WO2001057061A1 - Novel use of ribozymes to block gene expression - Google Patents

Abstract

The invention provides novel methods for determining the in vivofunction of genes of interest using ribozymes, as well as methods of determining new targets for treatment of disease. Further provide are methods and compositions for treating cancer using ribozymes specific for the polynucleotide products of one of the NFλB genes, an integrin gene, or a PECAM gene.

Description

Novel Use of Ribozymes to Block Gene Expression

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/180,586 filed 4 February 2000. The disclosure of this related application is hereby incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to the fields of cell biology, biochemistry, molecular genetics, medicine, oncology, and functional genomics. In particular, the invention provides methods which assay gene function by inhibiting gene expression with ribozymes in vivo. Also reported are methods which identify targets for the treatment of disease by inhibiting gene expression with ribozymes in vivo and screening for changes in disease symptoms. Further, the invention relates to methods of treating diseases, especially cancer and hyperplastic conditions, using ribozymes that have specificity for the polynucleotide products of the PECAM, integrins, and NFKB subunit genes and other therapeutically useful target genes and RNA species, among others. In addition, novel compositions for the systemic delivery of ribozymes are reported which comprise polynucleotides encoding ribozymes and cationic lipids or cationic polymers.

BACKGROUND OF THE INVENTION Functional genomics

Modern genome sequencing efforts have yielded the sequences of a staggering number of genes. The sequence of an entire genome from a multicellular eukaryotic species, Caenorhabditis elegans, has recently been completed. The human genome program is rapidly progressing towards the entire human sequence (see Jordan, 1999, J. Biomed. Sci 6:145-150). Currently, over a million partial sequences of human cDNA clones are publicly available representing perhaps one half of our estimated 100,000 unique genes (see Jordan). However, relatively few of these genes have known, well- characterized functions. Even those genes with known functions might have significant undiscovered functions. Future goals in the field of genomics must include ascribing function to the multitude of genes of humans and other species.

However, current efforts at identifying the function of a gene of interest, given the entire sequence or a partial sequence, are not adequate to identify the functions of each of the 100,000 or so human genes. One technique is sequence comparison. If a novel human sequence, for instance, shares significant homology with a well-characterized sequence from a simpler organism, then a guess at the function of the human sequence can be made (see Jordan). Genetic analysis of extended populations attempts to locate genetic polymorphisms linked to disease susceptibility as a first step to identify some of the genes involved in complex and multigenic diseases. Large scale expression studies might identify the tissue-specificity of the expression of a given gene. The yeast two-hybrid system searches for the interaction partners of a given protein among a few hundred entities at a time (see Jordan). Finally, a knockout mouse can be constructed but requires approximately one year for each gene (see Jordan).

Methods that screen a few hundred proteins at a time or that require one year per gene sequence studied are inadequate tools to identify the function of millions of sequences or sequence fragments from over 100,000 unique genes. Alternative methods are needed to rapidly and efficiently determine the functions of large numbers of genes. Ideally, such methods would need no more information than a partial sequence of a gene to generate functional information.

Ribozymes

RNA enzymes, or ribozymes, might provide an effective tool to identify the function of any gene. A ribozyme is an RNA molecule with the ability to specifically cleave a target RNA molecule. For virtually any gene sequence, a ribozyme can be designed to specifically cleave its transcription products. If such a ribozyme were effectively delivered into a cell, cleavage of those transcription products would reduce or block the expression of the products of the gene. A correlation of a change in gene expression and a change in the phenotype of the organism treated with the ribozyme would be indicative of the function of the gene. Ribozymes can even be designed to target the transcription products of a gene of which only a partial nucleotide sequence is known. However, current efforts have had limited success at effectively delivering a ribozyme into a cell even in vitro. The chemistry of ribozymes was discovered in the self-cleaving molecules in Tetrahymena thennophila (see Zaug and Cech, 1986, Science 231:470-475) and in ribonuclease P of Escherichia coli (see Guerrier-Takada et al., 1983, Cell 35:849-857). These ribozymes effect RNA splicing reactions in cis. Later work identified a catalytic domain in the self-cleaving RNA reaction in the virusoid from lucerne transient streak virus (see Forster and Symonds, 1987, Cell 49:849-857). The catalytic domain was termed "hammerhead" because of its secondary structure. Subsequent work demonstrated that the hammerhead domain could act in a truly catalytic manner by cleaving its target in trans (see Uhlenbeck 1987, Nature 328:596-600). Hammerhead ribozymes can be designed against virtually any gene transcript (see Haselhoff et al., 1988, Nature 334:585-591). Additionally, the design of gene-specific hairpin ribozymes is also feasible (see Hampel et al., 1990, Nuc. Acids. Res. 18:299- 304). The recently described axe-head motif of the human δ hepatitis agent provides a third motif for the design of a gene-specific ribozyme (see Branch and Robertson, 1991, Proc. Natl. Acad. Sci. USA 88:10163-10167). Other motifs include the RNAse P and Neurospora VS RNAs (see Rossi, Chemistry & Biology 6:R33-R37). Accordingly, methods of designing a variety of ribozymes are known. Ribozymes and disease

RNA catalysis might also offer a powerful tool for the treatment of human disease. Manipulation of gene-specific expression is emerging as an important option for the treatment of viral illnesses and cancer (see, for review, Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy 2(3):213-223).

One area of research is the use of ribozymes for the treatment of cancer. Cancer is viewed genetically as a multistep process which includes molecular alterations in tumor suppressor genes or oncogenes or both (see Kashani-Sabet and Scanlon). Tumor suppressor genes are inactivated by mutation or deletion, and the products of oncogenes are activated by mutation, gene amplification or overexpression, or chromosomal translocation. The use of ribozymes in the field of cancer has focused mainly on the inhibition of tumor-specific oncogene product expression.

Ribozyme-mediated approaches have largely targeted the expression products of oncogenes of the ras family of G proteins, mutated in 10% to 15% of human cancers, and the expression products of the chimeric bcr-abl gene found in the break point of the Philadelphia chromosome in more than 95% of chronic myelogenous leukemias (CMLs). For example, anti-ra-s^* ribozymes inhibited proliferation in vitro of human bladder cancer cells, human melanoma cells, and murine NIH3T3 cells. AnXi-bcr-abl ribozymes have been effective in reducing cell growth and cell proliferation when delivered in vitro to CML cell lines. Ribozymes that cleave RNA products of genes associated with drug-resistant phenotypes have also been effective in vitro. In the hope of developing a treatment for human immunodeficiency virus (HIV), ribozymes have been designed to target many of the genetic elements in the retroviral genome including those elements encoding functional proteins, leader sequences, and regulatory proteins and sequences. For instance, one study resulted in the inhibition of HIV replication in a cell line by using an anti-gag ribozyme combined with another therapy (see Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10802- 10806). A strategy using a hairpin ribozyme targeting the 5' leader sequence of HIV resulted in the inhibition of growth in culture of three different HIV strains (see Yu et al., Proc. Natl. Acad. Sci. USA 90:6340-6344). What is needed to further any possible HIV therapy with ribozymes is an improved delivery method. For example, retroviral delivery of ribozymes creates a risk of cancer related to insertional mutagenesis in a host whose immune system is already compromised.

Delivery of ribozymes

For the ribozyme to be effective, it must be delivered into an intracellular milieu. Prior efforts at delivery of ribozymes have suffered from a number of deficiencies. Endogenous delivery of ribozymes exploits cellular machinery for expression. A gene encoding a ribozyme is cloned into an available vector and delivered to cells by transfection of the plasmid or by retroviral infection. Other endogenous delivery systems under investigation for delivery of ribozymes including cationic liposomes, adenoviruses, and adeno-associated viruses have yet to achieve optimal vectors for effective delivery (see Kashani-Sabet and Scanlon, 1995, above). Detection of RNA cleavage by the transcribed ribozyme within the cell has been difficult in many studies due to the instability of the RNA cleavage products (see Kashani-Sabet and Scanlon, 1995, above).

Exogenous delivery has been attempted using naked ribozymes or ribozymes in complexes with cationic liposomes. A major limitation of prior methods of exogenous delivery of ribozymes has been the poor stability of ribozymes due to susceptibility to ribonuclease attack in serum or in the cell (see Kashani-Sabet and Scanlon, 1995, above). Chemically altered ribozymes incorporating 2'-fluoro and 2'-amino nucleotides at U and C positions do have improved stability, but the modifications were offset by a reduction in catalysis (see Pieken et al., 1991, Science 253:314-317). The incorporation of deoxynucleotides at selected positions of the hammerhead has yielded greater stability without as great a sacrifice in catalytic activity (see Kashani-Sabet and Scanlon, 1995, above).

What is needed is an effective method of delivering ribozymes to selected cells of an organism to alter the function of a gene of interest in either a generalized or a tissue specific manner. However, at present the in vivo delivery of ribozymes is ineffective because of difficulty in the synthesis of a sufficient amount of a synthetic ribozyme or the multiple existing limitations of viral vectors carrying ribozyme genes (see Rossi), and problems with maintaining prolonged duration of ribozyme expression.

SUMMARY OF THE INVENTION It is an object of the invention to overcome the noted deficiencies of the prior art by systemically delivering ribozymes to probe gene function. In one aspect, the present invention provides a method of quickly identifying a function of a gene of interest. The method entails systemically delivering a polynucleotide that encodes a ribozyme that has specificity for a polynucleotide product of the gene of interest into cells of a test animal; and comparing the phenotype of the test animal to the phenotype of a control animal, wherein a function of the gene of interest is correlated to a change in phenotype of the test animal.

In another aspect, the invention provides a method for evaluating a gene of interest as a target for the treatment of a disease. The method entails systemically delivering a polynucleotide that encodes a ribozyme that has specificity for a polynucleotide product of the gene of interest into cells of a test animal exhibiting symptoms of the disease thereby expressing the ribozyme in the cells of the test animal; and comparing the phenotype of the test animal to the phenotype of a control animal exhibiting the same symptoms as the test animal prior to delivery of the polynucleotide wherein the gene is identified as a target for the treatment of the disease if delivery of the ribozyme alters the symptoms or the pathophysiology of the disease in the test animal.

In a further aspect, the present invention provides methods for the treatment of a disease. A disease is treated in an animal by systemically delivering a therapeutically effective amount of a polynucleotide that encodes a ribozyme, thereby ameliorating a symptom of the disease. In a particularly preferred aspect, the disease is cancer and the ribozyme has specificity for polynucleotides that encode PECAM, an integrin subunit, an integrin β subunit, integrin β₃, or a subunit of NFKB (e.g., p65 or p50).

The invention also provides a method of preventing tumor growth or metastasis in a patient by reducing the activity in the patient of at least one protein subunit of NFKB. The activity of the protein subunit of NFKB can be reduced by, e.g., reducing levels of the RNA transcript encoding the protein subunit or treating the patient with a compound that inhibits the activity of NFKB or its subunits. In particular embodiments, the activity of the protein subunit of NFKB is reduced by delivering a ribozyme specific for an RNA encoding the protein subunit. The protein subunit of NFKB chosen as a target can be any one or more of Rel, RelB, NFκB2, p50 and p65.

Yet another aspect of the invention is a composition for systemic delivery of a ribozyme into an animal that has a disease comprising a polynucleotide that encodes a ribozyme; and optionally a cationic lipid or a cationic polymer, wherein the composition contains a therapeutically effective amount of the polynucleotide. In another embodiment of the invention, the plasmid vectors used to deliver and express the ribozymes contain an NRA element and/or a CRA element in order to produce prolonged expression of the delivered ribozymes.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. Generally, the term naturally- occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.

The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA". The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 12 nucleotides in length, frequently at least 20 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.

A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence.

The term "antineoplastic agent" is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.

As used herein "normal blood" or "normal human blood" refers to blood from a healthy human individual who does not have an active neoplastic disease or other disorder of lymphocytic proliferation, or an identified predisposition for developing a neoplastic disease. Similarly, "normal cells", "normal cellular sample", "normal tissue", and "normal lymph node" refers to the respective sample obtained from a healthy human individual who does not have an active neoplastic disease or other lymphoproliferative disorder.

As used herein, the term "transcriptional unit" or "transcriptional complex" refers to a polynucleotide sequence that comprises a structural gene (DNA encoding a ribozyme), a cis-acting linked promoter and other cis-acting sequences necessary for efficient transcription of the structural sequences, distal regulatory elements necessary for appropriate tissue-specific and developmental transcription of the structural sequences, and additional cis sequences important for efficient transcription and translation (e.g., polyadenylation site, mRNA stability controlling sequences). As used herein, "linked" means in polynucleotide linkage (i.e., phosphodiester linkage).

"Unlinked" means not linked to another polynucleotide sequence; hence, two sequences are unlinked if each sequence has a free 5' terminus and a free 3' terminus.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. Overview Cleavage of a gene product with a catalytic RNA allows the study of gene function with only a minimal knowledge of the sequence of the gene. However, application of such techniques in vivo has been hampered by lack of effective delivery and sufficiently high and/or sustained expression of the ribozymes. The present invention demonstrates, for the first time, systemic delivery of a polynucleotide that encodes a ribozyme that has specificity for a gene of interest into cells of an animal in vivo, such that the ribozyme is effectively expressed and a new phenotype is produced.

Additionally, because the polynucleotide that encodes the ribozyme is delivered non-virally, repeated administration is possible without generating an immune response against the vector delivery system. In another aspect of the invention, the plasmid vector containing the polynucleotide sequence confers both long term expression of the polynucleotide and the ability to repeatedly reexpress the polynucleotide in fully immunocompetent hosts, very long-term or sustained expression of the ribozyme can be produced using a non-integrating plasmid vector system.

The invention combines the catalytic activity of ribozymes with effective delivery to cells in vivo to provide improved methods of probing gene function, evaluating targets for disease treatment, and treating disease. The present invention allows observation of the effect of reduction of gene product expression in a native in vivo cell environment which encompasses the interactions between cell types and tissues. In addition, in vivo studies provide a more biologically and therapeutically relevant observation of gene function, whereas in vitro studies yield imperfect and often misleading indication of in vivo gene function by extrapolation from in vitro results.

