Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-06-26
2002-03-12
McGarry, Sean (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S455000, C536S023100, C536S024100, C536S024500
Reexamination Certificate
active
06355621
ABSTRACT:
BACKGROUND OF THE INVENTION
Negative-stranded nonsegmented RNA viruses include: paramixoviruses such as Newcastle's disease, mumps and parainfluenza; morbilliviruses such as measles, canine distemper and bovine rinderpest; pneumovirus or respiratory syncytial virus; vesiculovirus; and lyssaviruses such as rabies. The single-stranded negative sense genomic RNA of these viruses act as a direct template for both transcription and replication (Wunner et al.
Rev Infect Dis
1988, 10, S771-S784). In rabies virus, for example, during the process of transcription a positive strand leader RNA and five monocistronic mRNA are synthesized. These are then translated into individual structural proteins. In the process of replication, the full-length positive strand RNA is synthesized and then becomes the template for the synthesis of progeny negative-stranded nonsegmented genomic RNA.
Vaccination provides a means for preventing infection by several of these more prevalent viruses, i.e., mumps and measles. However, vaccinations are not available for all of these viruses. Further, post-infection treatments are oftentimes unsatisfactory.
For example, it is estimated that more than 50,000 people die of rabies each year. The postexposure treatment recommended by World Health Organization includes administration of rabies vaccine together with antirabies immunoglobulin. The treatment regimen is very effective providing it is initiated within 24-48 hours after exposure. However, such rapid treatment is not always possible. Treatment with rabies vaccines after neuronal infection and dysfunction have occurred may lead to exaggerated immunopathologic disease (Murphy, F. A.
Arch Virol
1977, 54, 279-297). Once clinical symptoms occur, the disease is usually fatal. There is no treatment or intervention currently available.
Oligodeoxynucleotides (ODN), which are complementary to certain message or viral sequences, have been reported to inhibit specific gene expression or virus infection (Zamecnik, P. C. and Stephenson, M. L.
Proc Nat'l Acad Sci USA
1978, 75, 280-284). ODN hybridization to complementary RNA sequences inhibits the processing, nuclear transport, or translation of mRNA by blocking the access of functional machinery to requisite mRNA sequences, and the formation of DNA-RNA hybrids leads to RNA cleavage by means of RNase H activities (Agrawal et al.
Proc Natl Acad Sci USA
1990, 87, 1401-1405). Thus, production of gene products is inhibited, reduced or shut off (Zamecnik, P. C. and Stephenson, M. L.
Proc Nat'l Acad Sci USA
1978, 75, 280-284). ODN have been used widely to study gene expression (Wickstrom, E.
Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS
, Wiley-Liss, New York, 1991) and to inhibit tumor growth (Agrawal, S.
Oligonucleotide Therapeutic Approach: Near Clinical Development
, Humana Press, New York, 1996). Antisense ODN to oncogenes, such as c-myb, c-Ha-ras, bcr-abl, or NF-
k
B, have been reported to inhibit the growth of tumor cells in vitro and in vivo (Kitajima et al.
J Biol Chem
1992, 267, 25881-25888; Gray et al. Cancer Res 1993, 53, 577-580; Skorski et al. Proc Natl Acad Sci 1994, 91, 4504-4508). One of the first successful demonstrations of using antisense to inhibit virus infection was performed in Rous sarcoma virus in cell culture (Zamecnik, P. C. and Stephenson, M. L.
Proc Nat'l Acad Sci USA
1978, 75, 280-284). Subsequently, it has been shown that ODN specific to viruses, when added into culture medium, can protect cultured cells to varying extents from infection by a variety of viruses, such as influenza virus (Leiter et al.
Proc Natl Acad Sci USA
1990, 87, 3430-3434), vesicular stomatitis virus (VSV) (Agris et al.
Biochemistry
1986, 25, 6268-6275), duck hepatitis virus (Offensperger et al.
EMBO J
1993, 12, 1257-1262), and human immunodeficiency virus type 1 (HIV-1) (Zaia et al.
J Virol
1988, 62, 3914-3917). Most of the ODN used to inhibit virus infections are antisense DNA complementary to different viral transcripts. However, ODN to other targets have also been shown to inhibit virus infection, such as the TAR element of HIV-1 (Vickers et al.
