Therapeutic applications of ribozymes

Pharmac.

I%rgamon

0163-7258(95)02008-H

ST. Vol. 68, No. 2, pp. 247-261, 1995 Copyright 0 1995 Ekvier Science Inc. Printed in Great Britain. All rights reserved 0163-7258195 $29.00

Associate Editor: P. K. CHIANG

THERAPEUTIC

APPLICATIONS

OF RIBOZYMES

HIROSHI KIJIMA, HIRONORI ISHIDA, TSUKASA OHKAWA, MOHAMMED KASHANI-SABET~ and KEVIN J. SCANLONT Biochemical Pharmacology, Department of Medical Oncology, Montana Buitiing, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010, USA Abstract -The demonstration that RNA can be cleaved by cis or tram ribozymes (catalytic RNAs, RNA enzymes) has potentially important therapeutic implications. Since their discovery in the 198Os,the biochemistry and conserved sequences of ribozymes have been well characterized. Ribozymes are effective modulators of gene expression because of their simple structure, sitespecific cleavage activity, and catalytic potential. The targets of ribozyme-mediated gene modulation have ranged from cancer cells to foreign genes that cause in&ious diseases. Additional target sites for ribozymes are in initial phases of development and design. Ribozymes have been targeted against a myriad of genes, including oncogenes (ras, BCR-ABL, c-fos) and drug resistance genes, as well as the human immunodeficiency virus-type 1 genome. These ribozymes have cleaved the target RNAs in virro and altered the cellular pathology. Currently, the therapeutic application of ribozymes to human diseases is limited by gene transfer systems. It is anticipated that ribozymes ultimately will play an important role in human gene therapy. Keywords-Ribozyme,

oncogene, cancer, antisense, gene therapy, drug resistance.

CONTENTS 1. Introduction 2. Biochemistry of Ribozymes 2.1. Hammerhead ribozyme 2.2. Hairpin ribozyme 2.3. Additional ribozymes 3. Applications of Ribozymes 3.1. Cancer 3.2. Human immunodeficiency virus-type 1 3.3. Other examples of ribozyme cleavage targets 3.4. Antisense versus ribozyme 4. Designing and Delivering Ribozymes 5. Conclusion Acknowledgements References

248 249 249 251 252 253 253 256 259 260 260 261 261 261

*Present address: Department of Dermatology, University of California at San Francisco, San Francisco, CA 94143, USA. tcorresponding author. Abbreviations-(-), minus strand; (+), plus strand; ACC, acetyl-CoA carboxylase; AD, actinomycin D; AP-1, activator protein-l; &APP, @-amyloid peptide precursor; CML, chronic myelogenous leukemia; CMV, cytomegalovirus; DHFR, dihydrofolate reductase; HBV, hepatitis B virus; HDV, hepatitis delta virus; HIV-l, human immunodeficiency virus-type 1; HPV, human papilloma virus; LCMV, lymphocytic choriomeningitis virus; LTR, long terminal repeat; MAP, mitogen-activated protein; MDR, multidrug resistance; mdr-I , multidrug resistance- 1; MMTV , mouse mammary tumor virus; MTX, methotrexate; 2’-OH, 2’-hydroxyl; PDGF, platelet-derived growth factor; P-glycoprotein, permeability-glycoprotein; PKC, protein kinase C; pMAMneo, plasmid, inducible gene expression; pol III, polymerase III; sTobRV, satellite RNA of tobacco ringspot virus; TAR, trans activation-response; TMQ, trimetrexate; TNF, tumor necrosis factor; uPAR, urokinase-type. plasminogen activator receptor. 247

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1. INTRODUCTION Ribozymes (catalytic RNAs, RNA enzymes) are RNAs that have site-specific RNA cleavage or ligation activities (for reviews, see Cech, 1990; Altman, 1987; Symons, 1992). Cech and colleagues described the first ribozyme, which was the 413-nucleotide Group I intervening sequence in the pre-rRNA of Tetrahymena thermophila; the intervening RNA sequence catalyzes its own excision, called selfsplicing (Cech et al., 1981; Kruger et al., 1982). Altman and collaborators reported the first truly catalytic ribozyme that could cleave other molecules with multiple turnover. It was the 400-nucleotide RNA component of bacterial RNase P (Guerrier-Takada et al., 1983). Some of the ribozymes originated from plant virus-like particles called viroids (Symons, 1992). Self-cleavage reactions have been demonstrated in vitro for the satellite RNA of tobacco ringspot virus (.$TobRV)(Buzayan et al. , 1986a,b), avocado sunblotch viroid (Hutchins et al., 1986), and luceme transient streak virus (Forster and Symons, 1987). Further studies have clarified the stmctures of ribozyme; the aforementioned selfcleaving RNAs of sTobRV, avocado sunblotch viroid, and luceme transient streak virus were shown to have hammerhead-shaped structures. In contrast, the plus strand (+) and minus strand (-) of sTobRV were shown to have either a hammerhead-shaped (former) or a hairpin-shape (latter). A biochemical difference between (+) and (-) of sTobRV is that a phosphorothioate strongly inhibited self-cleavage of the (+) sTobRV, but not of the (-) sTobRV, in the presence of Mg++ (Buzayan et al., 1988). Naturally occurring ribozymes are divided into six groups: (1) ribozymes derived from self-splicing of Etrahymena Group I introns, (2) RNA components of RNase P, (3) hammerhead ribozymes, (4) hairpin ribozymes, (5) genomic and antigenomic RNAs of hepatitis delta virus (HDV), and (6) RNA transcripts of the mitochondrial DNA plasmid of Neurospora (Symons, 1992, 1994; Castanotto et al. , 1994). Several studies have described tram site-specific cleavage by a ribozyme. Uhlenbeck (1987) has proposed that the ribozyme consensus sequence is derived from the sTobRV genome, and has shown a 19-nucleotide ribozyme to specifically cleave a 24-nucleotide RNA into its fragments. In vitro mutagenesis studies have defined ribozymes with the hammerhead structure that are capable of highly specific activities of RNA cleavage (Haseloff and Gerlach, 1988). Based on these studies, investigators have explored the potential of ribozymes to modulate gene expression inside the cell. Recent studies have defined the molecular basis of cell growth and differentiation (for reviews, see Cantley et al. , 1991; Hunter, 1991; Egan and Weinberg, 1993). The proliferation and differentiation of normal cells can be initiated by signal transduction cascades and regulated by a balance between the action of proto-oncogenes and suppressor genes. Among the proto-oncogene products, the Ras protein plays an important role in the signal transduction pathway. Ras transduces the signal from the cell surface to the nucleus, where genes are turned on or off in response to the incoming signal: upstream of Ras, such as the tyrosine kinase receptor, which is present at the cell membrane; downstream of Ras, the kinase cascade is followed by nuclear signaling. In cancer cells, alteration of these cascades and gene expression has been demonstrated (Seemayer and Cavenee, 1989). In addition, mutations of the ras oncogenes have been shown in a large number of human cancers (Bos, 1989; Barbacid, 1987). Therefore, these genes with their altered expression, as well as mutated ras genes, could become targets of ribozyme-mediated gene modulation. Investigators have also explored the molecular basis of resistance to anticancer drugs. The development of drug resistance significantly limits the effectiveness of chemotherapy, and is mechanically multifactorial and complicated. For instance, the c-fos gene has been shown to regulate cisplatin resistance (Scanlon et al., 1989). Overexpression of the multidrug resistance-l (n&-I) gene has been associated with multidrug resistance (MDR) phenotypes (Endicott and Ling, 1989; Shen et al., 1986; Chin et al., 1989; Gottesman and Pastan, 1993), and alteration of the dihydrofolate reductase (DHFR) gene plays an important role in methotrexate (MTX) resistance (Bertino, 1993; Ohnuma et al., 1985; Srimatkandada et al., 1989; Antony, 1992). This altered gene expression associated with drug resistance has also become a target for ribozyme strategies to reverse drug resistance. Ribozymes have the ability to modulate the altered expression of genes, such as oncogenes or genes associated with drug resistance, in various human cancers because of site-specific cleavage of target RNAs. This review focuses on the biochemistry of hammerhead and hairpin ribozymes and their application to human gene therapy.