For general methods relating to ribozymes, see U.S Patent 6,051,429, U.S. Patent 6,132,967, WOOO/74485, WOOO/66780, WOOO/61804, W099/31118, and WO99/67400; and for guidance on cationic lipid-DNA complexes refer to U.S. Patent 5,932,241, each of which is incorporated herein by reference.

Methods of Identifying Gene Function In one embodiment, the present invention provides methods of identifying the function of a product of a gene of interest (also known as the practice of functional genomics). The method takes advantage of a highly efficient and long-expressing plasmid-based delivery system and the power of catalytic ribozymes to cleave the transcription product of a gene of interest in cells of a test animal in vivo. Since a ribozyme can be designed from minimal sequence information (for example, a cDNA sequence fragment of an expressed gene), the sequence of the full length gene is not needed. Given only a short sequence of the transcribed region of a gene of interest, the method can be used to create a somatic cell "knockout" animal and thereby test for gene function in a period as brief as a few weeks. The resulting effects on the phenotypes of the test animal are used to deduce at least one function of the product encoded by the gene of interest. Typically, the presently described method for practicing functional genomics will express ribozymes specific for a product of the gene of interest at biologically relevant levels for prolonged periods without producing significant ongoing host toxicity and without producing a gene transfer vector-based phenotype based on host-immune, toxic, or transforming responses. Additionally, the present methods allow for the efficient re-expression of the ribozyme(s) after reinjection into immunocompetent hosts. Thus, expression can be maintained for very long periods if such periods are required in order to induce a phenotype. Also, the present methods allow the delivery and expression of very large DNA vectors, which allows the delivery and expression of multiple different ribozymes into a single animal. In this way, potential in vivo synergy of two or more genes can be readily assessed. For example, if a reduction in expression of two or more genes of interest causes a similar phenotype, and if co-expression of the ribozymes against those genes does not produce additive effects on the phenotypes, then one can determine that those particular genes of interest act within common or independent pathways to cause the observed phenotype. Thus, not only functions but also pathways can be deduced using the methods of the invention.

Moreover, ribozymes specific for the nucleotide products of large numbers of unknown genes can be expressed in a single animal, thus allowing the functional screening of very large numbers of unknown genes using relatively few animals. This feature of the presently described methods substantially increases the efficiency of screening, and significantly reduces the numbers of animals required to screen for the phenotypes of large numbers of genes.

In addition to the specifically exemplified mice, examples of mammalian species that can be used in the practice of the present invention include, but are not limited to: humans, non-human primates (such as chimpanzees), pigs, rats (or other rodents), rabbits, cattle, goats, sheep, and guinea pigs. Additionally, as the methods of gene delivery are not limited to specific species or animal types, the presently described methods are also suitable for use in the expression of ribozymes in non- mammalian somatic cell transgenic animals such as insects, arthropods, crustaceans, birds, and fish.

Genes of Interest

The method may be applied to any gene of interest from an animal provided that sufficient sequence information is available. Sequence information is sufficient if a ribozyme can be designed with specificity for the polynucleotide products of the gene of interest. A polynucleotide product of a gene of interest is defined for purposes of the invention as a primary RNA transcript of a gene of interest, or an RNA transcript that has been partially or completely processed by the cellular machinery (e.g., spliced, cleaved, or polyadenylated). Such polynucleotide products of a gene of interest include but are not limited to messenger RNAs (mRNAs) and structural RNA molecules. The design of the ribozyme is discussed below. Preferably, the binding sequence recognized by the ribozyme will be relatively unique within the cells of the organism so that the ribozyme uniquely cleaves a polynucleotide product of the gene of interest. Preferred genes of interest are those which are expressed in endothelial cells, macrophages, lung, heart, spleen, liver, stomach, blood, endotracheal, buccal, prostate, breast, brain, bone, neural, neuroglial, skin, and tumor cells thereof, among others. Given an animal disease, a gene of interest can also be chosen as a candidate target for the treatment of the disease. The gene may be chosen based on prior literature that associates the gene with the disease or based on a prior function that might be associated with the disease. However, the present invention is not limited to those genes which have previously been associated with the given disease. The present method requires so little knowledge about the function or even sequence of the gene and is so quick and inexpensive that any gene can be evaluated for function or as a target for a given disease.

For instance, in the non-limiting and illustrative examples discussed below, the function of several genes were assessed in a murine model of human melanoma, and surprising new functions for the expression products of several genes were discovered that could not be directly extrapolated from previous in vitro studies on these genes and their gene products. Essentially any gene which, when inhibited by a therapeutic dose of a functional ribozyme, results in a measurable dimunition of neoplasia or hyperplasia are suitable for use in the invention. These genes may be inhibited with the ribozyme-encoding compositions and methods of the invention either singly or in combination (multiplexed), with either single doses (which may be administered serially) each comprising multiple ribozyme-encoding species, often on a single encoding polynucleotide vector, or with multiple doses wherein different species of ribozyme-encoding polynucleotide are separate doses.

Genes that can be targeted by the ribozyme-encoding polynucleotides of the invention for antineoplastic or anti-hyperplastic benefit include, but are not limited to, the following: Cdc6,MDM2, E2F, cyclin 1, c-myc, N-myc, L-myc, IGF-1, K-ras, H-ras, p53, mutant p53, FHIT, c-ERB-2, BCRA-1, BCRA-2, Bcl-1, telomerase (RNA and/or protein subunit), BAX, and similar oncogenically active genes which, when inhibited, produce a detectable antineoplastic or antihyperplastic effect. For cardiovascular therapy of circulatory conditions (e.g., myocardial infarction, stroke, hypertension, atherosclerosis, arterial graft patency, stent patency, atheromagenesis, and the like), genes which can be targeted include but are not limited to p53, p21, p27 and/or BAX.

The ribozyme-encoding polynucleotides of the present invention and therapeutic compositions comprising them can be combined with other therapeutic modalities. For example and not limitation, the ribozyme-encoding polynucleotides can be combined with other antineoplastic therapeutic modalities, such as surgery, ionizing radiation, or chemotherapy (e.g., with an agent such as bleomycin, cisplatin, nitrogen mustard, doxyrubicin, daunirubicin, cyclophosphamide, nucleotide analogs, antiestrogens, 5-fluorouracil, taxol, taxotere, hydroxyurea, methotrexate, thioguanine, chlorambucil, leucovorin, adriamycin, myleran, vinblastine, vincristine, vindesine, nitrogen mustards (BCNU), DTIC, mitotane, mitomycin, leustatin, etoposide, asparaginase, antitumor antibodies (e.g., monoclonal antibodies and/or humanized anti-tumor antibodies and toxin or radioisotope conjugates thereof) telomerase inhibitors, gene therapy expression vectors, and the like).

In some embodiments, the ribozyme encoded by the ribozyme-encoding polynucleotide will specifically inhibit one or more gene products that confer resistance to one or more antineoplastic agent; such as for example aldehyde dehydrogenase (in conjunction with cyclophosphamide therapy), multidrug resistance protein(s) (MDR, P- glycoprotein), DHFR, and the like. In such embodiments the ribozyme-encoding polynucleotide composition can be given concomitantly or preferably prior to administration of the antineoplastic agent to which the ribozyme is intended to increase the sensitivity of the neoplastic cell. In some cases the ribozyme will target a gene product comprising an enzyme needed to activate a prodrug, a detoxification enzyme (a cytochrome P-450 species, a UDPGT enzyme, a glutathione S-transferase, and the like), an enzyme required for steroid hormone biosynthesis, or the like.

In certain embodiments intended to deliver ribozyme-encoding polynucleotides to cardiovascular cells, the gene of interest desired to be inhibited will be a gene involved in atherogenesis, restenosis, endothelial injury, endothelial proliferation, cardiomyocyte degeneration, loss of cardiomyocyte contractility, cardiomyocyte apoptosis, scar formation (fibroblastic invasion), and the like. Other medicaments and applications thereof comprising the compositions and methods of the present invention will be apparent to those skilled in the art in view of the present specification.

Polynucleotides Encoding Ribozymes Given a sequence or a sequence fragment of a gene of interest, a ribozyme is designed that has specificity for the polynucleotide transcription products of the gene of interest. If only a fragment of the gene sequence is available, a ribozyme is designed based on the sequence of a transcription product that would correspond to the available sequence. A ribozyme "has specificity for" a polynucleotide if the ribozyme' s binding site is complementary to a sequence in the polynucleotide and the ribozyme cleaves the polynucleotide at or near that sequence. The sequence in the target RNA must be chosen to allow the ribozyme to achieve maximum specificity in vivo. Preferably a nucleotide sequence unique to the target RNA will be chosen.

The sequence in the target RNA must also meet the requirements of RNA catalysis for the particular ribozyme chosen to be used. The chemistry of ribozymes was discovered in the self-cleaving molecules in Tetrahymena thermophila (see Zaug and Cech, 1986, Science 231:470-475) and in ribonuclease P of Escherichia coli (see Guerrier-Takada et al., 1983, Cell 35:849-857). These ribozymes effect RNA splicing reactions in cis. Later work identified a catalytic domain in the self- cleaving RNA reaction in the virusoid from lucerne transient streak virus (see Forster and Symonds, 1987, Cell 49:849-857). The catalytic domain was termed "hammerhead" because of its secondary structure. Subsequent work demonstrated that the hammerhead domain could act in a true catalytic manner by cleaving its target in trans (see Uhlenbeck 1987, Nature 328:596-600). In vitro mutagenesis studies of the plus strand of satellite RNA of tobacco ringspot virus (sTobRV) defined the consensus sequences required for catalytic cleavage of a target RNA (see Haselhoff et al., 1988, Nature 334:585-591). This information made feasible the design of hammerhead ribozymes against any gene. Additionally, the minus strand of sTobRV was shown to cleave target RNA using a "hairpin" catalytic domain, and the design of gene-specific hairpin ribozymes is also feasible (see Hampel et al., 1990, Nuc. Acids. Res. 18:299-304). The recently described axe-head motif of the human δ hepatitis agent provides a third motif for the design of a gene-specific ribozyme (see Branch and Robertson, 1991, Proc. Natl. Acad. Sci. USA 88:10163-10167). Other motifs include the RNAse P and Neurospora VS RNAs (see Rossi, Chemistry & Biology 6:R33-R37).

For instance, hammerhead ribozymes require a target sequence of XUN, and hairpin ribozymes appear to require a target sequence of BNGUC. A ribozyme's "binding site" is the sequence of nucleotides in the ribozyme that hybridizes to a target RNA.

The hammerhead ribozyme motif (see conserved sequences of the hammerhead ribozyme in Hasseloff and Gerlach, 1988, Nature 334:585-591; Ruffner et al., 1990, Biochemistry 29: 10695- 10702) shows remarkable specificity for the target sequence of its substrate. It is composed of a catalytic core (or hammerhead domain) region and three hybridizing helices or stems (see Kashani- Sabet and Scanlon). Stems I and III hybridize to sequences of the substrate that flank the cleavage site (see Kashani-Sabet and Scanlon). The following numbering system for the ribozyme and target sequences is according to Hertel et al., 1992, Nuc. Acids Res. 20:3252. In the hammerhead motif, the binding site comprises the nucleotide sequence N² 'N²²N²³ of stem I and nucleotides A^{15 *}N^{*5 2}N^{15 3} of stem III in the structure. Stem loop II is usually composed of eight complementary ribonucleotides with four ribonucleotides in the loop structure (see Kashani-Sabet and Scanlon).

The recognition sequence of helices I and III has an optimal length of about 12 bases with sequences rich in A or U favored over GC-rich sequences (see Bertrand, et al., 1994, Nuc Acids Res. 22:293-300; Herschlag, 1991, Proc. Natl. Acad. Sci. USA 88:6921-6925). The hammerhead ribozyme will tolerate some alterations in helix II with possible reductions in catalytic activity (see McCall et al., 1992, Proc. Natl. Acad. Sci. USA 89:5710-5714).

Mutational analysis revealed that ribozymes require a sequence of XUN at the substrate cleavage site, with X being any nucleotide and N being A, C, or U (see Haseloff, et al.; Ruffner, et al., 1990, Biochemistry 29:10695-10702; Hertel, et al., 1992, Nuc. Acids Res. 20:3253; Koizumi et al.,

1989, Nuc. Acids Res. 17:7059-7071). The ribozyme cleaves the substrate 3' to the N nucleotide. Targets with GUC, GUA, GUU, CUC, and UUC sequences are particularly well cleaved (see Perriman et al., 1992, Gene 113:157-163). A modified hammerhead ribozyme has been shown to cleave 3' to AUA sequences in trans (see Nakamaye and Eckstein, 1994, Biochemistry 33:1271-1277). The hammerhead ribozyme is able to discriminate between substrates with single base substitutions in the recognition site and between closely related RNAs (see Koizumi et al.; Bennett and Cullimore, 1992, Nuc. Acids Res. 20:831-837).

