Wisconsin Alumni Research Foundation

William M Sugden

Gregory Dean Kennedy

Jindong Wang

David M Mackey

Atsushi Jun Komano

Scott Eugene Lindner

Wolfgang Hammerschmidt

Wolfgang Friedrich Hammerschmidt

The invention provides a recombinant vector comprising a DNA segment having a synthetic origin of DNA synthesis that binds EBNA-1 and is capable of initiating DNA synthesis of sequences linked to the synthetic origin of DNA synthesis and maintaining the linked sequences when in the presence of EBNA-1. The synthetic origin of DNA synthesis comprises at least two binding sites for EBNA-1, wherein the two EBNA-1 binding sites are flanked by at least two half-binding sites for TRF2 or at least two binding sites for a protein that enhances the affinity of EBNA-1 for the synthetic origin of DNA synthesis. Further provided are host cells with the vector and methods of using the vector, for instance, ex vivo or in vivo.

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The invention provides a vector encoding a derivative of EBNA-1 that is not cytotoxic when expressed efficiently in cells, which supports extrachromosomal replication, maintenance and transcription from extrachromosomal oriP containing vectors but does not substantially activate transcription from host cell genes. Also provided is a vector having oriP and encoding a derivative of EBNA-1. The vectors of the invention may be employed in vitro and in gene therapy.

Non-cytotoxic oriP replicon

A method of inhibiting viral gene activities (and cellular gene activities) is disclosed. In one embodiment, the method comprises the step of delivering an effective amount of an inhibitor of a viral looping/linking factor to an infected patient.

Inhibition of viral gene activities

A method of assaying potential inhibitors of EBNA-1 is disclosed. In one aspect, this invention comprises the steps of (a) obtaining an EBV-positive cell line and an EBV-negative cell line; (b) exposing the cell lines to a test compound, and (c) observing the effect of the compound on induction of apoptosis in the cell lines, wherein significant apoptosis in the EBV-positive cell line and lack of significant apoptosis in the EBV-negative cell line indicates a compound that specifically inhibits EBNA-1.

Method of identifying inhibitors of EBNA-1

This invention is directed to construction of recombinant plasmids that contain the Epstein-Barr virus ('EBV') genetic information involved in the immortalization of human lymphocytes, but that lack one or more genes implicated in EBV lytic replication. The recombinant plasmids have the advantage of being able to immortalize human lymphocytes, while being incapable of generating infectious EBV particles. The invention also provides human lymphocytes that have been successfully infected and immortalized by the recombinant plasmids. These human lymphocyte clones can be used as cellular factories to produce desired proteins and as delivery vehicles for gene therapy. Additionally, the invention establishes a method by which desired proteins can be produced in immortalized cellular factories, without generating infectious EBV particles. These proteins can be the normal cellular products of immortalized human lymphocytes, or they can be proteins encoded by foreign genes, which have been cloned into the lymphocytes.

Immortalized lymphocytes for production of viral-free proteins

The invention provides lymphotrophic herpes virus (preferably EBV) recombinants. The portions of the virus that are responsible for the packaging and lytic phase replication have been isolated and cloned. When used with vectors and hosts containing a segment that controls plasmid replication, they provide a means of carrying foreign genes into B-lymphocytes.

Lytic origin of replication for Epstein-Barr virus (EBV)

A recombinant vector and a eukaryotic host transformed thereby are disclosed. In one embodiment, there is a eukaryotic host that has a recombinant plasmid comprising a plasmid backbone, a foreign eukaryotic gene component, and a first gene segment from a lymphotrophic herpes virus. There is also provided in the host a second gene segment from a lymphotrophic herpes virus. The first and second gene segments interact to assist the plasmid in maintaining itself as a plasmid when inserted into a eukaryotic host, and permit stable replication of the host. The preferred virus is the Epstein-Barr virus, and the two preferred viral segments are the EBV origin of replication and the segment coding for EBNA protein.

Growing community of inventors

Madison, WI, United States of America

William M Sugden