Two tales of Annexin A2 knock-down: One of compensatory effects by antisense RNA and another of a highly active hairpin ribozyme.

Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.

Trace of survivin in cancer.

Survivin is one of the most cancer-specific proteins overexpressed in almost all malignancies, but is nearly undetectable in most normal tissues in adults. Functionally, as a member of the inhibitor of apoptosis family, survivin has been shown to inhibit apoptosis and increase proliferation. The antiapoptotic function of survivin seems to be related to its ability to inhibit caspases directly or indirectly. Furthermore, the role of survivin in cell cycle division control is related to its role in the chromosomal passenger complex. Consistent with its determining role in these processes, survivin plays a crucial role in cancer progression and cancer cell resistance to anticancer drugs and ionizing radiation. On the basis of these findings, recently survivin has been investigated intensively as an ideal tumor biomarker. Thus, multiple molecular approaches such as use of the RNA interfering technique, antisense oligonucleotides, ribozyme, and small molecule inhibitors have been used to downregulate survivin regulation and inhibit its biological function consequently. In this review, all these approaches are explained and other compounds that induced apoptosis in different cell lines through survivin inhibition are also reported.

Development of Optimized Inhibitor RNAs Allowing Multisite-Targeting of the HCV Genome.

Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome's internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications.

Targeting of Receptor Activator of Nuclear Kappa B (RANK) in PC-3 Cells Increases Cell Proliferation and Matrix Adhesion In Vitro.

BACKGROUND: In Western societies, prostate cancer is the most frequently diagnosed cancer amongst men. Efforts to improve diagnosis and treatment remain a major focus and have been proven beneficial in the approach to localised disease. However, currently, metastatic disease management still remains palliative. Receptor activator of nuclear kappa B (RANK) has been extensively studied in bone biology and immunology, whilst several links have been made between RANK-positive breast cancer cells and disease progression. Its role in prostate cancer biology remains poorly understood, therefore the aim of this study was to explore the functional role of endogenously produced RANK in metastatic PC-3 prostate cancer cells in isolation and in response to hepatocyte growth factor (HGF). MATERIALS AND METHODS: RANK expression was targeted using hammerhead ribozyme technology in PC-3 prostate cancer cells, and verified by polymerase chain reaction and western blot. A variety of in vitro functional assays were conducted, including cell proliferation and matrix adhesion in the presence of HGF. RESULTS: Suppression of RANK expression was successfully targeted with anti-RANK hammerhead ribozyme transgenes, as verified by PCR and western blot. Reduced RANK expression resulted in significantly increased PC-3 cell proliferation (p<0.01) and cell-matrix adhesion (p<0.05) compared to control cells. CONCLUSION: Previous work into RANK and prostate cancer has focused on its interaction with the bone environment, particularly with regard to its receptor RANK ligand. This study has shown that endogenous RANK expression changes might also influence prostate cancer cell behaviour. Further work is now required to elucidate the signaling pathways involved in these processes.

Enhancing the pharmacokinetic/pharmacodynamic properties of therapeutic nucleotides using lipid nanoparticle systems.

Although activity has been reported in vivo, free nucleic acid-based drugs are rapidly degraded and cleared following systemic administration. To address these challenges and improve the potency and bioavailability of genetic drugs, significant efforts have been made to develop effective delivery systems of which lipid nanoparticles (LNP) represent the most advanced technology currently available. In this review, we will describe and discuss the improvements to the pharmacokinetic and pharmacodynamic properties of nucleic acid-based drugs mediated by LNP delivery. It is envisioned that the significant improvements in potency and safety, largely driven by the development of LNP encapsulated siRNA drugs, will be translatable to other types of genetic drugs and enable the rapid development of potent molecular tools and drugs.

Immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics.

INTRODUCTION: Nucleic acid-based therapeutics (NATs) are proven agents in correcting disorders caused by gene mutations, as treatments against cancer, microbes and viruses, and as vaccine adjuvants. Although many traditional small molecule NATs have been approved for clinical use, commercialization of macromolecular NATs has been considerably slower, and only a few have successfully reached the market. Preclinical and clinical evaluation of macromolecular NATs has revealed many assorted challenges in immunotoxicity, hematotoxicity, pharmacokinetics (PKs), toxicology and formulation. Extensive review has been given to the PK and toxicological concerns of NATs including approaches designed to overcome these issues. Immunological and hematological issues are a commonly reported side effect of NAT treatment; however, literature exploring the mechanistic background of these effects is sparse. AREAS COVERED: This review focuses on the immunomodulatory properties of various types of therapeutic nucleic acid concepts. The most commonly observed immunological and hematological toxicities are described for various NAT classes, with citations of how to circumvent these toxicities. EXPERT OPINION: Although some success with overcoming immunological and hematological toxicities of NATs has been achieved in recent years, immunostimulation remains the main dose-limiting factor challenging clinical translation of these promising therapies. Novel delivery vehicles should be considered to overcome this challenge.

Targeted anticancer effect through microRNA-181a regulated tumor-specific hTERT replacement.