The hairpin ribozyme (see Anderson et al., 1994, Nuc. Acids Res. 22:1096-1100, and Berzal- Herranz et al., 1993, EMBO J. 12:2567-2574) provides an alternate motif for the design of a catalytic

RNA that targets a polynucleotide product of a gene of interest. The hairpin ribozyme was derived from the minus strand of sTobRV RNA and site-specifically cleaves RNA substrates in trans (see Hampel and Tritz, 1989, Biochemistry 28:4929-4933; Feldstein et al., 1989, Gene 82:53-61). The original hairpin ribozyme consisted of 50-bases and cleaved corresponding 14 base RNA substrates (see Kashani-Sabet and Scanlon, as well as Anderson et al., 1994, Nuc. Acids Res. 22:1096-1100, and

Berzal-Herranz et al., 1993, EMBO J. 12:2567-2574). The ribozyme performed multiple substrate cleavages in the presence of Mg⁺⁺. A proposed secondary structure for the hairpin ribozyme-substrate complex consists of four helical regions separated by two internal loop sequences (see Hampel et al.,

1990, Nuc. Acids Res. 18:299-304; Haseloff and Gerlach, 1989, Gene 82:43-52). The substrate binds to the ribozyme through two helices, helix 1 and helix 2 (see Kashani-Sabet and Scanlon). Cleavage occurs to the 5' side of a guanosine within the internal loop, loop A, of the substrate separating helices 1 and 2. Loop B separates the two helices of the ribozyme (see Kashani-Sabet and Scanlon). Nucleotide sequences essential for catalytic activity have been described (see Chowira et al., 1991, Nature 354:320-322; Berzal-Herranz et al., 1993, EMBO J. 12:2567-2574). Within loop A, four bases of the ribozyme and one base of the substrate are essential. Within loop B, nine of the 11 bases of the ribozyme are essential, but only one base within the four helices of the ribozyme is essential for catalytic activity (see Joseph et al., 1993, Genes Dev. 7: 120-138). Cleavage occurs between the N⁵ and G⁶ of the substrate consensus sequence B⁴N⁵G⁶U⁷C⁸ (where B is C, U, or G and N is C, U, A, or G) (see Anderson et al., 1994, Nuc. Acids Res. 22:1096-1100 for numbering system). Since the particular mechanism of ribozyme cleavage chosen is not crucial to the methods, the present invention encompasses any catalytic RNA that can specifically cleave its target in vivo, whether that RNA includes a hammerhead motif, a hairpin motif, an axe-head motif, or any other ribozyme motif now known or later discovered.

Once the ribozyme is designed, a polynucleotide is constructed to express the ribozyme within cells of the test animal. The polynucleotide might further include any sequences known to one of skill in the art necessary for the effective delivery of the encoded ribozyme into target cells of the test animal, those sequences necessary for the effective expression of the ribozyme, and any sequences necessary for the stability of the polynucleotide. For example, the polynucleotide encoding a ribozyme will preferably include a promoter sequence and/or a promoter/enhancer sequence operatively linked to the polynucleotide that encodes the ribozyme. By "operatively linked" is meant that the promoter or promoter/enhancer is placed adjacent to the sequence encoding a ribozyme in such a manner that, when active, the promoter or promoter/enhancer directs the expression of the adjacent sequence encoding a ribozyme. The promoter or promoter/enhancer sequence should be active (i.e., direct transcription) in at least the cell type where expression is desired, and preferably is active at high levels in a broad spectrum of cell types. Appropriate promoters and promoter/enhancer sequences are well known in the art. A number of transcriptional promoters and enhancers can be used to express the gene of interest, including, but not limited to, the herpes simplex thymidine kinase promoter, cytomegalovirus enhancer/promoter, SV40 promoters, and retroviral long terminal repeat (LTR) enhancer/promoters, hormone response elements, including GREs, AP-1, SP-1, Ets, NF-1, CREBs, or NF-kB binding DNA sequences and the like, as well as any permutations and variations thereof, which can be produced using well established molecular biology techniques (see generally, Sambrook et al. (1989) Molecular Cloning Vols. I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and Current Protocols in Molecular Biology (1989) John Wiley & Sons, all Vols. and periodic updates thereof, herein incorporated by reference). Enhancer/promoter regions can also be selected to provide tissue- specific expression, including expression targeted to vascular endothelial cells, monocytes, macrophages, lymphocytes, various progenitor and stem cells, such as hematopoietic stem cells, and the like.

A preferred subgenus of ribozyme-encoding polynucleotides are polynucleotide vectors encoding a cellular retention activity (or nuclear retention activity) such as the EBV protein EBNA-1 and further comprising an EBV family of repeat (FR) sequence or an intact oriP sequence or functional equivalent, and a ribozyme-encoding sequence, wherein the cellular retention activity (or nuclear retention activity )and the ribozyme are expressible in the target cells of the patient to whom the polynucleotide vector is to be admnistered. In some embodiments, multiple ribozyme species are encoded on such a polynucleotide, typically on discrete expression cassettes. Exemplary embodiments of an illustrative vector suitable for hosting expression of a ribozyme-encoding sequence are disclosed herein in the Experimental Examples (infra), and in PCT publication W099/36514, incorporated herein by reference. These ribozyme-encoding polynucleotides are suitable for non- viral delivery to target cells of a host, such as a human or veterinary host organism, often via systemic administration such as intravenous or inhaled. For example and not to limit the invention, the following illustrative examples provide ribozyme targeting sequences for inhibition of the human p65 mRNA, p50 mRNA, pPECAM mRNA, and integrin β3 mRNA, respectively:

Ribozyme targeting human p65 mRNA: 3'- AG GTT CA AAG CAG GAG TGC CTG AGT AGT C GGA TAT -5' Cleaves 3' to GUU in position 428 of human p65 mRNA

Ribozyme targeting human p50 mRNA

3'- CC TCT CA AAG CAG GAG TGC CTG AGT AGT C CGA TGT -5' Cleaves 3' to GUU in position 173 of human p50 mRNA

Ribozyme targeting human pPECAM-1 mRNA

3'- AC TCC CA AAG CAG GAG TGC CTG AGT AGT C TTC TTT -5'

Cleaves 3' to GUC in position 223 of human PECAM-1 mRNA

Ribozyme targeting human integrin B3 mRNA

3'- ATGTA CA AAG CAG GAGTGC CTG AGTAGTC ATA GAG -5'

Cleaves 3' to GUA in position 420 of human integrin B3 mRNA Other ribozyme targeting sequences for these genes and others can be selected by the practitioner based on available sequence data fron GenBank, the scientific literature, and other sources.

Systemic delivery Once the polynucleotide is constructed, it can be delivered systemically into cells of a test animal where the encoded ribozyme is expressed and cleaves the polynucleotide products of the gene of interest in vivo. Ideal target cells include those cells that express the gene of interest. Cells that express the gene of interest can be identified by methods known to one of skill in the art. For instance, cells that are the source of a cDNA sequence of an expressed gene are potential targets for delivery of a ribozyme with specificity for the polynucleotide products of the expressed gene. Other cells might be chosen as targets to evaluate whether the gene of interest had any function in affecting a disease phenotype.

For purposes of the present invention, "systemically delivering" a polynucleotide that encodes a ribozyme means that the polynucleotide is delivered intravenously, intraarterially, or into the cardiac chambers in an animal. Preferably, systemic delivery results in delivery and expression of the polynucleotide encoding the ribozyme in macrophages, in vascular endothelial cells, in liver cells, in lung cells, and/or in tumor cells.

In a preferred embodiment, the polynucleotide is delivered in a non-viral delivery vector. For example, delivery can be accomplished intravenously in a complex with cationic liposomes or cationic polymers (such as, for example, DOTMA, DOTIM and polyethylimine (PEI)). For example, the polynucleotide is delivered in a complex with cationic liposomes according to Liu, et al., 1999, J. Biol. Chem. 274: 13338-13344, which is hereby incorporated by reference in its entirety. In preferred embodiments, polynucleotide DNA is complexed to l-[2-(9(2)-octadecenoyloxy)ethyl]-2-(8(2)- heptadecenyl)-3-(2-hydroxyethyl)-midizolinium chloride (DOTIM) :cholesterol multilamellar vesicles (MLVs), or pure DOTMA:MLVs. Preferably, the ratio of DNA to lipid (μg DNA/nmol of total lipid) for DOTIM: cholesterol MLVs is from around 1: 10 to around 1:38, more preferably from 1:12 to 1:20, and most preferably from around 1: 15 to around 1:17. In a typical embodiment, polynucleotide DNA is complexed to DOTIM holesterol MLV at a ratio (μg DNA/nmol of total lipid) of around 1: 16. The optimal ratio for DOTMA is preferably from around 1: 15 to around 1:35, more preferably from around 1:20 to around 1:30, and most preferably around 1:26. The optimal ratio for DOTAP is preferably from around 1:25 to around 1:45, more preferably from around 1:30 to around 1:40, and most preferably around 1:36.

In preferred embodiments of the invention, the polynucleotide encoding the ribozyme is administered on a vector that contains a cellular retention sequence (see PCT/US99/01036, the entire disclosure of which is expressly incorporated by reference). Such cellular retention vectors can be administered systemically, or indeed administered to an animal in any manner, such as, e.g., administered by inhalation, by subcutaneous (sub-q), intravenous (I.V.), intraperitoneal (I.P.), intracranial, intraventricular, intrathecal, or intramuscular (I.M.) injection, rectally, as a topically applied agent (transdermal patch, ointments, creams, salves, eye drops, and the like), in utero, or by direct injection into tissue such as tumors or other organs, or in or around the viscera.

Uses

The present invention also provides for the use of the compositions disclosed herein, including the described ribozyme-encoding polynucleotide encoding a cellular retention sequence (e.g., EBNA- 1 ), an EBV FR sequence and/oriP, and a ribozyme-encoding sequence operably linked to transcriptional control signals sufficient for expression of the ribozyme in target cells in the patient. The invention also provides for the use of a composition comprising said ribozyme-encoding polynucleotide in combination with cationic lipids, cationic liposomes, or cationic polymers. The invention also provides for the use of the aforesaid in a formulation comprising an excipient, stabilizer, or carrier. The invention also provides for such medicinal formulations in a delivery device, such as for example a hypodermic syringe, nasal sprayer, inhaler, intravenous delivery device, biolistics device, lavage or gavage device, vial, intracranial shunt, drug pump, or other suitable delivery device. The invention also provides for the use of the therapeutic method of treating neoplasia or hyperplasia by administration to a patient of a composition or medicinal formulation described herein.

Targeted Delivery and Function

In an aspect, the ribozyme-encoding polynucleotides of the invention are designed so as to preferentially express the ribozyme sequence in a desired cell type. This feature may be effected by a variety of approaches. One such approach is to employ an operably linked transcriptional regulatory sequence which drives transcription of the ribozyme sequence(s) and which provides a cell-type specific and/or inducible transcription effect. For example and not limitation, a lymphocyte-specific enhancer-promoter may be used as the operably linked transcriptional regulatory sequence (e.g., a granzyme promoter-enhancer immediately upstream of the granzyme A gene) driving the ribozyme expression if expression is desired in lymphocytes and not desired in non-lymphocytic cells. Similarly, a liver-specific albumin gene transcriptional regulatory sequence can be used to ensure expression in the liver but substantially lower expression in non-hepatic tissues. A lung-specific promoter can confer preferential expression in the lung, and so on. This feature permits a reduction in ribozyme expression in cells and cell types which are not relevant to the therapy, and which may provide a decreased alteration of gene expression and cellular homeostasis in non-targeted cells. A preferred embodiment for treating cancer provides a ribozyme-encoding sequence under the transcriptional control of a transcriptional regulatory sequence which is preferentially active in tumor cells and substantially less active in normal, non-neoplastic cells. It is possible to utilize the transcriptional regulatory sequences (typically the 0.5-5.0 kb segment immediately upstream of the genomic gene transcription start site including the promoter) of a gene which is substantially quiescent in non-neoplastic cells but which is transcriptionally activated in neoplastic cells. Examples of such genes expressed in liver tumors but substantially not expressed in normal hepatocytes are the TCDD-inducible aldehyde dehydrogenase gene (Dunn et al. (1989) J. Biol. Chem. 264: 13057) and the gamma-glutamyltranspeptidase gene (Goodspeed et al. (1989) Gene 76: 1). Other such tumor-induced genes are known in the art and also identifiable and obtainable by various methods, including differential RNA analysis, subtractive hybridization, GeneChip® array analysis, and the like. Comparisons of gene expression in different cell types or in a single cell type under different conditions provides a basis for analyzing the underlying biological processes controlling cell differentiation and metabolism. One way to compare gene expression between two cell populations is to identify mRNA species which are differentially expressed between the cell populations (i.e., present at different abundances between the cell populations). Current methods that distinguish mRNAs in comparative studies largely rely on subtractive hybridization (Lee et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 2825) or differential display employing arbitrary primer polymerase chain reaction (PCR) (Liang and Pardee (1992) Science 257: 967). Each of these methods has been used by various investigators to identify differentially expressed mRNA species, including mRNAs which have increased or decreased abundance in cancer cells (Salesiotis et al. (1995) Cancer Lett. 91 : 47; Jiang et al. (1995) Onco ene 10: 1855; Blok et al.

(1995) Prostate 26: 213; Shinoura et al. (1995) Cancer Lett. 89: 215; Murphy et al. (1993) Cell Growth Differ 4: 715; Austruy et al. (1993) Cancer Res. 53: 2888; Zhang et al. (1993) Mol. Carcinog. 8: 123; and Liang et al. (1992) Cancer Res. 52: 6966). The methods have also been used to identify mRNA species which are induced or repressed by drugs or certain nutrients (Fisicaro et al. (1995) Mol. Immunol. 32: 565; Chapman et al. (1995) Mol. Cell. Endocrinol. 108: 108; Douglass et al. (1995) L

Neuro ci. 15: 2471; Aiello et al. (1994) Proc. Natl. Acad. Sci. (U.S.A.) 91: 6231; Ace et al. (1994) Endocrinology 134: 1305. Similarly, inducible transcription regulatory sequences, such as estrogen- responsive promoter-enhancers, glucocorticoid-reponsive promoters-enhancers, gene silencers, tissue- specific enhancers, and the like can be used. Alternatively or in conjunction with the use of ribozyme-encoding polynucletoides having cell- specific transcriptional sequences, the cationic lipid delivery vehicle may incorporate features which target the composition so as to be preferentially taken into certain cells and cell types. A variety of approaches are known to those in the art, including but not limited to immunoliposomes employing immunoglobulin (e.g., Mab) which preferentially binds to a cell surface molecule on a desired cell type (e.g., a CD4 molecule or CD8 molecule on a lymphocyte, the hepatocyte asialoglycoprotein receptor, a cytokine receptor, a peptide hormone receptor, and the like). In a preferred embodiment, the targeting feature preferentially binds to a structure (e.g., a surface glycoprotein) on a target cell (e.g., a neoplastic cell) and thereby increases the effectively delivered fraction of the vector into the desired target cells as compared to undesired cells and cell types. Targeting features which bind to tumor-specific antigens (e.g., CALLA, etc.) are often preferred.

