Helper Virus-Free Herpesvirus Amplicon Particles and Uses Thereof
AssigneeUniversity of Rochester
InventorsHoward J. Federoff, William J. Bowers
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Abstract
The invention features new helper virus-free methods for making herpesvirus amplicon particles that can be used in immunotherapies, including those for treating any number of infectious diseases and cancers (including chronic lymphocytic leukemia, other cancers in which blood cells become malignant, lymphomas (e.g. Hodgkin's lymphoma or non-Hodgkin's type lymphomas). Described herein are methods of making helper virus-free HSV amplicon particles; cells that contain those particles (e.g., packaging cell lines or patients' cells, infected in vivo or ex vivo); particles produced according to those methods; and methods of treating a patient with an hf-HSV particle made according to those methods.
Claims42
A method of generating a herpesvirus amplicon particle, the method comprising providing a cell that has been stably transfected with a nucleic acid sequence that encodes an accessory protein;
- and
- transfecting the cell with (a) one or more packaging vectors that, individually or collectively, encode one or more HSV structural proteins but do not encode a functional herpesvirus cleavage/packaging site (b) an amplicon plasmid comprising a sequence that encodes a functional herpesvirus cleavage/packaging site, a herpesvirus origin of DNA replication, and a heterologous transgene and (c) an integration vector, wherein the integration vector encodes a Sleeping Beauty transposase mutant that catalyzes a reaction within the cell, the consequence of the reaction being that the transgene carried by the amplicon plasmid is inserted into the genome of the cell.
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A method of generating a herpes virus comprising a modified artificial chromosome, the method comprising
- (a) providing a cell that, optionally, comprises a nucleic acid sequence that encodes an accessory protein;
- (b) transfecting the cell with (i) one or more packaging vectors that, individually or collectively, encode one or more of the herpes virus structural proteins but do not include a functional herpes virus on and (ii) a modified artificial chromosome comprising a pair of cleavage sites that flank a packaging/cleavage site of a herpes virus, an on of a herpes virus, a first antibiotic resistance gene, and, optionally a sequence that encodes a detectable marker;
- a nucleic acid sequence of interest, and a second antibiotic resistance gene;
- and
- (c) culturing the cell for a time and under conditions that permit the cell to produce a herpes virus comprising the modified herpes virus.
A cell stably transfected by
- (a) a nucleic acid sequence that encodes an accessory protein;
- (b) one or more packaging vectors that, individually or collectively, encode one or more HSV structural proteins but do not encode a functional herpesvirus cleavage/packaging site;
- (c) an amplicon plasmid comprising a sequence that encodes a functional herpesvirus cleavage/packaging site, a herpesvirus origin of DNA replication, and a heterologous transgene;
- and
- (d) an integration vector, wherein the integration vector encodes a Sleeping Beauty transposase mutant that catalyzes a reaction within the cell, the consequence of the reaction being that the transgene carried by the amplicon plasmid is inserted into the genome of the cell.
Dependencies are inferred from the claim text.