Therapeutic Applications Of Transcleaving And Transsplicing Ribozymes

Ribozymes have the potential to become useful therapeutic agents, and currently they are being developed for a wide variety of clinical applications. The vast majority of effort has been expended in the development of trans-cleaving hammerhead and hairpin ribozymes as inhibitors of viral gene expression. In particular, these ribozymes have been targeted to cleave and destroy HIV-1 RNAs to inhibit viral replication in infected cells. The effectiveness of ribozymes as inhibitors of cellular gene expression in both prokaryotes and eukaryotes has been examined, and in a number of studies hammerhead ribozymes have been used to reveal particular gene functions. Trans-cleaving ribozymes are also being developed to target transcripts encoding oncogenes, such as the bcr/abl and cHa-ras. As mentioned earlier, new ribozyme applications are being developed for the group I trans-splicing ribozyme from T. thermophila, which can be used to repair defective cellular RNAs and to revise viral transcripts to give them antiviral activity. In the following section, we discuss the therapeutic applications of trans-cleaving and trans-splicing ribozymes. Because so much effort has been devoted to developing trans-cleaving ribozymes that target HIV-1 RNA, a more a detailed summary of the work performed in this area is presented.

A. Inhibition of Gene Expression by Trans-cleaving Ribozymes

1. Inhibition of HIV Replication by Ribozymes

Several different approaches have been described that employ RNA molecules to render cells resistant to HIV replication, including the use of antisense RNA and RNA decoys (80). Although these and other approaches have been reported to be effective in suppressing HIV replication in infected cells, ribozymes have 2 theoretical advantages as compared with other RNA-based HIV inhibition strategies: (1) cleavage of viral transcripts results in the direct, irreversible inactivation of the target RNA; and (2) fewer ribozymes may be required to inhibit a given target gene effectively because a single ribo-zyme can catalyze multiple cleavage reactions and thus destroy multiple viral transcripts. However, like many other anti-virals, ribozymes may also be sensitive to HIV sequence heterogeneity and effective inhibition may require the use of a combination of these strategies.

Ribozymes may be able to cleave viral target RNAs at a number of stages in the viral life cycle. Potential RNA targets include incoming genomic RNAs, early viral mRNAs, late viral mRNAs, and full-length genomic RNAs that are being encapsidated into virion (Fig. 3). Although cleavage of incoming RNAs would prevent viral integration and therefore be highly effective in protecting cells, the fact that HIV genomic RNAs are encapsidated within a viral core may make these transcripts difficult to access by ribozymes. Moreover, the viral polymerase may initiate reverse transcription before the ribozyme can base pair with and cleave the target sequence, setting up a race between the ribozyme and the reverse tran-scriptase machinery. Nevertheless, several reports suggest that ribozymes may be able to inhibit the initial step of the viral life cycle by cleaving incoming HIV genomic RNAs. However, in most these HIV inhibition studies, cleavage of incoming genomic RNAs has been assessed only semiquantitatively. Differences in the amount of proviral DNA (81) or gag mRNA (82) in ribozyme-expressing cells and controls cells has been only analyzed by PCR amplification reactions, which were not internally controlled. Unfortunately, no system has been developed to date that allows for the direct detection of cleavage products of incoming HIV genomic RNAs in mammalian cells.

Figure 3 HIV RNAs in the context of the viral life cycle. Trans-cleaving ribozymes can potentially inhibit HIV replication by cleaving and destroying viral RNA at a number of steps in the HIV life cycle. (A) Viral genomic RNAs can be targeted prior to reverse transcription into dsDNA and proviral integration. (B) During early gene expression, messenger RNAs encoding regulatory proteins are made. (C) During late gene expression, mRNAs encoding structural proteins are produced. (D) Full-length genomic RNAs are expressed for packaging into viral particles budding from the cell surface.

