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The androgen receptor (AR) plays a critical role in the development of prostate cancer (PCa) through the activation of androgen-induced cellular proliferation genes. Thus, blocking AR-mediated transcriptional activation is expected to inhibit the growth and spread of PCa. Using tailor-made splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we successfully redirected splicing of the AR precursor (pre-)mRNA and destabilized the transcripts via the introduction of premature stop codons. Furthermore, the SSOs simultaneously favored production of the AR45 mRNA in lieu of the full-length AR. AR45 is an AR isoform that can attenuate the activity of both full-length and oncogenic forms of AR by binding to their common N-terminal domain (NTD), thereby blocking their transactivation potential. A large screen was subsequently used to identify individual SSOs that could best perform this dual function. The selected SSOs powerfully silence AR expression and modulate the expression of AR-responsive cellular genes. This bi-functional strategy that uses a single therapeutic molecule can be the basis for novel PCa treatments. It might also be customized to other types of therapies that require the silencing of one gene and the simultaneous expression of a different isoform., Graphical Abstract, Blocking androgen receptor (AR)-mediated transcriptional activation can inhibit the growth and spread of PCa. Using splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we redirected splicing of the AR pre-mRNA, and destabilized the transcripts via introduction of premature stop codons. The selected SSOs silence AR expression and modulate the expression of AR-responsive cellular genes.

Oligonucleotide (ON) concentrations employed for therapeutic applications vary widely, but in general are high enough to raise significant concerns for off target effects and cellular toxicity. However, lowering ON concentrations reduces the chances of a therapeutic response, since typically relatively small amounts of ON are taken up by targeted cells in tissue culture. It is therefore imperative to identify new strategies to improve the concentration dependence of ON function. In this work, we have identified ammonium ion (NH4+) as a non-toxic potent enhancer of ON activity in the nucleus and cytoplasm following delivery by gymnosis. NH4+ is a metabolite that has been extensively employed as diuretic, expectorant, for the treatment of renal calculi and in a variety of other diseases. Enhancement of function can be found in attached and suspension cells, including in difficult-to-transfect Jurkat T and CEM T cells. We have also demonstrated that NH4+ can synergistically interact with arsenic trioxide (arsenite) to further promote ON function without producing any apparent increased cellular toxicity. These small, inexpensive, widely distributed molecules could be useful not only in laboratory experiments but potentially in therapeutic ON-based combinatorial strategy for clinical applications.

Oligonucleotides (oligos) have been under clinical development for approximately the past 30 years, beginning with antisense oligonucleotides (ASOs) and apatmers and followed about 15 years ago by siRNAs. During that lengthy period of time, numerous clinical trials have been performed and thousands of trial participants accrued onto studies. Of all the molecules evaluated as of January 2017, the regulatory authorities assessed that six provided clear clinical benefit in rigorously controlled trials. The story of these six is given in this review.

Approximately 15%–25% of men diagnosed with prostate cancer do not survive their disease. The American Cancer Society estimated that for the year 2016 the number of prostate cancer deaths will be 26,120. Thus, there is a critical need for novel approaches to treat this deadly disease. Using high-throughput small-molecule screening, we found that the small molecule 6-bromo-indirubin-3′-oxime (6BIO) significantly improves the targeting of antisense oligonucleotides (ASOs) delivered by gymnosis (i.e., in the absence of any transfection reagents) in both the cell cytoplasm and the nucleus. Furthermore, as a single agent, 6BIO had the unexpected ability to simultaneously downregulate androgen receptor (AR) expression and AR signaling in prostate cancer cells. This includes downregulating levels of the AR-V7, a drug-resistance-related AR splice variant that is important in the progression of prostate cancer. Combining 6BIO and an anti-AR oligonucleotide (AR-ASO) can augment the downregulation of AR expression. We also demonstrated that 6BIO enhances ASO function and represses AR expression through the inhibition of the two main glycogen synthase kinase 3 (GSK-3) isoforms: GSK-3α and GSK-3β activity. Our findings provide a rationale for the use of 6BIO as a single agent or as part of a combinatorial ASO-based therapy in the treatment of human prostate cancer.

