Expression of Small Interfering RNAs Targeted Against HIV-1 Rev Transcripts in Human Cells

Nature Biotechnology. May, 2002  |  Pubmed ID: 11981565

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.

Durability of Transgene Expression and Vector Integration: Recombinant SV40-derived Gene Therapy Vectors

Molecular Therapy : the Journal of the American Society of Gene Therapy. Aug, 2002  |  Pubmed ID: 12161189

Many applications of gene delivery require long-term transgene expression. In dividing cells, this result necessitates vector genome persistence, usually by integrating into cellular DNA. Since recombinant gene delivery vectors derived from tag-deleted, replication-incompetent simian virus-40 (SV40) provide for long-term transgene expression in resting and dividing cells, we tested whether such enduring transgene expression reflected integration into cellular genomes. Several lines of evidence suggested this likelihood. After transduction in vitro, continuously dividing cell lines and continuously stimulated primary cells uniformly showed transgene expression for many months. Mice whose livers were transduced in vivo, partially resected, and allowed to regenerate showed comparable levels of transgene expression in regenerated and preoperative livers. Thus, replicationincompetent SV40 vectors (rSV40) persist in vitro and in vivo despite extensive cell division. We tested the possibility that this persistence reflected integration directly. Southern blot analyses of genomic DNA from transduced 293 cells showed that vector genome incorporation into cell DNA happened within days of transduction. Episomal vector DNA was barely detectable 96 hours post-transduction. Inverted PCR, used to characterize vector integration points, showed vector DNA integrated randomly into the cell genome. The circular rSV40 genome opened at different points in each integrand. A significant proportion of the integrands did not contain the entire vector sequence, but rather only portions thereof. Quantitative Southern blot analysis showed approximately 3.05 transgene copies per cell. Therefore, recombinant SV40 gene delivery vectors integrate into the cellular DNA of both resting and dividing cells, and do so randomly and within days of transduction. This integration may explain long-term transgene expression.

Alteration of Branch Site Consensus Sequence and Enhanced Pre-mRNA Splicing of an NMDAR1 Intron Not Associated with Schizophrenia

American Journal of Medical Genetics. Aug, 2002  |  Pubmed ID: 12210277

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.

Interaction of MAPK and 12-lipoxygenase Pathways in Growth and Matrix Protein Expression in Mesangial Cells

American Journal of Physiology. Renal Physiology. Nov, 2002  |  Pubmed ID: 12372774

The lipoxygenase (LO) pathway of arachidonate metabolism and mitogen-activated protein kinases (MAPKs) can mediate cellular growth and ANG II effects in vascular smooth muscle cells. However, their role in renal mesangial cells (MC) is not very clear. ANG II treatment of rat MC significantly increased 12-LO mRNA expression and formation of the 12-LO product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE; P < 0.03]. ANG II-induced [(3)H]leucine incorporation was blocked by an LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (P < 0.02). 12(S)-HETE and ANG II directly induced cellular hypertrophy and fibronectin (FN) expression (P < 0.01) to a similar extent. ANG II and 12(S)-HETE led to activation of p38(MAPK) and its target transcription factor cAMP-responsive element-binding protein (CREB). ANG II- and 12(S)-HETE-induced CREB activation and [(3)H]leucine incorporation were blocked by the p38(MAPK) inhibitor SB-202190. A specific molecular inhibitor of rat 12-LO mRNA, namely, a novel ribozyme, could attenuate ANG II-induced FN mRNA. Thus p38(MAPK)-dependent CREB activation may mediate ANG II- and LO product-induced FN expression and cellular growth in rat MC. ANG II effects may be mediated by the LO pathway. These results suggest a novel interaction between LO and p38(MAPK) activation in MC matrix synthesis associated with renal complications.

A Nucleolar TAR Decoy Inhibitor of HIV-1 Replication

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2002  |  Pubmed ID: 12376617

Tat is a critical regulatory factor in HIV-1 gene expression. It mediates the transactivation of transcription from the HIV-1 LTR by binding to the transactivation response (TAR) element in a complex with cyclin T1. Because of its critical and early role in HIV gene expression, Tat and its interaction with the TAR element constitute important therapeutic targets for the treatment of HIV-1 infection. Based on the known nucleolar localization properties of Tat, we constructed a chimeric small nucleolar RNA-TAR decoy that localizes to the nucleoli of human cells and colocalizes in the nucleolus with a Tat-enhanced GFP fusion protein. When the chimeric RNA was stably expressed in human T lymphoblastoid CEM cells it potently inhibited HIV-1 replication. These results demonstrate that the nucleolar trafficking of Tat is critical for HIV-1 replication and suggests a role for the nucleolus in HIV-1 viral replication.

Enhanced Expression and HIV-1 Inhibition of Chimeric TRNA(Lys3)-ribozymes Under Dual U6 SnRNA and TRNA Promoters

Molecular Therapy : the Journal of the American Society of Gene Therapy. Oct, 2002  |  Pubmed ID: 12377189

We previously demonstrated that chimeric tRNA(Lys3)-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNA(Lys3) promoter variants and compared their expression levels from the internal tRNA(Lys3) promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNA(Lys3)-ribozyme containing a mutation in the tRNA B box. As detected by fluorescent in situ hybridization, transcripts from a construct with the tRNA promoter alone localized strictly to the cytoplasm, whereas transcripts from dual U6/tRNA promoter were present in both the cytoplasm and the nucleus. Inhibition of HIV-1 correlates well with expression levels of the chimeric constructs. The results presented demonstrate that U6 and tRNA promoters can be placed in tandem for high-level expression of small RNA therapeutic transcripts.