We previously generated a group I intron-based ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to stimulate transgene activity in cancer cells expressing the target RNA via an accurate and specific trans-splicing reaction. One of the major concerns of the hTERT RNA targeting anti-cancer approach is the potential side effects to hTERT(+) hematopoietic stem cell-derived blood cells. Thus, here we modified the ribozyme by inserting target sites against microRNA-181a, which is a blood cell-specific microRNA, downstream of its 3' exon. The specificity of transgene induction and anticancer activity in hTERT(+) cancer cells improved significantly with the modified ribozyme, resulting in selective targeting of hTERT(+) cancer cells, but not hematopoietic cells even if they are hTERT-positive. Importantly, the trans-splicing reaction of the microRNA-regulated ribozyme worked equally well in a nude mouse model of hepatocarcinoma-derived intrasplenic carcinomatosis, inducing highly specific expression of a therapeutic transgene and efficiently regressing hTERT-positive liver tumors with minimal liver toxicity when systemically delivered with an adenoviral vector encoding the ribozyme. These results suggest that a combined approach of microRNA regulation with targeted RNA replacement is more useful for effective anti-cancer treatment.

Design and characterization of a twin ribozyme for potential repair of a deletion mutation within the oncogenic CTNNB1-ΔS45 mRNA.

RNA repair is an emerging strategy for gene therapy. Conventional gene therapy typically relies on the addition of the corrected DNA sequence of a defective gene to restore gene function. As an additional option, RNA repair allows alteration of the sequence of endogenous messenger RNAs (mRNAs). mRNA sequence alteration is either facilitated by intracellular spliceosome machinery or by the intrinsic catalytic activity of trans-acting ribozymes. Previously we developed twin ribozymes, derived from the hairpin ribozyme, by tandem duplication and demonstrated their potential for patchwise RNA repair. Herein we describe the development of such a twin ribozyme for potential repair of a deletion mutation in the oncogenic CTNNB1-ΔS45 mRNA. We demonstrate that hairpin ribozyme units within the twin ribozyme can be adapted to efficiently cleave/ligate non-consensus substrates by introduction of compensatory mutations in the ribozyme. Thus, we show the twin ribozyme mediated repair of truncated CTNNB1 transcripts (up to 1000 nt length). Repair of the entire CTNNB1-ΔS45 mRNA, although apparently possible in general, is hampered in vitro by the secondary structure of the transcript.

[Determination of the in vitro antiviral activity of an engineered M1GS ribozyme that targets to the core gene of hepatitis C virus].

OBJECTIVE: Hepatitis C virus (HCV) is one of the major pathogens that lead to viral hepatitis. At present, Interferon treatment in combination with ribavirin is the first line clinical therapeutic approach. However, the responses are usually poor and the viral infection reoccurs. Therefore, exploring new antiviral agents and therapies is under urgent needs. METHODS: The sequence and structure of the core coding region of HCV genome were analyzed through the two computer software, DNAMAN and RNA Structure. The cytosine 52 nt downstream of the AUG initiation triplet was identified as the optimal target cleavage site. Based on the flanking sequence of this assumed cleavage site, a guide sequence (GS) was designed and covalently linked to the 3 prime terminus of the M1 RNA, which is catalytic subunit of the RNase P derived from Escherichia coli using PCR. We named this new targeting ribozyme M1GS-HCV/C52 and it antiviral activities were analyzed in cultured cells. RESULTS: In the in vitro cleavage assay, M1GS-HCV/C52 ribozyme could effectively cleave the HCV target RNA into two fragments at the specific cleavage site. Moreover, comparing to the blank control, this engineered M1GS ribozyme could reduce the core protein expression of more than 80% in the HCV-infected host cell and lead to a 1500-fold reduction of HCV RNA copies in the culture supernatant. An another M1GS ribozyme, M1GS-HCV/C52*, which has the same guide sequence but does not contain a 24nt-long bridge sequence, did not exhibit apparent inhibition for the expression of HCV core gene and viral proliferation in our paralleled assay. CONCLUSION: We successfully constructed an M1GS ribozyme showing affective and specific cleavage of target viral RNA. Further results showed that the engineering ribozyme had notably antiviral activity in cultured cells, thus provided a new promising approach for clinical anti-HCV therapeutic strategy.

[Construction of an engineered M1GS-HCV/C141 ribozyme and determination of its antiviral activity in vitro].

Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this M1IGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.

An engineered inhibitor RNA that efficiently interferes with hepatitis C virus translation and replication.

Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry site (IRES), mainly located at the 5'untranslatable region (5'UTR) of the viral genome. Viral protein synthesis clearly differs from that used by most cellular mRNAs, rendering the IRES an attractive target for novel antiviral compounds. The engineering of RNA compounds is an effective strategy for targeting conserved functional regions in viral RNA genomes. The present work analyses the anti-HCV potential of HH363-24, an in vitro selected molecule composed of a catalytic RNA cleaving domain with an extension at the 3' end that acts as aptamer for the viral 5'UTR. The engineered HH363-24 efficiently cleaved the HCV genome and bound to the essential IIId domain of the IRES region. This action interfered with the proper assembly of the translationally active ribosomal particles 48S and 80S, likely leading to effective inhibition of the IRES function in a hepatic cell line. HH363-24 also efficiently reduced HCV RNA levels up to 70% in a subgenomic replicon system. These findings provide new insights into the development of potential therapeutic strategies based on RNA molecules targeting genomic RNA structural domains and highlight the feasibility of generating novel engineered RNAs as potent antiviral agents.