Therapeutic

applications of ribozymes

2. BIOCHEMISTRY 2.1. Hammerhead

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OF RIBOZYMES Ribozyme

The hammerhead ribozyme is derived from the (+) strand of sTobRV, the (+) and (-) complementary strands of sTobRV replicate in tissues infected with tobacco ringspot virus, and have the capacity for self-catalyzed RNA cleavage (Gerlach er al., 1987). Several studies have demonstrated site-specific cleavage in the tram form of the hammerhead ribozyme (Uhlenbeck, 1987; Haseloff and Gerlach, 1988). The catalytic cleavage reaction has been shown to have multiple substrates and involve nonhydrolytic transesterification of the substrates in the presence of Mg++ (Buzayan et al., 1988; van To1 et al., 1990). Hertel and colleagues (1992) have proposed a unified numbering system for the hammerhead ribozyme (Fig. 1). Recent mutational analysis, structural studies and other biochemical experiments have described consensus sequences, as well as structural and kinetic characteristics, of the hammerhead ribozyme (Cech and Uhlenbeck, 1994). Pley and colleagues (1994) recently have demonstrated the threediiensional structure of a hammerhead ribozyme by X-ray crystallography. The catalytic core, consisting of the conserved C3U4G5A6, is involved in the sharp turn of the ribozyme strand at the base of Helix I (Fig. 1). This sharp turn is identical to the uridine turn of transfer RNA. These studies have led to a more fundamental understanding of the uniqueness of the hammerhead ribozyme. The consensus sequences of the hammerhead ribozyme and its target RNA substrate are shown in Fig. 1A (Herd et al., 1992; for a review, see Bratty et al., 1993). The secondary structure of the ribozyme-substrate complex consists of tlnee helical regions, a catalytic core region (i.e., hammerhead domain) and an internal loop sequence. The RNA substrate binds to the hammerhead ribozyme through two helices (Helix I and Helix III); the catalytic core region separates the two helices (Helix I and Helix II), as well as Helix III formed by four complementary base pairs (i.e., eight nucleotides) of the ribozyme; the internal loop (Loop 2) of four nucleotides is at the opposite end of Helix II. Cleavage occurs to the 3’ side of the N17 residue; the hammerhead ribozyme can significantly discriminate substrate RNAs with a single base mutation (Koizumi et al., 1989), as well as closely related RNAs (Bennett and Cullimore, 1992). Early studies of mutational analysis demonstrated that cleavage reaction requires the N16.2U16.1H17 triplet sequences 3’ of the cleavage site (N base is any nucleotide; H base is A, C, or U) (Haseloff and Gerlach, 1988; Koizumi et al., 1989; Ruffner et al., 1990). Usually, the substrates containing the GUC, GUA, GUU, UUC, or CUC triplets are efficiently cleaved, and the GAC, GUG, AUC, CGC, GGC, AGC, or UGC triplet sequences are poorly cleaved (Perriman et al., 1992). The AUA triplet is essentially not cleaved; however, a hammerhead ribozyme with G*@l-C1l.land a pyrimidine at position 7 has shown increased efficiency for AUA cleavage (Nakamaye and E&stein, 1994). Several biochemical conditions are required for effective cleavage of the hammerhead ribozyme. Studies using DNA/RNA chimeric nucleotides have demonstrated that cleavage reaction requires 2’-hydroxyl (2’-OH) groups in the catalytic core region, especially at the N17, as well as U16.’ (Perreault et al., 1990; Yang et al., 1990); importance of the 2’-OH groups at A15.1,G5, G*, and A9 have been shown for ribozyme catalysis (Perreault et al., 1991; Yang et al., 1992). Other studies have defined the significance of specific 2’-OH groups at G5 and G* using chemical modification of these groups (Williams et al., 1992; Fu and McLaughlin, 1992). Furthermore, other chemical modifications of the ribozymes and their cleavage activities have been examined (Heidenreich et al. , 1993), including the modification 2’-fluoro- and 2’-amino-nucleotides (Pieken et al., 1991), 2’-Oallyl- and 2’-O-methyl-nucleotides (Paolella et al., 1992), 2’-O-methylation of flanking sequences (Goodchild, 1992), 2’-pyrimidine modifications such as 2’-amino-2-deoxyuridines (Heidenreich et al., 1994), and isoguanosine substitution of conserved adenosines (AC, A9, A13, A’5.1)(Ng er al., 1994). Conditions of flanking sequences (i.e., sequences in Helices I and III) affect ribozyme-substrate kinetics. One study has reported that sequences of substrate RNA and their secondary structures are important for catalytic efficiency of the ribozyme (Fedor and Uhlenbeck, 1990). Another study has demonstrated that adding bases to the ribozyme’s flanking sequence increased site specificity. However, the dissociation step of the ribozyme-substrate duplex was much slower (HerschIag, 1991). The maximum discrimination was expected to be greater with A+U-rich sequences than with

H. Kijima et al.

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Fig. 1. Structureof ribozymes. The hammerhead ribozyme structure contains three helical stems. The target RNA substrate is cleav14 by the ribozyme to 3’ on NIT. The numbering system for the ribozyme and target sequences is according to Hertel el al. (1992). (A) The hammerhead ribozyme and the target RNA subsha&. @) Minizyme. (C) DNA/RNA chimetic ribozyme.

