Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-28
2004-06-29
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S975000, C536S023720
Reexamination Certificate
active
06756203
ABSTRACT:
1. INTRODUCTION
The present invention relates to a class of viruses called rhadino viruses, or gamma-2 herpes viruses. By way of example, but not by way of limitation, one commonly studied rhadino virus is Kaposi's Sarcoma-Associated Herpes Virus (KSHV), which is also known as Human Herpes Virus 8 (HHV8). More specifically, the invention relates to a rhadino virus protein known as LANA and to a segment of rhadino virus DNA known as the rhodino virus cis-acting element (RVCAE). The LANA protein is encoded by open reading frame (ORF) 73 and is expressed in mammalian cells that are latently infected with KSHV.
The present invention relates to the discovery that LANA is necessary and sufficient for the efficient presistence of rhadino virus DNA in mammalian cells. The invention encompases methods and assays for determining whether a compound of interest can modulate the ability of LANA to enable the efficient persistence of rhadino virus DNA in a mammalian cell, the binding of LANA to rhadino virus RVCAE DNA, the binding of LANA to mammalian chromosomes, or the tethering by LANA of rhadino virus RVCAE DNA to mammalian chromosomes, as well as such compounds themselves. Because persistent rhadino virus infection is linked to diseases such as Kaposi's Sarcoma (KS) and Primary Effusion Lymphoma (PEL), the invention also encompases methods and assays for determining whether a compound of interest can modulate rhadino virus-associated disease states, including but not limited to KS and PEL, as well as such compounds themselves.
The invention also relates to the discovery that the expression of LANA in mammalian cells along with the presence of RVCAE on a DNA plasmid is sufficient for the efficient maintenance of the plasmid in those cells. The invention additionally encompases heterologous DNAs that include RVCAE and a desired DNA such that the heterologous DNA effeciently persists as an episome in mammalian cells in which LANA is expressed. The invention also encompases heterologous DNAs that include RVCAE, a desired DNA, and an expression cassette for LANA, such that the heterologous DNA effeciently persists as an episome in mammalian cells. Because efficient persistance of a heterologous DNA in a mammalian cell is important for gene therapies and related techniques, the invention also encompases products and methods for gene therapies and related techniques that involve heterologous DNAs having RVCAE and a desired DNA.
The present invention is more fully described below.
2. BACKGROUND OF THE INVENTION
Kaposi's sarcoma (KS) associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) likely plays a central role in KS pathogenesis because KSHV seropositivity precedes KS and KSHV DNA is found in almost all KS lesions, whether or not there is coexisting human immunodeficiency virus (HIV) infection (Y. Chang, et al.,
Science
, 266, 1865 (1994); P. S. Moore, et al.,
J. Virol
., 70, 549 (1996); S. -J. Gao, et al.,
N. Engl. J. Med
., 335, 233 (1996); P. S. Moore, Y. Chang,
N. Engl. J. Med
., 332, 1181 (1995)). KSHV is also associated with lymphoproliferative disorders including primary effusion lymphomas (PELs) and multicentric Castleman's disease (E. Cesarman, et al.,
Blood
, 86, 2708 (1995); M. B. Rettig, et al.,
Science
, 276, 1851 (1997); J. Soulier, et al.,
Blood
, 86, 1276 (1995); E. Cesarman, et al.,
N. Engl. J. Med
., 332, 1186 (1995)).
Similar to the gamma-1 herpesvirus Epstein-Barr virus (EBV), KSHV infection in tumor tissue or lymphoma derived cell lines is predominantly latent. Latently infected cells have multiple copies of circularized KSHV DNA maintained as episomes (E. Cesarman, et al.,
Blood
, 86, 2708 (1995); M. B. Rettig, et al.,
Science
, 276, 1851 (1997); J. Soulier, et al.,
Blood
, 86, 1276 (1995); E. Cesarman, Y. Chang, P. S. Moore, J. W. Said, D. M. Knowles,
N. Engl. J. Med
., 332, 1186 (1995); L. L. Decker, et al.,
J. Exp. Med
., 184, 283 (1996). The EBV EBNA1 protein mediates efficient episome persistence through a cis-acting 1.8 kb EBV DNA sequence termed origin of plasmid replication (oriP) (J. Yates, N. Warren, D. Reisman, B. Sugden,
Proc. Natl. Acad. Sci. USA
, 81, 3806 (1984); J. L. Yates, N. Warren, B. Sugden,
Nature
, 313, 812 (1985); D. R. Rawlins, G. Milman, S. D. Hayward, G. S. Hayward,
Cell
, 42, 859 (1985)). The primate transforming herpes virus saimiri (HVS), which is a rhadino virus, also has a cis-acting sequence that enables efficient persistence of episomes in HVS infected cells (S.-H. Kung, P. G. Medveczky,
J. Virol
., 70, 1738 (1996)). However, KSHV has no obvious homology to the HVS cis-acting DNA and a trans-acting EBNA1 homolog or analog has not been identified in HVS or other gamma-2 herpesviruses.