Four decades have passed since oligonucleotides were first used to manipulate gene expression. There were few FDA approvals prior to 2016, mostly of drugs that eventually exhibited poor performance in the market. The aura of their younger siRNA relatives had also faded during the past 15 years. However, several FDA approvals have occurred in the past 4 years, restoring hope that a new era has dawned in oligonucleotide/siRNA clinical therapeutics. Here, we review the field of oligonucleotide therapeutics and provide an update on FDA approvals of oligonucleotides from 2017 until the second quarter of 2019. We take into consideration the ethical issues looming over the still somewhat limited efficacy of these molecules, the toxicity of treatment, and the exorbitant cost of these therapeutic agents, which limits accessibility for many.

Significance The deregulation of miRNA function is critical in the pathogenesis of cancer and other diseases. miRNAs and other noncoding RNAs (ncRNAs) tightly regulate gene expression, often in the cell nucleus. Heretofore, there has been no understanding that there exists a general shuttling mechanism that brings miRNAs, in addition to therapeutic oligonucleotides and siRNAs, from the cytoplasm into the nucleus. We have identified this shuttling mechanism, which occurs in response to cell stress. Nuclear imported miRNAs are functional, can potentially alter gene expression, and participate in cell stress response mechanisms. This shuttling mechanism can be augmented to target specific RNAs, including miRNA sponges, and long ncRNAs like Malat-1, which have been implicated in promoting tumor metastasis., Although some information is available for specific subsets of miRNAs and several factors have been shown to bind oligonucleotides (ONs), no general transport mechanism for these molecules has been identified to date. In this work, we demonstrate that the nuclear transport of ONs, siRNAs, and miRNAs responds to cellular stress. Furthermore, we have identified a stress-induced response complex (SIRC), which includes Ago-1 and Ago-2 in addition to the transcription and splicing regulators YB1, CTCF, FUS, Smad1, Smad3, and Smad4. The SIRC transports endogenous miRNAs, siRNAs, and ONs to the nucleus. We show that cellular stress can significantly increase ON- or siRNA-directed splicing switch events and endogenous miRNA targeting of nuclear RNAs.

Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.

On 1 April 2016, the Food and Drug Administration approved Defitelio, known for many years as Defibrotide (DF), for marketing in the United States. The indication is severe hepatic veno-occlusive disease (sVOD) following high-dose chemotherapy and autologous bone marrow transplantation, a toxicity of therapy with a high mortality. Defibrotide is not the kind of oligonucleotide drug beloved by molecular biologists and proponents of personalized medicine. Its very complicated mechanism of action, which is still elusive, is without question nonsequence specific, and almost certainly based on the charge-charge interactions of its constituents with biological macromolecules, which are almost certainly proteins.

We have identified the existence of a productive, PKC-α-dependent endocytotic silencing pathway that leads gymnotically-delivered locked nucleic acid (LNA)-gapmer phosphorothioate antisense oligonucleotides (ASOs) into late endosomes. By blocking the maturation of early endosomes to late endosomes, silencing the expression of PKC-α results in the potent reduction of ASO silencing ability in the cell. We have also demonstrated that silencing of gene expression in the cytoplasm is vitiated when PKC-α expression is reduced. Restoring PKC-α expression via a reconstitution experiment reinstates the ability of ASOs to silence. These results advance our understanding of intracellular ASO trafficking and activity following gymnotic delivery, and further demonstrate the existence of two distinct silencing pathways in mammalian cells, one in the cytoplasmic and the other in the nuclear compartment.

