Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-11-23
2004-08-17
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C536S023100, C536S024500, C435S069100, C435S320100, C435S325000
Reexamination Certificate
active
06776986
ABSTRACT:
This invention relates to inhibition of HIV-1 replication using antisense RNA expression.
BACKGROUND OF THE INVENTION
1. Field of the Invention
2. Description of the Related Art
HIV-1 infection is believed to be the primary cause of Acquired Immunodeficiency Syndrome (AIDS). HIV-1 is a retrovirus having a genome comprised of two copies of full length RNA. Without intending to be bound by a particular theory, it is hypothesized that the replication of the virus in the CD4+ host cell occurs as follows. When the host cell is infected, the viral genomic RNA is transcribed by reverse trascriptase into double stranded DNA. This double stranded DNA is then integrated into the host cell's chromosome(s). When this double stranded DNA is integrated iinto the genetic material of the host cell, it is called a provirus. Following activation of the host cell, the provirus is transcribed into RNA in two distinct phases. In the early phase of infection, RNA transcripts of the provirus produced in the nucleus are converted into multiple copies of short sequences by cellular splicing enzymes. These short RNA transcripts encode genes for proteins, e.g., tat, which regulate the further transcription, and rev, which is though to mediate the transition into the late phase transcription. This early phase dominates for about 24 hours. About 24 hours after activation of the cell, the transcription moves into the late phase. In late phase transcription, long unspliced RNA transcripts of about 9,200 bases and medium-length single-spliced transcripts of about 4,500 bases move out of the nucleus and into the cytoplasm. These unspliced and single-spliced transcripts encode the structural and enyzmatic proteins of the virus. These unspliced and single-spliced transcripts include, inter alia, the following regions: gag, which encodes the viral core proteins; pol, which encodes various enzymes; and env, which encodes the two envelope proteins.
FIG. 4
depicts the HIV-1 genomic structure. It will be noted that there is some overlap in the genes, because certain genes share some base sequences.
The unspliced and single-spliced transcripts are then further spliced, and the resulting mRNA is translated to produce the proteins necessary to make a new virus. The gag and pol regions are translated to produce the polyproteins gag and gag-pol, which are then cleaved by protease to form the mature proteins found in the virus. The env is spliced to generate a subgenomic messenger which encodes for the env polyproteins, which is likewise cleaved to produce the mature envelope proteins. Two strands of the viral RNA are then packaged into a core and surrounded with capsid protein, and the resulting virus is released from the cell together with a portion of the cell membrane.
Various antisense strategies to inhibit HIV-1 infection have been tried, including the use of trans-domninant proteins (Bevec, D., et al. 1992. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative rev trans-activator. Proc. Natl. Acad. Sci. USA 89:9870-9874 and Trono, D., et al. 1989. HIV-1 gag mutants can dominantly interfere with the replication of the wild-type virus. Cell 59:113-120), single chain antibodies (Levy-Mintz, P., et al. 1996. Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase. J. Virol. 70:8821-8832.), antisense RNAs (Chatterjee, S., et al. 1992. Dual-target inhibition of HIV-1 in vitro by means of adeno-associated virus antisense vector. Science 258:1485-1488., Choli, H., et al. 1994. Inhibition of HIV-1 multiplication in a human CD4+ lymphocytic cell line expressing antisense and sense RNA molecules containing HIV-1 packaging signal and rev response element(s). Antisense Res. and Dev. 4:19-29, Joshi, S., et al. 1991. Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression. J. Virol. 65:5524-5530, Kim, J. H., et al., 1996. Inhibition of HIV replication by sense and antisense Rev Response Elements in HIV-based retroviral vectors. J. Acquir. Immune Defic. Syndr. 12:343-351, Meyer, J., et al., 1993. Inhibition of HIV-1 replication by high-copy-number vector expressing antisense RNA for reverse transcriptase. Gene 129:263-268, Renneisen, K., et al 1990. Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region. J. Biol. Chem. 265:16337-16342 and Rhodes, A., et al. 1990. Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA. J. Gen. Virol. 71:1965-1974), RNA decoys (Lee, T., et al. 1994. Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev-response element decoy consisting of the 13-nucleotide minimal Rev-binding domain. J. Virol. 68:8254-8264 and Sullenger, B. A., et al 1990. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 63:601-608), and ribozymes (Ojwang, J. O., et al 1992. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc. Natl. Acad. Sci. USA. 89:10802-10806 and Zhou C., I. Bahner, et al 1994. Inhibition of HIV-1 in human T lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes. Gene. 149:33-39).
The trans-dominant HIV-1 protein RevM10 was first evaluated in a clinical trial using genetically modified peripheral blood lymphocytes (Woffendin, C et al. 1996. Expression of a protective gene prolongs survival of T cells in human immunodeficiency virus infected patients. Proc. Natl. Acad. Sci. USA. 93:2889-2894), although recently a ribozyme (Leavitt, M. C., et al 1996. Ex vivo transduction and expansion of CD4+ lymphocytes from HIV+ donors: prelude to a ribozyme gene therapy trial. Gene Ther. 3:599-606) and a transdominant Rev and antisense TAR based (Morgan R. A et al 1996. Clinical protocol: Gene therapy for AIDS using retroviral mediated gene transfer to deliver HIV-1 antisense TAR and transdominant Rev protein genes to syngeneic lymphocytes in HIV-1 infected identical twins. Hum. Gene Ther. 7:1281-1306.) approach have received RAC and FDA approval.
Intracellular expression of antisense RNAs offers an attractive, alternative gene therapy approach to inhibit HIV-1 replication. Antisense RNAs have been described as very specific and efficient inhibitors in both prokaryotic and eukaryotic systems . Viral replication has been successfully inhibited by addition of in vitro synthesized antisense oligonucleotides or intracellularly expressed antisense RNAs . Inhibition of HIV-1 replication has been shown previously using antisense RNAs targeted against several viral regulatory (Chatterjee et al 1992, Joshi et al 1991, Kim et al 1996, Sczakiel, G. et al 1991. Inhibition of human immunodeficiency virus type 1 replication in human T cells stably expressing antisense RNA. J. Virol. 65:468472 and Sczakiel, G et al 1992. Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analyses. J. Virol. 66:5576-5581) and structural gene products (Choli et al 1994, Gyotoky, et al 1991, Meyer et al 1993 and Rhodes et al 1990). A few reports described long antisense sequences expressed either intracellularly using retroviral vectors (Choli et al 1994, Gyotoky, et al 1991 and Rhodes et al 1990) or using antibody-targeted liposomal delivery (Renneisen et al). The different inhibition levels observed in these reports may reflect variation in antisense RNA expression levels, or secondary and tertiary RNA structures, which can influence the hybridization kinetics between two complementary RNAs (Sczakiel, G., M. Homann, and K. Rittner. 1993 Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1. Antisense Res. and Dev. 3:45-52), influencing the biological activity.
Generally, th
Boehnlein Ernst
Escaich Sonia
Ilves Heini
Veres Gabor
Hess Susan
Nguyen Dave T.
Novartis AG
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