Figure 3 HIV RNAs in the context of the viral life cycle. Trans-cleaving ribozymes can potentially inhibit HIV replication by cleaving and destroying viral RNA at a number of steps in the HIV life cycle. (A) Viral genomic RNAs can be targeted prior to reverse transcription into dsDNA and proviral integration. (B) During early gene expression, messenger RNAs encoding regulatory proteins are made. (C) During late gene expression, mRNAs encoding structural proteins are produced. (D) Full-length genomic RNAs are expressed for packaging into viral particles budding from the cell surface.

Early viral transcripts may prove to be the most attractive targets for conferring resistance to HIV-1 (Fig. 3). Viral RNAs expressed at this stage of the HIV-1 life cycle, such as those encoding tat, rev, and nef proteins, are not very abundant. Thus, fewer catalytic RNAs may be required to be protective. Cleavage of early transcripts that inhibit the expression of regulatory proteins such as rev should also result in the inhibition late gene expression.

Ribozyme-mediated cleavage of late viral transcripts may not be effective in inhibiting HIV replication. Although these RNAs may be accessible for ribozyme cleavage, their shear abundance would probably require that extremely high levels of ribozyme be expressed to reduce their levels in infected cells. In addition, the detrimental affects mediated by early viral regulatory proteins would not be inhibited, even if late viral gene function was eliminated. An alternative strategy may be to target highly conserved sequences in the long terminal repeats present in all viral RNAs. This approach could result in the inhibition of both early and late gene expression.

As mentioned above, much work has been performed to develop ribozymes for HIV gene therapy strategies. Hammerhead and hairpin ribozymes are particularly well suited for this purpose because of their small size, simple secondary structure, and the ease with which they can be manipulated to target specific HIV substrate RNAs for cleavage. In the first application of this approach to inhibit HIV replication, an anti-gag hammerhead ribozyme was generated that specifically cleaved gag-encoding RNAs in vitro and inhibited HIV-1 replication in a human T cell line (82). Subsequently, such trans-cleaving ribozymes have been designed to target a variety of highly conserved sequences throughout the HIV-1 genome and have been shown to inhibit HIV replication to varying degrees in a number of tissue culture studies [for extensive review, see (83,84)]. Moreover, certain trans-cleaving ribo-zymes have been shown to be able to inhibit the replication of diverse viral strains as well as clinical isolates in primary T cell cultures (81,83-85). Comparisons between catalytically active and inactive forms of these anti-HIV ribozymes have demonstrated that maximal inhibition of virus replication is usually associated with catalytic activity and not simply due to the antisense property of these anti-HIV ribozymes (86-88).

To assess the activity of ribozymes in more clinically relevant settings, human peripheral blood lymphocytes have been stably transduced with a hairpin ribozyme targeting U5 region of the HIV-1 genome. These cells were shown to resist challenge by both HIV-1 molecular clones and clinical isolates (89). More recently, macrophage-like cells, which were differentiated from hematopoietic stem/progenitor cells from fetal cord blood and which were stably transduced with a hairpin ribozyme targeted at the 5' leader sequence, resisted infection by a macrophage-tropic virus (90). Transduction of pluripo-tent hematopoietic stem cells with HIV-resistance genes may represent an avenue to continually generate cells that are resistant to HIV infection. Such stem cells differentiate into monocytes and macrophages, the major targets of HIV-1 infection.

The generation of sequence variants during HIV-1 replication has posed a major problem for immunization strategies and anti-HIV-1 drug therapies designed to suppress viral replication in infected patients. Frequent substitution of amino acids within the variable domains of the HIV-1 env gene has resulted in the emergence of neutralization escape mutants both in cell culture and in vivo. Similarly, the rapid emergence of resistant viral strains has limited the effectiveness of both nucleoside and nonnucleoside analog reverse transcriptase inhibitors. Selection for variants resistant to anti-HIV-1 ribo-zymes is also likely to occur because a single-point mutation at the cleavage site on the substrate RNA could inhibit ribozyme-mediated cleavage of viral transcripts. Unlike small molecule drugs that require substitutions at the protein level, even a silent-point mutation can generate a ribozyme-resistant strain. The affect of single-point mutations at the cleavage site of hairpin (5'NAGHY3', where H = U, C, or A, and Y = C or U) and hammerhead (5'NUXA3', where X = C, U, or A) ribozymes has not been studied, but the more stringent sequence requirement at the cleavage site of the hairpin ribo-zyme could enhance its sensitivity to mutations. Approaches that have been suggested to overcome sequence heterogeneity among HIV isolates include the development of multitargeted ribozymes that cleave a given RNA at multiple sites and to target either single or multiple ribozymes to highly conserved sequences within the HIV genome.