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G +C-rich sequences. A recent study has described a 12-base optimal length for the flanking sequences (Bertrand et al., 1994) The Stem II, consisting of Helix II and Loop 2, has been characterized in detail by McCall and colleagues (1992). They created a shortened ribozyme, called a “minizyme,” by reducing the Stem II size and replacing RNA nucleotides with DNA. Cleavage activity of the minizyme depended on the number and sequence of the nucleotides, and optimal activity was achieved with four or five deoxyribopyrimidines. This study suggested that Helix II may be dispensable to formation of the active structure and that the minizyme is active. Another study reported that Stem II with 2 bp rather than the conventional 4 bp maintained catalytic activity. However, when the size of Stem II was reduced to 1 bp or less, the ribozyme activity drastically decreased (Tuschl and Eckstein, 1993). Furthermore, this report demonstrated that inversion of the G1”l-C1’-r bp next to the invariant core led to a loss in activity even when the stem consisted of 4 bp. It has been suggested that they constructed an active “super minizyme” with an alternative G-C linker, which generated the simplest model for the hammerhead ribozyme (Fig. 1B). Studies substituting DNA for RNA in the various stems of the ribozyme have shown that DNA/RNA chimeric ribozymes with DNA in Helices I and III had a 6-fold greater &at value than the all-RNA ribozymes and that substitution of DNA in Stem II yielded a marked reduction in cleavage activity (Taylor et al. , 1992). Moreover, these chimeric ribozymes, when transfected by cationic liposomes into human T-lymphocytes, had a higher stability than their all-RNA counterparts. Another group demonstrated that cleavage ability of the DNA/RNA chimeric ribozymes with DNA in Helices I and III was enhanced more than 3-fold, compared with the all-RNA ribozyme (Hendry et al., 1992). The aforementioned minizyme with replacements of Helix II was as active as the full-size ribozyme (McCall et al., 1992). Shimayama and colleagues (1993) introduced deoxyribonucleotides with phosphorothiate linkages to the same regions of Helices I, III, and Stem II. They revealed that this thio-DNA/RNA chimeric ribozyme had 7-fold higher cleavage activity than the all-RNA ribozyme in a k,* value. This study also suggested that this tbio-DNA/RNA chimeric ribozyme had about IO-fold higher stability in human serum than in bovine serum. This thio-DNA/RNA chimeric ribozyme was more resistant to attack by nuclease than the all-RNA ribozyme in tivo. These DNA/RNA chimeric ribozymes with modified DNA for RNA have been shown to be one of the most suitable structures as transient therapeutic agents for hammerhead ribozymes (Fig. 1C). 2.2. Hairpin Ribozyme The hairpin ribozyme (Fig. 2) is derived from the 359-base (-) strand of sTobRV (Hampel and Trits, 1989; Feldstein et al. , 1989), and site-specifically cleaves RNA substrates in trans. The original hairpin ribozyme (Fig. 2) consisted of 50 bases (nucleotides) and cleaved corresponding 1Cbase RNA substrates. The cleavage reaction of the hairpin and hammerhead ribozymes is a multiple substrate cleavage event involving nonhydrolytic transesterification of the substrates in the presence of Mg++ (van To1 et al., 1990). A biochemical difference between the hammerhead and hairpin ribozymes of sTobRV is that a phosphorothioate strongly inhibited self-cleavage of the hammerhead ribozyme, but not of the hairpin ribozyme of sTobRV, in the presence of Mg++ (Buzayan et al., 1988). Limited mutational analysis, computer modeling, and phylogenetic studies have proposed secondary structure models for the hairpin ribozyme-substrate complex (Haseloff and Gerlach, 1989; Hampel et al., 1990). The ribozyme-substrate complex consists of four helical regions separated by two internal loop sequences. The substrate binds to the ribozyme through two helices (Helix I and Helix II). Cleavage occurs to the 5’ side of a guanosine within the internal loop (Loop A) of the substrate separating Helices I and II. A second internal loop (Loop B) separates the two helices (Helix III and Helix IV) of the ribozyme. Recent studies have described essential nucleotide sequences of the hairpin ribozyme for catalytic activity: most of the nucleotides within Loops A and B are necessary (Chowrira et al., 1991; BerzalHerranz et al., 1993). Within Loop A, there are four essential bases: three in the ribozyme (G*, A9, and Aro; base number beginning at the 5’ end of RNA) and one in the substrate (G6). Within Loop B, 9 of 11 bases (all except A*Oand U39) are essential sequences. In contrast, only one base (G”) within the four helices is important for catalytic activity (Joseph et al., 1993). A structural bend of the ribozyme occurs at or near the Ar3-Ai4 linkage (Feldstein and Bruening, 1993), and

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Loop B is three-dimensionally

2.3. Additional Ribozymes Apart from the aforementioned ribozymes, i.e., Group I introns, RNA components of RNase P, hammerhead ribozymes, and hairpin ribozymes, catalytic RNAs have been found in HDV and RNA transcripts of Neurosporu mitochondrial DNA (see Symons, 1992, 1994; and Section 1 of the present paper). HDV is a satellite virus of hepatitis B virus (HBV) and contains (-)-strand (single-stranded) RNA of about 1700 nucleotides; the (+)-strand (complementary) RNA has a coding region of 195 amino acids for the HDV antigen within the viral particle (Taylor, 1990; Wang ef aE., 1986). The HDV RNA is replicated by the rolling circle mechanism; self-cleaving domains play a significant role in the mechanism and require Mg++ (Chen et al., 1986; Wu et al., 1989; Wu and Lai, 1990). Branch and Robertson (1991) have reported axehead structures for the HDV self-cleaving domains; conserved sequences between negative (genomic) strand and positive (antigenomic) strand are demonstrated. Recently, several studies have described structural analyses in detail and cleavage in tmns of the ribozyme derived from the HDV sequence (Thill er al., 1993; Kumar et al., 1994; Perrotta and Been, 1992).

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A sixth type of self-cleavage RNA is the 881-base transcript of a circular mitochondrial plasmid DNA of Neurospora, Varkud-lc strain (Collins and Olive, 1993; SaviIle and Collins, 1990). Recently, the minimal sequence of the RNA transcript for self-cleavage activity has been investigated; however, computer analyses have proposed that its structure has shown some resemblance to that of hairpin ribozyme (Guo ef uZ., 1993). The ability of the Neurosporu self-cleaving RNA to cleave in frans has yet to be demonstrated.

3. APPLICATIONS

OF RIBOZYMES

3.1. Cancer Recent studies have defined the mechanisms of cellular proliferation, as well as the biology of human cancer in molecular forms (Fig. 3). Cell growth and differentiation can be initiated by signal transduction cascades (for reviews, see Cantley et al., 1991; Hunter, 1991; Roberts, 1992; Egan and Weinberg, 1993). The signal transduction pathway starts with binding of growth factors, such as epidermal growth factor and platelet-derived growth factor (PDGF), to their tyrosine kinase receptor, such as epidermal growth factor receptor and PDGF receptor; the binding results in phosphorylation of the receptor’s tyrosine residues. The phosphorylation recruits the Ras oncoprotein through other factors, such as Grb26em5 and SOS, which have the ability to convert inactive Ras @as-GDP) to active Ras (Ras-GTP). Downstream of the Ras oncoprotein, Ras-GTP activates the cytoplasmic phosphorylation cascade involving the Raf protein, mitogen-activated protien (MAP) kinase kinase, and MAP kinase; therefore, Ras plays an important role in the signal transduction pathway, although other signal transduction pathways exist to transduce a variety of other extracellular signals. These

Fig. 3. Signal transduction pathway. P, phosphatase; MAPK-K, MAP kinase kinase; MAPK, MAP kinase.