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Drosha-processed microRNAs (miRNAs) have been shown to be exported from the nucleus to the cytoplasm by Exportin 5, where they are processed a second time to generate mature miRNAs. In this work we show that miRNAs also use CRM1 for nuclear-cytoplasmic shuttling. Inhibition of CRM1 by Leptomycin B results in nuclear accumulation of miRNA guide sequences. Nuclear to cytoplasmic transport can be actively competed by synthetic small interfering RNAs, indicating that this pathway is shared by different classes of processed small RNAs. We also find that CRM1 coimmunoprecipitates with Ago-1, Ago-2, Topo2α, EzH2, and Mta, consistent with a role of Argonautes and small RNAs in chromatin remodeling.

Conventional si/shRNA designs are restricted to Watson/Crick pairing between guide strand and target RNA as this has been deemed crucial for potent Argonaute 2 based slicer activity. Despite individual publications partially challenging the requirement of sole Watson/Crick pairing these results have not yet found entry into RNAi designs.By rational incorporation of results from RNA crystallography structures and non-canonical and non-Watson/Crick pairings (nWCp) at certain positions we have developed a novel RNAi trigger design. Our approach allowed us to create shRNAs where a single guide strand suppresses a spectrum of target sequences through Argonaute 2 activity with target specificity, and similar or higher potency than a conventional design. Genomic off target effects were not observed in NGS analysis. In target/guide variant analysis several guide strands demonstrated potent RNAi activity even with G/U pairings at numerous positions or G/G pairings at position 10 or 11. Furthermore we demonstrate that this approach allows the application of RNAi against mutating targets as verified against HIV-1.Thus, our results demonstrate that an RNAi trigger can be applied to any therapeutic target (with an accessible RNA) prone to mutation-based resistance/escape offering significant improvement to the therapeutics of both infectious diseases and cancer. Additionally, we provide evidence that target and off-target prediction rules for microRNAs and shRNA/siRNAs may benefit from revisions to include a significantly wider set of pairings.

Purpose: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. Experimental Design: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. Results: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with “normal” levels of Bcl-2 protein expression expanded to be large, necrotic tumors. Conclusions: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.

Genetically manipulated T cells can be endowed with novel functions to obtain desired in vivo effects after adoptive transfer. This genetic approach is being used to introduce genes such as chimeric immunoreceptors and tumor-specific T cells are being evaluated in early phase clinic trials. However, the ability to alter the genetic programming of T cells also presents opportunities to remove unwanted T-cell functions in order to augment an anti-tumor effect or endow resistance such as to HIV infection. Specifically, the use of RNA interference (RNAi) to disrupt gene expression by targeting either the mRNA or the promoter, provides investigators with many new opportunities to genetically modify T cells that should prove useful in future applications of adoptive immunotherapy.

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.

The leukocyte- type 12/15-Lipoxygenase (12/15-LO) enzyme and its oxidized lipid products play important roles in vascular smooth muscle cell (VSMC) growth, migration, and matrix responses associated with hypertension, atherosclerosis, and restenosis. However, much less is known about their inflammatory effects. In this study, we showed that the 12/15-LO product of linoleic acid, 13-hydroperoxyocta decadienoic acid (13-HPODE) can transcriptionally upregulate the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in VSMC. We also observed reduced activation of the transcription factor, NF-kappa B and reduced expression of MCP-1/JE mRNA in VSMC from 12/15-LO knock-out mice relative to WT. To confirm the role of NF-kappa B in 13-HPODE-induced MCP-1 expression and to selectively block the induction of such inflammatory genes in VSMC, we designed novel molecular approaches to knockdown NF-kappa B with short interfering RNAs (siRNAs). We designed siRNAs to human NF-kappa B p65 transcriptionally active subunit by using a rapid PCR-based approach that generates sense and antisense siRNA separated by a hairpin loop downstream of the U6 promoter. siRNA PCR products targeting seven different sites on p65 cDNA could induce upto 92% reduction in HA-p65 protein levels. A six-fold decrease in NF-kappa B-dependent luciferase activity was also seen. Transfection of human VSMC with these siRNA PCR products resulted in 70% reduction in p65 protein levels. We cloned the PCR products into a pCR3.1 vector and these p65 siRNA expressing plasmids very effectively blocked 13-HPODE-induced expression of both MCP-1 and TNF-alpha genes. These results show for the first time that 13-HPODE can induce MCP-1 in the vasculature via activation of NF-kappa B.

