GB2342042A

GB2342042A - Combination gene therapy using the p53 gene

Info

Publication number: GB2342042A
Application number: GB9821206A
Authority: GB
Inventors: Christopher Barry Wood
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-09-30
Filing date: 1998-09-30
Publication date: 2000-04-05
Also published as: GB9821206D0

Abstract

The combination of wild type p53 gene and the E1B-deleted p53 gene are suitable for the treatment of human and animal cancers. The genes may be delivered sequentially or simultaneously using adenovirus or liposomal vectors. The combination may be administered intravenously, orally, intra-arterially, intraperitoneally or directly into the tumour. The combination is cytotoxic in cancer cell lines irrespective of their p53 genotype.

Description

2342042 The combination of Adenovirus encoding p53 and Adeno EIB-deleted

virus and the use of the combination to enhance and increase the cytotoxic effect on cancer cells.

it has been claimed (1) that the Adenovirus vector, ONYX-015, which has a deletion in the EIB region, can selectively replicate in, and kill, human cancer cells that lack functional p53, but Is non-cytotoXic to cells that have an intact (wild-type) p53.

Recent evidence (2) contradicts this claim and suggests that ONYX-O 15 can replicate in cancer cells with wild-type p53 and that cells lacking p53 function are resistant to adenovirus-induced cell death.

Functional p53 is deficient in over half of human cancers and its absence is thought to be a major factor in the pathogenesis of many malignant conditions. Many of the current cancer gene therapy protocols are using either wild-type p5_35 (Adwtp53) or the EIBdeleted variant (AdEIB), and usually only include patients with p5_3 mutations. However, these studies have only shown limited benefit, with many patients having stable disease, but few showing tumour regression. To improve the efficacy of gene therapy for cancer it is important to determine the true significance of the p5_3) status in cancer cells.

The present invention uses the combination of the wild-type p5_3 and the EIB-deleted p53 which,. hitherto, had not been considered scientifically justified because the overexpression of p53 would inhibit the AdEIBdeleted virus replication and, therefore, the killing of the cells.

The present invention also allows for the combination of the Adwtp5-') and the AdEIBdeleted p53 as a means of treating human and animal cancers with functional p53 and mutant p53.

The p53 gene is a very popular molecule in modem medicine, with extensive research being done to charactense the gene and numerous scientific articles written on the subject.

P53 is a 53 kilodalton phosphoprotein involved in several important mechanisms, such ascell cycle regulation, cell differentiation, DNA synthesis and repair, and apoptosis. It is a transcription factor that binds as a tetramer to a DNA sequence composed of two head-to-head repeats. The p53 gene is the most common target for genetic alteration across aU types of human cancers; it is a tumour suppressor gene, but in its mutated form it seems to act also as an oncogene.

The p53' gene is located on the short arm of chromosome 17 and contains 11 exons interrupted by 10 introns. In mammals its organization is similar, with the exception of the rat in which intron 6 is missing. In human, the messenger RNA codes for a 394 amino acid protein, which is expressed in all tissues in a relatively low level.

The gene is very rich in G/C (guamne:cytosine), which implies that the gene evolves under the base compositional restraints. In about one quarter of all p53 mutations in human cancers are transitions at the CpG nucleotides.

Onyx-O 15 is an attenuated E I B-deleted adenoVirus which does not produce a 55kD protein normally formed by the wild-type adenoVirUs. The protein inactivates wild-type p53 inside a host cell during infection. In this way the virus replicates and causes cytolysis of the infected cells. The EIB-deleted variant falls to inactivate the p5_3), no Viral replication can take place inside the cell, and an abortive infection results. If a cell lacks p53, or has a mutant, non- functioning p533, the 55kD protein is not required to inactivate p5_3) and the virus will replicate and produce cytolysis. This is the rationale for using Onyx-O 15 in gene therapy protocols. However, it is of no use in cancer cells that are expressing wild-type p53.

Alternative gene therapy strategies have used adenoViral vectors expressing wild type p53). This is based on studies that showed that transfection of tumour cells with plasmid DNA expressing wild-type p53 or infection with retrovirus expressing wild-type p53 was cytotoxic to cells expressing mutant p53.

Thus, the existing scientific evidence shows that gene therapy with either wildtype p5_3) or with the EIB-deleted p5_3) is active only against tumors that express the mutant or depleted p5 3 gene.