254

H. Kijirna et al.

“second messengers” include cyclic adenosine monophosphate, calcium ions, diacylglycerol, and phosphorylated forms of the sugar inositol. They can stimulate specific protein kinases, such as protein kinase C (PKC). PKC, as well as Ras, activates the Raf protein, which ultimately activates MAP kinase. MAP kinase translocates to the nucleus and may phosphorylate Jun and Fos oncoproteins. Activator protein-l (AP-1) plays an important role in transcription of DNA. AP-1 is a heterodimeric complex of several different proteins of the Jun and Fos family. The c-fos oncogene has been shown to modulate the expression of AP-1 responsive genes important in DNA synthesis, repair, and drug detoxification pathways, such as dTMP synthase, topoisomerase I, and metallothionein (Scanlon et al. , 1991). In cancer cells, alteration of these signal transduction cascades has been reported (Seemayer and Cavenee, 1989; Brunton and Workman, 1993). Mutations of the ras oncogene family have been shown in human cancers (Bos, 1989; Barbacid, 1987); specifically, rus gene mutations have been found in about 90% of pancreatic adenocarcinomas (Almoguera et al., 1988), in 40-50% of colon adenocarcinomas (Bos et al., 1987; Forrester et al., 1987), and in 30% of lung adenocarcinomas (Rodenhuis et al., 1988). Therefore, ras genes could become targets of ribozyme-mediated gene modulation. The nuclear oncogene c-fis has been targeted with ribozymes because of its important role in the nucleus, as well as its overexpression in cancer cells resistant to cancer chemotherapeutic agents (Scanlon et al., 1991). The mutant H-ras gene has become a significant target of ribozyme-mediated gene modulation. A hammerhead ribozyme against H-ras mutated at codon 12 (GUU) was shown to discriminate between the normal H-ras gene and the mutated H-rus gene in vitro (Koizumi et al., 1989, 1992). Scanlon and colleagues have made efforts to reverse the phenotype of several human cancers using ribozyme strategies. A hammerhead ribozyme was targeted against the H-ras mRNA mutated at codon 12 (GUC, mutated from GGC) (Kashani-Sabet et al., 1992). The anti-ras ribozyme was cloned into a pH/3 Apr-1 vector driven by the human @actin promoter, and transfected into the EJ bladder carcinoma cells. The ribozyme decreased H-ras gene expression and inhibited cell growth; the ribozyme transfectants exhibited reduced tumorigenicity, which resulted in noninvasive, nonmetastatic tumors in athymic mice. The effects of the anti-ras ribozyme were compared with those of a mutant ribozyme devoid of cleavage activity; the anti-ras ribozyme partially decreased H-rus gene expression and inhibited cell growth of the EJ transfectants (Tone et al., 1993). In addition, the anti-ras ribozyme has been transfected into FEM human melanoma cells; the transfectants exhibited not only decreased H-ras gene expression and inhibition of cell growth, but also induction of cellular differentiation associated with an increased synthesis of melanin, responsiveness to 12-0-tetradecanoyl phorbol13-acetate, and altered morphology (Ohta et al., 1994). In other studies with NIH3T3 cells, the cells were transformed with DNA from the FEMX-1 cells, which were a derivative of a human melanoma cell line (Kashani-Sabet et aI., 1994). The anti-ras ribozyme efficiently suppressed the transformed phenotype of NIH3T3 cells, such as H-r-as gene expression, growth characteristics in vitro, and tumorigenicity in nude mice. The NIH3T3 cells have been also transformed with pEJ6.6 plasmid containing activated H-r-as gene (Funato et al., 1994). The anti-ras ribozyme completely reversed H-r-as gene expression and cell growth; however, the mutant ribozyme devoid of cleavage activity did not suppress the transfected H-ras gene expression. The H-ras ribozyme also prevented transformation of NIH3T3 cells by the pEJ6.6 plasmid. The other oncogene targets of ribozyme cleavage in human carcinomas are the BCR-ABL hybrid gene, which is formed when the proto-oncogene ABL from chromosome 9 translocates to the breakpoint cluster region (BCR) on chromosome 22. The BCR-ABL gene generates a Philadelphia chromosome found in more than 95% of chronic myelogenous leukemia (CML), as well as in about half of acute lymphocytic leukemias (Kurzock et al., 1988). Most of the ribozyme targets are the GUU triplet at the junction site of the BCR-ABL fusion gene (Snyder et al., 1993; Shore et al., 1993). The 3’ flanking sequence of the designed ribozyme forms helices with BCR, while the 5’ flanking sequence forms duplexes with four bases of the BCR gene, as well as the ABL gene sequence; the ribozyme may specifically cleave the BCR-ABL mRNA transcripts. One study has demonstrated that the ribozyme targeting the BCR-ABL fusion gene theoretically may cleave not only leukemia-specific BCR-ABL transcripts, but also normal mRNA (Wright et al., 1993). In addition, another study has reported that the BCR-ABL fusion region targeted by the ribozyme could be an inauspicious secondary structure, and could be hardly cleavable by the ribozyme (Pachuk et al. , 1993). However, several reports have