Exposure Assessment and the Health of Deployed Forces

Drug and Chemical Toxicology. Nov, 2002  |  Pubmed ID: 12378949

The risk assessment process is a critical function for military Deployment Toxicology research objectives, emphasizing improved health protection of deployed forces. Reliable risk assessment methodology is essential for decision making related to risk reduction procedures during combat deployment, as well as during routine occupational activities. Such decision making must be based upon quality science that both guides sound judgments in risk characterization and management, and provides necessary health protection tools. The health and fitness of deployed forces must be considered for both acute and long-term issues. Exposure assessment specifies populations that might be exposed to injurious agents, identifies routes of exposure, and estimates the magnitude, duration, and timing of the doses that personnel may receive as a result of their exposure. Acute or short-term catastrophic risks for deployed forces are of immediate concern and must be addressed on a risk prioritization basis using Operational Risk Management (ORM) procedures. However, long-term effects of exposure to the same agents must be considered as part of the overall health concerns for deployed forces. In response to these needs, a number of military, federal government, academic and private sector organizations are currently developing new classes of biologically-based biosensors with the programmed capacity to detect the presence of virtually any environmental chemical or biological stressor with the capacity to induce health consequences in deployed personnel. A major objective of this engineering effort is development of biosensor systems that detect novel (previously unresearched) chemical or biological agents that might be used during international combat or terrorist attacks to induce acute or long-term health effects on military or civilian populations. A large portion of the discussion in this paper is devoted to describing the development, testing, and implementation of tissue-based biosensors (TBBs) that utilize small samples of living tissue from laboratory small animals for a wide range of human risk assessment applications.

Functional SiRNA Expression from Transfected PCR Products

RNA (New York, N.Y.). Nov, 2002  |  Pubmed ID: 12458798

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.

RNA-based Anti-HIV-1 Gene Therapeutic Constructs in SCID-hu Mouse Model

Molecular Therapy : the Journal of the American Society of Gene Therapy. Dec, 2002  |  Pubmed ID: 12498773

Effective suppression of HIV-1 replication requires inhibition of critical viral target molecules. Tat and Rev are indispensable regulatory factors for HIV-1 replication, whereas Env mediates virus entry by direct interaction with surface receptor CD4 and coreceptor CCR5 or CXCR4. Anti-HIV-1 tat-rev and env ribozymes and Rev aptamers were previously demonstrated to provide relatively long-term protection against HIV-1 infection in vitro. However, further improvements in these constructs for clinical application in a stem-cell-based gene therapy setting requires in vivo characterization. Toward this end, we introduced these constructs into CD34(+) hematopoietic progenitor cells by retrovirus-mediated gene transduction. Ribozyme- and aptamer-transduced CD34(+) cells differentiated normally into multiple lineages of erythroid and myeloid progenies in a colony-forming unit assay. Macrophages that differentiated from the transduced CD34(+) cells expressed anti-tat-rev and -env ribozymes and Rev aptamers and displayed their normal characteristic surface markers CD14, CD4, and CCR5. Using the SCID-hu mouse in vivo human thymopoiesis model, we demonstrated that ribozyme- and aptamer-transduced CD34(+) cells retained their normal capacity to reconstitute human fetal thymus and liver tissue (thy/liv) grafts. Reconstitution by ribozyme- and aptamer-transduced CD34(+) cells reached levels of up to 87% based on HLA surface marker staining. Differentiated thymocytes derived from reconstituted grafts expressed anti-tat-rev and -env ribozymes and Rev aptamers and showed significant resistance to HIV-1 infection upon challenge. Analysis of reconstituted thymocytes by hybridization revealed an average of 0.4 to 2 copies of vector sequences per cell. Southern analysis of proviral integration junctions in progeny thymocytes demonstrated that the human thy/liv grafts were reconstituted by a few primitive hematopoietic stem cells. These results highlight the utility of RNA-based anti-HIV-1 gene therapeutic approaches and their preclinical testing in a surrogate animal model harboring human tissue.

Localized Expression of Small RNA Inhibitors in Human Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Feb, 2003  |  Pubmed ID: 12597912

Several types of small RNAs have been proposed as gene expression repressors with great potential for use in gene therapy. RNA polymerase III (pol III) provides an ideal means of expressing small RNAs in cells because its normal products are small, highly structured RNAs that are found in a variety of subcellular compartments. We have designed cassettes that use human pol III promoters for the high-level expression of small RNAs in the cytoplasm, nucleoplasm, and nucleolus. The levels and subcellular destinations of the transcripts are compared for transcripts expressed using the U6 small nuclear RNA (snRNA), 5S ribosomal RNA (rRNA), and the 7SL RNA component of the signal recognition particle. The most effective location for a particular inhibitory RNA is not necessarily predictable; thus these cassettes allow testing of the same RNA insert in multiple subcellular locations. Several small interfering RNA (siRNA) inserts were tested for efficacy. An siRNA insert that reduces lamin expression when transcribed from the U6 snRNA promoter in the nucleus has no effect on lamin expression when transcribed from 5S rRNA and 7SL RNA-based cassettes and found in the nucleolus and cytoplasm. To test further the generality of U6-driven siRNA inhibitors, siRNAs targeting HIV were tested by co-transfection with provirus in cell culture. Although the degree of HIV-1 inhibition varied among inserts, results show that the U6 cassette provides a means of expressing an siRNA-like inhibitor of HIV gene expression.

