Targeted RNA degradation using nuclear ribozyme RNA

Details Targeted RNA degradation using nuclear ribozyme RNA Targeted RNA degradation using nuclear ribozyme RNA

1999-05-18

2001-07-24

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S320100, C435S325000, C536S023100, C536S024500

Reexamination Certificate

active

06265167

ABSTRACT:

BACKGROUND OF THE INVENTION
The polyoma virus genome is a small circular double-stranded DNA molecule whose early and late transcription units proceed in opposite directions from an intergenic regulatory region. In the early phase of a productive life cycle, the early genes (encoding the large, middle and small T antigens) are preferentially expressed. Before the initiation of viral DNA replication, few late-strand messages can be detected (Beard et al.,
J. Virol.
17:20-26 (1976)). Once the infection enters the late phase (after the onset of DNA replication), late gene expression increases rapidly while the relative level of early gene expression is dramatically reduced.
The observation that polyoma virus mutants with temperature sensitive large T antigens overexpress their early genes at the nonpermissive temperature has led to the proposal that the viral early genes are negatively regulated by their own products (Farmerie and Folk,
Proc. Natl. Acad. Sci. USA
81:6919-6923 (1984)). This autoregulation was thought to result from large T antigen binding to high affinity binding sites in the viral intergenic region and thereby inhibiting initiation from the early promoter (Fenton and Basilico,
Virology
121:384-392 (1982)). However, it has been shown using nuclear run-on assays that the relative transcriptional activities from the early and late promoters change little throughout infection (Hyde-DeRuyscher and Carmichael,
Proc. Natl. Acad. Sci. USA
85:8993-8997 (1988)). Regulation of early gene expression after transcription initiation is also consistent with the data of Farmerie and Folk (Ibid.), who observed early gene repression by large T antigen even in constructs where this transcription was driven by a heterologous promoter. Taken together these results suggest that regulation of early gene expression is at a post-transcriptional level.
It has been shown that transcription termination of the late genes is inefficient during the late phase of infection (Hyde-DeRuyscher and Carmichael, Ibid.), allowing RNA polymerase II to continue around the circular viral genome multiple times. The resulting giant primary transcripts are eventually processed by RNA splicing and polyadenylation, but unprocessed giant transcripts accumulate to high levels in the nucleus (Acheson,
Mol. Cell. Biol.
4:722-729 (1984)). Due to the circularity of the polyoma genome, giant late-strand transcripts contain sequences complementary to early-strand transcripts, serving as natural antisense RNA. These giant transcripts accumulate at the same time that early-strand RNAs become relatively less abundant.
There is a growing body of literature devoted to antisense technology; however, this work has related only to cytoplasmic antisense RNA. (Murray, J. A. H. and Crockett, N., “Antisense Techniques: An Overview, ” in
Antisense RNA and DNA,
(Wiley-Liss, Inc.) pp. 1-49 (1992)). There has been no work to date aimed at producing antisense RNA accumulated in the nucleus.
SUMMARY OF THE INVENTION
This invention pertains to the regulation of gene expression by nuclear antisense RNA. It is shown herein that the naturally occurring polyoma antisense early RNA generated from the late transcription unit is almost exclusively present in the nucleus. Furthermore, it is shown herein that polyoma early-strand RNAs are reduced by expression and accumulation in the nucleus of antisense-early RNAs. It is demonstrated that antisense RNAs can be expressed and accumulated in the nucleus using a construct comprising a promoter, antisense sequences, and sequences encoding a cis-ribozyme and optionally a stem loop structure. The cis-ribozyme is incorporated into the antisense construct in order to generate 3′-ends independently of the polyadenylation machinery and thereby inhibit transport of the RNA molecule to the cytoplasm. The construct may optionally contain a histone stem loop structure to aid in stabilizing the transcripts against exonucleolytic degradation. This invention has application to several areas. It may be used as a therapeutic agent, targeting and inhibiting harmful native gene expression. It may also be used therapeutically to inhibit viral gene expression, thereby making cells resistant to and curing viral infection. This invention also has application as a research tool with the ability to inhibit expression of any targeted gene.


REFERENCES:
patent: 5908779 (1999-06-01), Carmichael et al.

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