Therapeutic applications

of rihozymes

255

in vitro efficacy of anti-BCR-ABL ribozymes. The ribozyme cleaved BCR-ABL RNA in a cell-free in vitro system, and the chimeric DNA-RNA ribozyme in a liposome was transfected into EM-2 CML blast crisis cells (Snyder et al., 1993). The chimeric ribozyme decreased BCR-ABL mRNA levels, partially decreased the gene product ~210, and partially inhibited cell growth of the EM-2 transfectant. A second group cloned the anti-BCR-ABL ribozyme into a retroviral expression vector; it diminished ~210 protein-kinase activity, which is encoded by the BCR-ABL gene, in the K562 human CML blast crisis cell line (Shore et al., 1993). A third group has also demonstrated the efficacy of the anti-BCR-ABL ribozyme transfected into K562 cells by lipofection; the ribozyme inhibited proliferation of the K562 cells and produced a 3- to 5fold reduction of BCR-ABL mRNA molecules per single cell (Lange et al., 1993). In addition, efficient cleavage by the different antiBCR-ABL ribozymes have been studied using the K562 cells; the ribozyme with flanking sequence of 7 bases at the 5’ side (BCR) and 9 bases at the 3’ side (3 BCR and 6 ABL) had the most efficiency to cleave the BCR-ABL mRNA (Lange et al., 1994). Gene expression of PDGFP and PDGFP receptor have been shown in several mesothelioma cell lines, and could contribute to cellular transformation; the mesothelioma cells may be stimulated by PDGF/PDGF receptor autocrine mechanisms (Versnel et al., 1988, 1991). One group has cloned anti-PDGFP ribozyme into the pHfi Apr-1-neo plasmid, and expressed it in the VAMT-1 human mesothelioma cell line, which overexpresses both PDGFP and PDGFP receptor (Dorai et al., 1994). The anti-PDGF ribozyme decreased approximately 30% of PDGFP mRNA level, as well as 40% of cell growth of the VAMT-1 cells. Tumor necrosis factor (TNF)-a plays an important role in immune responses or inflammatory rheumatic diseases, and modulates the expression of cytokines, such as interleukin 1 and interleukin 6, as well as proteins including Class I antigens of the major histocompatibility complex (Beutler and Cerami, 1988, 1989). One study has described down-regulating the expression of TNF-a by a hammerhead ribozyme (Sioud et al., 1992). The anti-TNF ribozyme was delivered by cationic liposome-mediated transfection, and reduced 90% and 80% of TNF-CYmRNA in the HL60 human promyelocytic leukemia cells and peripheral blood mononuclear cells, respectively. Human papilloma virus (HPV) could be one target of human gene therapy because HPV has been implicated as an important cofactor in the development of human anogenital malignancies, especially cervical carcinomas (Lowy et al., 1994). One group has reported in vitro cleavage of HPV-16 E6 and E7 open reading frames, which are associated with viral DNA replication or gene regulation, by hammerhead ribozymes (He et al., 1993). Another group has demonstrated in vitro cleavage of HPV-16 E6 and E7 mBNA by ribozyme sequences that were encoded by the adenoassociated virus-based vector (Lu et al., 1994). This suggests application in the future of a ribozymemediated gene therapy for HPV-associated diseases. Meanwhile, many investigators have studied molecular mechanisms of anticancer drug resistance, as development of drug resistance still remains one of the most serious limitations in the treatment of human cancers. Cisplatin is one of the most widely used anticancer agents, and its multifactorial mechanisms of resistance pose serious clinical problems in cancer chemotherapy (for a review, see Ishida et al. , 1995). However, studies in cisplatin-resistant cell lines have suggested the importance of the C-$X oncogene in maintaining the drug-resistant phenotype (Scanlon et al., 1989). The Fos protein, in interaction with the Jun protein, affects cell proliferation, apoptosis and drug resistance through transcriptional activation of genes containing AP-1 elements in their regulatory regions (reviewed by Ransone and Verma, 1990). The A2780 ovarian carcinoma cell line resistant to cisplatin has been shown to exhibit C-$X overexpression, as well as that of c-myc, H-ras, thymidylate synthase, DNA polymerase /3 and topoisomerase I (Scanlon et al., 1990, 1991; Kashani-Sabet et al., 1990b). Tumor tissues from a patient with colon carcinoma failing cisplatin/5-fluorouril treatment revealed a similar pattern of gene expression to the resistant A2780 subclone (Kashani-Sabet et al., 1990a). These data have suggested that the c-&s gene regulates downstream enzymes associated with DNA synthesis and repair and plays a principal role in cisplatin resistance. The hammerhead ribozyme against the C-$X gene has been investigated in the cisplatin-resistance field (Scanlon et aZ., 1991; Funato et al., 1992). The cisplatin-resistant A2780 subclone (lo-fold) was transfected with an anti-&s ribozyme driven by the plasmid, inducible gene expression (pMAMneo) vector containing the mouse mammary tumor virus (MMTV) dexametbasone-inducible promoter, and was rendered sensitive to the antineoplastic effects of cisplatin. The ribozyme-mediated transfectant has shown down-regulation described

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Of c-$9~gene expression, as well as expression of c-fos responsive genes, such as DNA polymerase P, topoisomerase I, and metallothionein IIA. Although the ribozyme was able to cleave about 80% of the substrate (c-fos mRNA), the mutant ribozyme did not cleave the substrate, thus being capable of only antisense activity. Another major drug-resistant phenotype, partly responsible for resistance to drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, and actinomycin D (AD), is well known as MDR and is associated with overexpression of the r&r-I gene (Gottesman and Pastan, 1993). The &r-l gene encodes a membrane phosphoglyooprotein (P-glycoprotein), which plays a role in energy-dependent drug efflux. Overexpression of mdr-I has been shown in the MDR phenotypes of human cancers. Four groups have demonstrated efficacy of an anti-r&r-I ribozyme. The first group has reported efficacy of an anti-r&r-I hammerhead ribozyme driven by the P-actin promoter in MOLT-3 acute leukemia cell line, as well as in vitro cleavage of the ribozyme (Kobayashi et al., 1993, 1994a). The anti-r&--l ribozyme down-regulated mdr-1 gene expression, as well as P-glycoprotein expression in the MOLT-3 cells resistant to trimetrexate (TMQ), and decreased their vincristine resistance from 700-fold to ZO-fold. The second group has demonstrated anti-&r-l ribozyme efficacy in PXF1118 mesothelioma cells using a liposome-mediated transfer system (Kiehntopf et al., 1994). The antimdr-1 ribozyme by chemical synthesis reversed both overexpression of P-glycoprotein and drugresistance levels in the PXF118 subclones resistant to vindesine or adriamycin. The third group has described the reversal of daunorubicin resistance by the anti-r&r-l ribozyme driven by the @-actin promoter (Holm et al., 1994). The ribozyme down-regulated rndr-l gene expression in EPP85-181 pancreatic carcinoma cells resistant to daunorubicin; the ribozyme-expressing EPP85-181 cells were 300~fold more sensitive to daunorubicin compared with the parental-resistant cells. The fourth group has cloned the anti-r&r-l hammerhead ribozyme into the pH@ Apr-1-neo plasmid and transfected it into the A2780 ovarian carcinoma cell line resistant to AD; the A2780 subclone was 16.6-fold resistant to AD and overexpressed r&r-I (Scanlon et al., 1994). The anti-&r-l ribozyme reduced m&-I gene expression in the A2780 cells resistant to AD, and almost completely reversed their AD resistance to the sensitive level after 18 weeks in culture. The promoter of the mfr-l gene has an AP-1 binding site (Teeter et al., 1991), suggesting that the c-fos gene, as well as rndr-1, may play an important role in regulating MDR. In the A2780 cells, the cells expressing MDR overexpress c-fos, as well as rndr-I. Moreover, the anti-&r ribozyme down-regulates not only c-fis expression, but also expression of mdr-I, c-jun, topoisomerase I, and mutant ~53; the anti-@ ribozyme is more efficacious than the n&-l ribozyme (Scanlon et al., 1994). MTX is an important folate antagonist used in cancer chemotherapy; its mechanism of action is the competitive inhibition of folate metabolism (Bertino, 1993). Alteration of DHFR gene expression has been shown in several human tumor cell lines resistant to antifolates (Ohnuma et al., 1985; Srimatkandada et al. , 1989; Antony, 1992). One group has designed a hammerhead ribozyme against mutated DHFR mRNA, and demonstrated cleavage of mutated DHFR RNA derived from human leukemia cell sublines resistant to both MTX and TMQ (Kobayashi et al., 1994b). The ribozyme cleaved not only mutated DHFR RNA, but also normal DHFR RNA; this suggests that reaction conditions could change cleavage specificity of the ribozyme. However, pharmacological studies such as the reversal of TMQ and MTX resistance by the ribozyme were not demonstrated. 06-methylguanine-DNA methyl transferase is a DNA repair enzyme, active in removing alkyl adducts from the O6 position of guanine (Lindahl, 1982). One study has demonstrated both in vitro and in vivo ribozyme-mediated cleavage of 06-methylguanine-DNA methyl transfemse mRNA using human HeLa CCL2 cells (Potter et al., 1993). However, this report examined the consequences of ribozyme action on DNA repair activity. In conclusion, the studies mentioned above suggest the presence of many targets for ribozyme design in the field of cancer gene therapy. However, problems of ribozyme delivery to appropriate targets in humans have to be overcome for the success of ribozyme-mediated gene therapy.

