Efficacy Of Ribozyme Research Articles

Intracellular efficacy of tumor‐targeting group I intron‐based trans‐splicing ribozyme

Group I intron-based trans-splicing ribozyme, which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA, could be a useful tool for tumor-targeted gene therapy. In the present study, the therapeutic feasibility of this ribozyme was investigated by analyzing trans-splicing efficacy in vivo as well as in cells. We assessed transgene activation, degree of ribozyme expression, targeted hTERT mRNA level, or the level of trans-splicing products in hTERT(+) cells or in human tumor nodules xenografted in animals after ribozyme administration. The activity and efficacy of the trans-splicing ribozyme in cells was dependent on the amount of endogenous hTERT mRNA and/or the accumulation of ribozyme RNA in cells. Intracellular activity of the ribozyme reached a plateau when no more targetable substrate mRNA was available or the ribozyme RNA level was fully saturated. In addition, the efficacy of ribozyme in xenografted tumor tissues was dependent on the dose of the delivered ribozyme-encoding adenoviral vector, indicating the potential of the ribozyme expression level as a determining factor for the in vivo efficacy of the trans-splicing ribozyme. On the basis of these results, we enhanced the intracellular ribozyme activity by increasing the ribozyme expression level transcriptionally and/or post-transcriptionally. We analyzed ribozyme efficacy and determined the most influential factors of its trans-splicing reaction in mammalian cell lines as well as in vivo. The present study could provide insights into the optimization of the trans-splicing ribozyme-based RNA replacement approach to cancer treatment.

Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons: Inhibition of endogenous caspase‐3 activity and protection from apoptotic cell death

Antisense technology, including ribozyme and small interfering RNA, is being developed to mediate the down-regulation of specific intracellular genes. It was observed in this study that both antiluciferase ribozymes and short hairpin RNAs (shRNAs) could significantly reduce the activity of exogenously expressed luciferase in primary hippocampal neurons in a viral titer-dependent manner. shRNAs were more effective gene-silencing agents than ribozymes, although they exhibited some nonspecific gene-silencing effects at high viral titers. We also attempted to increase ribozyme efficacy by using a woodchuck hepatitis posttranscriptional regulatory element (WPRE) in the ribozyme expression cassette. The results showed that adenoviral vectors encoding specific ribozymes could silence the cellular expression of luciferase and endogenous procaspase-3 significantly. Furthermore, the antiprocaspase-3 ribozyme was shown to inhibit staurosporine-mediated cell death. The addition of a WPRE did not, however, increase or decrease ribozyme activity. As far as we are aware, this is the first example of adenovirally mediated delivery of hammerhead ribozymes being used to manipulate gene expression in primary neurons. The results therefore suggest that hammerhead ribozymes may be useful tools for studying neuronal gene function and have potential as therapeutic agents to treat CNS diseases.

547. Antiviral RNA Interference Strategies That Resist HIV Escape

The ribozyme 9456, a small RNA molecules having catalytic activity, has been designed to specifically cleave target sequences in the U3 region of SIV. Recent developments have indicated that lentiviral vectors may transduce quiescent cells and therefore provide more efficient gene transfer to important target populations for AIDS gene therapy. However, the optimal position within the lentiviral vector for expression of small RNA inhibitors, especially using internal polymerase III promoters, has not been determined. To compare expression of the tRNAval Pol III promoter/SIV-specific ribozyme 9456 inhibitor cassette, we generated a murine leukemia virus (MLV) vector with the inhibitor in the antisense orientation and a series of a self-inactivating lentiviral vectors (HRST) with the inhibitor in different positions in both the sense and antisense orientations (before RRE: P1/invP1, after CMV-GFP: P2/invP2, in the ΔU3 of the 3′ HIV LTR: P3/invP3). The CD4+ CEMx174 cell line was transduced with the MLV vector before selecting individual transduced clones or with the HRST vectors before sorting by green fluorescent protein (GFP) expression. Total RNA was isolated from transduced cells for analysis by quantitative, real-time RT-PCR for the expression of ribozyme 9456, MLV vector, HRST vector and β-actin sequences. To assess inhibition of viral replication, transduced cells were challenged with an MOI of 10−2 TCID50/cell of SIVmac239 and followed over fourteen days for viral replication. Expression of GFP from the internal CMV promoter with the HRST vectors was assessed by flow cytometry and transduced cells were >90% positive. Expression of the ribozyme 9456 inhibitor relative to expression of the β-actin gene in the MLV-transduced clones and the HRST-transduced populations ranged between 1.1 × 103 to 9.0 × 104 and 3.0x103 to 1.3 × 105 units, respectively. Expression of the ribozyme 9456 was highest in the sense orientation of P2 and P3 of the HRST vectors. The MLV vector backbone showed high levels of expression ranging from 2.2 × 106 to 2.9 × 107 units, while the HRST vector was lower but detectable ranging between 3.8 × 103 to 1.4 × 104 units, indicating that the ΔU3 promoter in the HIV LTR of the HRST vectors is not completely inactive. The MLV clones with the highest levels of 9456 expression also provided the strongest inhibition of viral replication. Many of the HRST vectors (P3, invP1, invP2, invP3) showed strong inhibition (>80%). Inhibition with P1 and P2 was modest even though P2 expressed the 9456 at the highest level. Inhibition with the HRST vectors was consistently stronger than in the MLV clones. These vectors demonstrate variable expression of the ribozyme inhibitor from a Pol III promoter in HRST and MLV vector backbones and may provide valuable insight into the expression of other small RNA molecules in transduced cells. Optimization of delivery of ribozymes by lentiviral vectors should facilitate analysis of the efficacy of ribozymes for stem cell gene therapy for AIDS in a nonhuman primate model.