Hammerhead Ribozyme-mediated Destruction of Nuclear Foci in Myotonic Dystrophy Myoblasts

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2003  |  Pubmed ID: 12718910

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG expansion in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase gene (DMPK). Transcripts from this altered gene harbor large CUG expansions that are retained in the nucleus of DM1 cells and form foci. It is believed that the formation of these foci is closely linked to DM1 muscle pathogenesis. Here we investigated the possibility of using a nuclear-retained hammerhead ribozyme expressed from a modified tRNAmeti promoter to target and cleave mutant transcripts of DMPK. Accessible ribozyme target sites were identified in the 3'UTR of the DMPK mRNA and a hammerhead ribozyme was designed to cut the most accessible site. Utilizing this system, we have achieved 50 and 63% reductions, respectively, of the normal and CUG expanded repeat-containing transcripts. We also observed a significant reduction in the number of DMPK mRNA-containing nuclear foci in human DM1 myoblasts. Reduction of mutant DMPK mRNA and nuclear foci also corroborates with partial restoration of insulin receptor isoform B expression in DM1 myoblasts. These studies demonstrate for the first time intracellular ribozyme-mediated cleavage of nuclear-retained mutant DMPK mRNAs, providing a potential gene therapy agent for the treatment of myotonic dystrophy.

Biological and Health Effects of Exposure to Kerosene-based Jet Fuels and Performance Additives

Journal of Toxicology and Environmental Health. Part B, Critical Reviews. Jul-Aug, 2003  |  Pubmed ID: 12775519

Over 2 million military and civilian personnel per year (over 1 million in the United States) are occupationally exposed, respectively, to jet propulsion fuel-8 (JP-8), JP-8 +100 or JP-5, or to the civil aviation equivalents Jet A or Jet A-1. Approximately 60 billion gallon of these kerosene-based jet fuels are annually consumed worldwide (26 billion gallon in the United States), including over 5 billion gallon of JP-8 by the militaries of the United States and other NATO countries. JP-8, for example, represents the largest single chemical exposure in the U.S. military (2.53 billion gallon in 2000), while Jet A and A-1 are among the most common sources of nonmilitary occupational chemical exposure. Although more recent figures were not available, approximately 4.06 billion gallon of kerosene per se were consumed in the United States in 1990 (IARC, 1992). These exposures may occur repeatedly to raw fuel, vapor phase, aerosol phase, or fuel combustion exhaust by dermal absorption, pulmonary inhalation, or oral ingestion routes. Additionally, the public may be repeatedly exposed to lower levels of jet fuel vapor/aerosol or to fuel combustion products through atmospheric contamination, or to raw fuel constituents by contact with contaminated groundwater or soil. Kerosene-based hydrocarbon fuels are complex mixtures of up to 260+ aliphatic and aromatic hydrocarbon compounds (C(6) -C(17+); possibly 2000+ isomeric forms), including varying concentrations of potential toxicants such as benzene, n-hexane, toluene, xylenes, trimethylpentane, methoxyethanol, naphthalenes (including polycyclic aromatic hydrocarbons [PAHs], and certain other C(9)-C(12) fractions (i.e., n-propylbenzene, trimethylbenzene isomers). While hydrocarbon fuel exposures occur typically at concentrations below current permissible exposure limits (PELs) for the parent fuel or its constituent chemicals, it is unknown whether additive or synergistic interactions among hydrocarbon constituents, up to six performance additives, and other environmental exposure factors may result in unpredicted toxicity. While there is little epidemiological evidence for fuel-induced death, cancer, or other serious organic disease in fuel-exposed workers, large numbers of self-reported health complaints in this cohort appear to justify study of more subtle health consequences. A number of recently published studies reported acute or persisting biological or health effects from acute, subchronic, or chronic exposure of humans or animals to kerosene-based hydrocarbon fuels, to constituent chemicals of these fuels, or to fuel combustion products. This review provides an in-depth summary of human, animal, and in vitro studies of biological or health effects from exposure to JP-8, JP-8 +100, JP-5, Jet A, Jet A-1, or kerosene.

Small Interfering RNAs Expressed from a Pol III Promoter Suppress the EWS/Fli-1 Transcript in an Ewing Sarcoma Cell Line

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2003  |  Pubmed ID: 12788655

The EWS/Fli-1 fusion gene encodes an oncogenic fusion protein. The fusion is a product of the translocation t(11;22) (q24;q12), which is detected in 85% of Ewing sarcoma and primitive neuroectodermal tumor cells. Utilizing intracellularly expressed 21- to 23-nucleotide small interfering RNAs (siRNAs) targeting the EWS/Fli-1 fusion transcript in an Ewing sarcoma cell line, we achieved a greater than 80% reduction in the EWS/Fli-1 transcript. The reduction in transcript levels was accompanied by growth inhibition of an Ewing cell line. In addition to quantitating the reduction of the fusion transcript, we carefully monitored reduction of the endogenous EWS and Fli-1 mRNAs as well. One of the two siRNAs targeted to the fusion transcript also partially downregulated the Fli-1 mRNA, further potentiating the growth inhibition. These results highlight both the power of siRNAs and the potential side reactions that need to be carefully monitored. In addition, these results provide the first demonstration of expressed siRNAs downregulating an oncogenic fusion transcript. The results and observations from these studies should prove useful in targeting other fusion transcripts characteristic of sarcomas and erythroleukemias.

Inhibition of HIV-1 by Lentiviral Vector-transduced SiRNAs in T Lymphocytes Differentiated in SCID-hu Mice and CD34+ Progenitor Cell-derived Macrophages

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jul, 2003  |  Pubmed ID: 12842429

The phenomenon of RNA interference mediated by small interfering RNAs (siRNAs) is a potent gene-silencing mechanism. A number of recent studies demonstrated inhibition of HIV-1 replication in cultured cells using this approach. To make further progress and harness this technology for HIV-1 gene therapy in a stem cell setting, in vivo studies using primary hematopoietic cells are needed. Using an HIV-based lentiviral vector we introduced an anti-Rev siRNA construct into CD34(+) hematopoietic progenitor cells. The siRNA-transduced progenitor cells were allowed to mature into macrophages in vitro and T cells in vivo in SCID-hu mouse thy/liv grafts. Phenotypically normal T cells and macrophages displaying characteristic surface markers were obtained. In vitro HIV-1 challenge of the siRNA-expressing macrophages and T cells with macrophage-tropic and T-cell-tropic HIV-1, respectively, showed marked viral resistance. These experiments demonstrate the utility of siRNAs delivered into hematopoietic stem cells via lentiviral vectors for future in vivo applications.