In some variations, inclusion of a polycation (e.g., polylysine, polyarginine) and/or a lysosomotrophic compound is preferred. The skilled artisan will select the appropriate combination and calibrate for optimal function by varying the component ratios within typical ranges known in the art.

Comparison of Phenotypes and Determination of Gene Function

Effective delivery of the polynucleotide encoding a ribozyme results in the expression of the ribozyme in the target cells of the test animal. The ribozyme is thereby able to cleave the polynucleotide products of the gene of interest in the target cells. Effective cleavage of the polynucleotide products of the gene of interest results in a partial or complete elimination of the expression of the gene of interest. If the expression of the gene in the target cells functions to establish or affect a phenotype of the test animal, then a partial or complete elimination of the expression of the gene of interest should alter the phenotype of the test animal. The test animal is carefully observed for changes in phenotype that might be a result of the altered expression of the gene of interest. A phenotype is any identifiable biologic and/or disease trait exhibited by a living organism.

The use of ribozymes as described herein is significant for the study of functional genomics. Specifically, the presently described methods and compositions provide the ability to significantly reduce the expression of essentially any mRNA expressed in a cell for extended periods of time in animals. This feature of the presently described invention enables one to assess the (previously unknown) function of a given gene product by creating, in effect, a somatic cell knockout animal system. Additionally, the progression, amelioration, or prevention of disease states can be monitored using suitable genetically modified somatic cell knockout animal models. Using this approach, a variety of parameters are monitored in the somatic cell knockout animal, including appearance (skin, hair, etc.), full blood counts and blood chemistries, cytokine levels, full histopathologic analysis, including monitoring for possible organ changes of injury, inflammatory responses and/or the induction of disease states, including cancer, heart disease, atherosclerosis, hypertension, diabetes, asthma, maintenance of body weight, etc. Alternatively, this approach can be used to reduce expression of genes whose function is unknown in animal models of cancer, heart disease, atherosclerosis, hypertension, diabetes, asthma, etc. in order to determine whether in vivo reduction in expression of one or more of these genes can produce significant therapeutic effects in animal models directly relevant to common human diseases. By correlating the observed phenotypic changes with the introduction of polynucleotides encoding specific ribozymes (by comparing the treated animals with both mock-treated and untreated control animals) the specific function(s) of these uncharacterized DNA sequences can be assessed. The test system is chosen to evaluate the effect of the disruption of expression of the target gene on the phenotype of an animal. Test animals and control animals (including mock-treated control animals) should both exhibit the same or similar phenotypes prior to delivery of the ribozyme. Test animals receive the polynucleotide encoding the ribozyme with specificity for a product of the gene of interest. After sufficient time, the phenotypes of test animals are compared with those of control animals. Statistically significant differences between the phenotypes of the test animals and those of the control animals are identified. The function of the gene is thereby identified as contributing to the expression of those phenotypes showing statistically significant changes from the test animals to the control animals. The statistical significance of a change in phenotype is measured by techniques known to those skilled in the art. For example, in one validating embodiment of the invention, the gene function investigated was the effect, if any, of the genes of interest on tumor metastasis. Test animals and control animals were both inoculated with a tumor cell line. Test animals then received intravenous injections of CLDC MLVs containing a polynucleotide expressing a ribozyme with specificity for a product of a gene of interest. After a period of time, the mice were sacrificed and the number of tumors in the test mice were compared with the number of tumors in the control mice. For certain genes tested, the mice showed statistically significant reductions in the number of metastatic tumors.

Methods of Evaluating Target Genes for the Treatment of Disease

The present invention also provides methods that exploit the power of ribozymes to evaluate genetic targets for the treatment of disease. Since many diseases are the result of the abnormal expression of a number of gene products, therefore altering or inhibiting the expression of one gene might nevertheless ameliorate the symptoms of a given disease. In addition, the identification of a gene whose function contributes to the progression of a disease provides a basis for further therapies by screening for new drugs that inhibit the polypeptide products of the gene and rational drug design. The present invention provides methods for identifying genetic targets for treatment of disease by inhibiting the expression of a gene of interest in a test animal with signs and symptoms of the disease, and observing the effect on the progress of the disease. If the signs and/or symptoms of a disease are significantly altered by inhibiting the expression of a gene of interest, the gene of interest is identified as a target for the treatment of the disease. The present method has a number of advantages over prior methods of identifying genetic targets for the treatment of disease. The present method requires minimal to no knowledge of the function of the gene. A partial nucleotide sequence is all that is necessary. Since the method is actually carried out in vivo, no extrapolation is necessary to postulate the in vivo function of the gene. Finally, the method can be carried out in a relatively short period of time at a relatively minimal expense. The present method requires as little as a few weeks and can reduce the number of test animals used, particularly when multiple different ribozymes are efficiently expressed in a single test organism.

Disease Models

The present method is applicable to potentially any mammalian, particularly human, disease. As long as a suitable animal model is available for observing a given disease, genes of interest can be evaluated as targets for the disease. Such animal models include but are not limited to: cancer, heart disease, atherosclerosis, hypertension, diabetes, asthma, Alzheimer's disease, maintenance of body weight, prostatic hypertrophy, stroke, multiple sclerosis, senescence, progeria, scleroderma, systemic lupus erythematosis, inflammation, arthritis, viral infection (e.g., HIV disease, HBV, herpes, and the like), etc.

A preferred disease for investigating new gene product targets using the methods of the invention is cancer. In illustrative embodiments of the invention, genes were evaluated as targets for cancer therapy by observing the progression of melanoma in a mouse testing system. However, other testing models for animal diseases, and especially other cancer models, known or yet to be discovered can also be useful in the present methods.

Given a disease in an animal, a gene is chosen as a candidate target for treatment of the disease. One sequence or several sequences from the nucleotide sequence of the gene is used to create a ribozyme that has specificity for the polynucleotide products of the gene. The design of such a ribozyme is described above. Preferably, sequences in the candidate target gene are chosen so that the ribozyme has unique specificity for the polynucleotide products of the candidate target gene in the cells of the animal. A polynucleotide is constructed to express the ribozyme in the animal as described above. The polynucleotide expressing the ribozyme is systemically delivered to cells of a test animal as described above. The test animal(s), and control animal(s), must exhibit some measurable symptom of the disease. The polynucleotide expressing the ribozyme is systemically delivered to cells in an animal that exhibit symptoms of the disease or that cause the animal to exhibit symptoms of the disease.

After a sufficient time, the disease symptoms of the test animals are compared with those of the control animals. Statistically significant differences between the disease symptoms of the test animals and those of the control animals are identified. The statistical significance of a change in phenotype is measured by techniques known to those skill in the art. If disrupting the expression of a gene with a ribozyme creates a statistically significant change in the symptoms of the disease in the test animal, the gene is chosen as a target for the treatment of the disease.

Examples of specific disease models and phenotypes that can be assayed in each disease model include but are not limited to:

Table 1

New Gene Functions Determined In Vivo

In several validating embodiments of the invention, the effect of a reduction in expression of particular genes on the phenotypes observed in an animal model of cancer was investigated. The genes of interest targeted in these experiments were those for the p65 and p50 subunits of NFKB, PEC AM, integrin β₃, and the FLK- 1 receptor, and apoptin. A cDNA coding for the potent anti-angiogenic antitumor agent angiostatin served as a positive control. Test animals and control animals were inoculated with a melanoma tumor cell line. Test animals received a polynucleotide expressing a ribozyme with specificity for the polynucleotide product of a gene of interest. After a period of time, the mice were sacrificed and the number of tumors in the test mice were compared with the number of tumors in the control mice. A gene was determined to be a target for the treatment of cancer if disruption of the product of the gene with a ribozyme resulted in a statistically significant change in the number of tumor cells in a test mouse.

One of the most surprising discoveries using the methods and compositions of the invention was that NFKB plays a role in carcinogenesis. NFKBS are a family of transcription factors that function in multiple cellular signaling pathways (see Sha, 1998, J. Exp. Med. 187:143-146 for a review). Currently five mammalian NFKB proteins are known: Rel, p65 (or RelA), RelB, p50 (or NFKB I ), and p53 (or NFκB2). Each NFKB transcription factor is a dimer of two NFKB proteins. The dimers are held latently in the cytoplasm of the cell in a complex with IκB proteins. There are five known IκB proteins: IκBα, IκBβ, IκBe, IκBγ, and bcl-3. The pl05 precursor of p50 and the plOO precursor of p52 contain domains that function as IKBS. NFKB transcription factors are activated when a signaling pathway results in the phosphorylation and degradation of IKBS, thereby unmasking a nuclear localization signal that leads to translocation of the NFKB dimer into the nucleus. A number of signaling pathways involved in immune function and development can activate the NFKB transcription factors. For example, they are activated by the cytokines TNF-α and ILl , the chemotactic peptide flvlet-Leu-Phe, and various bacterial and viral products through receptors such as antigen receptors, CD28, and CD40.

NFKB transcription factors also appear to function in pathways outside the immune system including apoptosis. Apoptosis, or programmed cell death, is a cell-killing mechanism activated by cytokines such as tumor necrosis factor (TNFα), chemotherapy, and radiation. Although NFKB transcription factors appear to regulate cell death, the function of NFKB appears to be very dependent upon the system and type of stimulus examined, and can be pro- or anti-apoptotic (see Schneider et al., 1999, Nat. Med. 5:554-559; and Baichwal et al., 1997, Curr. Biol. 7:R94-96). For example, transgenic mice that are "knocked-out" for the gene for NFKB transcription factor subunit RelA (p65) die between days 14 and 15 of gestation due to massive liver destruction; mice lacking both the genes for RelA and TNFα develop normally with normal livers (Doi et al, 1999, Proc. Natl. Acad. Sci. USA 96:2994- 2999). On the other hand, a recent study indicated that NFKB transcription factors may mediate a mechanism by which tumor cells become resistant to chemotherapy and radiation. NFKB transcription factors were inhibited by overexpression of an IκB in tumor cells, and the sensitivity to CPT-11, an analog of the chemotherapeutic camptothecin, was observed. Neither CPT- 11 alone nor IκB expression alone had any effect on tumor growth (see Wang, et al., 1999, Nature Med. 5:412-417).

The authors of the study concluded that NFKB activation in response to chemotherapy is a mechanism of tumor chemoresistance. Further, activation of the NFKB transcription factors in hippocampal neurons can protect against oxidative stress induced apoptosis (Mattson et al, 1997, J. Neurosci. Res. 49:681-697). Thus, the NFKB transcription factors appear to function in a wide variety of pathways. Using the methods of the invention, expression of the gene for either the RelA subunit (p65), or the p50 subunit of the NFKB transcription factors was inhibited by systemically delivering a polynucleotide encoding a ribozyme specific for the polynucleotide product of the gene encoding the respective subunit in a mouse model of melanoma. Inhibition of expression of the polynucleotide product of either the p65 gene or the p50 gene demonstrated striking and consistent anti-tumor effects. Such effects could not be predicted from previous in vitro and in vivo experiments.

PECAM is a 130 kDa immunoglobulin that is a major constituent of the endothelial cell intercellular junction (see Newman, 1997, J. Clin. Invest. 99:3-8 for a review). PECAM is a glycoprotein that functions as an adhesion molecule that contributes to cell migration (see Watt, et al., 1999, Leuk. Lymphoma 17:229-244). When endogenous PECAM-1 levels were down regulated by anti-sense transfection of Polyoma middle T transformed mouse brain endothelial cells, the transfected cells turned on expression of endogenous TSP1 and its angioinhibitory receptor, CD36 (see Sheibani and Frazier, 1999, Histol Histopathol 14:285-294). Therefore, it appears that PECAM acts to promote angiogenesis in these cells (Id.). PECAM interactions can also suppress apoptosis (see Bird et al., 1999, J. Cell Sci. 112:1989-1997). Using the methods of the invention, expression of the gene for PECAM was inhibited by systemically delivering a polynucleotide encoding a ribozyme to a mouse model of human melanoma. Inhibition of expression of the polynucleotide product of the PECAM gene also demonstrated clear and consistent anti-tumor effects. This result demonstrates the first function elucidated for PECAM by blocking its expression in vivo. Integrins are heterodimers composed of noncovalently linked α and β subunit transmembrane glycoproteins (see Seftor, 1998, Am. J. Pathol. 153: 1347-1351 for a review). The subunits contain large extracellular domains, short transmembrane domains, and carboxy -terminal cytoplasmic domains. Currently, seventeen subunits and eight β subunits are known, and over 20 integrin heterodimers have been identified. Additional alternately spliced integrin subunits have also been observed. The eight β subunits share 40 to 80% amino acid sequence homology while the α subunits are more heterogeneous. The cell surface distribution of different integrin subunit pairs varies; for example, when the α_v subunit associates with the β₃ subunit, the resulting integrin can localize to focal adhesions. However, when the α_v subunit associates with the β₅ subunit, this homologous integrin remains randomly distributed over the cell surface. Integrins have been speculated to play roles in a number of cellular processes including signal transduction, gene expression, cell proliferation, apoptosis, metastasis, tumor progression, and angiogenesis. A recent experiment has shown that the integrin α_vβ₃ plays a role in the progression of a human primary cutaneous melanoma from the nontumorigenic, nonmetastatic radial growth phase to the tumorigenic, metastatically competent vertical growth phase (see Hsu et al., 1998, Am. J. Pathol. 153: 1435-1442). Thus, expression of integrin α_vβ₃ promoted tumor invasiveness in this model system.

