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Methods and compositions comprising DNA damaging agents and p53

a technology of dna damaging agents and compositions, applied in the direction of dna/rna fragmentation, peptide/protein ingredients, viruses, etc., can solve the problems of cancer development, lung cancer mortality rate will remain high well into the 21st century, and current treatment methods for cancer, including radiation therapy, surgery, chemotherapy, etc., to inhibit the tumorigenicity of h358 cells, great therapeutic

Inactive Publication Date: 2006-08-17
ROTH JACK +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for killing cells using a combination of a tumor suppressor gene or protein and a DNA damaging agent. This method can be used to treat various types of cancer by targeting the growth and division of cancer cells. The invention also provides a method for delivering a p53 gene to cells to induce programmed cell death. The invention has been tested in malignant cells and has shown promising results in reducing cell growth and inducing cell death. The invention can be applied to a wide range of target cells, including benign and malignant cells. The method involves contacting cells with both the p53 protein and a DNA damaging agent in a combined amount effective to kill the cell. The two agents can be delivered separately or simultaneously, and the delay between their delivery should not exceed about 12 hours. The invention also provides a variety of DNA damaging agents that can be used in combination with p53, including radiation and chemical compounds. Overall, the invention provides a novel and effective way to kill cells and treat cancer.

Problems solved by technology

Current treatment methods for cancer, including radiation therapy, surgery, and chemotherapy, are known to have limited effectiveness.
Although implementation of smoking-reduction programs has decreased the prevalence of smoking, lung cancer mortality rates will remain high well into the 21st century.
For example, in many cases, the expression of oncogenes is known to result in the development of cancer.
Several factors may contribute to an imbalance in these two forces, leading to the neoplastic state.
One of the most challenging aspects of gene therapy for cancer relates to utilization of tumor suppressor genes, such as p53.
However, major problems are associated with using retroviral vectors for gene therapy since their infectivity depends on the availability of retroviral receptors on the target cells, they are difficult to concentrate and purify, and they only integrate efficiently into replicating cells.
Tumor cell resistance to chemotherapeutic drugs represents a major problem in clinical oncology.
The stability and duration of expression of the introduced gene are still controversial, however.
A variety of treatment protocols, including surgery, chemotherapy, and radiotherapy, have been tried for human NSCLC, but the long-term survival rate remains unsatisfactory.

Method used

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  • Methods and compositions comprising DNA damaging agents and p53
  • Methods and compositions comprising DNA damaging agents and p53
  • Methods and compositions comprising DNA damaging agents and p53

Examples

Experimental program
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Effect test

example 1

Construction of p53 Expression Vector

[0124] This example describes the construction of a p53 expression vector. This vector is constructed as indicated and is used to replace the E1 region (1.3-9.2 m.u.) of the Adenovirus strain Ad5 genome and employed to construct the Adenovirus virion described in Example 2.

[0125] The p53 expression cassette shown in FIG. 1, which contains human cytomegalovirus (CMV) promoter (Boshart, et al., 1985), p53 cDNA, and SV40 early polyadenylation signal, was inserted between the Xba I and Cla I sites of pXCJL1 (provided by Dr. Frank L. Graham, McMaster University, Canada).

[0126] The genome size is about 35.4 kb, divided into 100 map units (1 m.u. 0.35 kb). The p53 expression cassette replaced the E1 region (1.3-9.2 m.u.) of the Ad5 genome.

[0127] Primer 1 has the sequence 5′-GGCCCACCCCCTTGGCTTC-3′ (SEQ ID NO:1) and is located in the first intron downstream of the human CMV major IE gene promoter (Boshart, et al., 1985). Primer 2 has the sequence 5′-T...

example 2

Generation and Propagation of Recombinant p53 Adenovirus

[0128] This example describes one method suitable for generating helper-independent recombinant adenoviruses expressing p53. The molecular strategy employed to produce recombinant adenovirus is based upon the fact that, due to the packaging limit of adenovirus, pJM17 cannot form virus on its own. Therefore, homologous recombination between the p53 expression vector plasmid and pJM17 within a transfected cell results in a viable virus that can be packaged only in cells which express the necessary adenoviral proteins.