SiRNAs, Ribozymes and RNA Decoys in Modeling Stem Cell-based Gene Therapy for HIV/AIDS

Anticancer Research. May-Jun, 2003  |  Pubmed ID: 12894572

Gene therapy strategies for HIV infection require gene transduction of hematopoietic stem cells with effective therapeutic constructs. Here we summarize our studies on anti-HIV ribozymes, RNA decoys and the newly described siRNAs. The therapeutic constructs consisted of an anti-CCR5 ribozyme to down-regulate the HIV-1 cell surface co-receptor and ribozymes targeted to viral mRNAs coding for the tat, rev and env proteins. The RNA decoy targeted rev and the siRNA was directed against a sequence common to rev and tat mRNAs. CD34 hematopoietic progenitor cells were transduced with retroviral or lentiviral vectors containing these constructs. They were differentiated into macrophages in vitro and T cells in vivo in a SCID-hu mouse thymopoiesis model. The transgene-containing macrophages and T cells were found to be phenotypically normal. When challenged in vitro with HIV-1, they showed significant anti-viral resistance. These proof-of-concept studies demonstrated the utility of RNA-based anti-HIV constructs for gene therapy.

Inhibition of HIV-1 Infection by Lentiviral Vectors Expressing Pol III-promoted Anti-HIV RNAs

Molecular Therapy : the Journal of the American Society of Gene Therapy. Aug, 2003  |  Pubmed ID: 12907142

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.

Treatment of Spontaneous and Chemically Induced EEG Paroxysms in the Fischer-344 Rat with Traditional Antiepileptic Drugs or AED+CGP 35348 Polytherapy

Progress in Neuro-psychopharmacology & Biological Psychiatry. Aug, 2003  |  Pubmed ID: 12921919

Twenty-four Fischer-344 rats implanted with cortical electroencephalograph (EEG) electrodes were evaluated for frequency, duration, and mean duration (duration/frequency) of both spontaneous and chemically induced spike-and-wave discharges (SWDs) during earlier and later adulthood. Fischer-344 rats, exhibiting a low level of spontaneous SWDs during earlier adulthood, progressed to exhibit a significantly larger number and duration of SWDs during later adulthood. The convulsant trimethylolpropane phosphate (TMPP), known to antagonize the GABAA inhibitory system through blockade of the chloride channel, induced or increased SWDs in every rat tested. Spontaneous or chemically induced SWDs in Fischer-344 rats were eliminated or reduced by several drugs used to control human absence seizures. The gamma-aminobutyric acid (GABA)B antagonist CGP 35348 reduced spontaneous EEG paroxysms; CGP 35348 as a polytherapy with several traditional antiabsence treatments improved control of TMPP-induced seizures. It would appear that the Fischer-344 rat provides an excellent research alternative to use of genetically selected rodent strains for investigation of progression of absencelike epilepsy with aging, induction of SWDs by toxicant exposures, and treatment of spontaneous and chemically induced paroxysms.

Coupling of RNAi-mediated Target Downregulation with Gene Replacement

Antisense & Nucleic Acid Drug Development. 2003  |  Pubmed ID: 12954115

Short interfering RNA (siRNA)-mediated knockdown of deleterious endogenous transcripts has potential applications for the treatment of hereditary diseases. In situations where the mutant and wildtype transcripts cannot be discriminated from one another by siRNAs, it may be necessary to simultaneously carry out gene replacement with a modified form of the target RNA that is resistant to siRNA activity. To test this possibility, we have taken advantage of a potent siRNA that knocks down EGFP mRNA. In this system, wild-type EGFP expression is suppressed by the siRNA, whereas an EGFP construct with codon modifications in the target region that is otherwise fully functional is not downregulated. When expression of the wild-type message is inhibited, EGFP expression can be simultaneously restored by transfecting these cells with the codon-modified version of EGFP. These studies provide a detailed methodology and system for testing this strategy with RNA interference (RNAi).

Approaches for the Sequence-specific Knockdown of MRNA

Nature Biotechnology. Dec, 2003  |  Pubmed ID: 14647331

Over the past 25 years there have been thousands of published reports describing applications of antisense nucleic acid derivatives for targeted inhibition of gene function. The major classes of antisense agents currently used by investigators for sequence-specific mRNA knockdowns are antisense oligonucleotides (ODNs), ribozymes, DNAzymes and RNA interference (RNAi). Whatever the method, the problems for effective application are remarkably similar: efficient delivery, enhanced stability, minimization of off-target effects and identification of sensitive sites in the target RNAs. These challenges have been in existence from the first attempts to use antisense research tools, and need to be met before any antisense molecule can become widely accepted as a therapeutic agent.

RNA-mediated Inhibition of HIV in a Gene Therapy Setting

Annals of the New York Academy of Sciences. Dec, 2003  |  Pubmed ID: 14751823

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.