RNase P ribozyme as an antiviral agent against human cytomegalovirus.

Human cytomegalovirus (HCMV) represents one of the most medically important human viruses and causes a wide spectrum of human diseases, including birth defects and mental retardation in newborns, common opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients (e.g., CMV-associated retinitis and pneumonia), and possibly cardiovascular diseases such as atherosclerosis. This chapter describes the utilization of RNase P ribozyme-specifically, M1GS ribozyme, as a gene-targeting agent for blocking HCMV gene expression and growth. The target for the RNase P ribozyme is the overlapping region of the mRNAs that code for HCMV major transcription factors IE1 and IE2, which are essential for viral gene expression and replication. The methods described in this chapter focus primarily on i) construction of the retroviral vector for expression of M1GS ribozymes in cultured cells, ii) generation of stable cell lines expressing ribozymes, iii) determination of the expression of M1GS RNAs in human cells, and iv) evaluation of the efficacy of ribozymes in inhibiting HCMV IE1/IE2 expression and viral growth. Using these methods, we successfully constructed M1GS RNAs against the IE1/IE2 mRNA sequence and recently showed that a reduction of up to 150- to 3000-fold in HCMV growth is found in cells that express the ribozymes.

Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes.

The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.

Nuclease-resistant synthetic ribozymes: developing a new class of therapeutics.

The discovery of ribozymes by Cech and Altman has fundamentally changed the view of the function of RNA in chemistry, biology, and medicine (1–3). Prior to this discovery, RNA had been viewed as a passive molecule that only carried information or provided structure to RNA-protein complexes. It is now clear that RNA can act as an enzyme and is capable of catalyzing RNA splicing and cleavage, as well as several other chemical reactions. These novel activities of RNA now permit the development of enzymatic RNA molecules as therapeutic agents that can suppress the expression of deleterious proteins by catalyzing the trans-cleavage of the corresponding mRNAs (4). RNA targets for ribozyme-based therapeutics may encode oncoproteins, growth factors, proinflammatory cytokines and their corresponding cell-surface receptors, and signal transduction molecules; viral and microbial mRNAs or genomic RNAs are also readily cleaved by this approach. RNA-cleaving ribozymes gain their target specificity from Watson-Crick base-pairing between the ribozyme’s binding-arm sequences and sequences that flank the cleavage site of the target RNA. Once bound, their mechanism of cleavage involves attack of the 2′-OH that is 5′ to the scissile bond in the target, thus destabilizing the target RNA’s phosphate backbone. Upon cleavage, the resultant products dissociate from the ribozyme complex and the ribozyme is released and may bind and cleave other targets again. The cleavage event renders the mRNA untranslatable and leads to further degradation of the target by cellular ribonucleases. With the recent completion of the sequencing of the human genome and several viral and bacterial pathogens, the number of potential ribozyme drug targets is enormous. This number will increase as the functions of all the genes in the human genome become clearer and the number of pathogens being sequenced increases. However, if they are to reach the clinic, ribozymes directed at these targets will need to be designed so that they can survive in nuclease-rich biological tissues and fluids, have a favorable pharmacokinetic (PK) profile, and prove effective in preclinical cell culture and animal models. Several groups have demonstrated the efficacy of ribozymes in cell culture and animal models. To date, only three ribozymes — one vector-expressed and two synthetic — have been reported in clinical trials. The first, a retrovirally expressed ribozyme that targets the HIV tat and rev exons, entered clinical testing in late 1996 and is currently in phase II testing for patients with AIDS-related lymphoma. The second, ANGIOZYME, is directed against the mRNA for Flt-1 (VEGF-R1), the high-affinity receptor for VEGF. This antiangiogenic ribozyme entered phase I trials in late 1998, phase I/II trials in late 1999, and is currently in phase II trials for several tumor types. The third, HEPTAZYME, a ribozyme targeting the 5′–untranslated region (5′-UTR) of the hepatitis C virus (HCV) RNA genome, has recently completed a phase I/II clinical trial in patients with chronic hepatitis C. This perspective will focus on nuclease-resistant, chemically synthesized ribozymes, as exemplified by ANGIOZYME and HEPTAZYME. These ribozymes can be readily synthesized, can be administered subcutaneously (s.c.) or intravenously (i.v.), have excellent specificity, are well tolerated, and function well in several in vitro and in vivo model systems. The clinical efficacy of these two ribozymes is being assessed currently. If they, or others in preclinical development, show a clinical effect, then the potential utility of nuclease-resistant ribozymes in medicine will be realized, with many more applications to follow.

Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes.

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

Dose-response resistance to HIV-1/MuLV pseudotype virus ex vivo in a hairpin ribozyme transgenic mouse model.

We have investigated the efficacy of a hairpin ribozyme targeting the 5' leader sequence of HIV-1 RNA in a transgenic model system. Primary spleen cells derived from transgenic or control mice were infected with HIV-1/MuLV pseudotype virus. A significantly reduced susceptibility to infection in ribozyme-expressing transgenic spleen cells (P = 0.01) was shown. Variation of transgene-expression levels between littermates revealed a dose response between ribozyme expression and viral resistance, with an estimated cut off value below 0.2 copies of hairpin ribozyme per cell. These findings open up possibilities for studies on ribozyme efficacy and anti-HIV-1 gene therapy.

MRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes.

Subcellular localization signals for several mRNAs are positioned in their 3' untranslated regions (UTR). We have utilized the human alpha- and beta-actin 3' UTRs as signals for colocalizing hammerhead ribozymes with a lacZtarget mRNA. Ribozyme and target genes containing matched or unmatched 3' UTRs were cotransfected into 12-day-old chicken embryonic myoblast and fibroblast (CEMF) cultures and assayed by in situ hybridization (ISH) using a dual label, antibody sandwich procedure, and dual fluorescence microscopy to monitor intracellular colocalization. Beta-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl beta-D-galactopyranoside (ONPG) assay. We found that the percentage of colocalization using the matched alpha- or beta-actin 3' UTR (alpha-alpha or beta-beta) was enhanced approximately threefold relative to unmatched 3' UTRs. The increase in ribozyme-mediated inhibition of beta-galactosidase activity observed when matched 3' UTRs were used was consistent with the observed percentage of colocalization. These results represent the first direct demonstration that mRNA localization signals (zipcodes) can be utilized to enhance intracellular ribozyme efficacy.

Efficient Hammerhead Ribozymes Targeted to the Polycistronic Sendai Virus P/C mRNA: STRUCTURE-FUNCTION RELATIONSHIPS

The Sendai virus polycistronic P/C mRNA encodes the P and C proteins from alternate overlapping reading frames. To determine the functions of these proteins in virus replication, hammerhead ribozymes were targeted to cleave the 5'-untranslated region of the P/C mRNA. Both cell-free and intracellular assays were employed to determine ribozyme efficacy. To appropriately compare activities between cell-free and intracellular assays, identical ribozymes were synthesized in vitro as well as expressed in cells. Ribozyme parameters, namely hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES), were found to have rate-determining properties. In cell-free reactions, ribozymes with 13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL. Ribozymes with 9-mer HAL were relatively ineffective in transfected cells. Minimizing the number of NES increased ribozyme efficiency in vitro. However, ribozymes with minimal NES were essentially inert intracellularly. The NES at the termini of the most effective intracellular ribozyme, Rz13st ( approximately 95% inhibition of the p gene expression), were predicted to fold into stem-loop structures. These structures most likely increase ribozyme stability as evidenced by the 8-fold higher resistance to ribonuclease T2 digestion of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of productive ribozyme:substrate duplexes and that increase RNA stability should be optimized.