Again using the methods of the invention, expression of the gene for the integrin β₃ subunit was inhibited by systemically delivering a polynucleotide encoding a ribozyme to a mouse model of human melanoma. Inhibition of expression of the polynucleotide product of the gene for the integrin β₃ subunit demonstrated clear and consistent anti-tumor effects. Accordingly, using the methods of the invention, new and important proximal functions for known genes have been identified by generating, in effect, somatic cell knock-outs of genes of interest. Such methods can be expanded to determine functions of a gene of interest in virtually any animal model. If down-regulation of expression of a gene of interest causes an advantageous change in disease phenotype, such gene products are targets for ribozyme-mediated treatment. Further, any change in phenotype observed upon down regulation of a gene product from a gene of interest using the methods of the invention indicates that the gene product is a target for small molecules that are either agonists or antagonists of the gene products in drug development for the disease model.

Methods for the Treatment of Disease Using Ribozymes In another embodiment, ribozymes are used directly to treat the symptoms of disease by blocking the expression of a gene associated with the disease. Prior efforts at the therapeutic application of ribozymes have been limited by ineffective gene delivery systems or by the use of synthetic ribozymes which often show inadequate efficacy and/or non-specific activity in vivo. The present method demonstrates that one can systemically deliver ribozymes to target cells with therapeutic effect against a target gene. Such a gene might be identified by the method of the present invention that uses ribozymes to evaluate potential genetic targets for the treatment of a disease. Once a gene is identified as a target for treatment, a ribozyme is designed to specifically cleave the mRNA products of the gene according to the consensus sequences discussed above. A polynucleotide is constructed to express the ribozyme in the target cells. Accordingly, the present invention also encompasses compositions for the delivery of polynucleotides encoding ribozymes. The compositions comprise a polynucleotide encoding a ribozyme specific for the polynucleotide product of the gene of interest which is optionally in a complex with cationic lipids or cationic polymers. In one embodiment, the composition comprises polynucleotide DNA complexed to l-[2-(9(2)-octadecenoyloxy)ethyl]-2-(8(2)-heptadecenyl)-3-(2- hydroxyethyl)-midizolinium chloride (DOTIM): cholesterol multilamellar vesicles (MLVs). Preferably, the ratio of DNA to lipid (μg DNA/nmol of total lipid) is from around 1:10 to around 1:38, more preferably from 1:12 to 1:20, and most preferably around 1: 16. In another embodiment, the composition comprises DNA complexed with pure DOTMA at a ratio of DNA to lipid (μg DNA/nmol of total lipid) from around 1:10 to around 1:40, more preferably around 1:18 to around 1:30, and preferably around 1:26. The polynucleotide can encode a ribozyme with specificity for a product of a gene that was identified as a target for the treatment of a disease by the methods of the present invention or which has been previously or otherwise identified as a target gene for therapeutic ribozyme intervention to treat or prevent a hyperplastic or neoplastic disease or condition.

Given the contemplated in vivo use of the described lipid/polynucleotide complexes, it is important that, to the extent possible, all components and materials used during the assembly and production of the lipid/polynucleotide complexes be biocompatible or biotolerable. As such, the lipid complexes of the present invention are preferably formed using solutions and compounds that are routinely used in the treatment of patients. Examples of such solutions and compounds include, but are not limited to: Ringers lactate, 5 percent dextrose, buffered saline, dextran 40, serum proteins, protamine sulfate, albumin, purified lipoproteins or lipoprotein fragments, human serum, transferrin, albumin, or mixtures thereof.

A therapeutically effective amount of the polynucleotide is systemically delivered to the target cells as described above. An amount of a polynucleotide encoding a ribozyme that is "therapeutically effective" is an amount sufficient to ameliorate signs or symptoms of an animal that has the disease. One of ordinary skill will appreciate that, from a medical practitioner's or patient's perspective, virtually any alleviation or prevention of an undesirable symptom (e.g., symptoms related to disease, sensitivity to environmental factors, normal aging, and the like) would be desirable. Thus, for the purposes of this Application, the terms "therapy", "treatment", "preventative treatment", "therapeutic use", or "medicinal use" used herein shall refer to any and all uses of the claimed compositions which remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

When used in the therapeutic or preventative treatment of disease, an appropriate dosage of polynucleotide delivery complex, or a derivative thereof, can be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD, of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Before human studies of efficacy are undertaken, Phase I clinical studies in normal subjects help establish safe doses. For example, a typical dosage of a CLDC complex of the invention (polynucleotide DNA encoding a ribozyme specific for a polynucleotide product of a gene of interest, complexed to DOTMA:cholesterol at a ratio of 1 : 16) would be in the range of about 0.01 to 20 mg per kilogram of body weight, and more preferably about 0.1 to 2 mg per kilogram of body weight. Such dosages have been shown to be effective in providing maximal levels of gene expression without significant toxicities in animal models (results shown herein).

Inhibition Of NFKB To Treat Cancer

Another aspect of the invention is the surprising discovery that inhibiting NFKB in an animal model of melanoma will prevent both tumor growth and metastasis. This effect was observed in the absence of other anticancer treatments such as chemotherapy. Thus, the invention also provides a method of preventing tumor growth or metastasis in a patient with cancer by reducing the activity in the patient of at least one protein subunit of NFKB. The protein subunit of NFKB chosen as a target can be any one or more of Rel, RelB, NFκB2, p50 and p65.

The activity of the protein subunit of NFKB can be reduced by, e.g., treating the patient with a compound that specifically inhibits the activity of NFKB or its subunits. For example, one can use a compound that specifically binds to and/or specifically interferes with dimerization of NFKB protein subunits, phosphorylation of NFKB protein subunits, or transcriptional activity of activated NFKB protein. By a compound that "specifically binds to" or "specifically interferes with" or "specifically inhibits" is meant a compound that interacts preferably with the NFKB protein or subunits or RNA transcripts, e.g., has an affinity for NFKB or its transcripts of at least three orders of magnitude greater than other cellular constituents or proteins. Thus, general tyrosine kinase inhibitors are excluded from this definition. Such compounds can be identified by well known screening assays using NFKB proteins and/or subunits. Examples of screening assays include but are not limited to binding assays, scintillation proximity assays, tyrosine kinase assays, and yeast two-hybrid assays. Alternatively, one can use a dominant negative mutant polypeptide that is a variant of an

NFKB polypeptide. Such dominant negative mutant polypeptides can be deficient for: (1) the ability to form protein: protein interactions with proteins in a signaling pathway of NFKB; (2) the ability to bind a ligand of NFKB; or (3) the ability to bind to an intracellular target or target protein of NFKB. The polypeptides can be delivered to the tumor cells by way of gene therapy vectors, a wide variety of which are known in the art. Preferred gene therapy vectors are the systemic delivery vectors described herein and in application PCT/US99/01036.

In an alternative approach, one can reduce levels of the RNA transcript, or reduce transcription of the transcript, encoding one of the NFKB protein subunits, thereby reducing the amount of NFKB protein in a cell. In one embodiment of the invention, one can administer an antisense RNA that is complementary to an mRNA transcript encoding the NFKB protein subunit chosen as a target for inhibition, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding the target protein. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

The antisense nucleic acid molecules are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. In addition, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. An antisense nucleic acid molecule can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330). The invention also encompasses the use of nucleic acid molecules which form triple helical structures with sequences at the chromosomal locus encoding the NFKB protein subunit gene chosen for inhibition. For example, expression of a polypeptide can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.

In various embodiments, the nucleic acid molecules can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.

In particularly preferred embodiments, the activity of the protein subunit of NFKB is reduced by delivering a ribozyme specific for an RNA encoding the protein subunit, especially delivering the ribozyme systemically as described herein.

The invention having been described, the following examples are offered by way of illustration and not limitation.

EXPERIMENTAL EXAMPLES

Overview of Examples

We used cationic liposome: DNA complex (CLDC)-mediated systemic delivery of plasmid-based ribozymes targeting NF-kB to assess 1) its overall role in the metastatic phenotype, and 2) the critical functional pathway (apoptosis, mitosis, angiogenesis or invasion) through which NF-kB regulates metastasis. Furthermore, we used CLDC-based ribozyme targeting to identify

NF-kB-regulated genes that mediate the effects of NF-kB on metastasis in tumor-bearing hosts. Systemic delivery of plasmid-based ribozymes targeting NF-kB-p65 into adult mice blocked NF-kB expression in metastatic tumor cells, as well as in vascular endothelial cells, a critical normal cell type that regulates both tumor angiogenesis and tumor invasion . Conversely, p65-knockout mice die in utero, thereby precluding their use to evaluate phenotypes manifested primarily in adult life, such as tumor metastasis. The systemic, plasmid-based approach for expressing ribozymes was facilitated by the development of iv-injected, CLDC that can transfect the majority of both lung vascular endothelial cells and melanoma cells metastatic to lung, as well as of an Epstein-Barr Virus (EB V)-based expression plasmid that substantially prolongs the expression of delivered genes at therapeutic levels in adult animals.

Hammerhead ribozymes were designed complementary to sequences containing cleavage sites in the p65 and p50 subunits of murine NF-kB, and the 35 bp ribozyme sequences were inserted into an HCMV-IE1 -driven expression plasmid containing both the Epstein-Barr nuclear antigen-1 (EBNA-1) cDNA and the EBV family of repeats. The resulting plasmids, p65-R and p50-R, respectively, expressed the corresponding ribozyme sequences in metastatic tumors, following CLDC-based iv injection into tumor-bearing mice, as determined by RT-PCR. The effects of iv, CLDC-based injection of p65-R on both target gene expression and tumor metastasis were then assessed in C57B16 mice bearing syngeneic B16-F10 melanoma tumors.

Cationic liposomes complexed to either p65-R, or to the same expression plasmid lacking the 35 bp ribozyme insert, pVector, were injected intravenously into mice bearing metastatic B 16-F10 tumors. Iv injection of p65-R significantly reduced the levels of p65 protein in both metastatic tumor cells and in three different normal lung cell types. Specifically, immunohistochemistry for p65 revealed significant reductions in p65-immunoreactivity in: A) metastatic B16-F 10 lung tumors (p < 0.005), B) vascular endothelial cells (p < 0.0005), C) alveolar epithelial cells (p < 0.05) and D) bronchial lining cells (p < 0.05) from mice iv-injected with CLDC-p65-R, when compared to those treated with

CLDC-pVector. Furthermore, despite the fact that some cells retained p65-immunoreactivity in the p-65R-treated group, the intensity of p65-immunoreactivity was significantly reduced in all cell types from mice injected with CLDC-p65-R, when compared to those from mice treated with CLDC-pVector. p65 staining did not differ significantly between CLDC-pVector-treated mice, CLDC-pLUC (the same plasmid as pVector, but containing the luciferase cDNA)-treated mice or tumor-bearing mice not treated with CLDC. The ability of iv, plasmid-based ribozymes to reduce NF-kB expression most significantly in metastatic B16-F10 tumor cells and in lung vascular endothelial cells is consistent with prior observations showing that these are the two cell types most efficiently transfected in the lung, following iv injection of CLDC. Immunohistochemical determination of p50 immunoreactivity served as an internal control, as the expression of intratumoral p50 protein did not differ significantly between the lungs of mice treated with CLDC-p65-R (76.0 + 5.3 %) versus CLDC-pVector (86.2 + 3.3 %) (p > 0.1). The use of systemic, plasmid-based ribozymes allowed down-regulation of target gene expression in critical normal cells, as well as in tumor cells, thereby permitting a more comprehensive assessment of gene function in vivo than does the inoculation of stably-transfected tumor cell lines, which alters target gene expression only in tumor cells.

We then assessed whether CLDC-mediated systemic delivery of plasmids coding for hammerhead ribozymes targeting NF-kB could alter the metastatic spread of B 16-F10 melanoma cells in syngeneic C57B16 mice. Iv, CLDC-based delivery of plasmid p65-R, which significantly reduced p65 expression in the lung, also significantly reduced the metastatic spread of B16 melanoma tumors in tumor-bearing mice. Specifically, a single intravenous injection of CLDC containing p65-R, seven days after iv injection of 25,000 B 16-F10 cells, significantly reduced the total number of lung metastases (p < 0.025), when compared to tumor-bearing mice treated with CLDC containing pVector. The anti-p65 ribozyme was as effective as iv, CLDC-based delivery of the murine angiostatin gene, whose overexpression produces significant anti-metastatic effects against B 16-F10. There was no apparent toxicity produced by iv, CLDC-based injection of the anti-p65 ribozyme construct in tumor bearing mice.

Previously, the use of synthetic oligonucleotide-based anti-sense constructs has been shown to produce significant non-sequence-specific effects in biologic systems. Therefore, we compared the anti-metastatic activity produced by systemic delivery of CLDC containing p65-R to that produced by CLDC containing p65-R-mut, a mutated anti-p65 ribozyme plasmid that differed from p65-R by a single base substitution in its catalytic core, required for cleavage activity. This single base pair substitution abolished the anti-metastatic activity of the anti-p65 ribozyme, reducing its activity to that associated with CLDC-pVector . Intravenous, CLDC-based delivery of p50-R also significantly reduced (p < 0.025) the metastatic spread of B16-F10 (22 + 7 lung metastases), when compared with p50-R-mut, its single-base mutant counterpart, (55 + 16 lung metastases). Thus, plasmid-based ribozyme activity appeared to be highly sequence-specific in mice.