Specific Killing of Ph+ Chronic Myeloid Leukemia Cells by a Lentiviral Vector-delivered Anti-bcr/abl Small Hairpin RNA

Oligonucleotides. 2003  |  Pubmed ID: 15000831

Chronic myeloid leukemia (CML) is characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 t(9;22)(q34;q11) that causes fusion of the bcr and abl genes. Transcription and splicing of the fusion gene generate two major splice variants of the bcr/abl transcript that encode an oncoprotein with tyrosine kinase activity. We have taken advantage of lentiviral vectormediated delivery of anti-bcr/abl short hairpin RNAs (shRNA) to downregulate the bcr/abl transcript in Philadelphia chromosome-positive (Ph+) K562 leukemia cells. This downregulation caused complete inhibition of proliferation of these cells and was accompanied by >90% inhibition of the bcr/abl transcript and p210 protein. These results demonstrate the feasibility of using a lentiviral vector to stably transduce therapeutic shRNAs into leukemia cells for the potential ex vivo purging of Ph+ cells in an autologous hematopoietic cell transplant setting. Furthermore, the robust expression of the shRNAs from our lentiviral vector suggests that this system could be generally useful for the expression of other shRNAs.

In Vivo Detection of Ribozyme Cleavage Products and RNA Structure by Use of Terminal Transferase-dependent PCR

Methods in Molecular Biology (Clifton, N.J.). 2004  |  Pubmed ID: 15017045

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.

Construction and Transfection of PCR Products Expressing SiRNAs or ShRNAs in Mammalian Cells

Methods in Molecular Biology (Clifton, N.J.). 2004  |  Pubmed ID: 15017076

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.

Cyclophilin A is Required for Retinoic Acid-induced Neuronal Differentiation in P19 Cells

The Journal of Biological Chemistry. Jun, 2004  |  Pubmed ID: 15047706

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Control of HIV-1 Replication by RNA Interference

Virus Research. Jun, 2004  |  Pubmed ID: 15068880

Small interfering RNAs (siRNAs) have been shown to direct sequence-specific inhibition of gene expression in mammalian cells. siRNAs are RNA duplexes of 21-23 nucleotides (nts) with approximately 2nt 3' overhangs that can induce degradation of their homologous target mRNAs without interferon responses in mammalian cells. The degradation of the target occurs at the post-transcriptional level, meaning a post-transcriptional gene silencing (PTGS) mechanism called as RNA interference (RNAi). RNAi has emerged as an efficient method to inhibit gene expression in mammalian cells with increasingly successful cases of knockdown of many specific genes. Recent works have shown that the use of RNAi could inhibit HIV-1 replication by targeting viral or cellular genes. RNAi can be considered as a gene-specific therapeutic option for controlling HIV-1 replication. However, the control of HIV-1 replication has become complex because of the limited effectiveness of existing anti-HIV-1 agents and the high speed mutation rate of the HIV-1 genome. Careful assessments are required for the potential of RNAi as a gene therapy approach for controlling HIV-1 replication. This review will discuss the status of the science using RNAi for controlling HIV-1 replication and will describe possible problems for therapeutic applications of RNAi-mediated technologies for HIV-1 behind this novel mechanism.

Anti-HIV-1 Gene Expressing Lentiviral Vectors As an Adjunctive Therapy for HIV-1 Infection

Current HIV Research. Apr, 2004  |  Pubmed ID: 15078182

Lentiviral based gene therapy may provide a valuable addition to the current anti-HIV arsenal. Many lentiviral vector systems have been described including those based on feline immunodeficiency virus (FIV), human immunodeficiency virus 1 (HIV) and 2 (HIV-2/SIV) as well as replication incompetent, self-inactivating (sin) vs. conditionally replicating (mobilizable) vectors. Lentiviral vectors offer promise in treating HIV-1 infection as they are capable of stably transducing both dividing and nondividing cells, specifically those cells involved in HIV-1 replication and immune restoration: T-cells, hematopoietic stem cells, and dendritic cells. Moreover, some of the HIV-1 and 2 based vectors can be mobilized by wildtype HIV-1 in vivo and spread to those cells targeted by the virus as well as can compete with viral RNA for packaging and access to viral proteins such as Tat and Rev required for viral replication. Finally, lentiviral vectors can be designed to express therapeutic anti-HIV-1 genes, which specifically target various stages of viral replication. Many candidate RNA based anti-HIV-1 genes have been expressed from lentiviral vectors including ribozymes and anti-sense RNA [1]. Recently, small interfering RNAs (siRNAs) have been shown to potently suppress HIV replication [2-6]. This review will focus on the current status of lentiviral vector development and the feasibility of using lentiviral vectors in delivering anti-HIV genes, specifically ribozymes, and siRNAs as a therapeutic approach to employ in conjunction with current anti-retroviral therapies.

Regulation of Monocyte Chemoattractant Protein-1 by the Oxidized Lipid, 13-hydroperoxyoctadecadienoic Acid, in Vascular Smooth Muscle Cells Via Nuclear Factor-kappa B (NF-kappa B)

Journal of Molecular and Cellular Cardiology. Apr, 2004  |  Pubmed ID: 15081318

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.

RNAi Applications in Mammalian Cells

BioTechniques. Apr, 2004  |  Pubmed ID: 15088371

Gene Expression Profiles in the Rat Central Nervous System Induced by JP-8 Jet Fuel Vapor Exposure

Neuroscience Letters. Jun, 2004  |  Pubmed ID: 15182950

Jet propulsion fuel-8 (JP-8) is the predominant fuel for military land vehicles and aircraft used in the US and NATO. Occupational exposure to jet fuel in military personnel has raised concern for the health risk associated with such exposure in the Department of Defense. Clinical studies of humans chronically exposed to jet fuel have suggested both neurotoxicity and neurobehavioral deficits. We utilized rat neurobiology U34 array to measure gene expression changes in whole brain tissue of rats exposed repeatedly to JP-8, under conditions that simulated possible occupational exposure (6 h/day for 91 days) to JP-8 vapor at 250, 500, and 1000 mg/m(3), respectively. Our studies revealed that the gene expression changes of exposure groups can be divided into two main categories according to their functions: (1). neurotransmitter signaling pathways; and (2). stress response. The implications of these gene expression changes are discussed.

