Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-07-12
2001-06-05
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C424S094100, C435S006120, C435S070100, C435S183000, C435S091500, C435S412000, C435S091500, C435S023000
Reexamination Certificate
active
06242175
ABSTRACT:
The present invention relates to the processes whereby retroviruses and retrotransposons (retroposons) insert their genetic material into the genome of a eukaryotic host cell in order to carry out a productive infection cycle. More specifically, it relates to proteins of the host cell that have now been found to be required for efficient retrotransposition, which are highly conserved throughout the eukaryotic kingdom but which are not required for cell functioning under most normal conditions. These proteins represent novel targets for anti-retroviral drugs. In addition, assay systems are provided with which anti-retroviral drugs can be screened and tested in vivo and in vitro.
The invention is based on the surprising discovery, contrary to prior teachings, that Ku-associated DNA repair mechanisms are involved in retrovirus and retrotransposon nucleic acid integration. Retrovirus and/or retrotransposon activity is shown by experimental work described herein to be inhibited in both yeast and mammalian cells where Ku function in the cells is reduced.
Retroviruses and Retrotransposons
Retroviruses are RNA viruses that must insert a DNA copy (cDNA) of their genome into the host chromosome in order to carry out a productive infection. When integrated, the virus is termed a provirus (Varmus, 1988). Some eukaryotic transposable DNA elements are related to retroviruses in that they transpose via an RNA intermediate. These elements, termed retrotransposons or retroposons, are transcribed into RNA, the RNA is copied into double-stranded (ds) DNA, then the dsDNA is inserted into the genome of the host cell.
The available evidence indicates that the integration of retroviruses and retrotransposons occurs through entirely analogous mechanisms, and that retroviruses can be viewed as retrotransposons with an extracellular phase of their life cycle. For example, the Ty1 and Ty5 retrotransposons of the yeast
Saccharomyces cerevisiae
have been shown to integrate into the host yeast genome by the same type of mechanism that is employed by mammalian retrotransposons and retroviruses to integrate into mammalian host cell DNA (Boeke et al., 1985; Garfinkel, 1985; Grandgenett and Mumm, 1990; Boeke and Sandmeyer, 1991).
Retroviruses are of considerable risk to human and animal health, as evidenced by the fact that retroviruses cause diseases such as acquired immune deficiency syndrome (AIDS; caused by human immunodeficiency virus; HIV-1), various animal cancers, and human adult T-cell leukaemia/lymphoma (Varmus, 1988); also retroviruses have been linked to a variety of other common disorders, including Type I diabetes and multiple sclerosis (Conrad et al., 1997; Perron et al. 1997 and Benoist and Mathis, 1997). In many but not all cases, cancer formation by certain retroviruses is a consequence of them carrying oncogenes. Furthermore, retroviral integration and retrotransposition can result in mutagenic inactivation of genes at their sites of insertion, or can result in aberrant expression of adjacent host genes, both of which can have deleterious consequences for the host organism. Retroviruses are also becoming used more and more commonly for gene delivery and are likely to play increasingly important roles in gene therapy. An understanding of how retroviruses function and how they can be controlled is therefore of great commercial and medical importance.
Over recent years, a vast amount of effort has been directed towards identifying inhibitors of retroviral infection because these agents have potential use in combatting retrovirally-borne diseases. To date, most drug development programmes have focused on virally-encoded products. However, given the short life cycle of retroviruses and their inherently high rates of genetic change, it is anticipated that a frequent problem with such strategies will be that drug resistant virus derivatives will arise through alterations of the virally-encoded target molecule (for example, Sandstrom and Folks, 1996 and references therein). Thus, most anti-retroviral drugs that interfere with virally-encoded proteins may only have a limited useful life-span. Another limitation of drugs that target virus proteins is that many will not have a broad applicability and will be inherently highly specific to a particular virus or even a certain strain of a particular virus.
Retroviral Integration
Given what is known about the retroviral life cycle, an attractive target for anti-retroviral therapeutics is to interfere with the integration of the viral cDNA into the host genome. Most importantly, this event is essential for efficient viral propagation (for example, Sakai et al., 1993; for reviews, see Varmus, 1988; Grandgenett and Mumm, 1990). In addition, since similar types of process are not believed to be essential for the functioning of most normally growing host cells, inhibitors of retroviral integration would not be expected to be particularly toxic to the host.
In light of these and other considerations, retroviral reverse transcriptases and integrases have been targeted for drug development. Although this has met with some success, high rates of genetic change by the targeted virus and variations between different viral strains is likely to limit the scope for anti-reverse transcriptase and anti-integrase drugs, particularly in the long term.
One way to surmount the problems outlined above would be to identify host cell proteins that are required for efficient retroviral integration and derive drugs that inhibit these molecules. First, it would be very difficult or impossible for the virus to mutate in such a way that it could evade drug action. Second, such host cell proteins would be expected to be necessary for the propagation of most retroviruses, meaning that drugs that interfere with them would be effective against a wide spectrum of retrovirus types.
Until now, the idea of there being a host factor (or host factors) that is required for retroviral integration but is not necessary for normal host cell growth seemed unlikely. This is because several lines of research have indicated that all the steps needed for covalently linking retrovirus or retrotransposon cDNA to the target DNA molecule can be performed in vitro by purified retroviral integrase protein (for example, Craigie et al., 1990; Bushman et al., 1990; Katz et al., 1990; Grandgenett and Mumm, 1990). In addition, although host factors have been conceived to help with viral integration, it was assumed that these would correspond to “housekeeping proteins” that are essential for host cell viability. Thus, if host “helper” proteins did exist, it was expected that inhibiting them with drugs would not be worthwhile in a therapeutic context because this would also kill the cells of the host.
In spite of these predictions, the present invention is surprisingly founded on the discovery that a series of host cell proteins are essential for efficient retrotransposon integration despite being unnecessary for host cell viability under most circumstances. These factors, which are components of a system termed the Ku-associated DNA repair apparatus, are therefore highly attractive targets for anti-retroviral therapy.
The Ku-Associated DNA Repair System
Previous work has revealed that the protein Ku is an essential component of the DNA repair apparatus in organisms ranging from humans, to
Drosophila melanogaster
, to
S. cerevisiae
(Jackson and Jeggo, 1995; Boulton and Jackson, 1996; Boulton and Jackson, 1996 and references therein). Specifically, the type of DNA repair process in which Ku is involved is termed illegitimate DNA end-joining or DNA double-strand break (DSB) repair. Since Ku binds to DNA DSBs in vitro, it has been proposed that Ku binds to DNA DSBs as they arise in vivo and helps to promote their efficient ligation. In addition, Ku is required for V(D)J recombination, a DNA “cut-and-paste” process that generates the antigen-binding molecules of the immune system of vertebrates (for reviews, see Lewis, 1994; Jackson and Jeggo, 1995).
In all organisms in which it has been identified, Ku exists as a
Downs Jessica Anne
Jackson Stephen Philip
Bozicevic Field & Francis LLP
KuDos Pharmaceuticals Limited
Sherwood Pamela
Stucker Jeffrey
Winkler Ulrike
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