To assess the effects of tumor cell-specific blockade of NF-kB expression on the metastatic phenotype, B16-F10 clones were stably transfected with either the p65-R plasmid or with (pLUC). The resultant stably-transfected clones were screened for p65 protein levels by immunohistochemistry, using anti-p65-specific monoclonal antibodies. Two clones: B16-p65-R, stably transfected with plasmid p65-R that showed 0% of cells positive for p65, and B16-pLUC, stably transfected with pLUC that showed 50-80+% of cells strongly positive for p65, (similar to the level of p65 expression in wildtype B 16-FlO melanoma cells), were selected for further analysis. Elimination of p65 expression in the B16-p65-R clone significantly reduced (p < 0.0005) the ability of B16-F10 cells to metastasize in C57B16 mice (53 + 10.2 lung tumor metastases), when compared to the B16-pLUC clone (117.6 + 12.8 metastases). Thus, blocking NF-kB expression within tumor cells alone was also able to significantly reduce tumor metastasis. Several different functional pathways essential to the metastatic phenotype, including tumor angiogenesis, apoptosis and mitosis were evaluated in p65-R-treated and control mice to assess how ribozymes blocking NF-kB may suppress the metastatic spread of B16-F10 cells. When compared to CLDC-pLUC-treated control mice, mice treated with CLDC-p65-R did not show significantly different levels of tumor cell apoptosis (2.4 + 0.5 (pLuc) versus 2.5 + 0.6 (p65-R) per 1000 cells), tumor cell mitosis (3.5 + 0.4 (pLuc) versus 3.2 + 0.7 (p65-R) per 1000 cells) or tumor angiogenesis (16.8 + 1.3 (pLuc) versus 16.1 + 3.5 (p65-R) total blood vessels per tumor). The inability of p65-R to significantly alter either tumor apoptosis or mitosis was further demonstrated in cells stably transfected with either p65-R or pLUC. Specifically, B16-p65-R cells showed 2.8 + 0.7 apoptotic cells per 1000 cells counted versus 1.5 + 0.5 for B16-pLUC, and 6.4 + 1.7 mitotic cells per 1000 cells counted versus 5.8 + 1.5 for B16-pLUC. In contrast, tumor invasiveness was significantly inhibited by suppression of NF-*B expression. Tumor cell invasion, as assessed by both invasion into matrigel, and by Boyden chamber analysis, was significantly reduced (p < 0.0005) in B16-p65-R cells, when compared to either B16-pLUC cells, or to wildtype B16-F10 cells. When plated on extracellular matrix, the migrating and invasive B16-pLUC cells displayed an elongated morphology, compared to the rounded B16-p65R cells. The short 16h incubation time of the Boyden chamber invasion assay ensured that the fraction of cells stained corresponded to invasive and migratory cells and not proliferative cells. Thus, blocking NF-kB expression altered the ability of tumor cells to invade their surrounding micro-environment, but did not significantly alter the level of tumor cell apoptosis or mitosis, nor did it decrease tumor angiogenesis.

Based on these results, CLDC-based systemic delivery of ribozymes in tumor-bearing mice was used to identify genes whose NF-kB-regulated expression could play a role in promoting tumor invasiveness. Integrin vβ3 and PECAM- 1 were targeted because they form a ligand-receptor pair, and are each regulated by NF-kB. In addition, vβ3 has been shown to participate in the progression of human melanoma, in part by virtue of its effects on melanoma cell invasion. Finally, given the role of vascular endothelial growth factor-mediated signaling in tumor invasion, CLDC-based, iv delivery of a HCMV-driven plasmid expressing an anti-FLK- 1 ribozyme was also investigated. Systemic delivery of the anti-β3 and anti-PECAM-1 ribozymes each reduced the metastasis of B16-F10 melanoma in C57B16 mice at least as effectively as did p65-R, the anti-p65 ribozyme, suggesting that these adhesion molecules may participate in the invasive phenotype generated by activation of NF-kB. Conversely, the anti-FLK- 1 ribozyme failed to significantly reduce metastatic spread, consistent with results achieved in studies utilizing the continuous infusion of a synthetic oligonucleotide-based ribozyme targeting FLK-1.

NF-kB can transactivate both genes known to promote metastasis, and genes known to inhibit metastasis in tumor cells themselves, as well as in critical normal cell types. Therefore, the ability to selectively block the expression of NF-kB, or important NF-kB -regulated genes, in both tumor cells and vascular endothelial cells was used to dissect out specific genes and functional pathways that regulate the metastatic phenotype in tumor-bearing hosts. Systemic, plasmid-based ribozymes targeting either the p65 or p50 sub-units of NF-kB, as well as iv injection of tumor cells stably transfected with an anti-p65 ribozyme, demonstrated that the expression of NF-kB plays an essential role in promoting the metastatic spread of melanoma in tumor-bearing mice. The loss of NF-kB expression in tumor cells also significantly decreased their capacities to invade the extracellular matrix. In addition, ribozymes targeting integrin vβ3 or PECAM- 1, NF-kB-regulated genes involved in cell adhesion, also significantly reduced metastatic spread, suggesting that this ligand-receptor pair may in part mediate the role of NF-kB in promoting both invasion and metastasis. These studies demonstrate the utility of gene targeting in adult animals, via systemic, plasmid-based ribozymes to dissect out the functional genomics of complex biologic phenotypes such as tumor metastasis.

EXAMPLE 1: In Vivo Function of FLK-1, PECAM, and the p65 subunit of NFKB The functions of FLK-1, PECAM, and the p50 and p65 subunits of NFKB in vivo in a mouse model of melanoma were evaluated by systemically delivering ribozymes directed against the products of those genes to the tumor cells in mice. Ribozymes with specificity for the polynucleotide products of the PECAM gene and ribozymes with specificity for the polynucleotide products of the gene encoding p65 were effective in reducing tumor metastasis in an animal cancer model.

MATERIALS AND METHODS Plasmids

Plasmid constructions began with plasmid p4379 (HCMF-luc-FR-2), which is described in PCT US99/01036. p4379 contains the approximately 900 bp family of repeats fragment (bp 3157- 4043), isolated from p985 by Bam HI digestion followed by insertion into the Bam HI site of vector pVR1255. Thus, the FR is located downstream from the luciferase coding sequence. p4378 is identical to p4379 except that it lacks an Xbal site in the 3' end of the multiple cloning site. p4549 was made by annealing together and inserting the anti-p65 ribozyme oligos (5'- ACT TGA TAT CGG TAC CGT GAA ACT GAT GAG TCC GTG AGG ACG AAA CAC CTC TCT AG A GAT C and 5 ' - GAT CTC TAG AG A GGT GTT TCG TCC TC A CGG ACT CAT CAG TTT

CAC GGT ACC GAT ATC AAG T) into the EcoR V- Xba I cutting site of p4378 (replacing the luciferase cDNA).

The anti-FLK- 1 ribozyme oligos ( 5' - ACT TGA TAT CGG TAC CAT TTA ACT GAT GAG TCC GTG AGG ACG AAA CAA GTT TTC TAG AGA TC and 5' - GAT CTC TAG AAA ACT TGT TTC GTC CTC ACG GAC TCA TCA GTT AAA TGG TAC CGA TAT CAA GT ) were annealed and used to replace the luciferase cDNA in p4378, creating p4562.

In the same way, anti-PECAM-1 ribozyme oligos ( 5' - ACT TGA TAT CGG TAC CTC TCT TCT GAT GAG TCC GTG AGG ACG AAA CCA CTT TTC TAG AGA TC and 5' - GAT CTC TAG AAA AGT GGT TTC GTC CTC ACG GAC TCA TCA GAA GAG AGG TAC CGA TAT CAA GT ) and Integrin B 3 ribozyme oligos ( 5' - ACT TGA TAT CGG TAC CCC TGG ACT GAT GAG TCC GTG AGG ACG AAA CCC ATC TCT AGA GAT C and 5' - GAT CTC TAG AGA TGG GTT TCG TCC TCA CGG ACT CAT CAG TCC AGG GGT ACC GAT ATC AAG T ) were annealed and replaced the luciferase cDNA in p4378, creating p4565 and p4567, respectively. p461, the vector only control, is based on p4378, but the luciferase cDNA was excised, leaving only the vector alone.

Construction of a plasmid used to direct the expression of angiostatin is described in Liu et al., 1999, J. Biol. Chem. 274:13338-13444.

Preparation of cationic liposomes and CLDC A protocol for systemic delivery of generic DNA sequences via cationic liposome-DNA complexes (CDLC) can be found in Liu et al., 1999, J. Biol. Chem. 274:13338-13344. DOTMA MLVs were prepared as described in Liu et al., 1999, which is hereby incorporated by reference in its entirety. Cationic liposome-DNA complexes were prepared as described in Liu et al., 1995, J. Biol. Chem. 270:24864-24870, which is hereby incorporated by reference in its entirety. Tumor cells and tumor inoculation

Murine B16-F10 melanoma cells were grown in RPMI 1640 with 5% fetal bovine serum at 37°C with 5% C0₂. For tumor cell inoculation, B16-F10 cells were trypsinized, and then 25,000 cells/mouse in 200μl of culture medium were injected by tail vein into 25-g female C57B16 mice (Simonson, Gilroy, CA). B16-F10 melanoma is a highly metastatic subclone of B16 melanoma (see Fidler and Nicolson, 1976, J. Natl. Cancer Inst. 57:1199-1202) that kills mice approximately 35 days following intravenous inoculation of 25,000 cells.

In vivo transfections and analysis of anti-tumor activity

Each mouse received 25 μg of plasmid DNA complexed to DOTMA MLV. The DNA ipid ratio (μg of DNA/nmol of total lipid) was 1: 16. This DNAdipid ratio has been determined to produce maximal levels of gene expression following intravenous injection of CLDC. CLDC were injected into tumor-bearing mice 3 and 10 days after tumor cell inoculation. Mice were sacrificed from 21-30 days after tumor cell inoculation, and lungs from each mouse were dissected out, infused transtracheally with 10% neutral buffered formalin (Fisher), and then fixed in 10% neutral buffered formalin. The number and size of the black-appearing tumor nodules were counted two times under a dissecting microscope by an individual blinded to the identity of the groups. The total number of tumors and the number of tumors greater than 2 mm in diameter were included in the analysis. The statistical significance of differences between various groups was assessed using an unpaired, two- sided Student's t test.

RESULTS

The size and number of lung metastases in the ribozyme-treated mice and control vector- treated mice were compared 21 days after intravenous injection of 25,000 B16-F10 melanoma cells/mouse. Individual mice in groups of eight received 650 nmol of DOTMA MLV complexed to 25 μg of a vector plasmid (mock-treated controls), 25 μg of a plasmid encoding a ribozyme specific for p65, 25 μg of a plasmid encoding a ribozyme specific for platelet endothelial cell adhesion molecule (PECAM), 25 μg of a plasmid encoding a ribozyme specific for FLK-1, or 25 μg of an expression plasmid encoding the murine angiostatin gene on day 3 and again on day 10 follow tumor inoculation.

One group of test mice was inoculated intravenously with the angiostatin gene in a CLDC. These mice showed significant reductions in the number of lung tumors (p < 0.0005). CLDC-mediated, intravenous delivery of the plasmid encoding the ribozyme with specificity for a polynucleotide that encodes p65 also showed significant anti-metastatic effects as determined by the total number of lung metastases when compared to the vector control (p < 0.025) by a two-sided Student's t test (see Table 1). The CLDC-delivered anti-p65 ribozyme plasmid showed even stronger anti-metastatic effects as determined by the number of lung metastases greater than 2mm in diameter when compared to the vector control (p < 0.0005). Metastases >2mm are both clinically significant and angiogenesis dependent, whereas those <2mm generally are not.

The plasmid encoding the ribozyme with specificity for a polynucleotide that encodes PECAM also showed surprising anti-metastatic effects as determined by the total number of lung metastases versus vector control (p < 0.025) and by the number of lung metastases greater than 2 mm versus vector control (p < 0.0005).

The plasmid encoding the ribozyme with specificity for a polynucleotide that encodes FLK-1 did not show statistically significant anti-metastatic effects as determined by the total number of metastases (p < 0.375) or those greater than 2 mm (p < 0.375). Table 2

CLDC Injected Tumors p vs. vector Tumors > 2 mm p vs. vector

Vector 200.6 ± 20.6 (SEM) - 44.1 ± 3.0 (SEM) - anti p65 108.6 + 27.6 p < 0.025 10.4 ± 3.4 p < 0.0005 anti PECAM 140.8 ± 10.0 p < 0.025 23.4 ± 5.9 p < 0.0005 anti FLK-1 163.6 ± 40.3 p < 0.375 27.0 ± 10.4 p < 0.375 angiostatin 98.6 ± 9.5 p < 0.0005 15.7 ± 1.7 p < 0.0005

DISCUSSION In this example we probed the functions of the gene encoding the p65 subunit of NFKB, the

PECAM gene, and the FLK-1 gene by designing ribozymes with specificity for their mRNA products. DNA encoding the ribozymes were systemically delivered to tumor cells in a mouse model to evaluate a possible function for the genes in cancer. Finally, this example demonstrates that cancer can be treated by inhibiting expression of the genes with ribozymes specific for the mRNA products of the gene encoding the p65 subunit of NFKB and the ribozymes with specificity for the mRNA products of the PECAM gene.

The results of systemic delivery of the ribozymes of the present example on the metastatic phenotype were compared to systemic delivery of the angiostatin gene. Angiostatin is an internal fragment of plasminogen and a potent inhibitor of angiogenesis which selectively inhibits endothelial cell proliferation. When given systemically, angiostatin inhibits tumor growth and can maintain metastatic and primary tumors in a dormant state (see O'Reilly, 1997, EXS 79:273-294). Previous results have shown that CLDC-mediated intravenous delivery of the angiostatin gene has antimetastatic effects (see Liu et al., 1999, J. Biol. Chem. 274:13338-13444). In the present experiment, CLDC- mediated delivery of the angiostatin gene also had antimetastatic effects as shown by the reduction in the total number of lung tumors and the total number of tumors greater than 2 mm in diameter. The statistical significance of the reduction in the number of tumors for the known antitumor gene (p < 0.0005) provides a standard for comparing the effect of the novel ribozyme treatments.

This experiment assayed the function of NFKB in tumor growth and metastasis. Starting with the oligonucleotide sequence of relA gene which encodes the p65 NFKB subunit, a ribozyme was designed to specifically cleave the mRNA transcription product of the gene. A DNA sequence encoding the ribozyme was placed in a plasmid under the control of the cytomegalovirus promoter. A CLDC was prepared with the plasmid and inoculated into a mouse bearing a highly metastatic melanoma cell line. After 21 days the total number of tumor cells in the test mice and the control mice were counted. Surprisingly, the mice inoculated with the anti p65 ribozyme showed significantly fewer tumors than the control mice. In fact, treatment with the anti p65 ribozyme (p < 0.0005) was comparably effective as treatment with the known antitumor gene angiostatin (p < 0.0005) when tumors greater than 2 mm in diameter were counted.