Antisense-mediated Inhibition of Human Immunodeficiency Virus (HIV) Replication by Use of an HIV Type 1-based Vector Results in Severely Attenuated Mutants Incapable of Developing Resistance

Journal of Virology. Jul, 2004  |  Pubmed ID: 15194784

We have constructed a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector expressing a 937-base antisense sequence against the HIV-1 envelope gene. Transduction of CD4(+) T lymphocytes with this vector results in expression of the therapeutic antisense sequence and subsequent inhibition of productive HIV-1 replication. In this report, we examined the effect of antisense-mediated suppression on the potential development of virus escape mutants using a permissive T-cell line cultured under conditions that over serial passages specifically allowed for generation and amplification of mutants selected for by antisense pressure. In the resulting virus clones, we found a significant increase in the number of deletions at the envelope target region (91% compared to 27.5% in wild-type HIV). Deletions were most often greater than 1 kb in length. These data demonstrate for the first time that during antisense-mediated suppression of HIV, mutants develop as a direct result of selective pressure on the HIV genomic RNA. Interestingly, in clones where deletions were not observed, there was a high rate of A-G transitions in mutants at the antisense target region but not outside this region, which is consistent with those mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA by the enzyme double-stranded RNA-specific adenosine deaminase. These clones were not found to be escape mutants, as their replicative ability was severely attenuated, and they did not replicate in the presence of vector.

Ribozyme Diagnostics Comes of Age

Chemistry & Biology. Jul, 2004  |  Pubmed ID: 15271347

Biosensing ribozymes could soon be used to diagnose viral infection. The Kossen group from Sirna Therapeutics have developed a sensitive, high-throughput means of screening for hepatitis C virus, using their target activated half-ribozyme technology, as reported in the June issue of Chemistry & Biology.

Recent Applications of RNAi in Mammalian Systems

Current Pharmaceutical Biotechnology. Aug, 2004  |  Pubmed ID: 15320766

RNAi is a powerful cellular mechanism that involves targeted destruction of mRNAs. Although the phenomenon was first discovered in plants and lower eukaryotic organisms, it was later discovered as an important genetic regulatory mechanism in mammalian cells. RNAi is triggered by double stranded RNAs that are cleaved into short 21-23 base pair duplexes by an RNAse III type enzyme called Dicer. The short RNAs, termed small interfering RNAs (siRNAs), act as triggers for targeted RNA degradation. One of the two strands is selectively incorporated into a complex of proteins called the RNA induced silencing complex, or RISC. The incorporated small RNA guides the complex to the complementary target sequence, and this event is followed by endonucleolytic cleavage of the target and recycling of RISC. In mammalian cells, siRNAs do not activate interferon pathway genes, thereby making these powerful tools for sequence specific knockdown of RNAs. In this article we review the methods for programming mammalian cells with siRNAs, and overview a number of applications ranging from targeting oncogenes to inhibiting viral replication. The article also summarizes some important biological conclusions that can be drawn from selective downregulation of certain mRNA targets and addresses potential uses of RNAi as a new therapeutic modality.

Rapid Assessment of Anti-HIV SiRNA Efficacy Using PCR-derived Pol III ShRNA Cassettes

Molecular Therapy : the Journal of the American Society of Gene Therapy. Sep, 2004  |  Pubmed ID: 15336659

Identification of sequences within a target mRNA that are susceptible to potent siRNA knockdown often requires testing several independent siRNAs or shRNA expression cassettes. Using RNAi against HIV RNAs is further complicated by the length of the viral genome, the complexity of splicing patterns, and the propensity for genetic heterogeneity; consequently, it is most important to identify a number of siRNA targets that potently block viral replication. We previously described a facile PCR-based strategy for rapid synthesis of si/shRNA expression units and their testing in mammalian cells. Using this approach, which is rapid and inexpensive, it is possible to screen a number of potential RNAi targets in HIV to identify those that are most susceptible to RNAi. We report that shRNA expression cassettes constructed by PCR and cotransfected directly into mammalian cells with HIV proviral DNA express shRNAs that are inhibitory to HIV-1 replication. Our results also demonstrate that there is a wide range of efficacies among shRNAs targeting different sites throughout the HIV genome. By screening several different targets we were able to identify a sequence in a common tat/rev exon that is exquisitely sensitive to RNAi. Furthermore we relate the efficacies of our PCR product expressed shRNAs to the relative stabilities of the siRNA duplexes and the accessibilities of the target sites to antisense base pairing in cell extracts.

Unlocking the Potential of the Human Genome with RNA Interference

Nature. Sep, 2004  |  Pubmed ID: 15372045

The discovery of RNA interference (RNAi) may well be one of the transforming events in biology in the past decade. RNAi can result in gene silencing or even in the expulsion of sequences from the genome. Harnessed as an experimental tool, RNAi has revolutionized approaches to decoding gene function. It also has the potential to be exploited therapeutically, and clinical trials to test this possibility are already being planned.

Therapeutic Applications of RNA Interference: Recent Advances in SiRNA Design

Advances in Genetics. 2004  |  Pubmed ID: 15522731

Medicine: a Cholesterol Connection in RNAi

Nature. Nov, 2004  |  Pubmed ID: 15538347

Negative Feedback Inhibition of HIV-1 by TAT-inducible Expression of SiRNA

Nature Biotechnology. Dec, 2004  |  Pubmed ID: 15568018

Here we demonstrate that an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. Our strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and hematopoietic stem cell-derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection.