At present, treatment for HIV-1 infection employs highly active anti-retroviral therapy (HAART), which utilizes a combination of RT and protease inhibitors. Unfortunately, HIV can escape many therapies because of its high mutation rate and the complexity of its pathogenesis. HIV-1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. As an alternative or adjuvant to chemotherapy we have been developing an RNA-based gene therapy approach for the treatment of HIV-1 infection. This article summarizes the various RNA based technologies that we have developed for potential application in a gene therapy setting.

At present, treatment for human immunodeficiency virus (HIV)-1 infection employs highly active anti-retroviral therapy (HAART), which utilizes a combination of retroviral therapy (RT) and protease inhibitors [1] . Unfortunately, HIV can escape many therapies because of its high mutation rate and the complexity of its pathogenesis. HIV-1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. As an alternative or adjuvant to chemotherapy we have been developing a ribonucleic acid (RNA) based gene therapy approach for the treatment of HIV-1 infection. Dr. Narva Sarver was a visionary and an activist who saw the potential for gene therapy as a long term treatment for HIV-1 infection [2] . She was a strong proponent of RNA based gene therapy, in particular ribozyme gene therapy for HIV-1 treatment. Working in close communication with Dr. Sarver over the past several years we have investigated gene therapy approaches that employ the use of anti-HIV ribozymes to control viral replication in acquired immunodeficiency syndrome (AIDS) patients. This article summarizes our past work, as well as describing new technologies being developed for application in a gene therapy setting.

Aberrant splicing of pre-mRNA is recognized to account for a significant minority of disease-causing mutations. The N-methyl-D-aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African-American male near a weak putative branch-site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in-frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer-extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African-American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease.

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA–aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA–aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications.

Creation of inhibitors targeted to a gene of interest is key both to the study of gene function and to drug development, and RNA interference (RNAi) is an attractive approach that complements the development of standard small-molecule drugs. A key factor for the successful application of this technology is the design of potent triggers of RNAi that can be employed at sufficiently low concentrations to avoid or minimize off-target or toxic effects. In an article that appeared recently in Molecular Cell,1 Fellmann et al. report the development of a library selection screen for the generation of potent RNAi triggers that is likely to work for virtually any target gene transcript. The selection method screens every possible overlapping sequence stretch for a particular target gene, thereby providing complete coverage of a given target to identify the most efficacious RNAi triggers.

Hammerhead ribozymes have been proposed as potential therapeutic agents for the treatment of viral and other diseases. However, a clear understanding of the cleavage reaction in vivo is not available at present. In these studies, we chose the yeast Saccharomyces cerevisiae as a model system to study the effects of hammerhead cleavage on gene expression in vivo. Several reporter genes were employed to monitor the self-cleaving characteristics of three different ribozymes. We show that these ribozymes decrease expression of some reporter genes by interfering with splice site selection or translation initiation and not by in vivo cleavage of the RNA transcripts. In fact, it appears that although these ribozymes can efficiently self-cleave the RNA in vitro, they are not able to function in vivo. We have identified a yeast splicing protein that interacts in vivo with our cis-ribozyme by specifically recognizing the ribozyme structure (Lin and Rossi, 1996). This interaction does not occur if different secondary structures are used in place of the ribozyme. The binding of this protein to the ribozyme can account for the inability of ribozymes to efficiently cleave in yeast. Remarkably, when yeast extracts are added to in vitro trans-cleavage reactions, the cleavage ability of the ribozyme is hampered, whereas the addition of mammalian extracts yields an enhancement of the reactions. These results confirm the presence of factor(s) that can block ribozyme function in the yeast intracellular environment.