Thus, this experiment demonstrated a surprising new function for p65 and, more generally, NFKB. Prior literature suggested that NFKB functions in the induction of chemoresistance in tumors, but does not have a direct effect on tumor growth. Since inhibiting the expression of the NFKB subunit p65 reduced metastasis, these results established a direct link between NFKB and metastasis. Inhibiting expression of a gene with a ribozyme is a much more sensitive assay of gene function than the probe with a protein inhibitor of prior NFKB research. The results of this experiment also showed that the p65 subunit of NFKB is an effective genetic target for the treatment of melanoma and that ribozymes with specificity for the mRNA products of the gene encoding p65 provide effective treatment for cancer.

Since other proteins that influence angiogenesis or apoptosis have a role in the growth and metastasis of tumors, PECAM might also have a significant effect on tumor progression. To probe the function of PECAM in metastasis, a ribozyme was designed to cleave specifically the mRNA transcription product of the gene encoding PECAM. A DNA sequence encoding the ribozyme was placed under the control of the cytomegalovirus promoter in an expression plasmid, and CLDC were prepared with the plasmid. Mice were inoculated with the CLDC and the total number of lung tumors in the mice was counted after 21 days. Because treatment with the anti PECAM ribozyme resulted in a significant reduction in the total number of lung tumors (p < 0.0005), there is a significant link between PECAM expression and metastasis. Furthermore, PECAM was demonstrated to be a potential target for the treatment of melanoma, and this example shows that ribozymes with specificity for the mRNA products of the PECAM gene provide effective treatment for cancer.

Finally, a ribozyme was designed to specifically cleave the mRNA transcription product of the FLK-1 gene, and a DNA sequence encoding the ribozyme was placed in a plasmid under the control of the cytomegalovirus promoter. A CLDC was prepared with the plasmid and inoculated into a mouse bearing a highly metastatic melanoma cell line. After 21 days the total number of tumor cells in the test mice and the control mice were counted. The test mice did not show a significant reduction in the number of tumors (p < 0.375) or the number of tumors greater than 2 mm in diameter (p < 0.375). Thus, under the particular conditions of this assay, reducing FLK-1 expression did not inhibit turmor metastasis in this mouse model of cancer.

Overall, the results of this experiment revealed a new and unexpected function for p65 and PECAM in tumor cells and metastasis. Ribozymes systemically delivered to the tumors showed that inhibiting the expression of p65 or PECAM significantly reduces the metastasis of tumors. Treatment with the ribozyme against p65 was as effective as treatment with the known anti-tumor protein, angiostatin.

EXAMPLE 2: In Vivo Function of FLK-1, PECAM, p65 subunit of NFKB, and Integrin β₃ In this example, we investigated the in vivo function of FLK-1, PECAM, the p65 subunit of

NFKB, and integrin β₃ by targeting their gene products. Systemic delivery of polynucleotides encoding ribozymes with specificity for the gene products demonstrates that the genes encoding PECAM. the p65 subunit of NFKB, and integrin β₃ each function in tumor metastasis. Accordingly, these results identify PECAM, NFKB, and integrin β₃ as targets for the treatment of cancer. Further, this animal model demonstrates that cancer can be treated in vivo using ribozymes with specificity for the polynucleotide products of the PECAM gene, the polynucleotide products of the gene encoding p65, or the polynucleotide products of the gene encoding integrin β₃.

MATERIALS AND METHODS In vivo transfections and analysis of anti-tumor activity

CLDC and B16-F10 cells were prepared as described in Example 1 above. Each mouse received 25 μg of plasmid DNA complexed to DOTIM: cholesterol DOTMA MLV. Mice were transfected with CLDC as described in Example 1 above and sacrificed 18 days after tumor cell inoculation. Lung tumors were quantified as described in Example 1 above.

RESULTS The size and number of lung metastases in the ribozyme-treated mice and control vector- treated mice were determined 18 days after intravenous injection of 25,000 B16-F10 melanoma cells/mouse. Individual mice in groups of eight received 650 nmol of DOTMA MLV complexed to 25 μg of a vector plasmid (mock-treated controls), 25 μg of a plasmid encoding a ribozyme specific for p65, 25 μg of a plasmid encoding a ribozyme specific for PECAM, 25 μg of a plasmid encoding a ribozyme specific for FLK- 1 , or 25 μg of a plasmid encoding a ribozyme with specificity for a polynucleotide that encodes integrin β₃.

Surprisingly, CLDC-mediated, intravenous delivery of the plasmid encoding the ribozyme with specificity for a polynucleotide that encodes p65 showed significant anti-metastatic effects as determined by the total number of lung metastases when compared to the vector control (p < 0.025) (See Table 2). The plasmid encoding the ribozyme with specificity for a polynucleotide that encodes PECAM also showed significant anti-metastatic effects as determined by the total number of lung metastases versus vector control (p < 0.025) and by the number of lung metastases greater than 2 mm versus vector control (p < 0.005). The plasmid encoding a ribozyme with specificity for a polynucleotide that encodes integrin β₃ showed significant anti-metastatic effects as determined by the total number of lung metastases (p < 0.005). The plasmid encoding the ribozyme with specificity for a polynucleotide that encodes FLK-1 did not show statistically significant anti-metastatic effects as determined by the total number of metastases (p < 0.375) or those greater than 2 mm (p > 0.4).

Table 3

CLDC Injected Tumors p vs. vector Tumors > 2 mm p vs. vector

Vector 70.0 ± 8.6 - 13.0 ± 1.8 - anti p65 45.5 ± 6.5 p < 0.025 8.1 ± 2.3 p < 0.1 anti PECAM 45.6 ± 6.5 p < 0.025 6.0 ± 0.9 p < 0.005 anti FLK-1 60.0 ± 15 p < 0.375 12.4 ± 4.0 p > 0.4 anti integrin β₃ 31.8 ± 9.5 p < 0.0005 8.6 ± 3.1 p < 0.375

DISCUSSION The functions of the gene products from the genes encoding the p65 subunit of NFKB, the PECAM gene, the FLK- 1 gene, and the integrin β₃ gene were probed by designing ribozymes with specificity for their mRNA products. The ribozymes were systemically delivered to tumor bearing mice to evaluate a possible function for the genes in cancer.

Inhibiting the expression of p65 with a systemically delivered ribozyme showed inhibition of tumor metastasis although the reduction in the number of lung tumors was not as significant in this experiment with p65 as it was in the experiment described in Example 1 above. Treatment with systemic delivery of the ribozyme against PECAM also showed significant, but reduced, effects on the total number of lung tumors. The reduction in the statistical significance of the effects of the ribozyme against p65 and the ribozyme against PECAM might be due to the small number of tumors seen in the experiment. In the experiment described in Example 1, the control mice had an average of 200 tumors while the control mice in the experiment described in this example only had 70 tumors. Treatment with the ribozyme against FLK-1 did not show significant antitumor effects.

Given the prior associations between integrin α_vβ₃ and tumor growth and metastasis, we also examined the effects on tumor metastasis of inhibiting the expression of cell surface adhesion receptor subunit integrin β₃. Inhibiting the expression of the integrin β₃ gene in tumor cells with a ribozyme yielded a significant reduction in the total number of lung tumors in the mouse model (p < 0.0005) but not as significant a reduction in the number of tumors greater than 2 mm in diameter (p < 0.375). These results demonstrate that integrin β₃ does function in tumor metastasis. These results also demonstrate that integrin β₃ is a target for the treatment of cancer and that ribozymes with specificity for the mRNA products of the integrin β₃ gene can provide an effective treatment for cancer. EXAMPLE 3: Delivery of Polynucleotide Encoding Ribozyme Inhibits Metastasis

In this example, we demonstrate the effectiveness of the methods of the present invention for treating disease. Specifically, the effects of ribozymes with specificity for the products of the PECAM gene and those with specificity for the products of the gene encoding the p65 subunit of NFKB are compared with the effects of delivering genes known to retard tumor metastasis. Systemic delivery of a ribozyme with specificity for the polynucleotide products of the gene encoding the p65 subunit of NFKB according to the methods of the present invention is at least as effective as systemic delivery of the known anti-metastatic gene angiostatin in inhibiting tumor growth in a mouse model.

MATERIALS AND METHODS

Plasmids p4557 directs the expression of an anti-p50 ribozyme. This plasmid was constructed by annealing the following anty-p50 ribozyme oligos (5' - ACT TGA TAT CGG TAC CTC TGT TCT GAT GAG TCC GTG AGG ACG AAA CAG TGG TCT AGA GAT C and 5' - GAT CTC TAG ACC ACT GTT TCG TCC TCA CGG ACT CAT CAG AAC AGA GGT ACC GAT ATC AAG T ) and using this synthetic DNA to replace the luciferase cDNA in p4378 at the same cutting site as p4549. Ribozyme oligos of anti-p65,( 5' - ACT TCT GCA GAT ATC GGT ACC GTG AAA CTG ATG AGT CCG TGA GGA CGA AAC ACC TCT CTA GAG CGG CCG CGA TC and 5' - GAT CGC GGC CGC TCT AGA GAG GTG TTT CGT CCT CAC GGA CTC ATC AGT TTC ACG GTA CCG ATA TCT GCA GAA GT); anti-mutant p65,( 5' - ACT TCT GCA GAT ATC GGT ACC GTG AAA CTG ATG AGT CCG TGA GGA CGA AAC ACC TCT CTA GAG CGG CCG CGA TC and 5' - GAT CGC GGC CGC TCT AGA GAG GTG TTT CGT CCT CAC GGA CTC ATG AGT TTC ACG GTA CCG ATA TCT GCA GAA GT); anti-p50, ( 5' - ACT TCT GCA GAT ATC GGT ACC AAA TGA CTG ATG AGT CCG TGA GGA CGA AAC ATT TGT CTA GAG CGG CCG CGA TC and 5' - GAT CGC GGC CGC TCT AGA CAA ATG TTT CGT CCT CAC GGA CTC ATC AGT

CAT TTG GTA CCG ATA TCT GCA GAA GT) and anti-mutant p50 ( 5' - ACT TCT GCA GAT ATC GGT ACC AAA TGA CTG ATG AGT CCG TGA GGA CGA AAC ATT TGT CTA GAG CGG CCG CGA TC and 5' - GAT CGC GGC CGC TCT AGA CAA ATG TTT CGT CCT CAC GGA CTC ATG AGT CAT TTG GTA CCG ATA TCT GCA GAA GT) were annealed and inserted into the Pst I - Not I cutting site of p4486, a plasmid that contains the FR, plus the EBNA- 1 cDNA in a separate CMV-driven expression cassette (see PCT US99/01036), creating p4653, p4660, p4701, p4702, respectively.

The apoptin expression plasmid, which was used as a positive control, was constructed by inserting the coding sequence for apoptin (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843-5847) in place of the luciferase coding sequence in plasmid p4378. In vivo transfections and analysis of anti-tumor activity CLDC and B16-F10 cells were prepared as described in Example 1 above. Mice were transfected with CLDC as described in Example 1 above and sacrificed 21 days after tumor cell inoculation, a time when greater numbers of tumors were present than the time points used in Example 2. Lung tumors were quantified as described in Example 1 above.

RESULTS The size and number of lung metastases in the ribozyme-treated mice and control vector- treated mice were determined 18 days after intravenous injection of 25,000 B16-F10 melanoma cells/mouse. Individual mice in groups of eight received 650 nmol of DOTIM:cholesterol DOTMA MLV complexed to 25 μg of a plasmid encoding luciferase (mock-treated controls), 25 μg of a plasmid encoding a ribozyme specific for p65, 25 μg of a plasmid encoding a ribozyme specific for PECAM, 25 μg of plasmid encoding apoptin, 25 μg of a plasmid encoding angiostatin, or 12.5 μg of a plasmid encoding apoptin together with 12.5 μg of a plasmid encoding angiostatin.

The plasmids encoding the angiostatin gene showed significant anti-metastatic effects as expected (see Liu et al., 1999, J. Biol. Chem. 274: 13338-13344). A plasmid encoding the apoptosis- inducing protein Apoptin also showed significant anti-metastatic effects. However, co-injection of the combination of the apoptin gene and the angiostatin gene showed slight, if any, synergistic effects. Surprisingly, CLDC-mediated, intravenous delivery of the plasmid encoding the ribozyme with specificity for a polynucleotide that encodes p65 showed significant anti-metastatic effects as determined by the total number of lung metastases when compared to the vector encoding luciferase (p < 0.025) and by the number of lung metastases greater than 2 mm versus the luciferase control (p < 0.005) (See Table 2). In fact, treatment with the anti-p65 ribozyme showed comparable reduction in lung tumor metastases (p < 0.025) as treatment with the angiostatin gene (p < 0.025)and treatment with the apoptin gene (p < 0.01). The plasmid encoding the ribozyme with specificity for a polynucleotide that encodes PECAM also showed significant anti-metastatic effects as determined by the total number of lung metastases greater than 2 mm versus the luciferase control (p < 0.05).

Table 4

CLDC Injected Tumors p vs. control Tumors > 2 mm p vs. control

Luciferase 174.0 ± 16 - 59 ± 9 - anti p65 104 ± 12 p < 0.005 27 ± 3 p < 0.005 anti PECAM 133 ± 24 p < 0.1 35 ± 8 p < 0.05 apoptin 110 ± 21 p < 0.025 32 ± 6 p < 0.01 angiostatin 100 ± 8 p < 0.005 35 ± 6 p < 0.025 apoptin + angiostatin 99 ± 13 p < 0.005 26 ± 5 p < 0.005 DISCUSSION

In this example, we demonstrate the effectiveness of the methods of the present invention for the treatment of disease. Specifically, ribozymes with specificity for the mRNA products of the gene encoding p65 and ribozymes with specificity for the mRNA products of the PECAM gene were used to treat a murine melanoma model. The results of treatment with these ribozymes were compared with treatment with the known anti-metastatic gene angiostatin and apoptin.

