indeed between virus particles of a single infected individual. There are a few “conserved” regions of the HIV genome which tend not to mutate. These regions are presumed to encode portions of proteins essential for virus function which can thus withstand very few mutational events.
The HIV env gene encodes the glycoprotein, gpl60, which is normally processed by proteolytic cleavage to form gp120, the external viral glycoprotein, and gp41, the viral transmembrane glycoprotein. The gpl120remains associated with HIV virions by virtue of noncovalent interactions with gp41. These noncovalent interactions are weak, consequently most of the gpl120 is released from cells and virions in a soluble form.
Like most viruses, HIV often elicits the production of neutralizing antibodies. Unlike many other viruses and other infectious agents for which infection leads to protective immunity, however, HIV specific antibodies are insufficient to halt the progression of the disease. Therefore, in the case of HIV, a vaccine that elicits the immunity of natural infection could prove to be ineffective. In fact, vaccines prepared from the HIV protein gp160 appear to provide little immunity to HIV infection although they elicit neutralizing antibodies. The failure to produce an effective anti-HIV vaccine has led to the prediction that an effective vaccine will not be available until the end of the 1990's. Therapeutic agents currently used in treatment of AIDS often cause severe side-effects which preclude their use in many patients. It would, thus, be useful to have alternative methods of treating and preventing the disease that do not entail vaccination and currently available pharmaceutical agents.
Recently, attempts have been made to moderate protein production associated with viral infections by interfering with the mRNA molecules that direct their synthesis. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such a therapeutic approach to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression which is believed to have promise is the “antisense” approach. Single-stranded nucleic acid, primarily RNA, is the target molecule for ODNs that are used to inhibit gene expression by an antisense mechanism. A number of workers have reported such attempts: Stein and Cohen (1988)
Cancer Res
., 48:2659-2668; Walder (1988)
Genes & Development
, 2:502-504; Marcus-Sekura (1988)
Anal. Biochem
., 172:289-295; Zon (1987)
J. Pro. Chem
., 6:131-145; Zon (1988)
Pharm. Res
., 5:539-549; Van der Krol et al. (1988)
Biotechniques
, 6:958-973; and Loose-Mitchell (1988)
TIPS
, 9:45-47. Antisense ODNs are postulated to exert an effect on target gene expression by hybridizing with a complementary RNA sequence. The hybrid RNA-ODN duplex appears to interfere with one or more aspects of RNA metabolism including processing, translation and metabolic turnover. Chemically modified ODNs have been used to enhance nuclease stability and cell permeability.
Duplex DNA can be specifically recognized by oligomers based on a recognizable nucleomonomer sequence. The motif termed “GT” recognition has been described by Beal et al. (1992)
Science
, 25:1360-1363; Cooney et al. (1988)
Science
, 241:456-459; and Hogan et al., EP Publication 375408. In the G-T motif, the ODN is oriented antiparallel to the target purine-rich sequence and A-T pairs are recognized by adenine or thymine residues and G-C pairs by guanine residues.
Sequence-specific targeting of both single-stranded and duplex target sequences has applications in diagnosis, analysis, and therapy. Under some circumstances wherein such binding is to be effected, it is advantageous to stabilize the resulting duplex or triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one approach to prolong stabilization. Sequence-specific recognition of single-stranded DNA accompanied by covalent crosslinking has been reported by several groups. For example, Vlassov at al. (1986)
Nuc. Acids Res
., 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleomonomers complementary to target sequences. A report of similar work by the same group is that by Knorre et al. (1985)
Biochimie
, 67:785-789. It has also been shown that sequence-specific cleavage of single-stranded DNA can be mediated by incorporation of a modified nucleomonomer which is capable of activating cleavage. Iverson and Dervan (1987)
J. Am. Chem. Soc
., 109:1241-1243. Covalent crosslinking to a target nucleomonomer has also been effected using an alkylating agent complementary to the single-stranded target nucleomonomer sequence. Meyer et al. (1989)
J. Am. Chem. Soc
., 111:8517-8519. Photoactivated crosslinking to single-stranded ODNs mediated by psoralen has been disclosed. Lee et al. (1988)
Biochem
., 27:3197-3203. Use of crosslinking in triple-helix forming probes has also been disclosed. Horne et al. (1990)
J. Am. Chem. Soc
., 112:2435-2437.
Use of N
4
, N
4
-ethanocytosine as an alkylating agent to crosslink to single-stranded and double-stranded oligomers has also been described. Webb and Matteucci (1986)
J. Am. Chem. Soc
., 108:2764-2765; (1986)
Nuc. Acids Res
., 14:7661-7674; and Shaw et al. (1991)
J. Am. Chem. Soc
., 113:7765-7766. These papers also describe the synthesis of ODNs containing derivatized cytosine. The synthesis of oligomers containing N
6
, N
6
-ethanoadenine and the crosslinking properties of this residue in the context of an ODN binding to a single-stranded DNA has been described. Matteucci and Webb (1987) Tetrahedron Letters, 28:2469-2472.
In a recent paper, sequence-specific binding of an octathymidylate conjugated to a photoactivatable crosslinking agent to both single-stranded and double-stranded DNA is described. Praseuth et al. (1988)
Proc. Natl. Acad. Sci. (USA)
, 85:1349-1353. In addition, targeting duplex DNA with an alkylating agent linked through a 5′-phosphate of an ODN has been described. Vlassov et al. (1988)
Gene
313-322; and Fedorova et al. (1988)
FEBS Lett
., 228:273-276.
In effecting binding to obtain a triplex, to provide for instances wherein purine residues are concentrated on one chain of the target and then on the. opposite chain, oligomers of inverted polarity can be provided. By “inverted polarity” is meant that the oligomer contains tandem sequences which have opposite polarity, i.e., one having polarity 5′→3′ followed by another with polarity 3′→5′,

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