The discovery that gene expression can be controlled by the Watson–Crick base-pairing of small RNAs with messenger RNAs containing complementary sequence — a process known as RNA interference — has markedly advanced our understanding of eukaryotic gene regulation and function. The ability of short RNA sequences to modulate gene expression has provided a powerful tool with which to study gene function and is set to revolutionize the treatment of disease. Remarkably, despite being just one decade from its discovery, the phenomenon is already being used therapeutically in human clinical trials, and biotechnology companies that focus on RNA-interference-based therapeutics are already publicly traded.

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.

The synthesis and transfection of PCR short interfering/short hairpin RNA (si/shRNA) expression cassettes described in this chapter can be used to rapidly test siRNA targeting and function in cells. One critical element in the design of effective siRNAs is the selection of siRNA-target sequence combinations that yield the best inhibitory activity. This can be accomplished by using synthetic siRNAs and transfection procedures, but these can be costly and time consuming. By using the PCR strategy, it is possible to create several expression cassettes and simultaneously screen for the best target sites on any given mRNA. This PCR strategy allows a rapid and inexpensive approach for intracellular expression of si/shRNAs and subsequent testing of target site sensitivity to downregulation by RNA interference (RNAi).

The synthesis and transfection of PCR short interfering⧸short hairpin RNA (si⧸shRNA) expression cassettes described in this chapter can be used to rapidly test siRNA targeting and function in cells. One critical element in the design of effective siRNAs is the selection of siRNA–target sequence combinations that yield the best inhibitory activity. This can be accomplished by using synthetic siRNAs and transfection procedures, but these can be costly and time consuming. By using the PCR strategy, it is possible to create several expression cassettes and simultaneously screen for the best target sites on any given mRNA. This PCR strategy allows a rapid and inexpensive approach for intracellular expression of si⧸shRNAs and subsequent testing of target site sensitivity to downregulation by RNA interference (RNAi).

Methylation of cytosines in CpG motifs is an important mechanism for epigenetic regulation of gene expression in mammalian cells. The initiating event(s) for de novo methylation in mammalian cells, particularly in cancer, is unknown. In plants, short RNAs homologous to DNA sequences are known to initiate de novo methylation. To investigate whether short hairpin RNAs (shRNAs) may also serve as initiators for de novo methylation in human cells we have expressed short hairpin RNAs complementary to the CpG island including the promoter and early transcribed regions of the human RASSF1A gene. RASSF1A encodes a putative tumor suppressor that is hypermethylated in a variety of human cancers, whereas in some human cell lines, such as HeLa, RASSF1A is unmethylated and transcriptionally active. We demonstrate that shRNAs complementary to the RASSF1A promoter or early transcribed regions can direct low levels of de novo DNA methylation and partial gene silencing in HeLa cells. In contrast, an shRNA harboring four central mismatches with the target cannot direct such methylation. The results presented suggest provocative potential mechanisms for transcriptional gene silencing via DNA methylation in cancer cells.

This study analysed the level of adherence to anti-HIV therapies in illegal drug users compared to non-users. Out of 214 patients interviewed, 60 (28%) reported current use of one or more illegal drugs (heroin, cocaine), alcohol (>6 U/day), psychoactive drugs and others. Within the two groups reporting use of heroin and cocaine, the percentage of patients achieving good levels of compliance was higher than among patients who reported not using any substance (50 and 46% vs. 42.3%), among the users of psychoactive pharmaceuticals and alcohol the percentage of high-compliant patients tended to be lower (respectively 26.6 and 30%), but the difference was not significant.

Terminal transferase-dependent PCR (TDPCR) can be used after reverse transcription to analyze RNA. This method (RT-TDPCR) is able to provide in vivo information at nucleotide-level resolution, and has been used for study of ribozymes, RNA size, RNA structure, and RNA-protein interactions. A detailed protocol of RT-TDPCR is presented here with examples of its use in detecting ribozyme cleavage intermediates in yeast and a RNA transcription start site in mammalian cells.