Interferon Induction by SiRNAs and SsRNAs Synthesized by Phage Polymerase

Nature Biotechnology. Mar, 2004  |  Pubmed ID: 14990954

Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silencing and a major new genetic tool for investigating mammalian cells. When synthetic siRNAs are used for gene silencing, the costs can be substantial because of variations in siRNA efficacies. An alternative to chemically synthesized siRNAs are siRNAs produced by bacteriophage T7 RNA polymerase. We found that siRNAs synthesized from the T7 RNA polymerase system can trigger a potent induction of interferon alpha and beta in a variety of cell lines. Surprisingly, we also found very potent induction of interferon alpha and beta by short single-stranded RNAs (ssRNAs) transcribed with T3, T7 and Sp6 RNA polymerases. Analyses of the potential mediators of this response revealed that the initiating 5' triphosphate is required for interferon induction. We describe here an improved method for T7 siRNA synthesis that alleviates the interferon response while maintaining full efficacy of the siRNAs.

Lentiviral Transduction of Tar Decoy and CCR5 Ribozyme into CD34+ Progenitor Cells and Derivation of HIV-1 Resistant T Cells and Macrophages

AIDS Research and Therapy. Dec, 2004  |  Pubmed ID: 15813986

BACKGROUND: RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys), and small interfering RNAs (siRNAs). Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages. RESULTS: High efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection. CONCLUSIONS: Viral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS.

Helping RNAi Deliver

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2005  |  Pubmed ID: 15851004

Amplification of RNAi--targeting HLA MRNAs

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2005  |  Pubmed ID: 15851019

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.

Small Nuclear RNAs Encoded by Herpesvirus Saimiri Upregulate the Expression of Genes Linked to T Cell Activation in Virally Transformed T Cells

Current Biology : CB. May, 2005  |  Pubmed ID: 15916956

Seven small nuclear RNAs of the Sm class are encoded by Herpesvirus saimiri (HVS), a gamma Herpesvirus that causes aggressive T cell leukemias and lymphomas in New World primates and efficiently transforms T cells in vitro. The Herpesvirus saimiri U RNAs (HSURs) are the most abundant viral transcripts in HVS-transformed, latently infected T cells but are not required for viral replication or transformation in vitro. We have compared marmoset T cells transformed with wild-type or a mutant HVS lacking the most highly conserved HSURs, HSURs 1 and 2. Microarray and Northern analyses reveal that HSUR 1 and 2 expression correlates with significant increases in a small number of host mRNAs, including the T cell-receptor beta and gamma chains, the T cell and natural killer (NK) cell-surface receptors CD52 and DAP10, and intracellular proteins--SKAP55, granulysin, and NKG7--linked to T cell and NK cell activation. Upregulation of three of these transcripts was rescued after transduction of deletion-mutant-HVS-transformed cells with a lentiviral vector carrying HSURs 1 and 2. These changes indicate an unexpected role for the HSURs in regulating a remarkably defined and physiologically relevant set of host targets involved in the activation of virally transformed T cells during latency.

Current Status of Gene Therapy Strategies to Treat HIV/AIDS

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2005  |  Pubmed ID: 15922953

Progress in developing effective gene transfer approaches to treat HIV-1 infection has been steady. Many different transgenes have been reported to inhibit HIV-1 in vitro. However, effective translation of such results to clinical practice, or even to animal models of AIDS, has been challenging. Among the reasons for this failure are uncertainty as to the most effective cell population(s) to target, the diffuseness of these target cells in the body, and ineffective or insufficiently durable gene delivery. Better understanding of the HIV-1 replicative cycle, host factors involved in HIV-1 infection, vector biology and application, transgene technology, animal models, and clinical study design have all contributed vastly to planning current and future strategies for application of gene therapeutic approaches to the treatment of AIDS. This review focuses on the newest developments in these areas and provides a strong basis for renewed optimism that gene therapy will have an important role to play in treating people infected with HIV-1.

Behavioral Test Methods Workshop

Neurotoxicology and Teratology. May-Jun, 2005  |  Pubmed ID: 15939202

A one and a half day workshop on behavioral testing was conducted in order to discuss experimental procedures and practices that may help enhance the utility of behavioral data as a reliable index of neurotoxicity and in the safety evaluation of chemical substances. The workshop was open to participation by all sectors of the neuroscience community including academia, government, testing laboratories, and industry. The level of confidence with which changes in behavior can reliably signal adverse effects on the nervous system depends, in part, on the scientific quality of the data generated. With an emphasis on education and problem solving, the workshop focused on the practical aspects and scientific rationale underlying valid and high quality testing. In behavioral testing, there are numerous experimental factors that may impact on the quality of data. These include such elements as experimental design, selection of test methods, the care and precision in the conduct of behavioral testing, procedures to minimize bias and potential confounds, appropriateness of statistical analyses, and data interpretation. In plenary session investigators experienced in behavioral testing discussed the significance of these various experimental factors to data quality, outlined problematic issues, and presented a synopsis of approaches for addressing each of the factors as outlined in a draft of a primer developed by the Interagency Committee on Neurotoxicology (ICON). During the remainder of the workshop, open discussions in small breakout groups were used to address the problematic issues identified by the plenary speakers and explore alternative approaches for dealing with them. Finally, all workshop participants were reconvened in plenary session for summation of breakout group discussions and final recommendations. Information from the workshop was used to form the basis of this manuscript and will be used to help finalize a behavioral test methods primer being drafted by the ICON. The overall conclusions from the workshop were that consensus can be reached on the fundamentals of behavioral assessment, and that aspects of behavioral assessment including experimental design, test method selection, training, validation, control of confounds, data variability, data analysis, and data interpretation need to be carefully considered in the planning and conduct of behavioral safety assessments.