Correlation between hammerhead ribozyme-mediated eggshell protein gene cleavage and reproduction inhibition of Schistosoma japonicum.

Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting the eggshell protein gene of S. japonicum (SjESG). The cleavage activities of these three ribozymes were determined using cleavage experiments. The in vitro cleavage results showed that among the three synthesized ribozymes (Rz1, Rz2 and Rz3), Rz1 and Rz3 cleaved their target RNAs effectively. However, Rz2 did not cleave its target RNA detectably. The putative therapeutic roles of these three ribozymes to inhibit the reproduction of S. japonicum in mice were studied in vivo. Compared with the negative controls, Rz1 and Rz3 treatments resulted in increased levels of IFN-γ but decreased levels of IL-4 in mice. Rz2 affected levels of IFN-γ and IL-4 to degrees similar with those caused by the vector controls. In addition, Rz1 and Rz3 reduced the amounts of adult worms and eggs in the livers of mice more extensively than Rz2 and the vector controls. Altogether, these results suggest a correlation between the in vitro cleavage abilities of Rz1 and Rz3 and their roles in reproduction inhibition of S. japonicum.

Effective inhibition of human immunodeficiency virus 1 replication by engineered RNase P ribozyme.

Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G(83) -> U(83) and G(340) -> A(340)) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.

A novel ribozyme-based prophylaxis inhibits influenza A virus replication and protects from severe disease.

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses.

Therapeutic oligonucleotides.

A brief historical introduction describes early attempts to silence specific genes using the antisense oligonucleotides that flourished in the 1980s. Early aspirations for therapeutic applications were almost extinguished by the unexpected complexity of oligonucleotide pharmacology. Once the biochemistry and molecular biology behind some of the pharmacology was worked out, new approaches became apparent for using oligonucleotides to treat disease. The biochemistry of small nucleic acids is outlined in Section 2. Various approaches employing oligonucleotides to control cellular functions are reviewed in Section 3. These include antisense oligonucleotides and siRNA that bind to RNA, antigene oligonucleotides that bind to DNA, and aptamers, decoys, and CpG oligonucleotides that bind to proteins.

Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice.

Safe, effective, and tissue-specific delivery is a central issue for the therapeutic application of nucleic-acid-based gene interfering agents, such as ribozymes and siRNAs. In this study, we constructed a functional RNase P-based ribozyme (M1GS RNA) that targets the overlapping mRNA region of M80.5 and protease, two murine cytomegalovirus (MCMV) proteins essential for viral replication. In addition, a novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little cytotoxicity and pathogenicity in mice, was constructed and used for delivery of anti-MCMV ribozyme. In MCMV-infected macrophages treated with the constructed attenuated Salmonella strain carrying the functional M1GS RNA construct, we observed an 80-85% reduction in the expression of M80.5/protease and a 2,500-fold reduction in viral growth. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into spleens and livers, leading to substantial expression of the ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV-infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers, and improved survival compared to the untreated mice or mice treated with Salmonella containing control ribozyme sequences. Our results provide direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene-targeting applications in vivo.

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations.

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ∼ 10-fold higher than physiologic concentrations of Mg(2+) ions. Extended versions containing native loop-loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg(2+) concentrations, for reasons that are still ill-understood. Here, we use Mg(2+) titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop-loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop-loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop-loop interactions in extended hammerhead ribozymes-and Mg(2+) ions that bind to minimal ribozymes-may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state.

Ribozyme-mediated inhibition of 801-bp deletion-mutant epidermal growth factor receptor mRNA expression in glioblastoma multiforme.

The epidermal growth factor receptor (HER1/EGFR) is known to be disregulated in a large subgroup of glioblastoma multiforme cases. Disregulation of HER1/EGFR is related to malignant transformation and tumor growth in various human cancers, including malignant glioma. One mechanism that may lead to disregulated HER1/EGFR signaling is the intrinsic alteration of the receptor structure due to mutational changes. The most common mutant form of HER1/EGFR, named variant III (EGFRvIII), results from an 801 bp in-frame deletion in the DNA sequence encoding the extracellular ligand-binding domain. Independent of ligand-binding, EGFRvIII is constitutively activated and beyond external control. Since its cellular expression was shown to relate enhanced tumorigenicity, various therapeutic strategies were developed to target EGFRvIII, including monoclonal antibodies, vaccination therapies and small-molecule tyrosine kinase inhibitors. In this review, we focus on ribozyme-mediated inhibition of EGFRvIII messenger RNA expression as a gene therapeutic approach for EGFRvIII-expressing glioblastoma multiforme.

Inhibition of HIV-1 replication and dimerization interference by dual inhibitory RNAs.

The 5'-untranslated region (5'UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5' UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5'UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.