[0129] The method of this example utilizes 293 cells as host cells to propagate viruses that contain substitutions of heterologous DNA expression cassettes at the E1 or E3 regions. This process requires cotransfection of DNA into 293 cells. The transfection largely determines efficiency of viral propagation. The method used for transfection of DNA into 293 cells prior to the present invention was usually calcium-ph...

example 3

Confirming the Identity of Recombinant Adenovirus

[0132] This example illustrates a new polymerase chain reaction (PCR) assay for confirming the identity of recombinant virions following cotransfection of the appropriate cell line.

[0133] Aliquots of cell culture supernatants (50 to 370 μl) were collected from the test plates, treated with proteinase K (50 μg / ml with 0.5% SDS and 20 mM EDTA) at 56° C. for 1 hour, extracted with phenol-chloroform, and the nucleic acids were ethanol precipitated. The DNA pellets were resuspended in 20 μl dH2O and used as template for PCR amplification. The relative locations of the PCR primers and their sequences are depicted in FIG. 1 and are SEQ ID NOS:1, 2, 3 and 4, respectively. The cDNA insert-specific primers define a 1.4 kb PCR product and the viral genome-specific primers define a 0.86 kb PCR product. The PCR reactions were carried out in a 50 μl volume containing 4 mM MgCl2, 50 mM KCl, 0.1% triton X-100, 200 μM each of dTPs, 10 mM Tris-Cl (pH...

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Abstract

The present invention relates to the use of tumor suppressor genes in combination with a DNA damaging agent or factor for use in killing cells, and in particular cancerous cells. A tumor suppressor gene, p53, was delivered via a recombinant adenovirus-mediated gene transfer both in vitro and in vivo, in combination with a chemotherapeutic agent. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into tumors subcutaneously, followed by intraperitoneal administration of a DNA damaging agent, cisplatin, induced massive apoptotic destruction of the tumors. The invention also provides for the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

Description

[0001] The present application is a continuation-in-part of co-pending U.S. patent application Ser. No. 08 / 145,826, filed Oct. 29, 1993; which is a continuation-in-part of U.S. patent application Ser. No. 07 / 960,543, filed Oct. 13, 1992; which is a continuation-in-part of U.S. Ser. No. 07 / 665,538, filed Mar. 6, 1991; the entire text and figures of which disclosures are incorporated herein by reference without disclaimer.[0002] The government owns rights in the present invention pursuant to NIH grants ROI CA 45187 and CA 16672, and Training Grants CA 09611 and CA 45225.BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates generally to the area of novel strategies for the improvement of chemotherapeutic intervention. In other aspects, the present invention provides novel methods and compositions that combine the potency of DNA damaging agents with the combined delivery of a tumor suppressor. The combination of DNA damaging factors with the h...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00A61K31/7072A61K31/7048A61K31/704A61K31/513A61K31/4745A61N5/00A61K33/24C12N15/09A61K9/08A61K31/277A61K31/40A61K31/407A61K31/505A61K31/70A61K31/711A61K33/243A61K35/76A61K38/00A61K38/17A61K45/00A61K51/00A61P35/00C07K14/47C07K14/82C12N15/113C12N15/861
CPCA61K31/4745A61K31/513A61K31/704A61K31/7048A61K31/7072A61K33/24A61K45/06A61K48/00C07K14/4746C07K14/82C12N15/1135C12N15/86C12N2710/10332C12N2710/10343C12N2799/022A61K38/00A61P35/00A61K33/243A61K38/17
Inventor ROTH, JACKFUJIWARA, TOSHIYOSHIGRIMM, ELIZABETHMUKHOPADHYAY, TAPASZHANG, WEI-WEIOWEN-SCHAUB, LAURIE
Owner ROTH JACK
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