Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1995-06-06
2003-01-21
Whisenant, Ethan C. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C536S023100, C536S024300, C514S04400A
Reexamination Certificate
active
06509149
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the human papillomavirus. More specifically, this invention relates to the inhibition, treatment, and diagnosis of human papillomavirus-associated lesions using synthetic oligonucleotides complementary to human papillomavirus nucleic acid.
BACKGROUND OF THE INVENTION
Human papillomaviruses (HPV) comprise a group of at least 70 types, based on DNA sequence diversity as measured by liquid hybridization (Pfister et al. (1994)
Intervirol.
37:143-149). These nonenveloped DNA viruses infect epithelial cells resulting in a range of lesions from benign skin and genital warts (condyloma acuminata) and epidermodysplasia verruciformis (EV) to respiratory or laryngeal papillomatosis and cervical carcinoma. Each virus type exhibits host specificity.
Several HPV types infect genital epithelia and represent the most prevalent etiologic agents of sexually transmitted viral disease. The genital HPV types can be further subdivided into “high-risk” types that are associated with the development of neoplasms, most commonly HPV-16 and HPV-18; and “low-risk” types that are rarely associated with malignancy, most commonly HPV-6 and HPV-11. The malignant types may integrate into the genome of the host cell, thereby eliminating the requirement for viral DNA replication gene products. In contrast, the benign types, most commonly HPV6 and HPV11, rely on viral proteins E1 and E2 for replication of the episomal genome.
Current treatment for HPV infection is extremely limited. There are at present no approved HPV-specific antiviral therapeutics. Management normally involves physical destruction of the wart by surgical, cryosurgical, chemical, or laser removal of infected tissue. Topical anti-metabolites such as 5-fluorouracil and podophyllum preparations have also been used. (Reichman in
Harrison's Principles of Internal Medicine,
13th Ed. (Isselbacher et al., eds.) McGraw-Hill, Inc., NY (1993) pp.801-803). However, reoccurrence after these procedures is common, and subsequent repetitive treatments progressively destroy healthy tissue. Interferon has so far been the only treatment with an antiviral mode of action, but its limited effectiveness restricts its use (Cowsert (1994)
Intervirol.
37:226-230; Bornstein et al. (1993)
Obstetrics Gynecol. Sur.
4504:252-260; Browder et al. (1992)
Ann. Pharmacother.
26:42-45).
Two HPV types, HPV-6 and HPV-11 are commonly associated with laryngeal papillomas, or benign epithelial tumors of the larynx. Neonates may be infected with a genital papillomavirus at the time of passage through their mother's birth canal. By the age of two, papillomas will have developed, and infected juveniles will undergo multiple surgeries for removal of benign papillomas which may occlude the airway To date there is no method of curing juvenile onset laryngeal papillomatosis. There is consequently a serious need for a specific antiviral agent to treat human papillomavirus infectica.
New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression (see, Zamecnik et al. (1978)
Proc. Natl. Acad. Sci.
(USA) 75:280-284). These agents, called antisense oligonucleotides, bind to target single-stranded nucleic acid molecules according to the Watson-Crick rule or to double stranded nucleic acids by the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (
Proc. Natl. Acad. Sci.
(USA) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (
Proc. Natl. Acad. Sci.
(USA) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having ( more than one type of internucleotide linkage within the oligonucleotide have been developed. Pederson et al. (U.S. Pat. Nos. 5,149,797 and 5,220,007) discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Agrawal et al. (PCT US93/06884) discloses hybrid oligonucleotides having regions of deoxyribonucleotides and 2′-O-methyl-ribonucleotides.
A limited number of antisense oligonucleotides have been designed which inhibit the expression of HPV. For example, oligonucleotides specific for various regions of HPV E1 and E2 mRNA have been prepared (see, e.g., U.S. Pat. No. 5,364,758, WO/91/08313, WO 93/20095, and WO 95/04748).
A need still remains for the development of oligonucleotides that are capable of inhibiting the replication and expression of human papillomavirus whose uses are accompanied by a successful prognosis and low or no cellular toxicity.
SUMMARY OF THE INVENTION
It has been discovered that specific oligonucleotides complementary to particular portions of nucleic acid encoding the translational start site of human papillomavirus E1 gene can inhibit HPV replication and expression. This discovery has been exploited to provide synthetic oligonucleotides complementary to regions spanning the translational start site of mRNA encoding the HPV E1 protein.
As used herein, a “synthetic oligonucleotide” includes chemically synthesized polymers of about five and up to about 50, preferably from about 15 to about 30 ribonucleotide and/or deoxyribonucleotide monomers connected together or linked by at least one, and preferably more than one, 5′ to 3′ internucleotide linkage.
For purposes of the invention, the term “oligonucleotide sequence that is complementary to nucleic acid or mRNA” is intended to mean an oligonucleotide that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means, including in the case of an oligonucleotide binding to RNA, causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen base pairing under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence.
In a first aspect, the invention provides synthetic oligonucleotides complementary to a nucleic acid spanning the translational start site of human papillomavirus gene E1, and including at least 15 nucleotides. In preferred embodiments, the oligonucleotides of the invention are from about 15 to about 30 nucleotides in length.
In some embodiments, these oligonucleotides are modified. In preferred embodiments, these, modifications include at least one alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester internucleotide linkage or a combination of such linkages, as in a chimeric oligonucleotide. In one preferred embodiment, an oligonucleotide of the invention includes phosphorothioate internucleotide linkages. In yet another preferred embodiment, the oligonucleotide includes at least one methylphosphonate internucleotide linkage.
The oligonucleotides of the invention may also include at least one deoxyribonucleotide, at least one ribonucleotide, or a combi
Frank Bruce L.
Goodchild John
Greenfield Isobel M.
Kilkuskie Robert E.
Mills John S.
Hybridon, Inc.
Keown & Associates
Whisenant Ethan C.
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