Systemic delivery of the angiostatin gene and systemic delivery of the apoptin gene both yielded significant reductions in the number of lung tumors in the mouse model. Systemic delivery of the ribozymes according to the methods of the present invention yielded comparable amelioration of the cancer symptoms in the mouse model. Treatment with the ribozymes against the PECAM gene products showed a significant reduction in the number of tumors. Treatment with the ribozymes against the p65 gene products yielded more significant reductions. The results for both the ribozymes against the PECAM gene product and the ribozymes against the p65 gene products demonstrated a reproducible and significant reduction in metastatic spread. In fact, treatment with the ribozymes against the p65 gene products was as effective as treatment with the angiostatin gene and even as effective as combined treatment with the angiostatin gene and the apoptin gene.

EXAMPLE 4: Ribozymes Are Catalytically Effective In Vivo

In this example, we demonstrate that the ribozymes were catalytically effective in vivo. Systemic delivery of a polynucleotide encoding an intact ribozyme with specificity for an mRNA target was compared to systemic delivery of a polynucleotide encoding an antisense RNA that complements the same mRNA target. Delivery of the ribozyme is also compared to delivery of a mutated version of the same ribozyme that lacks catalytic ability. The intact ribozyme yields a significantly greater reduction of tumor metastasis in a mouse model than the antisense RNA and the catalytically defective version of the same ribozyme.

MATERIALS AND METHODS In vivo transfections and analysis of anti-tumor activity CLDC and B16-F10 cells were prepared as described in Example 1 above. Mice were transfected with CLDC as described in Example 1 above and sacrificed 21 days after tumor cell inoculation. Lung tumors were quantified as described in Example 1 above.

RESULTS The size and number of lung metastases in the ribozyme-treated mice and control vector- treated mice were determined 18 days after intravenous injection of 25,000 B16-F10 melanoma cells/mouse. Individual mice in groups of eight received 650 nmol of pure DOTMA MLV complexed to 25 μg of a vector plasmid encoding the apoptin gene in the 3' - 5' direction (mock-treated control) or 25 μg of a plasmid encoding the apoptin gene. Individual mice in additional groups of eight received 650 nmol of DOTIM holesterol MLV complexed to 25 μg of a plasmid encoding the luciferase gene (mock treated control), 25 μg of a plasmid encoding a ribozyme with catalytic specificity for a polynucleotide that encodes p65, 25 μg of a plasmid encoding a RNA complementary to the p65 mRNA transcription product, or 25 μg of a plasmid encoding a mutant ribozyme lacking catalytic activity but having specificity for a polynucleotide that encodes p65.

Apoptin is a protein derived from an avian virus that is a potential cancer therapeutic because it induces programmed cell death, or apoptosis, in certain mammalian cells including tumor cells (see Noteborn et al., 1998, Mutat. Res. 400:447-55). Tissue-specific delivery of the apoptin gene shows that apoptin has a pronounced effect on tumorigenesis. In three separate groups of mice, those treated with the apoptin gene showed a significant reduction of the number of tumors (p < 0.05 or better) and the number of tumors greater than 2 mm in diameter (p < 0.025 or better).

To distinguish the catalytic effect of the p65 ribozyme from its antisense effects, two new vectors were constructed. One encoded an RNA with an antisense sequence complementary to the mRNA transcription product of the p65 gene to quantify the effects of antisense p65 RNA therapy against B 16-F10 tumors. Another encoded a mutant version of the ribozyme against the p65 mRNA that lacked catalytic activity. Significant anti-tumor effects of the active ribozyme when compared to the mutant ribozyme indicate that the ribozyme has effective catalytic activity within the cell. In fact, the catalytically defective ribozyme mutant (which had only a single base pair change in the catalytic sequence) showed little anti-metastatic effects when compared to the mock-treated control for the total number of tumors (p < 0.375) and for the number of tumors greater than 2 mm in diameter (p < 0.1). The antisense treatment did show some anti-metastatic effects, in keeping with the established potential activity of antisense RNA. However, the ribozyme treatment was significantly more effective both for the total number of tumors (p < 0.0005 for the ribozyme vs. p < 0.025 for antisense RNA) and for the number of tumors greater than 2 mm in diameter (p < 0.0005 for the ribozyme vs. p < 0.025 for antisense RNA). The loss of anti-metastatic effect in the catalytically defective mutant and the improvement of the ribozyme over antisense treatment both indicate that the ribozyme cleaves the p65 mRNA in the cell with significant effects on tumor progression and metastasis- Table 5

CLDC Injected Tumors p vs. control Tumors > 2 mm p vs. control apoptin 5' - 3' 30.9 ± 4.6 p < 0.05 8.7 ± 1.9 p < 0.025 apoptin 3' - 5' 45.0 + 4.9 - 14.6 ± 1.5 anti p65 20.2 + 2.5 p < 0.0005 6.4 ± 1.0 p < 0.0005 anti p65 (antisense) 29.5 ± 3.9 p < 0.025 10.3 ± 1.3 p < 0.025 anti p65 (mutant) 38.5 ± 4.9 p < 0.375 13.5 ± 2.2 p < 0.1 luciferase 35.0 ± 6.8 - 13.0 ± 3.0 p < 0.375 apoptin - 25 μg 24.8 ± 3.5 p < 0.005 6.3 ± 1.4 p < 0.0005 apoptin - both 5' - 3' 27.6 ± 2.9 p < 0.005 p < 0.005

DISCUSSION

These results demonstrate that the phenotypes observed are indeed the result of specific catalysis of the ribozymes on the intended targets. Mice inoculated with melanoma cells were treated with a ribozyme against the products of the p65 gene. Mice were also treated with an antisense RNA complementary to the products of the p65 gene. While the antisense RNA did show some reduction in the number of tumors, the ribozyme yielded significantly improved reductions in the number of tumors. Other mice were treated with a version of the ribozyme against the products of the p65 gene that had been altered. The altered ribozyme retained the binding sequence of the anti p65 ribozyme, but lacked the ability to cleave its target RNA. The catalytically defective mutant showed a much poorer reduction in the number of tumors in the mouse model compared to the intact anti p65 ribozyme and was comparable to the luciferase control. These results indicate that the catalytic properties of the ribozyme are responsible for the observed changes in phenotypes.

EXAMPLE 5: Ribozymes Against p50 Are Catalytically Effective In Vivo

In this example, we demonstrate that a ribozyme directed against the polynucleotide product of the p50 subunit of NFKB was also catalytically effective in vivo. As a control, a mutant ribozyme (with a single base pair deletion) against the p50 gene polynucleotide product was used.

MATERIALS AND METHODS In vivo transfections and analysis of anti-tumor activity

CLDC and B16-F10 cells were prepared as described in Example 1 above. Mice were transfected with CLDC as described in Example 1 above and sacrificed 25 days after tumor cell inoculation. Lung tumors were quantified as described in Example 1 above.

RESULTS The number of lung metastases in the p50 ribozyme-treated mice and control p50 mutant ribozyme-treated mice was determined 25 days after intravenous injection of 25,000 B16-F10 melanoma cells/mouse. Table 6

CLDC Injected Tumors p vs. control anti p50 101.1 ± 9.3 mutant anti p50 (control) 145.4 + 12.0 p < 0.01

DISCUSSION

The results demonstrated that inhibition of expression of the p50 subunit of NFKB also demonstrated significant anti-tumor effects.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

CLAIMS What is claimed is:

1. A method of identifying a function for a gene of interest comprising: a) delivering a non- viral ribozyme-encoding polynucleotide that encodes and expresses a ribozyme that has specificity for a polynucleotide product of the gene of interest into cells of a test animal; and b) comparing the phenotype of the test animal to the phenotype of a control animal, wherein a function of the gene of interest is correlated to a detectable change in phenotype of the test animal, and c) denoting said detectable change of phenotype as a function of said gene.

2. The method of claim 1 wherein said ribozyme-encoding polynucleotide comprises an EBNA- 1 gene expression cassette and a EBV FR sequence.

3. The method of claim 1 wherein the ribozyme-encoding polynucleotide encodes a ribozyme which inhibits metastasis of a neoplasm in said animal.

4. The method of claim 2 wherein the encoded ribozyme inactivates a mRNA for PECAM, an NF-kB subunit, integrin β3, or FLK-1.

5. The method of claim 4 wherein the disease is neoplasia.

6. The method of claim 1 wherein the ribozyme-encoding polynucleotide further encodes a second ribozyme that is specific for a second gene of interest.

7. The method of claim 1 wherein the ribozyme-encoding polynucleotide is delivered by systemic administration in a complex with cationic lipids or cationic polymers.

8. The method of claim 1 wherein the ribozyme-encoding polynucleotide is delivered in a complex with cationic lipids or cationic polymers.

9. The method of claim 1 wherein the encoded ribozyme has specificity for polynucleotides that encode FLK-1, PECAM, an integrin subunit, an integrin β, integrin β₃, a subunit of NFKB, p50 or p65.

10. A method of evaluating a gene of interest comprising: a) systemically delivering a non-viral ribozyme-encoding polynucleotide that encodes a ribozyme that has specificity for a polynucleotide product of the gene of interest into cells of a test animal exhibiting symptoms of a disease; and b) comparing the phenotype of the test animal to the phenotype of a control animal exhibiting the symptoms as the test animal prior to delivery of the polynucleotide wherein the gene is identified as a target for the treatment of the disease if delivery of the ribozyme alters the symptoms of the disease in the test animal.

11. The method of claim 10 wherein the disease is cancer.

12. The method of claim 10 wherein the ribozyme-encoding polynucleotide comprises an EBNA-1 encoding sequence, an EBV FR sequence, and a ribozyme-encoding sequence.

13. The method of claim 10 wherein the polynucleotide sequence is delivered in a complex with cationic lipids or cationic polymers.

14. A composition comprising a non-viral ribozyme-encoding polynucleotide comprising a EBNA- 1 expression cassette, an EBV FR sequence, and a ribozyme-encoding sequence operably linked to a transcriptional regulatory sequence.

15. The composition of claim 14, wherein the ribozyme-encoding sequence encodes a ribozyme having a targeting sequence that cleaves a mRNA species encoding a protein that confers oncogenic or metastatic effects to a cell.

16. The composition of claim 15 wherein the ribozyme-encoding sequence encodes a ribozyme that inhibits the expression of PECAM, an integrin, NF-kB, FLK-1.

17. The composition of claim 14, wherein said ribozyme-encoding polynucleotide is complexed with cationic lipids or cationic polymers.

18. The composition of claim 14, wherein said ribozyme-encoding polyncucleotide encodes a first species of ribozyme and a second species of ribozyme.

19. The composition of claim 14 which further comprises a polynucleotide sequence encoding an expressible cDNA, minigene, or genomic gene encoding a protein which confers a phenotype on a mammalian cell expressing said protein.

20. The composition of claim 14 which comprises a first ribozyme-encoding polynucleotide species and a second ribozyme-encoding polynucleotide species.

21. A delivery device for systemic administration of a DNA-lipid complex wherein said delivery device contains the composition of claim 17.

22. A method of treating a disease in an animal comprising delivering a therapeutically effective amount of a non- viral ribozyme-encoding polynucleotide comprising an expression cassette that when transcribed encodes a ribozyme.

23. The method of claim 22 wherein the ribozyme inhibits the expression of an mRNA encoding a protein that confers an oncogenic or metastatic phenotype on a cell.

24. The method of claim 23 wherein the ribozyme inhibits the expression of a mRNA for PECAM, an NF-kB subunit, integrin β3, or FLK-1.

25. The method of claim 22 wherein the non-viral ribozyme-encoding polynucleotide comprises a EBNA-1 expression cassette and a EBV FR sequence.

26. The method of claim 22 wherein the ribozyme-encoding polynucleotide is complexed with a cationic lipid or a cationic polymer.

27. The method of claim 26 wherein the ribozyme has specificity for polynucleotides that encode PECAM, an integrin subunit, an integrin β, integrin β₃, a subunit of NFKB, p50 or p65.

28. The method of claim 22 wherein the polynucleotide sequence is delivered in a non- viral vector.

29. The method of claim 22 wherein the polynucleotide is introduced in a complex with cationic lipids or cationic polymers.

30. A method of preventing tumor growth or metastasis in a patient, the method comprising reducing the activity in the patient of at least one protein subunit of NFKB.

31. The method of claim 30 wherein the activity of the protein subunit of NFKB is reduced by reducing steady state levels of an RNA encoding the protein subunit.

32. The method of claim 31 wherein the activity of the protein subunit of NFKB is reduced by delivering a ribozyme specific for an RNA encoding the protein subunit.

33. The method of claim 32 wherein the protein subunit of NFKB is selected from the group consisting of Rel, RelB, NFκB2, p50 and p65.

34. The method of claim 32 wherein the ribozyme is delivered by administration of a composition comprising a complex of a cationic lipid or cationic polymer with a ribozyme-encoding polynucleotide comprising a EBNA- 1 expression cassette, a ENV FR sequence, a ribozyme- encoding sequence operably linked to a transcriptional regulatory sequence for intracellular expression of said ribozyme in a neoplastic cell.

35. A composition for systemic delivery of a ribozyme into an animal that has a disease comprising: a) a polynucleotide that encodes the expression of a ribozyme in an animal cell; and b) a cationic lipid or a cationic polymer wherein the composition, when systemically delivered into the animal, directs the expression of an amount of the ribozyme that is therapeutically effective against the disease.

36. The composition of claim 35 wherein the disease is cancer and wherein the ribozyme has specificity for polynucleotides that encode PECAM, an integrin subunit, an integrin β, integrin β₃, a subunit of NFKB, p50 or p65.

37. The use of the composition of claim 14 or claim 35, the method of claim 1, 10, 22, or 30, and the delivery device of claim 21.