Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-08
2002-09-10
Martinell, James (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S440000, C435S325000, C435S252300, C536S023100, C536S023500
Reexamination Certificate
active
06448000
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention provides methods of identifying cellular genes used for viral growth or for tumor progression. Thus, the present invention relates to nucleic acids related to and methods of reducing or preventing viral infection and for suppressing tumor progression. The invention also relates to methods for screening for additional such genes.
2. Background Art
Various projects have been directed toward isolating and sequencing the genome of various animals, notably the human. However, most methodologies provide nucleotide sequences for which no function is linked or even suggested, thus limiting the immediate usefulness of such data.
The present invention, in contrast, provides methods of screening only for nucleic acids that are involved in a specific process, i.e., viral infection or tumor progression, and further, for nucleic acids useful in treatments for these processes because by this method only nucleic acids which are also nonessential to the cell are isolated. Such methods are highly useful, since they ascribe a function to each isolated gene, and thus the isolated nucleic acids can immediately be utilized in various specific methods and procedures.
For, example, the present invention provides methods of isolating nucleic acids encoding gene products used for viral infection, but nonessential to the cell. Viral infections of the intestine and liver are significant causes of human morbidity and mortality. Understanding the molecular mechanisms of such infections will lead to new approaches in their treatment and control.
Viruses can establish a variety of types of infection. These infections can be generally classified as lytic or persistent, though some lytic infections are considered persistent. Generally, persistent infections fall into two categories: (1) chronic (productive) infection, i.e., infection wherein infectious virus is present and can be recovered by traditional biological methods and (2) latent infection, i.e., infection wherein viral genome is present in the cell but infectious virus is generally not produced except during intermittent episodes of reactivation. Persistence generally involves stages of both productive and latent infection.
Lytic infections can also persist under conditions where only a small fraction of the total cells are infected (smoldering (cycling) infection). The few infected cells release virus and are killed, but the progeny virus again only infect a small number of the total cells. Examples of such smoldering infections include the persistence of lactic dehydrogenase virus in mice (Mahy, B. W. J.,
Br. Med. Bull.
41: 50-55 (1985)) and adenovirus infection in humans (Porter, D. D. pp. 784-790 in Baron, S., ed.
Medical Microbiology
2d ed. (Addison-Wesley, Menlo Park, Calif. 1985)).
Furthermore, a virus may be lytic for some cell types but not for others. For example, evidence suggests that human immunodeficiency virus (HIV) is more lytic for T cells than for monocytes/macrophages, and therefore can result in a productive infection of T cells that can result in cell death, whereas HIV-infected mononuclear phagocytes may produce virus for considerable periods of time without cell lysis. (Klatzmann, et al.
Science
225:59-62 (1984); Koyanagi, et al.
Science
241:1673-1675 (1988); Sattentau, et al.
Cell
52:631-633 (1988)).
Traditional treatments for viral infection include pharmaceuticals aimed at specific virus derived proteins, such as HIV protease or reverse transcriptase, or recombinant (cloned) immune modulators (host derived), such as the interferons. However, the current methods have several limitations and drawbacks which include high rates of viral mutations which render anti-viral pharmaceuticals ineffective. For immune modulators, limited effectiveness, limiting side effects, a lack of specificity all limit the general applicability of these agents. Also the rate of success with current antivirals and immune-modulators has been disappointing.
The current invention focuses on isolating genes that are not essential for cellular survival when disrupted in one or both alleles, but which are required for virus replication. This may occur with a dose effect, in which one allele knock-out may confer the phenotype of virus resistance for the cell. As targets for therapeutic intervention, inhibition of these cellular gene products, including: proteins, parts of
DETAILED DESCRIPTION OF THE INVENTION
The present invention utilizes a “gene trap” method along with a selection process to identify and isolate nucleic acids from genes associated with a particular function. Specifically, it provides a means of isolating cellular genes necessary for viral infection but not essential for the cell's survival, and it provides a means of isolating cellular genes that suppress tumor progression.
The present invention also provides a core discovery that virally infected cells become dependent upon at least one factor present in serum for survival, whereas non-infected cells do not exhibit this dependence. This core discovery has been utilized in the present invention in several ways. First, inhibition of the “serum survival factor” can be utilized to eradicate persistently virally infected cells from populations of non-infected cells. Inhibition of this factor can also be used to treat virus infection in a subject, as further described herein. Additionally, inhibition of or withdrawal of the serum survival factor in tissue culture allows for the detection of cellular genes required for viral replication yet nonessential for an uninfected cell to survive. The present invention further provides several such cellular genes, as well as methods of treating viral infections by inhibiting the functioning of such genes.
Furthermore, the present invention provides a method for isolation of cellular genes utilized in tumor progression.
The present method provides several cellular genes that are necessary for viral growth in the cell but are not essential for the cell to survive. These genes are important for lytic and persistent infection by viruses. These genes were isolated by generating gene trap libraries by infecting cells with a retrovirus gene trap vector, selecting for cells in which a gene trap event occurred (i.e., in which the vector had inserted such that the promoterless marker gene was inserted such that a cellular promoter promotes transcription of the marker gene, i.e., inserted into a functioning gene), starving the cells of serum, infecting the selected cells with the virus of choice while continuing serum starvation, and adding back serum to allow visible colonies to develop, which colonies were cloned by limiting dilution. Genes into which the retrovirus gene trap vector inserted were then isolated from the colonies using probes specific for the retrovirus proteins (modification enzymes that include, but are not restricted to glycosylation, lipid modifiers [myriolate, etc.]), lipids, transcription elements and RNA regulatory molecules, may be less likely to have profound toxic side effects and virus mutation is less likely to overcome the ‘block’ to replicate successfully.
The present invention provides a significant improvement over previous methods of attempted therapeutic intervention against viral infection by addressing the cellular genes required by the virus for growth. Therefore, the present invention also provides an innovative therapeutic approach to intervention in viral infection by providing methods to treat viruses by inhibiting the cellular genes necessary for viral infection. Because these genes, by virtue of the means by which they are originally detected, are nonessential to the cell's survival, these treatment methods can be used in a subject without serious detrimental effects to the subject, as has been found with previous methods. The present invention also provides the surprising discovery that virally infected cells are dependent upon a factor in serum to survive. Therefore, the present invention also provides a method for treating viral inf
Dubois Raymond N.
Organ Edward L.
Rubin Donald H.
Martinell James
Needle & Rosenberg PC
Vanderbilt University
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