Nucleic Acid Res
1991, 19, 3359-3368). Further, in a brief abstract made available to the public in May of 1996, Fu et al. disclosed that ODNs complementary to rabies virus genomic RNA blocked almost completely rabies virus infection at concentrations as low as 2 &mgr;M, while ODNs complementary to vial transcripts did poorly even at concentrations as high as 20 &mgr;M (Fu et al., Abstract W15-9, page 110, presented at the 15th Annual Meeting of the American Society for Virology on Jul. 13-17, 1996). This abstract also teaches that the antigenomic ODNS inhibited cell-to-cell spread of the rabies virus and it is suggested that ODNS complementary to rabies virus genomic RNA may have potential to be used for therapy in clinical rabies.
It has now been found that antigenomic ODN targeted to negative strand genomic RNA of nonsegmented RNA viruses are effective inhibitors of viral transcription and may be useful in the treatment and prevention of infection by negative-stranded nonsegmented RNA viruses.
SUMMARY OF THE INVENTION
An object of the present invention is to provide oligodeoxynucleotides complementary to genomic RNA of negative-stranded nonsegmented RNA viruses.
Another object of the present invention is to provide a method of inhibiting infection of cells by a negative-stranded nonsegmented RNA virus which comprises exposing cells to an effective amount of an oligodeoxynucleotide complementary to genomic RNA of a negative-stranded nonsegmented RNA virus so that infection of the cells by the virus is inhibited.
Yet another object of the present invention is to provide a method of treating animals infected with a negative-stranded nonsegmented RNA virus which comprises administering to an animal infected with a negative-stranded nonsegmented RNA virus an effective amount of an oligodeoxynucleotide complementary to genomic RNA of the negative-stranded nonsegmented RNA virus.
DETAILED DESCRIPTION OF THE INVENTION
Oligodeoxynucleotides (ODN) complementary to the genomic RNA of negative-stranded nonsegmented RNA viruses have now been found to block virus infection. Further, the oligodeoxynucleotides of the present invention have been found to be more effective inhibitors of viral transcription and replication than antisense oligodeoxynucleotides targeted to mRNA of the viruses.
The effects of antigenomic ODNs in blocking infection by negative-stranded nonsegmented RNA virus were studied with eight 16 mer ODNs targeted to rabies virus. The sequences and targets of these ODNs are listed in the following Table 1.
TABLE 1
SEQUENCES AND TARGETS OF THE ODNS
SEQ ID
ODN
SEQUENCE
NO
TARGET
RH+1
ATCAAAGAAAAAACAG
1
nts 16-31 of 3′ end
of negative strand
RE−1
TTCTCATTTTTGTTGT
2
nts 16-31 of 3′ end
of positive strand
RL−1
CAGGTTGAGAAGTGTT
3
upstream noncoding
region of rabies
virus L mRNA
RL−2
TCCAGGATCGAGCATC
4
initiation region of
rabies virus L mRNA
RL+2
GATGCTCGATCCTGGA
5
sense ODN to RL−2
RNS−1
CGAAAGGAGGGGTGTT
6
upstream noncoding
region of rabies
virus NS mRNA
RNS−2
CAAAGATCTTGCTGAT
7
initiation region of
rabies virus NS mRNA
RNS+2
ATGAGCAAGATCTTTG
8
sense ODN to RNS−2
Inhibitory activities of these ODNs were tested in baby hamster kidney (BHK) and mouse neuroblastoma (NA, clone 1300) cells. BHK and NA cells grown in 24-well plates were infected with rabies virus ERA strain (derived from the same isolate as SAD B19, Sacramento et al.
J Gen Virol
1992, 73, 1149-1158) at a multiplicity of infection (moi) of approximately 5 focus-forming units (ffu) per cell to ensure that 100% of the cells were infected. After a one hour incubation, the viral inoculum was removed and fresh medium was added with different ODNs to a final concentration of 20 &mgr;M. ODNs were further added to culture medium at the same concentration at 3 and 5 hours post infection (p.i.). The cells were cultured for another 16 hours before har
Dietzschold Bernhard
Fu Zhen Fang
Koprowski Hilary
Wickstrom Eric
McGarry Sean
Thomas Jefferson University
University Thomas Jefferson
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