3.2. Human Immunodeficiency

Virus-Type

1

Human irnmunod&ciency virus-type 1 (HIV-l) is the retroviral etiologic agent of acquired immunodeficiency syndrome @arm-Sinoussi et at. ,1983; Gall0 et al. ,1984). Recently, seveml molecular strategies to inhibit HIV-l infection have been developed (for reviews, see Buchschacher, 1993; Sarver

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1993; Sarver and Rossi, 1993; Vaishnav and Wong-Staal, 1991). Ribozymes may be a potential therapeutic agent for the treatment of HIV-l infection (for reviews, see Rossi et al., 1992; Altman, 1993; YU et al., 1994). Chang et al. (1990) first demonstrated that an anti-HIV-l gag ribozyme driven by constitutive human @-actin promoter was effective in CD4+ cells. Consequently, using the same anti-gag ribozyme, Sarver et al. (1990) have shown that the ribozyme could protect the cells from HIV-l infection. When human HeLa CD4+ cells expressing the ribozyme were challenged with HIV-l, gag gene expression, as well as p24 antigen levels, were reduced in the ribozyme-expressing cells compared with the control. Anti-HIV-l ribozymes have been investigated in several target sites for HIV-l, such as long terminal repeat (LTR) RNA, which includes the 5’ leader sequence, the R region containing TAR sequence and U5 leader sequence, the retrovirus packaging ($) sequence, gag RNA, integrase RNA, vifRNA, tat RNA, env RNA, and rev RNA (Vaishnav and Wong-Staal, 1991); RNA viruses such as HIV-l continuously undergo mutational changes due to the error-prone nature of reverse transcriptase (Holland et aZ. , 1982; Preston et al., 1988; Roberts et al., 1988). The 5’ leader sequence is recognized as a transcription initiation site and is conserved among most HIV-l isolates. Weerasinghe et al. (1991) have shown that the retroviral vector, driven by Herpes Simplex Virus thymidine kinase-trans activation-response (TAR) fusion promoter, expressing an HIV-l 5’ leader sequence-specific hammerhead ribozyme, conferred resistance to HIV-l infection in human CD4+ MT4 cells for 22 days. Another group used the HIV-l S-leader-sequence-specific hairpin ribozyme driven by human fl-actin promoter to demonstrate that HIV expression was inhibited as measured by p24 antigen levels and reduced Tat activity in vitro and in vivo (Ojwang et al., 1992). Consequently, the authors designed a vector plasmid containing the same ribozyme driven by the strong RNA polymerase III (pol III) promoter, and showed that its expression rate was 88% higher than the @-actin promoter, and that p24 levels were decreased by over 90% compared with control cells in vivo. Moreover, inhibition of replication of several HIV-l strains (even MN, a typical Northern American HIV-l strain, which has a single point mutation in the target sequence) has been shown by this ribozyme (Yu et al., 1993). The S-TAR region of HIV-l RNA is also highly conserved in different HIV-l isolates and is an important regulatory region involved in viral expression. Ventura et al. (1994) have demonstrated that the hammerhead ribozymes targeting the 5’-TAR region driven by the pol III promoter could inhibit 50-60% of the HIV-1-LTR-dependent chloramphenicol acetyltransferase expression in vivo. This lower inhibition rate (50~60%), compared with the study of Yu et al. (1993), might depend on the hairpin structure of the TAR region. Several studies have shown that the U5 hammerhead ribozyme cleaved its target site of HIV-l in vitro (Goodchild and Kohli, 1991; Heidenreich and E&stein, 1992; Dropulic et al. , 1992). Dropulic et al. (1992) demonstrated that HIV-l replication was suppressed in vivo by this cis and tram ribozymes when placed into the HIV-l at the nefgene, but its inhibition only persisted for 1 week due to development of escape mutants. Yamada et al. (1994) have reported that a hairpin ribozyme driven by the pol III promoter target in the U5 leader sequence showed long-term resistance to challenge with diverse HIV-l viral clones and clinical isolates. Subsequently, in this study, Leavitt et al. (1994) have established a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes, and revealed the same efficacy to protect from HIV-l infection for long-term periods. Moreover, the ribozyme in this system had no inhibition of HIV-type 2 clones, and no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T-cells of resistance to HIV-l infection conferred by gene transfer, and this system shows promise as a preclinical model. The retroviral packaging region is an obviously important region for packaging of HIV-l genomic RNA into virus particles (Man and Baltimore, 1985; Lever et al., 1989), such that its sequence has been conserved among 18 published HIV-l isolates (Harrison and Lever, 1992). Sun et al. (1994) have designed an anti-HIV1 packaging site ribozyme (hammerhead), and demonstrated that the ribozyme reduced p24 levels by 80-90% on days 6, 9, 12, and even day 22, and allowed to significantly delay the initiation of syncytia formation in the human T-cell line SupTl. The integrase gene encodes a protein required for the integration of viral DNA into the host chromosome (Donehower and Varmus, 1984; Schwartzberg et al., 1984). Sioud and Drlica (1991)

