Cancer constitutes one of the greatest health threats in the world, responsible for over one-half million deaths each year in the U.S. alone. Unfortunately, current treatment methods for cancer, including radiation therapy, surgery, and chemotherapy, are known to have limited effectiveness. For example, non-small-cell lung cancer (xe2x80x9cNSCLCxe2x80x9d), which includes squamous cell carcinoma, adenocarcinoma and large-cell carcinoma, accounts for 75%-80% of all lung cancers (Gould and Warren, 1989). Multimodality therapeutic strategies have been applied to regionally advanced NSCLC but the overall cure rate, which is approximately 10%, remains unsatisfactory (Belani, 1993; Roth et al., 1994).
Increased understanding of the molecular pathogenesis of cancer has profoundly changed the view of the pathogenesis of the disease, as the development of cancer is considered to result from multiple genetic alterations (Goyette et al., 1992; Klein et al., 1987). It now is well established that a variety of cancers are caused, at least in part, by genetic abnormalities that result in either the overexpression of one or more genes, or the expression of an abnormal or mutant gene or genes. For example, in many cases, the expression of oncogenes is known to result in the development of cancer. xe2x80x9cOncogenesxe2x80x9d are defined as genetically altered genes whose mutated expression product somehow disrupts normal cellular function or control (Spandidos at al., 1989). From melanomas to lymphomas, these mutations are believed to effect the neoplastic growth of cells derived from every tissue.
Most oncogenes studied to date have been found to be xe2x80x9cactivatedxe2x80x9d as the result of a mutation, often a point mutation, in the coding region of a normal cellular gene, i.e., a xe2x80x9cproto-oncogenexe2x80x9d. The mutation results in amino acid substitutions in the expressed protein product. This altered expression product exhibits an abnormal biological function that takes part in the neoplastic process. The underlying mutations can arise by various means, such as by chemical mutagenesis or ionizing radiation. A number of oncogenes and oncogene families, including ras, myc, neu, raf, erb, src, fms, jun and abl, have now been identified and characterized to varying degrees (Travali et al., 1990; Bishop, 1987).
Another gene of interest involved in the regulation of cell growth is the tumor suppressor p53. Mutations of the p53 gene span several coding regions and are the most common yet described for human cancer (Hollstein et al., 1991; Lane and Benchimol, 1990). These mutations not only eliminate the tumor suppressor activity but also stimulate growth of malignancies. In addition, the mutant p53 protein may possess transforming ability and can cooperate with other oncogenes in the transformation of normal cells (Parada et al., 1984; Jenkins et al., 1984; Elihayu et al., 1984; Hinds et al., 1989). The mutant p53 protein also has a prolonged half-life of 2 to 12 hours, resulting in higher intracellular concentrations than the wild-type protein (Reihsaus et al., 1990). Loss of the ability to suppress transformation and gain of transforming potential are properties of the mutant p53 gene product.
Importantly, the malignant phenotypes of certain cancer cells can be reversed by the introduction of a recombinant construct that reverses a single genetic lesion, a single normal cell-derived chromosome, or a copy of a wild-type tumor suppressor gene (Goyette et al., 1992; Takahashi et al., 1992; Anderson and Stanbridge, 1993; Mukhopadhyay et al., 1991). This finding suggests that correction of a single oncogene or tumor suppressor gene abnormality may overcome the effect of multiple genetic changes in the cancer cell (Goyette et al., 1992; Takahashi et al., 1992; Anderson and Stanbridge, 1993; Mukhopadhyay et al., 1991). It also may be desirable to develop enzymes that inactivate a particular oncogene.
Although proteins traditionally have been targeted for biological catalysis or enzyme mimics, other biological macromolecules, such as RNA molecules (commonly known as xe2x80x9cribozymesxe2x80x9d), are also capable of accelerating chemical transformations. Ribozymes may be particularly promising because many of these enzymes have a specific catalytic domain that possesses endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (xe2x80x9cIGSxe2x80x9d) of the ribozyme prior to chemical reaction.
Despite the broad range of chemical functionalities present within RNA, ribozyme catalysis mainly has been limited to sequence specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Ribozyme-mediated inhibition of gene expression may be particularly useful in therapeutic applications if the catalytic sequence can be designed to cleave a specific target RNA sequence (Scanlon et al., 1991; Sarver et al., 1990; Sioud et al., 1992).
Recently, it was reported that ribozymes elicited genetic changes in some cell lines to which they were applied; the altered gene included H-ras, c-fos, and genes of HIV virus. Most of this work involved the modification of mRNA based on a specific mutant codon that is cleaved by a specific ribozyme.
Despite these advances, there remains a need for improved methods of using ribozymes in the treatment of malignant cells. In particular, there is a need for specific application of ribozyme-mediated intervention in p53 transformation of human cancer cells.