In mammalian cells, the RNA interference (RNAi) effect has been observed through expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. Here, we describe a facile polymerase chain reaction (PCR)-based strategy for rapid synthesis and evaluation of small interfering RNAs (siRNA) expression units in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells for functional testing. This method is fast and inexpensive, allowing several different siRNA gene candidates to be rapidly screened for efficacy.

Terminal transferase-dependent PCR (TDPCR) can be used after reverse transcription to analyze RNA. This method (RT-TDPCR) is able to provide in vivo information at nucleotide-level resolution, and has been used for study of ribozymes, RNA size, RNA structure, and RNA-protein interactions. A detailed protocol of RT-TDPCR is presented here with examples of its use in detecting ribozyme cleavage intermediates in yeast and a RNA transcription start site in mammalian cells.

In mammalian cells, the RNA interference (RNAi) effect has been observed through expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. Here, we describe a facile polymerase chain reaction (PCR)-based strategy for rapid synthesis and evaluation of small interfering RNAs (siRNA) expression units in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells for functional testing. This method is fast and inexpensive, allowing several different siRNA gene candidates to be rapidly screened for efficacy.

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. In mammalian systems, the sequence-specific RNAi effect has been observed by expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. We describe here a facile PCR based strategy for rapid synthesis of siRNA expression units and their testing in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells resulting in functional expression of siRNAs. This approach should prove useful for identification of optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.

We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin [J. Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)]. When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step. The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer. This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR. Band patterns obtained are reproducible and quantifiable. We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage. Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain.

Publisher Summary Ribozymes represent a relatively new approach for specific inactivation of gene expression. To date, ribozymes have been reported to be effective in cell culture against a variety of RNA targets, including viral, endogenous, and transfected sequences. Ribozymes have been utilized to functionally destroy mRNAs as well as for the repair of mutant RNAs. These RNA catalysts have progressed to the stage where both hammerhead and hairpin ribozymes are currently being tested in human clinical trials. The effective use of ribozymes in vivo depends on effective target-site selection, knowledge of the rules governing intracellular localization, effective delivery to target cells, and maintaining a high and long-term intracellular expression of the engineered gene. Over the past decade, ribozymes have been widely employed in a variety of systems. Their use as tools to control gene expression is both enticing and simple in concept. Ribozymes can be designed to specifically base pair with and cleave any given RNA target, with the result of producing an irreversible inactivation of the targeted RNA. Because these catalytic RNAs can be designed to have exquisite specificity and are inherently devoid of coding capacities, there is little likelihood for undesired cellular toxicities. The combination of specificity, the possibilities of endogeneous synthesis or exogenous delivery, and the capability to be adapted for the destruction or modification of any RNA species make ribozymes extremely attractive genetic tools and potential therapeutic agents. The major limitations to effective intracellular ribozyme use are colocalization of ribozymes with target RNAs and target-site accessibility. These specific problems are discussed in this chapter. In addition, strategies and protocols for expression and delivery of ribozyme genes are addressed.

In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood. In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing. Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation. This activation is sequence-specific and unidirectional. However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG. Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals. Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC.

Publisher Summary This chapter discusses the use of ribozymes as therapeutic agents and tools for gene analysis. Ribozymes are RNA molecules with enzymatic activity. These RNAs can be engineered to base pair with and cleave targeted RNAs, whether they are of cellular or viral origins. Ribozymes have been effectively used in cell cultures against viral targets, endogenous transcripts, and transfected genes. They have also been demonstrated to function effectively in vivo in transgenic animals (mouse and Drosophila ), allowing the generation of phenotypes that are difficult to achieve by conventional genetic approaches. Because of their high target specificity, coupled with the possibility of being delivered either exogenously or endogenously, ribozymes have potential for use in both basic studies and therapeutic applications. There are five catalytic motifs that have been successfully adapted for use in ribozyme applications. These are the group I introns, RNase P, the hammerhead and hairpin motifs, and the self-cleaving domain of the hepatitis delta virus. The chapter presents the catalytic and base pairing domains in a schematic fashion and focuses on the hammerhead ribozyme motif because it has received the most attention from those interested in developing ribozymes as surrogate genetic tools.