In summary, several studies have suggested that ribozymes can inhibit HIV replication in cell culture experiments when cells are challenged with very low innoculums of HIV. It remains to be tested if this first generation of ribozymes can also inhibit virus replication in HIV-infected patients under conditions of active viremia and where a multitude of quasi-species of the virus preexist. Ultimately, this question will be answered as ribozymes begin to be evaluated in clinical trials in HIV-infected individuals (91-93).

2. Inhibition of Hepatitis C Virus by Ribozymes

In addition to its use as an anti-HIV-1 antiviral, hammerhead ribozymes are also being used to target hepatitis C virus (HCV) infection (94). Fifteen different hammerhead ribo-zymes were designed to target different sites in the conserved 5' untranslated region (UTR) present in all HCV RNAs, and tested for their ability to reduce expression of a luciferase reporter gene downstream of the HCV 5' UTR. Cotransfection of OST7 cells with target and ribozyme plasmids resulted in a 40% to 80% reduction in luciferase activity. In a more realistic setting, inhibition of polio viral replication in HeLa cells was ascertained by a chimeric construct that contains the HCV 5'UTR fused to the polio virus. Several HCV 5' UTR targeted ribozymes inhibited the chimeric HCV-PV replication by 90%, suggesting that hammerhead ribozymes may be useful in reducing viral burden in HCV infection.

3. Inhibition of Tumor Growth and Metastasis by Ribozymes

The use of hammerhead ribozymes targeted to vascular endo-thelial growth factor (VEGF) receptors was investigated to prevent angiogenesis and subsequent tumor growth and metastasis in vivo (95). Ribozymes targeted to Flt-1 (VEGF-1) and KDR (VEGF-2) receptors reduced tumor growth in a meta-static variant of Lewis lung carcinoma, but only the ribozyme targeted to Flt-1 inhibited lung metastasis. In addition, ribozyme inhibition of Flt-1 mRNA expression also reduced liver metastasis in a human metastatic colorectal cancer model. These data suggest that ribozyme inhibition of gene expression in vivo is possible and that inhibition of VEGF receptor expression can reduce tumor growth and metastasis.

B. Trans-cleavage of mRNAs Encoding Dominant Oncogenes

Neoplastic transformation is often associated with the expression of mutant oncogenes. Because ribozymes can be designed to inhibit the expression of specific gene products, their potential as antineoplastic agents is currently being evaluated. For example, hammerhead ribozymes have been reported to be able to suppress the tumorigenic properties of cells harboring an activated human ras gene (96-98). More recently, the bcr/ abl fusion transcript has been the target of many ribozyme studies (99-102). This abnormal mRNA is transcribed from the Philadelphia chromosome, which is present in 95% of patients with chronic myelogenous leukemia (CML) and in many patients with acute lymphocytic leukemia (ALL). In vitro experiments have shown that the 8500-nucleotide long bcr/abl transcript was efficiently cleaved by an anti-bcr/abl ribozyme. In CML blast crisis cell lines, expression of ribo-zymes targeted at bcr/abl mRNA was reported to be able to reduce the production of p210bcr/abl, and bcr/abl transcripts and reduce cell proliferation.

C. RNA Revision by Trans-splicing Ribozymes

Recently, it has been argued that ribozymes may also be able to alter the sequence of targeted RNAs not just destroy them and that such RNA revision may be useful for treating a variety of diseases via gene therapy (12). As described earlier, the Tetrahymena group I ribozyme can catalyze a trans-splicing reaction (20,53). Such targeted trans-splicing can potentially be used to repair mutant transcripts and to alter viral RNAs to give them antiviral activity.