It is, therefore, a goal of the present invention to provide compositions and methods relating to the control of oncogenesis as it relates to cancer cells having transforming mutations in the p53 gene. While conventional gene replacement therapy may be sufficient in certain situations, e.g., where the p53 mutant simply has lost tumor suppressing activity, it may not suffice in others where the altered p53 actively contributes to the malignant phenotype. In such a case, it will be desirable to inactivate the mutant gene while, at the same time, restoring normal p53 function. The present invention seeks to provide gene therapeutic constructs and methods that inactivate a mutant p53 and also provide for restoration of wild-type p53 function.
This invention generally relates to expression constructs that express a ribozyme that inactivates pre-mRNA of the mutant p53 and methods for their use. More specifically, the present invention provides a retroviral vector-mediated system that can be used to transduce various hammerhead ribozymes into cancer cells, such as human lung cancer cells.
This invention also relates to the design of ribozymes that will interrupt the pre-mRNA splicing process of p53 transcripts. An advantage of this method for modifying pre-mRNA is that joint sequences between introns and exons can be used to develop ribozyme target sequences. Such a ribozyme cleaves the target sequence and interrupts the process of splicing from pre-mRNA to mRNA. At the same time, the ribozyme would not affect a cDNA provided to the same cell.
It also is contemplated, as part of the present invention, to provide a replacement p53 gene that exhibits wild-type p53 activity. This replacement gene is engineered to avoid the action of the ribozyme, for example, by being provided in the form of a cDNA or a construct otherwise lacking the ribozyme target.
In one embodiment of the present invention, an expression construct is provided comprising a first promoter functional in eukaryotic cells and a first nucleic acid encoding a p53-specific ribozyme, where the first nucleic acid is under transcriptional control of the first promoter. Preferred embodiments of this aspect of the invention include a retrovirus promoter or an SV40 promoter being most preferred. In a specific embodiment, the ribozyme of the expression construct targets a p53 intron-exon splice junction, particularly the p53 codon 187.
In another embodiment of the present invention, an expression construct further codes for a second nucleic acid, preferably a cDNA, encoding a functional p53, where the second nucleic acid transcript is not cleaved by the ribozyme. As used herein the term xe2x80x9csecond nucleic acid transcriptxe2x80x9d refers to the wt-p53 mRNA that is expressed by the construct and encoded by the second nucleic acid. The second nucleic acid transcript is not cleaved because the ribozyme specifically cleaves a target site absent from that transcript. In certain applications, it may be preferable to have the second nucleic acid under the transcriptional control of a second, separate promoter which also is functional in eukaryotic cells.
In another embodiment of the present invention there is provided a pharmaceutical composition comprising (i) an expression construct comprising a first promoter functional in eukaryotic cells and a first nucleic acid encoding a p53-specific ribozyme, where the first nucleic acid is under transcriptional control of said first promoter and (ii) a pharmaceutically acceptable buffer, solvent or diluent. It may be preferable to have the expression construct further comprise a second nucleic acid encoding a functional p53, wherein the second nucleic acid transcript is not cleaved by the ribozyme. It also may be preferable to have the expression construct further comprise a second promoter functional in eukaryotic cells, wherein the second nucleic acid is under the transcriptional control of the second promoter.
In yet another embodiment, the present invention encompasses a method for inhibiting mutant p53 function in a cell comprising the steps of (i) providing an expression construct comprising a promoter functional in eukaryotic cells and a nucleic acid encoding a p53-specific ribozyme, where the nucleic acid is under transcriptional control of the promoter; and (ii) contacting the expression construct with the cell.
A preferred expression construct of the present invention is a retrovirus. It is also preferred to have the ribozyme target a p53 intron-exon splice junction, particularly the p53 codon 187.
A further embodiment of the present invention includes a method for restoring p53 function to a cell lacking a functional p53 comprising the steps of (i) providing a first expression construct comprising a first promoter functional in eukaryotic cells and a first nucleic acid encoding a p53-specific ribozyme, where the first nucleic acid is under transcriptional control of the first promoter; (ii) providing a second expression construct comprising a second promoter functional in eukaryotic cells and a second nucleic acid encoding a functional p53 lacking the target site for the p53-specific ribozyme, where the second nucleic acid is under transcriptional control of the second promoter and the second nucleic acid transcript is not cleaved by the ribozyme; and (iii) contacting the first and the second expression constructs with the cell.
Particular embodiments of the present invention are provided for restoring p53 function to a cell lacking a functional p53 utilizing retrovirus expression constructs.
Also provided are methods for treating a mammal with cancer comprising the steps of (i) identifying a mammal having a cancer characterized by cells expressing a mutated, transforming p53 product; (ii) providing an expression construct, preferably a retrovirus containing a first promoter functional in eukaryotic cells and a first nucleic acid encoding a p53-specific ribozyme, where the first nucleic acid is under transcriptional control of the first promoter and a second nucleic acid encoding a functional p53 lacking the target site for the p53-specific ribozyme, preferably a cDNA, wherein the second nucleic acid transcript is not cleaved by said ribozyme; and (iii) contacting the expression construct with the cells.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.