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have shown that an anti-HIV-l integrase hammerhead ribozyme inhibited integrase RNA and completely blocked integrase protein synthesis in Escherichiu coli. However, the mutant ribozyme, which was devoid of catalytic activity, only showed low efficiency inhibition of these targets in vitro and in viva. These results might suggest that ribozyme has a greater potential as an inhibitor of HIV-l infection than antisense RNA. The vifgene of HIV-l is required to enhance viral infectivity (Fisher et al., 1987; Strebel et al., 1987). In the in vitro cleavage reaction, efficient cleavage of vif RNA by the hammerhead ribozyme (but not a mutant ribozyme) occurred at pH 7.5 and 37°C in the presence of Mg++ targeted to the vifgene (Lorentzen et al., 1991). No in vivo results have been reported using the anti-vifribozyme. The tat RNA sequence is conserved in various HIV-l isolates and its product is essential for viral replication (Sodroski et al., 1985; Fisher et al., 1986). Several groups have reported that the ribozyme targeted to the tat gene inhibited HIV-l replication in vitro and/or in vivo (Lo et al., 1992; Crisell et al. , 1993; Ohkawa et al., 1993; Zhou et al., 1994). Ohkawa et ul. (1993) have developed a shotguntype ribozyme expression vector by combining cis- and truns-acting ribozymes targeted to LTR-gag or tat HIV-l RNA. The shotgun ribozyme was shown to have greater cleavage activity compared with tandem-type ribozymes in vitro. Zhou et al. (1994) have constructed anti-tat or anti-common exon for tut and rev hammerhead ribozymes cloned into a retroviral vector driven by viral LTR promoter. The vectors were transfected into PA317 amphotropic packaging cells, and then transduced human T-lymphocytes. All transfectants showed resistance to HIV-l replication for 12- 15 days after challenge with HIV-l. Lo et al. (1992) have designed a Moloney retroviral vector containing an anti-tat ribozyme, which targeted the first coding exon of the tat gene of HIV-l. The anti-tat ribozyme-producing Jurkat transfectant was shown to inhibit HIV-l replication for 7 days, but the inhibition was less effective when compared with tat-antisense. However, this ribozyme contained 48 bases of flanking sequence, and may not have achieved optimal cleavage. In order to improve the efficacy of anti-HIV-l ribozyme, several new strategies have been developed. One approach has used multi-targeted ribozymes that were categorized into either shotgun or tandem ribozymes. As mentioned above, the shotgun ribozyme expression vector contained &-acting ribozymes to independently separate each tandem truns-acting ribozyme. The activity of the ribozyme increased in proportion to the number of connected units (range l-10 units) in vitro (Ohkawa et al., 1993). The tandem ribozyme connected several sequences of ribozymes that are specific for different target sites in tandem, and a few groups have reported in vitro and in vivo results (Chen et al., 1992; Ohkawa et al., 1993; Zhou et al., 1994). Chen et al. (1992) have developed mono-, di-, penta-, and nonahammerhead ribozymes targeted to HIV-l env RNA. The nona (9) ribozyme, when co-expressed with the infectious HIV-l clone pNL4-3 in HeLa T4 cells under the control of the HIV-l LTR, dramatically inhibited HIV-l replication. On the other hand, Zhou et al. (1994) have shown that a vector containing both anti-tat and anti-tat/rev ribozymes (tandem type) was equally inhibitory to the vectors that contained either of the ribozymes singly in vivo. Ohkawa et al. (1993) have shown that the activity of the tandem ribozymes reached a plateau at three ribozymes in vitro. Although these tandem ribozymes targeted different sites in the HIV-l genome, the shotgun ribozyme may have greater potential to inhibit HIV-l infection than the tandem ribozyme. A second approach has combined an anti-HIV gene ribozyme with RNA decoys. Lisziewicz et al. (1993) constructed a retroviral vector (TAR-Rib) that contained both anti-gag ribozymes and 50 tandem copies of polymeric Tat activation response elements. The Molt 3 cell line was transduced with the vector and challenged with HIV-l. Surprisingly, TAR-Rib inhibited HIV-l replication by 99%, and the inhibition was maintained over a 14-month period, suggesting the circumvention of escape mutants. This strategy might be one of the most effective to prevent the mutant escape phenomenon. However, it is unclear what role the ribozyme played in this setting, as there was no direct comparison between the effects of the TAR-Rib versus either alone. A third approach has utilized an anti-HIV ribozyme with a long antisense RNA (Crisell et al., 1993; Homann et al., 1993). Homann et al. (1993) have designed a hammerhead ribozyme with 413 nucleotides, a long antisense RNA targeted to the 5’-leader/gag region (2asRz12), the corresponding unmodified antisense RNA (2as) and a mutated ribozyme (2as-Rz-15), and each of these RNAs was co-transfected into human SW480 cells with infectious complete proviral HIV-l DNA. 2asRz12 transfectants showed 4- to 7-fold stronger inhibition of HIV-l replication compared with 2as and 2asRz15. Meanwhile, Crisell et al. (1993) have determined the optimal number of flanking sequence

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nucleotides (range 18-609; the number indicates the sum of Helices I and III) in anti-tat ribozyme in vivo, and established that the optimal length was over 66 nucleotides. Thus, the optimal length of flanking sequence still remains unclear in vivo for inhibition of HIV-l. In another study, chimeric RNA/DNA ribozymes have been developed to increase the resistance of ribozymes to ribonuclease degradation without decreasing their catalytic efficiency (Pieken et al. , 1991; Heidenreich and E&stein, 1992). This motif might be useful for transient exogenous delivery for HIV-l infection. In conclusion, the ribozyme has been employed to inhibit HIV-l infection in numerous studies and encouraging results have been reported, although several problems still remain to be resolved (i.e., optimal target site(s) and delivery system of the ribozyme). Ribozymes may be an effective strategy to inhibit HIV-l infection, and ribozyme gene therapy may exploit HIV-l infection (for a review, see Yu et al. , 1994). 3.3. Other Examples of Ribozyme

Cleavage Targets

Apart from HIV, several pathogenic viruses have also become targets for ribozyme cleavage. One group has described ribozymes against the influenza virus; hammerhead and hairpin ribozymes have been designed to cleave viral RNA segment 5 of influenza A virus (Tang et at., 1994). They have reported that the hammerhead ribozyme was more efficient than the hairpin ribozyme in their in vitro study. The hammerhead ribozyme driven by the Simian virus 40 early promoter or Simian virus 40 early and late promoters was transfected into monkey COS cells; high levels of inhibition (up to 70-80%) of influenza A virus were demonstrated in the COS cells transfected with the ribozyme, which correlated with levels of ribozyme expression. Another group has reported ribozymes targeted against lymphocytic choriomeningitis virus (LCMV); LCMV is a prototype of the arenavirus family of enveloped RNA viruses (Xing and Whitton, 1992). The anti-LCMV ribozymes cleaved different target sites of LCMV RNAs. Furthermore, expression of the anti-LCMV hammerhead ribozyme by the pMAMneo vector containing the MMTV enhancer/promoter reduced levels of LCMV RNA, as well as infectious virus production (Xing and Whitton, 1993). Another target of ribozyme cleavage against a pathogenic virus is the pregenomic RNA of HBV. The pregenomic RNA is transcribed from HBV genomic DNA (von Weizsticker et al., 1992). A triple ribozyme in tandem was designed and cleaved three target sites within the HBV pregenomic RNA; however, in vivo studies have not yet been reported. Investigators have been exploring application of ribozymes to metabolic modulation. One group has described inhibition of fatty acid synthesis by ribozymes against acetyl-CoA carboxylase (ACC) (Ha and Kim, 1994). The anti-ACC ribozymes were cloned into the pCMV-Rb mammalian vector driven by the cytomegalovirus (CMV) promoter, and transfected into 3OA5 preadipocyte cells. The ribozymes decreased ACC mRNA, as well as ACC enzyme activity, and reduced fatty acid synthesis 30-70% in the transfectant cells; this was associated with a reduction of lipid accumulation in the transfected cells. Alzheimer’s disease is characterized by the histopathological presence of senile plaques in the brain. The major component of the plaque cores consist of the Alzheimer amyloid peptide precursor (/3-APP) (Haass et al., 1992; Seubert ef al., 1992). A hammerhead ribozyme was designed against /?-APP and characterized in vitro; the ribozymes cleaved the target mRNA (Denman and Miller, 1993; Denman, 1993). Furthermore, anti-&APP hammerhead ribozymes and anti-P-APP hairpin ribozymes were cloned into the pMEP4 eukaryotic episomal expression vector system and also into the steroid-inducible pMAMneo vector. These vectors were transfected into COS-7 cells (Denman et al., 1994). Hammerhead ribozymes in the pMAMneo vector reduced 25-30% of fl-APP mRNA; meanwhile, both hammerhead and hairpin ribozymes in the pMEP vector decreased 67-80% of the target mRNA in the COS-7 cells. To demonstrate an animal model for diabetes (matmity-onset diabetes of the young), anti-glucokinase ribozyme has,been expressed in transgenic mice (Efrat et al., 1994). The transgenic mice had about 30% of glucokinase activity of the pancreatic islets; insulin release in response to glucose from the pancreas was reduced; however, the plasma glucose and insulin levels of the mice were within normal range.