Enhancement of Hammerhead Ribozyme Catalysis by Glyceraldehyde-3-phosphate Dehydrogenase

A specific tumour necrosis factor alpha ribozyme (TNF-α-Rz) binding activity has been purified and identified by N-terminal microsequencing as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The purified protein as well as commercial GAPDH binds tightly to TNF-α ribozyme compared to a variety of other ribozymes and RNAs. Binding of GAPDH to the TNF-α-Rz and its derivatives was inhibited by NAD+ and ATP, suggesting that the GAPDH Rossmann fold structure is a part of the ribozyme binding site. Interestingly, GAPDH increased the in vitrocleavage rates of hammerhead ribozymes by up to 25-fold, while no significant stimulation was observed with the lactate dehydrogenase (LDH). This effect was found to be due to the unfolding activity of GAPDH. In fact, pulse – chase experiments demonstrate directly that GAPDH has the capacity to accelerate the ribozyme/substrate association, especially of ribozymes and/or substrates whose predicted secondary structure might interfere with the association step. Under our conditions, the presumed unfolding activity of GAPDH also enhances the turnover of ribozymes by increasing the rate of product dissociation, although only for short cleavage products. Longer duplexes required more incubation time to dissociate. In vitronon-specific interaction of the GAPDH with hammerhead ribozymes and RNA substrates was found to be adequate for the cleavage enhancement effect to occur. However, an analysis of the ability of various prototypical ribozymes to inhibit the expression of interleukin-2 suggests that the addition of a sequence having a high affinity for GAPDH improves the efficacy of ribozymes in the cells. Thus, the characterization of cellular proteins with unfolding activity, which specifically bind to hammerhead ribozyme, should facilitate the design of a more effective ribozyme in vivo.

Comparison of the antiviral efficacy of ribozymes and antisense RNA directed against bovine leukemia virus rex/tax.

Despite the theoretical attraction of ribozymes, which cleave their target RNA, as compared with antisense RNA, which only blocks translation, few studies have assessed the relative efficacy of ribozymes and antisense RNA directed against the identical target sequence. Previously, we described the efficacy of a hammerhead ribozyme targeted against rex/tax, a regulatory gene of bovine leukemia virus (BLV). In this study, we asked whether antisense RNA targeted against the same site would also be efficacious. BLV-infected bat lung cells were transfected with an antisense RNA-encoding plasmid under the control of sarcoma virus (RSV) promoter, and stable cell lines were selected. No inhibition of viral p24 expression was demonstrated in seven cell lines transfected with the antisense RNA targeted at the same site as the ribozyme or in three cell lines transfected with an antisense sequence targeted against the tax 5' initiation codon. Although in previous experiments antisense DNA oligonucleotides inhibited Tax expression in vitro, stably transfected plasmids encoding antisense RNA of the same sequence did not inhibit viral expression in BLV-infected cells in this study. These results suggest that in persistently infected cells producing high levels of BLV, the ribozyme is more effective than antisense RNA directed at rex/tax transcripts.

Alternative Approaches for the Application of Ribozymes as Gene Therapies for Retroviral Infections

Publisher Summary Ribozymes (ribonucleotide enzymes) are RNA molecules derived from these self-splicing introns that function as sequence-specific endoribonucleases. The ability to target ribozymes against specific RNA molecules has led to the development of strategies in which ribozymes are employed to specifically destroy gene transcripts and thereby inhibit gene expression. There are at least five ribozyme motifs that show promise as sequence specific inhibitors of gene expression. The function, structure, and possible applications of these ribozymes vary greatly, and some ribozymes may be better suited for a particular application than others. Although theoretically ribozymes could be used in gene therapy approaches to a number of infectious, oncologic, rheumatic, and cardiovascular diseases, most of the work in this field till date has focused on the use of ribozymes to inhibit replication of human immunodeficiency virus type 1 (HIV-1). In tissue culture, ribozymes have shown promise in gene therapy approaches to the inhibition of HIV-1 replication, but the major limitation of this application of ribozymes is the development of systems to efficiently deliver ribozymes to HIV-1 host cells. The efficacy of ribozymes in the treatment of HIV-1 infection ultimately can be determined in clinical trials in HIV-1-infected individuals. Lastly, ribozymes are capable of more than just cleavage of other RNA molecules.

Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA.

The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV-1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect.

Practical Ribozymes: Making ribozymes work in cells

Ways are being found to increase the efficacy of ribozymes in cells, offering hope that they may soon begin to fulfill their promise as new reagents for inactivating specific RNAs in cells.