In the targeted trans-splicing reaction, the Tetrahymena ribozyme recognizes and binds to its substrate RNA (the 5' exon) by base pairing between the IGS and a sequence in the substrate. Following cleavage, the ribozyme splices its 3' exon onto the cleaved substrate RNA (Fig. 2c). Because the ribo-zyme cleaves after the sequence N5U, the only sequence requirement for the substrate is to have a uridine residue preceding the cleavage site. Thus, any uridine nucleotide in an RNA molecule can in principle serve as a target for the ribozyme if the target sequence is accessible for ribozyme binding. Moreover, because there are no sequence requirements for the 3' exon in the trans-splicing reaction (2), almost any sequence can be spliced onto the 5' target transcript.

The lack of sequence requirements for the 3' exon suggests that it could be manipulated so that trans-splicing can be employed to replace a defective portion of an RNA transcript with a functional sequence (Fig. 4). Trans-splicing ribozymes could be designed that would cleave defective transcripts upstream of point mutations or small insertions or deletions. A 3' exon consisting of the wild-type sequence could then be spliced onto the cleaved target, resulting in a corrected mRNA. Trans-splicing ribozymes can be employed to repair defective RNA messages. In the first example of this application, the group I ribozyme from T. thermophila was reengineered to repair truncated lacZ transcripts via targeted trans-splicing in E. coli (103) and in mammalian cells (104). In both settings, the ribozyme was shown to be able to splice restorative sequences onto mutant lacZ target RNAs with high fidelity and thus maintain the open reading frame for translation of the repaired transcripts. In a subsequent study, the efficiency of RNA repair was monitored and the ribozyme was shown to be able to revise up to 50% of the truncated lacZ transcripts when ribozyme and lacZ substrate- encoding plasmids were cotransfected into mammalian fibroblasts (105).

More recently, several groups have demonstrated that group I ribozymes can be employed to amend faulty transcripts that are associated with common genetic diseases. Phy-lactou et al. demonstrated that a trans-splicing ribozyme could be employed to amend transcripts associated with myotonic dystrophy (106), Lan et al. employed this RNA repair approach to correct mRNAs associated with sickle-cell disease (Fig. 4) (107), Watanabe et al. corrected mutant p53 tran-scripts,(108) and Rogers et al. repaired mutant canine skeletal muscle chloride channel (109). In the myotonic dystrophy case, a trans-splicing ribozyme was employed to shorten the trinucleotide repeat expansion found in the 3' untranslated region of the human myotonic dystrophy protein kinase transcript in cell culture studies (106). In the sickle-cell experiments, trans-splicing was employed to convert sickle p-globin transcripts into 7-globin-encoding mRNAs in erythrocyte precursors isolated from patients with sickle-cell disease (107). In both studies, sequence analysis of the amended RNAs demonstrated that the ribozyme had accomplished such repair with high fidelity, forming the proper splice junctions between the targeted transcript and the corrective sequences. In addition, a trans-splicing ribozyme was employed to repair mutant p53 in human cancer cells. After trans-splicing, repaired functional p53 was monitored by the induction of luciferase gene under the control of a p53-dependent promoter (108). These data suggest that ribozyme repaired mRNAs are translated in mammalian cells and retain wild-type activity. Finally, Rogers et. al. used a trans-splicing ribozyme to correct a mutant canine skeletal muscle chloride channel (cCIC-1). Although repair efficiency was low (1.2%) when ascertained by quantitative reverse transcription and polymerase chain reaction (RT-PCR) in a population of cells, patch-clamp analysis of individual cells yielded a wide range of repair efficiency with 18% of cells showing some restoration and several cells showing complete restoration of wild-type function (109).