KSHV open reading frame (ORF) 73 encodes the latency-associated nuclear antigen (LANA, LNA, or LNA1) which is predicted to be 1162 amino acids and lacks a known function (Y. Chang, et al.,
Science
, 266, 1865 (1994); P. S. Moore, et al.,
J. Virol
., 70, 549 (1996); S.-J. Gao, et al.,
N. Engl. J. Med
., 335, 233 (1996); P. S. Moore, Y. Chang,
N. Engl. J. Med
., 332, 1181 (1995); J. J. Russo, et al.,
Proc. Natl. Acad. Sci. USA
, 93, 14862 (1996); P. Kellam, et al.,
J. of Human Virol
., 1, 19 (1997); L. Rainbow, et al.,
J. Virol
., 71, 5915 (1997); D. H. Kedes, M. Lagunoff, R. Renne, D. Ganem,
J. Clin. Invest
., 100, 2606 (1997); F. Neipel, J. C. Albrecht, B. Fleckenstein,
J. Virol
., 71, 4187 (1997)). A homologous open reading frame exists in other gamma-2 herpesviruses such as, but not limited to, HV saimiri and MHV68. (J. C. Albrecht, et al.,
J. Virol
., 66, 5047 (1992); H. W. Virgin IV, et al.,
J. Virol
., 71, 5894 (1997)). LANA is reactive with most KSHV-immune sera which detect LANA in KSHV infected PEL cells and KS spindle cells (P. Kellam, et al.,
J. of Human Virol
., 1, 19 (1997); L. Rainbow, et al.,
J. Virol
., 71, 5915 (1997); D. H. Kedes, M. Lagunoff, R. Renne, D. Ganem,
J. Clin. Invest
., 100, 2606 (1997); F. Neipel, J. C. Albrecht, B. Fleckenstein,
J. Virol
., 71, 4187 (1997); S. J. Gao, et al.,
Nature Medicine
, 2, 925 (1996); D. H. Kedes, et al.,
Nature Medicine
, 2, 918 (1996); D. Jones, et al.,
N. Engl. J. of Med
., 339, 444 (1998); R. Renne, et al.,
Nature Medicine
, 2, 342 (1996); L. Szekely, et al.,
J. of General Virology
, 79, 1445 (1998)).
3. SUMMARY OF THE INVENTION
The present invention encompases methods and assays for determining whether a compound of interest can modulate the ability of LANA to enable the efficient persistence of rhadino virus in a mammalian cell, the binding of LANA to rhadino virus RVCAE DNA, the binding of LANA to mammalian chromosomes, the tethering by LANA of rhadino virus RVCAE DNA to mammalian chromosomes, or any rhadino virus-associated disease states, including but not limited to KS and PEL, as as well as such compounds themselves. The present invention also encompases products and methods for gene therapies and related techniques involving the use of heterologous DNAs that include the rhadino virus cis-acting element (RVCAE) and a desired DNA such that the heterologous DNA effeciently persists as an episome in mammalian cells in which LANA is expressed, including heterologous DNAs that carry an expression cassette for LANA.
The invention is based, in part, on the discovery that LANA is necessary and sufficient for the efficient presistence of rhadino virus DNA in mammalian cells. The examples described infra provide the first identification of a trans-acting factor that supports episome persistence of gamma-2 herpesvirus DNA. The factor is identified as RVCAE. According to one embodiment of the invention the RVCAE element includes at least one copy of the rhodino virus terminal repeat (TR). According to another embodiment of the invention, the RVCAE element includes at least three copies of the rhodino virus terminal repeat (TR) and 0.6 kb of the rhodino virus adjacent unique DNA.
It is shown that LANA associates with KSHV episomes in interphase and on chromosomes in mitotic cells. Cell lines stably expressing LANA are shown to support episo
Ballestas Mary E.
Kaye Kenneth M.
Kieff Elliott D.
Kieff Elliott D.
Kieff F. Scott
Salimi Ali R.
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