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The other target of ribozyme cleavage is urokinase-type plasminogen activator receptor (uPAR) mRNA uPAR, which is associated with normal and pathologic processes of cell migration and invasion (Pollanen et aZ., 1991). A hammerhead ribozyme against human uPAR has been designed, and transfected by lipofection into human osteosarcoma cells (Karik6 et al., 1994). The study has suggested that lipofectin enhances rapid transport and stable expression of the ribozyme in an in vivo model system. 3.4. Antisense Versus Ribozyme Antisense deoxynucleotides, as well as ribozymes, have been a mainstay of gene therapeutic agents, and many reports have supported their efficacy for inhibition of targeted gene expression (for reviews, see Neckers et al., 1992; Stein and Cheng, 1993; Mercola and Cohen, 1995). Several studies have compared the efficacy of ribozymes with antisense. These reports suggested that ribozymes were more effective than antisense (Cameron and Jennings, 1989; Scanlon et al., 1991; Sioud and Drlica, 1991; Homann et al., 1993). For instance, a hammerhead ribozyme targeted to TNF-(Y RNA reduced its expression by 90% in HL60 cells, as opposed to 40% achieved by antisense RNA (Sioud et al., 1992). Moreover, mutant ribozymes had no catalytic action and were also less effective suppressors of c-&r (Scanlon et al., 1991; Funato et al., 1992), H-ras (Funato et at., 1994; Tone et al., 1993; Kashani-Sabet et al., 1994), BCR-ABL (Lange et al., 1993), m&-l (Kobayashi et al., 1994a), or HIV-l RNA (Yu et aZ., 1993). In contrast, only a few studies were reported that showed an antisense RNA to be more effective than a ribozyme (Cotten et al., 1989; Lo et al., 1992). Thus, ribozymes have an advantage over antisense for the following reasons: (1) the ribozyme has a potential of cleavage reaction for targeted genes, (2) the process is catalytic and lower concentrations are required than antisense, and (3) long-length antisense may have its own inherent structural limitations. 4. DESIGNING

AND DELIVERING

RIBOZYMES

For application of gene therapy, one has to comprehend determinants for an effective ribozyme, such as the target gene (Sullenger and Cech, 1994), cleavage sites, flanking sequences, ribozyme stability, and delivery systems (Thompson et al., 1995). With the exception of the delivery system, these factors could be determined by optimal ribozyme cleavage conditions in cell-free systems. If ribozyme activities are expressed in cultured cell lines, in vivo studies, or therapeutic application, it will be necessary to discuss viral or nonviral delivery systems. In order to express ribozymes in cultured cancer cell lines, many investigators have cloned them into the pH/3 Apr-1-neo plasmid driven by the human @-actin promoter (Gunning et al. , 1987). Some ribozymes, such as the anti-&s ribozyme, have been cloned into the pMAMneo vector containing the MMTV dexamethasone-inducible promoter (Lee et al., 1981). The steroid-inducible vector expresses transient amounts of ribozyme RNA compared with pHfl Apr-1-neo. The anti-@ ribozyme does not completely cleave all of the c-fis mRNA; a minimal amount of the c-fos gene expression is necessary to maintain cell growth of the cancer cells. Thus, a transiently expressed ribozyme is effective against one type of gene, while a constitutively expressed ribozyme would be active against a mutant oncogene or drug resistance gene (Scanlon et al. , 1991; Kashani-Sabet et al. , 1992). Viral vectors have been used as a gene transfer system with several classes of genes. The vector systems are divided into three categories: (1) vectors previously used in patients (retrovirus, adenovirus); (2) vectors under development (adeno-associated virus); and (3) future viral vectors (Jolly, 1994). Retroviral vectors associated with the murine leukemia virus and family are one of the oldest transfer systems with extensive clinical experience (Anderson et al., 1990). These vectors are characterized by stable integration, potentially long-term expression, and restriction of infection to proliferating cells. Adenoviral vectors, in the form of viruses causing human respiratory infection, are another system recently used in clinical trials. These vectors am characterized by transient (episomal) expression with high-level titers and infecting both dividing and nondividing cells. Adeno-associated virus Type 2 is not an adenovirus, but a nonautonomous parvovirus (Muzyczka, 1992). The parental vector is characterized by stable integration, long-term expression and infecting both dividing and nondividing cells. In general, the delivered genes in the infected cells are driven by promoters, such as CMV promoter, RNA pol III, and Rous sarcoma virus promoter (for a review, see Castanotto et al., 1994).

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However, tissue-specific promoters such as the tyrosinase promoter in melanoma could have specificity and efficacy within the target cells (Vile and Hart, 1993a,b). Lipofectin (cationic liposome) is another gene transfer system (for a review, see Jolly, 1994). Two studies have reported that liposome-encapsulated ribozymes were delivered into human leukemia cell lines (Sioud et al., 1992; Snyder et al., 1993). However, a problem of clinical applications is the high toxicity of the lipofectin formulation; new lipid formulations should be explored in the future.

5. CONCLUSION Since their discovery, the biochemistry of ribozymes has been characterized extensively. Ribozymes are effective modulators of gene expression because of their site-specific cleavage activity. Ribozymes have been targeted to oncogenes (ras, BCR-ABL., c-$x), drug resistance genes, as well as HIV-l. Ribozymes have altered the phenotypes of the target cell lines in culture and in murine model systems. Future studies are required to advance ribozymes as therapeutic agents in clinical application for gene therapy. At present, it is necessary to investigate optimal vector systems for ribozyme expression in animal model systems. Efficacy of modified ribozymes with clinically relevant delivery systems need to be examined. Ribozymes offer minimal toxicity when targeted to tumor-selective genes and tissue-specific promoters. It is anticipated that ribozymes will make an impact in advancing human gene therapy in the near future. Acknowledgements-We would like to thank Ms. Carol Polchow for preparing the manuscript. The research on ribozymes from this laboratory was supported by the National Institutes of Health (CA50618), Tobacco Related Disease Research Program grant from the State of California, and Gene Shears Ltd., Sydney, Australia.

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