3 35 This present invention is unique in that it combines the wild-type p53 adenovirus and the EIB deleted adenovirus and that this combination is cytotoxic to turnour cells that express wild-type p53 and to those with mutant p5_3). Thus, this present invention provides a method of enhancing and increasing the cytotoxic effects of gene therapy with adenoviral vectors. 40 The status of p53 expression within cancer cells is of considerable clinical significance because a large number of cancers do not express mutant p53 or are p5_3) deleted. Such cancers do not respond well to current gene therapy regimens. Furthermore, most cancers have a mixture of cells, some expressing mutant or deleted p53, and others expressing wild-type p5_3). Thus, gene therapy protocols 45 which are aimed at mutant p53 will be only partially successful. A series of hepatocellular cancer cell lines were transfected with Adwtp53 and it produced a cytotoxic effect in the Hep3B cells (deleted p5_33), partially cytotoxic in the PLVC cells (mutated p5_3)), but there was no inhibition of HepG2 cells (Wildtype p5_33). The doses used were not excessive. In these tumour cell lines the use of 50 wtp53 was only effective in those cells expressing mutant or deleted p5_3). The same cell line types, as above, were transfected, in separate experiments, with AdElB. The EIBdeleted adenovirus selectively inhibited Hep3B (deleted p5_3)) andPLC (mutatedp5_35), but failedto inhibitHepG2 (wild-type p53). In a third series of experiments, a combination of Adwtp53 and AdEIB-deleted 55 virus was used to transfect the hepatocellular cancer cell lines. The combination led to complete cell death in all cell lines, irrespective of the p53 status. In addition, when all three cell lines were grown together, mimicking the cell mixture typically expected in human cancers, there was a complete cytotoxic effect, with total cell death. Furthermore, the cytotoxic effect was seen with a viral concentration 2 log less than that required for each viniS individually. This invention allows for the combination of a combination of an adenovirus vector expressing wild-type p53 and an adenovirus expressing EIB-deleted p53. This combination is effective in killing cancer cells that express mutant or deleted p53) and also cells expressing wild-type p53). Therefore, the invention allows a 65 new therapeutic approach to cancer treatment, based on the simultaneous use of both viruses for the treatment of cancer in humans and animals.

S

Claims

I A combination of wild-type p53 gene and the ElMeleted p53 gene given simultaneously to produce a cytotoxic effect in human and animal cancer cells. 2 A combination of wild-type p53 gene and the E I B-deleted p53 gene given sequentially to produce a cytotoxic effect in human and animal cancer cells. A combination of wild-type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1 &2, using an adenovirus. vector to transport the combination of genes. 4 A combination of wild- type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1 &2, using a liposomal vector to transport the combinati on of genes. 5 A combination of wild-type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1&2, using any vector carrying the E I A gene to transport the combination of genes. 6 A combination of wild- type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by the intravenous route. 7 A combination of wild-type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1 &2, using a vector, as in 3,4,&5, administered by the oral (cochleate) route.

8 A combination of wild-type p53 and the EIB-deleted p53 given sequentially or 3 simultaneously, as in 1 &2, using a vector, as in 5,4,&5, administered by the intraperitoneal route.

9 A combination of wild-type p53 and the EIB-deleted p5_3) given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, admimstered directly into the tumour.

A combination of wild-type p5_3) and the EIB-deleted p53) given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by the intra arterial route.

I I A combination of Wild-type p5_3) and the F I B-deleted p53 given sequentially or simultaneously, as in 1 &2, using a vector, as in 3,4,& 5, administered by any of the routes described in 6,7,8,9,& 10, and used to kill human and animal cancer cells expressing a mutant p53 gene.

12 A combination of wild-type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by any of the routes described in 6,7,8,9,& 10, and used to kill human and animal cancer cells with a deleted p53 gene.

I A comb' p53 and the E I B-deleted p53 given sequentially or ination of NNII 1.1 1 simultaneously, as in 1 &2, using a vector, as in _3),4,&5, administered by any of the routes described in 6,7,8,9,& 10, and used to kill human and animal cancer cells expressing the wild-type p53 gene.

14 A combination of wild-type p53) and the ElB-deleted p5_3) given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by any of the routes described in 6,7,8,9,&10, and used to kill human and animal cancer cells expressing a combination of forms and expressions of p53 gene, including but not exclusively, a combination of the forms and expressions described in 11, 12, &13.

A method of using the invention whereby the adenovirus E I B-deleted form is used first andwild-type p53) is added to the vector 16 A method of using the invention, as in 1& 2, whereby plasnud p53) and the gene encoding RIA are used in combination or sequentially.

17 A method of using the invention, as in 1,2, 11,12,13, & 14, and administered in ways described in Claims 6-10, whereby plasmid wild-type p53 and the gene encoding E I A are used in combination or sequentially vAth the E I A gene either as a plasmid or in a vector. 18 A combination of wild-type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1,2, 11,12,13, & 14, and administered in ways described in Claims 6- 10, using a retrovirus vector to transport the combination of genes or the genes sequentially. 19 A combination of wildtype p5_3) and the EIB-deleted p53 given sequentially or simultaneously, as in 1,2, 11,12,13, & 14, and administered in ways described in Claims 610, using a Herpes virus vector to transport the combination of genes or the genes sequentially. 20 A combination of wild-type p53 and the EIBdeleted p53 given sequentially or simultaneously, as in 1,2, 11,12,13), & 14, and administered in ways described in Claims 6- 10, using fusion proteins to transport the combination of genes or the genes sequentially,