Trans-splicing ribozymes have recently been employed to deliver new gene activities to E. coli (110), yeast (111), and mammalian cells (112). Kohler et al. (110) designed a trans-splicing ribozymes that contain a 3' LacZ exon and targets either chloramphenicol acetyltransferase, HIV-1, or cucumber mosaic virus sequences. After induction of target and ribo-zyme expression in E. coli, trans-splicing was monitored by LacZ expression. In addition, Ayre et al. (111) investigated the utility of using a ribozyme that targets the coat protein of cucumber mosaic virus to inhibit viral propagation while selectively destroying virally infected cells. A trans-splicing ribozyme was developed that contains the diptheria toxin A chain (DTA) as a 3' exon, which is spliced in frame to the viral target sequence. After trans-splicing, yeast cells that express the spliced viral target RNA fail to grow. Finally, this new genetic approach was developed to combat HVC infection in human cells (112). A trans-splicing ribozyme was targeted to a site in the HCV internal ribosome entry site (IRES) present in all viral RNAs and used for cap-independent translation of viral genes in mammalian cells. The 3' exon contained the IRES sequence after the splice site fused in-frame to the DTA. After trans-splicing and IRES-mediated translation of the DTA, virally infected cells were destroyed by apoptosis. These data suggest that trans-splicing ribozymes can be used specifically and selectively to deliver new gene activities to a variety of cell types.

These results demonstrate that a trans-splicing group I ribozyme can be employed to repair pathogenic transcripts in clinically relevant, cellular settings. However, as with the development of almost every novel therapeutic approach, several technical issues must be addressed before ribozyme-mediated repair of mutant RNAs can become useful in the clinic. First, it remains to be determined whether repair of any pathogenic transcript can proceed efficiently enough in primary human cells to be therapeutically beneficial. In the case of sickle-cell disease, conversion of as little as 5% to 10% of the sickle p-globin transcripts into mRNAs encoding 7-globin is expected to greatly reduce cell sickling and thus the severity of the disease. Whether this relatively modest level of repair can be achieved in erythrocyte precursors from individuals with sickle cell disease is unclear but results demonstrating that 50% of the mutant lacZ transcripts expressed in mammalian cells can be revised by ribozymes (105) is at least encouraging in this regard. Second, the specificity of trans-splicing may have to be increased because in mammalian cell experiments the Tetrahymena

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Figure 4 Ribozyme-mediated repair of mutant transcripts. A trans-splicing ribozyme (green) binds to a mutant RNA transcript that contains a point mutation, deletion, or insertion. The IGS on the ribozyme recognizes a uridine residue (red) 5' of the mutation (indicated by Xm). The ribozyme cleaves the mutant RNA and releases the downstream, mutation-containing cleavage product (light blue). Next, the ribozyme ligates the wild-type sequence (Xwt and yellow) of the target RNA onto the upstream cleavage product to yield a repaired transcript. See the color insert for a color version of this figure.

Figure 4 Ribozyme-mediated repair of mutant transcripts. A trans-splicing ribozyme (green) binds to a mutant RNA transcript that contains a point mutation, deletion, or insertion. The IGS on the ribozyme recognizes a uridine residue (red) 5' of the mutation (indicated by Xm). The ribozyme cleaves the mutant RNA and releases the downstream, mutation-containing cleavage product (light blue). Next, the ribozyme ligates the wild-type sequence (Xwt and yellow) of the target RNA onto the upstream cleavage product to yield a repaired transcript. See the color insert for a color version of this figure.

group I ribozyme was shown to react not only with intended lacZ target RNAs, but also with other cellular transcripts (104). Such limited reaction specificity is fully anticipated from knowledge about the energetics of substrate binding by this ribozyme. This biochemical knowledge is now being used to redesign the ribozyme to enhance its specificity (51).

In summary, the ability to employ trans-splicing ribozymes to revise genetic instructions embedded in targeted RNAs represents a broad new approach to genetic therapy. Because defective RNAs can only be repaired in the cells in which they are present and only when they are expressed, RNA repair may become an effective means of recapitulating the natural expression pattern of therapeutic genes. Moreover, RNA repair may be especially useful in the treatment of genetic disorders that are associated with the expression of dominant or deleterious mutant RNAs and proteins. In these cases, RNA repair should simultaneously engender wild-type protein production and eliminate production of the deleterious gene product. For these reasons, the concept of RNA repair is likely to continue to attract increased interest from gene therapists.

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