As an initial step towards developing a widely applicable system for expressing small ribozymes (Rz) in various cell types using T7 RNA polymerase, we have replaced the loop domain of a natural prokaryotic antisense RNA (RNA out from Tn10), with hammerhead (Hh) Rz. RNA out was chosen, because the stem of its secondary structure gives it an unusually long half life in Escherichia coli which should also confer in vivo stability to small RNA sequences expressed within its loop domain. In order to define the 3′ end of the Rz-RNA out chimeric RNAs, a poly(U) tract was inserted just 3′ of the RNA out stem. Molecular analysis indicates that these RNAs function both as transcription terminators and Rz. In addition, the RNAs are stable in E. coli and can be expressed in mammalian cells. These results show that certain characteristics of a naturally evolved RNA, RNA s in this case, can be used to provide additional functions to short RNAs containing Hh Rz without disrupting the enzymatic activity of the Rz.

Saccharomyces cerevisiae RAP1 ( Sc RAP1) is an essential protein which interacts with diverse genetic loci within the cell. RAP1 binds site-specifically to the consensus sequence, 5′-AYCYRTRCAYYW ( UAS RPG , where R = A or G, W = A or T, Y = C or T). In Kluyveromyces lactis (Kl ) ribosomal protein-encoding genes ( rp ) retain functional RAP1-binding elements, suggesting the presence of a RAP1-like factor. Kl extracts display an activity capable of specifically binding to rp fragments bearing UAS RPG ,. We subsequently isolated the Kl RAPl-encoding gene by homology to a subfragment which encodes the N terminus of the DNA-binding domain of Sc RAP1. The predicted amino acid (aa) sequence of Kl RAP1 indicates it is smaller than Sc RAP1 (666 vs. 827 aa) with the N terminus being truncated. The DNA-binding domain is virtually identical between the two RAP1 proteins, while the RIF1 domain is moderately conserved. The region between these two domains and the N-termini are highly divergent. Two potential UAS RPG were identified in the 5' flanking region, suggesting an autoregulatory role for RAP1. Despite the similarities between the two proteins, KI RAP1 is unable to complement Sc rap 1 ts mutants, suggesting that domains essential for function in Sc are absent from the KI protein.

Publisher Summary This chapter presents an overview of well-characterized ribozymes with an emphasis on the problems that remain to be overcome before ribozyme therapy can be made a useful form of medical treatment. The chapter illustrates that many techniques are currently being used to enhance ribozyme activity. Chemical modifications can be used to assess the sequence requirements for cleavage. In one case, nucleotides that may naturally inhibit the cleavage reaction have been identified. The in vitro evolution has also been used to isolate mutant forms of a group I intron, which cleave DNA up to 100-fold more efficiently than the wild-type intron. In vitro evolution takes advantage of the known ligation activities of the hairpin and group I ribozymes. Unfortunately, the hammerhead is less amenable to such in vitro selection as its ligation reaction is extremely inefficient. In conclusion, ribozymes hold considerable promise for future therapeutic applications and will play a key role in the arsenal of pharmacotherapy.

The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.

Eight pHIV-7-GFP lentiviral vectors encoded anti-HIV-1 small hairpin RNA (shRNA) were constructed. Three target sites in the HIV genome were selected to construct the shRNAs. The first site (SI) resides in a region of rev that overlaps with the adjacent tat gene, while the second (SII) and third sites (SIII) are selected in conserved regions that reside exclusively in rev or tat. The expression and functionality were compared using snRNA Pol III U6 versus Pol II U1 promoters in the following constructs