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

Patent metadata

Field	Value
Publication number	US-2016153000-A1
Application number	US-201414905708-A
Country	US
Kind code	A1
Filing date	Jul 17, 2014
Priority date	Jul 17, 2013
Publication date	Jun 2, 2016
Grant date	—

Abstract

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.

First claim

1 . 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.

2 . The HSV vector of claim 1 , wherein the vector is capable of expression of a transgene for at least 28 days post infection (dpi) in non-complementing cultured human dermal fibroblast (HDF) cells.

3 - 4 . (canceled)

5 . The HSV vector of claim 1 , wherein a transgene is inserted within said genome in operable connection with an insulator sequence, and wherein the vector does not express ICP0, ICP4, ICP22, ICP27, and ICP47 as immediate early genes.

6 . The HSV vector of claim 5 , wherein the insulator sequence comprises a genetic element that protects against heterochromatin formation.

7 . (canceled)

8 . The HSV vector of claim 5 , wherein a transgene is inserted within about 5 kb of the insulator sequence within said genome.

9 - 14 . (canceled)

15 . The HSV vector of claim 1 , wherein transgene is flanked by insulator sequences within said genome.

16 . The HSV vector of claim 1 , wherein said genome comprises chromatin boundary elements CTRL1 and CTRL2 and wherein a transgene is within about 4 kb of both of said CTRL1 and CTRL2 elements.

17 - 20 . (canceled)

21 . The vector of claim 1 , which comprises the following genetic perturbations relative to wild-type HSV:
(a) an inactivating deletion of the gene encoding ICP0, (b) an inactivating deletion of the gene encoding ICP4, (c) a deletion of the joint, (d) the gene encoding ICP22 being under the control of an early promoter, (e) an inactivating deletion of the gene encoding ICP27, and (f) a deletion of the promoter and start codon of the gene encoding ICP47.

22 . The vector of claim 21 , which further comprises an inactivating deletion in UL41.

23 . The vector of claim 21 , wherein said genome comprises a LAT gene region comprising CTRL1 and CTRL2 sequences and a LATP2 or LAP2 enhancer element and wherein a first transgene is inserted in said LAT gene region between the CTRL1 and CTRL2 sequences.

24 . The vector of claim 23 , wherein said first transgene is inserted in said LAT gene region between the LATP2 and CTRL1 elements.

25 . The vector of claim 23 , wherein said first transgene is inserted in said LAT gene region between LATP2 and CTRL2 elements.

26 - 39 . (canceled)

40 . An HSV vector, which is JΔNI5 or JΔNI8 with the addition of one or more transgene expression cassettes inserted within a latency-associated transcript (LAT) gene region and wherein the mCherry transgene in ICP4 is substituted with a different coding sequence.

41 . An HSV vector, which is JΔNI5 or JΔNI8 with the addition of one or more transgene expression cassettes including an insulator sequence, which is inserted at a site other than a latency-associated transcript (LAT) gene region and wherein the mCherry transgene in ICP4 is substituted with a different coding sequence.

42 . A complementing cell for the production of the vector of claim 1 , wherein the complementing cell is a U2OS cell that comprises an ICP4 expression cassette that is expressed in trans when the cell is infected with HSV.

43 - 46 . (canceled)

47 . A stock comprising at least 10 7 pfu/ml, of the vectors of claim 1 .

48 . A method of expressing a transgene, comprising infecting a non-complementing cell with the HSV vector of claim 1 so that one or more of said transgenes within the vector is expressed within the cell.

49 - 54 . (canceled)

55 . A method of treating a disease or condition in a subject, comprising administering the vector of claim 1 to the subject, in an amount and at a location sufficient to infect cells of the subject such that one or more of said transgenes is expressed within the cells of the subject, and wherein the expressed transgene(s) encodes one or more prophylactically or therapeutically active proteins, polypeptides or ncRNA.

56 - 57 . (canceled)

58 . The method of claim 55 , wherein the subject is a human.

59 . A composition comprising the vector of claim 1 and a pharmaceutically acceptable carrier.

Assignees

• Glorioso Iii Joseph C
• Cohen Justus
• Miyagawa Yoshitaka
• Krisky David
• Wechuck James
• Wolfe Darren
• Univ Pittsburgh

Inventors

• Glorioso Joseph C
• Cohen Justus
• Miyagawa Yoshitaka
• Krisky David
• Wechuck James
• Wolfe Darren

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P25/04

  Centrally acting analgesics, e.g. opioids · CPC title