Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-10-01
2002-11-19
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C424S009200, C435S007210, C435S325000, C530S350000
Reexamination Certificate
active
06482587
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to novel compounds and methods for the detection of compounds that are agonistic or antagonistic for the binding of viral genetic material to genomic host DNA. Additionally, the inventions generally relates to compounds and methods related to gene transfer and gene therapy, as well as therapeutics for virally based diseases.
BACKGROUND
In 1872, Moritz Kaposi described a multifocal vascular tumor affecting elderly men of Mediterranean or Eastern European origin. More recently, this neoplasm has become prevalent in immunocompromised patients, such as transplant recipients on immunosuppressive therapy, and AIDS patients, where it has become the most common cancer (Beral, V. “Epidemiology of Kaposi's sarcoma”
Cancer Surv
10:5-22, 1991). The relationship of the disease to geography and immunocompromised patients led to the suspicion of an infectious agent in Kaposi's sarcoma pathogenesis. This suspicion was supported when KSHV or human herpesvirus 8 (HHV8) was identified through PCR based studies of tumor samples from AIDS patients with Kaposi's sarcoma (Chang, Y. et al. “Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma”
Science
266:1865-1869, 1994). Subsequent studies have shown that the virus is of the gammaherpesviridae family, bearing sequence similarity to herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) (Russo, J. “Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)”
PNAS
93:14862-14867, 1996). Although there is increasing epidemiologic data associating the virus with human disease, little is known about the biology of this new gammaherpesvirus.
Indirect immunofluorescence studies of the latently infected BCBL cell line with serum from KS patients reveals a characteristic punctate pattern of nuclear immunofluorescence due to the presence of what was termed the latency-associated nuclear antigen (LANA) (Moore, P. S., et al. “Primary characterization of a herpesvirus agent with Kaposi's sarcoma”
J Virol
70:549-558, 1996; Simpson, G. R. et al. “Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen”
Lancet
348:1133-1138, 1996). LANA is detected in the majority of cells in a KS lesion as well as in cell lines derived from body cavity lymphomas (Simpson, G. R. et al. “Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen”
Lancet
348:1133-1138, 1996; Rainbow, L., et al. “The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpes 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen”
J Virol
71:5915-5921, 1997). Studies based on the detection of antibodies to LANA have shown that KSHV infection precedes onset of KS and other associated lymphoproliferative diseases (Gao, S. J., et al. “Seroconversion to antibodies against Kaposi's sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi's sarcoma”
N Engl J Med
335:223-241, 1996). LANA is encoded by orf73 of KSHV and is expressed as a latency-associated protein in the infected cell (Rainbow, L., et al. “The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpes 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen”
J Virol
71:5915-5921, 1997; Kedes, D. H., et al. “Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus”
J Clin Invest
100:2606-2610, 1997). An analysis of the LANA amino acid sequence reveals several acidic and proline/glutamine rich regions as well as a zinc finger DNA binding domain (Neipel, F., et al. “Fleckenstein Cell-homologous genes in the Kaposi's sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity?”
J Virol
71:4187-4192, 1997; Ganem, D. “KSHV and Kaposi's sarcoma: the end of the beginning of the end?”
Cell
91:157-160, 1997). In spite of this suggestion that LANA may act as a transcription factor, a specific function is yet to be assigned for this viral protein.
Little is known regarding the mechanism and establishment of KSHV latency. However, the persistence of the viral genome through generations of host cell divisions potentiates the host's propensity of contracting the disease encoded by the virus. What is needed is a drug screen for agents that would disrupt the ability of a viral genome (e.g. the KSHV genome) to bind to host DNA thereby eliminating the viral genome in the host.
SUMMARY OF THE INVENTION
The present invention generally comprises novel compounds and methods to screen for compounds that interfere with the ability of viral genomic DNA or RNA to bind to host genomic DNA. Additionally, the present invention relates to the targeting of genes to host genomes in gene therapy applications. Furthermore, the present invention relates to compositions and methods for the treatment of viral infections and tumors.
Genomic DNA from latent viruses is able to persist for multiple generations of the host cell by binding to a tethering protein that is encoded by the viral DNA. We have discovered the mechanism of binding of the viral DNA to the host cell. For example, genomic DNA from the Kaposi sarcoma virus (KSHV) is able to persist for multiple generations of the host cell by binding to a tethering protein, the latency-associated nuclear antigen (LANA). LANA tethers the KSHV viral DNA to the chromosomal structural protein, histone 1 (H1). LANA is encoded by the viral DNA, therefore it will only be present in a host cell after infection. Likewise, the lack of LANA in a host cell would indicate the lack of viral infection by viruses that utilize this or similar proteins to ensure persistence. LANA binds to specific locations on the KSHV genomic DNA designated Z6, Z8 and Z2. We have defined three other smaller binding regions that partially overlap with Z6, Z8 and Z2, which we have named LBR1 (LANA binding region 1), LBR2, LBR3 and LBR4. These regions are located at approximately 22-27 kb, 109-111 kb, 127-132 kb and a region at the left 1.8 kb of the KSHV genome including one copy of the terminal repeat, respectively. LBR1 is located within the Z1 binding region, LBR2 is located immediately 3′ to the Z8 binding region and LBR3 is located within the Z2 binding region. The Epstein Bar Virus (EBV) persists in host cells by a similar mechanism in that it utilizes a tethering protein (EBNA1) to bind the viral genomic DNA to host histone H1 proteins. These discoveries will permit (among other things) the screening of agents that interfere with viral DNA binding to host DNA.
As noted above, the present invention also contemplates screening assays to identify drugs that inhibit or potentiate the binding of tethering proteins (e.g. LANA and EBNA1) to host histone H1 proteins. A variety of assay formats are contemplated for testing the potential of compounds suspected of modulating tethering protein binding. In one embodiment, cells are pretreated with the compound suspected of modulating the binding of the tethering protein, followed by the addition of viral DNA or viruses that encode the tethering protein. A cell free assay for the screening of drugs that inhibit or potentiate the binding of tethering proteins (e.g. LANA and EBNA1) to host histone H1 proteins is contemplated by the present invention. For example, providing i) histone H1 proteins and ii) LANA or EBNA1; adding a compound or compounds suspected of inhibiting or potentiating the binding of LANA or EBNA1 to histone H1; and detecting said binding (e.g., by Western blot).
The invention is not limited to any particular measurement technique to measured bound tethering protein. Various methods are envisioned. In one embodimen
Cotter Murray A.
Robertson Erle S.
Medlen & Carroll LLP
Stucker Jeffrey
The Regents of the University of Michigan
Winkler Ulrike
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