University of Pittsburgh

The United States Government As Represented by the Department of Veterans Affairs

David Krisky

Joseph C Glorioso, Iii

Joseph C Glorioso

Darren Wolfe

Justus B Cohen

David J Fink

Michael Cascio

James R Goss

Yoshitaka Miyagawa

James Wechuck

The invention provides a method of modulating electrophysiological activity of an excitable cell. The method involves causing exogenous expression of a glycine receptor (GlyR) protein in an excitable cell of a subject. Thereafter, the excitable cell is exposed to an allosteric modulator of the GlyR protein. Modulation of the exogenous GlyR protein (an ion channel) in response to the allosteric modulator modulates the electrophysiological activity of the excitable cell. The method can be used to control pain in a subject. The invention further provides a replication-defective HSV vector comprising an expression cassette encoding a GlyR protein, stocks and pharmaceutical compositions containing such vectors, and a transgenic animal.

Targeted delivery of glycine receptors to excitable cells

The invention provides a method of modulating electrophysiological activity of an excitable cell. The method involves causing exogenous expression of a glycine receptor (GlyR) protein in an excitable cell of a subject. Thereafter, the excitable cell is exposed to an allosteric modulator of the GlyR protein. Modulation of the exogenous GlyR protein (an ion channel) in response to the allosteric modulator modulates the electrophy-stological activity of the excitable cell. The method can be used to control pain in a subject. The invention further provides a replication-defective HSV vector comprising an expression cassette encoding a GlyR protein, stocks and pharmaceutical compositions containing such vectors, and a transgenic animal.

The invention provides a herpes simplex virus (HSV) vector that does not express toxic HSV genes in non-complementing cells and which comprises a genome comprising one or more transgenes, wherein the vector is capable of expression of a transgene for at least 28 days in non-complementing cells. The disclosed vectors include vectors having deletions in the genes ICP0, ICP4, TCP22, TCP27 and TCP47, or alternative inactivating mutations, or vectors which express one or more of these genes with modified kinetics. The invention also relates to viral stocks of the inventive vectors, compositions thereof suitable for use therapeutically or for in vitro applications, and methods relating thereto. In another aspect, the invention provides a complementing cell, in particular a U20S cell, engineered to express ICP4 and ICP27 when the cell is infected with HSV for the production of the inventive vector. Said cells are disclosed as naturally complementing ICP0.

Non-toxic HSV vectors for efficient gene delivery applications and complementing cells for their production

The invention provides a cell line complementing HSV ICP4, ICP27, and UL55 genes.

Cell line

The invention provides a herpes simplex virus vector comprising deletions in ICP4, ICP27, and UL55.

Herpes simplex virus vector

The present invention provides a method for preparing HSV vectors. The method comprises co-transfecting a source vector and a mutating cassette together into a population of appropriate host cells, such that homologous recombination occurs between the mutating cassette and the source vector whereby the mutating cassette replaces a region of the HSV genome. The mutating cassette has a unique restriction site not present in the sequence of the vector. The method further comprises plaquing the co-transfected host cells, selecting plaques in which recombination has occurred between the source vector and the mutating cassette, and isolating the viral DNA from the plaques. The isolated viral DNA is digested with a restriction endonuclease appropriate for cleaving the viral DNA at the unique restriction site within the mutating cassette to produce two viral polynucleotides. Following purification, the two viral polynucleotides can be ligated to form an HSV vector comprising the two viral polynucleotides. Alternatively, the two isolated viral polynucleotides can be recombined with an insertion cassette to form an HSV vector comprising the insertion cassette at the former locus of the unique restriction site. The present invention further provides a mutant vector, particularly an HSV vector constructed in accordance with the method for the present invention. The present invention further provides a multigene HSV vector, particularly a multigene HSV vector for cancer therapy.

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Sewickley, PA, United States of America

David Krisky