Receptor-targeted SiRNAs

Nature Biotechnology. Jun, 2005  |  Pubmed ID: 15940237

Short Hairpin RNA-directed Cytosine (CpG) Methylation of the RASSF1A Gene Promoter in HeLa Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jul, 2005  |  Pubmed ID: 15963934

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.

Martin Tabler, Ph.D. June 20, 1956-April 3, 2005

Oligonucleotides. 2005  |  Pubmed ID: 15989420

A Society of Our Own

Oligonucleotides. 2005  |  Pubmed ID: 15989421

Lentiviral Vector Delivery of SiRNA and ShRNA Encoding Genes into Cultured and Primary Hematopoietic Cells

Methods in Molecular Biology (Clifton, N.J.). 2005  |  Pubmed ID: 15990406

RNAi and the P-body Connection

Nature Cell Biology. Jul, 2005  |  Pubmed ID: 15990892

HnRNP H Inhibits Nuclear Export of MRNA Containing Expanded CUG Repeats and a Distal Branch Point Sequence

Nucleic Acids Research. 2005  |  Pubmed ID: 16027111

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder associated with a (CUG)n expansion in the 3'-untranslated region of the DMPK (DM1 protein kinase) gene. Mutant DMPK mRNAs containing the trinucleotide expansion are retained in the nucleus of DM1 cells and form discrete foci. The nuclear sequestration of RNA binding proteins and associated factors binding to the CUG expansions is believed to be responsible for several of the splicing defects observed in DM1 patients and could ultimately be linked to DM1 muscular pathogenesis. Several RNA binding proteins capable of co-localizing with the nuclear-retained mutant DMPK mRNAs have already been identified but none can account for the nuclear retention of the mutant transcripts. Here, we have employed a modified UV crosslinking assay to isolate proteins bound to mutant DMPK-derived RNA and have identified hnRNP H as an abundant candidate. The specific binding of hnRNP H requires not only a CUG repeat expansion but also a splicing branch point distal to the repeats. Suppression of hnRNP H expression by RNAi rescued nuclear retention of RNA with CUG repeat expansions. The identification of hnRNP H as a factor capable of binding and possibly modulating nuclear retention of mutant DMPK mRNA may prove to be an important link in our understanding of the molecular mechanisms that lead to DM1 pathogenesis.

Functional Polarity is Introduced by Dicer Processing of Short Substrate RNAs

Nucleic Acids Research. 2005  |  Pubmed ID: 16049023

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

Long-term Inhibition of HIV-1 Infection in Primary Hematopoietic Cells by Lentiviral Vector Delivery of a Triple Combination of Anti-HIV ShRNA, Anti-CCR5 Ribozyme, and a Nucleolar-localizing TAR Decoy

Molecular Therapy : the Journal of the American Society of Gene Therapy. Nov, 2005  |  Pubmed ID: 16115802

Combinatorial therapies for the treatment of HIV-1 infection have proven to be effective in reducing patient viral loads and slowing the progression to AIDS. We have developed a series of RNA-based inhibitors for use in a gene therapy-based treatment for HIV-1 infection. The transcriptional units have been inserted into the backbone of a replication-defective lentiviral vector capable of transducing a wide array of cell types, including CD34+ hematopoietic progenitor cells. The combinatorial therapeutic RNA vector harbors a U6 Pol III promoter-driven short hairpin RNA (shRNA) targeting the rev and tat mRNAs of HIV-1, a U6 transcribed nucleolar-localizing TAR RNA decoy, and a VA1-derived Pol III cassette that expresses an anti-CCR5 ribozyme. Each of these therapeutic RNAs targets a different gene product and blocks HIV infection by a distinct mechanism. Our results demonstrate that the combinatorial vector suppresses HIV replication long term in a more-than-additive fashion relative to the single shRNA or double shRNA/ribozyme or decoy combinations. Our data demonstrate the validity and efficacy of a combinatorial RNA-based gene therapy for the treatment of HIV-1 infection.

Approaches for Chemically Synthesized SiRNA and Vector-mediated RNAi

FEBS Letters. Oct, 2005  |  Pubmed ID: 16199038

Successful applications of RNAi in mammalian cells depend upon effective knockdown of targeted transcripts and efficient intracellular delivery of either preformed si/shRNAs or vector expressed si/shRNAs. We have previously demonstrated that 27 base pair double stranded RNAs which are substrates for Dicer can be up to 100 times more potent than 21mer siRNAs. In this mini-review we elaborate upon the rationale and design strategies for creating Dicer substrate RNAs that provide enhanced knockdown of targeted RNAs and minimize the utilization of the sense strand as RNAi effectors. Expression of shRNAs or siRNAs in mammalian cells can be achieved via transcription from either Pol II or Pol III promoters. There are certain constrictions in designing such vectors, and these are described here. Additionally, we review strategies for inducible shRNA expression and the various viral vectors that can be used to transduce shRNA genes into a variety of cells and tissues. The overall goal of this mini-review is to provide an overview of available approaches for optimizing RNAi mediated down regulation of gene expression in mammalian cells via RNA interference. Although the primary focus is the use of RNAi mediated cleavage of targeted transcripts, it is highly probable that some of the approaches described herein will be applicable to RNAi mediated inhibition of translation and transcriptional gene silencing.

Mammalian Dicer Finds a Partner

EMBO Reports. Oct, 2005  |  Pubmed ID: 16222240

Enhancing SiRNA Effects in T Cells for Adoptive Immunotherapy

Hematology (Amsterdam, Netherlands). Dec, 2005  |  Pubmed ID: 16321811

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.

Uncoupling of RNAi from Active Translation in Mammalian Cells

RNA (New York, N.Y.). Jan, 2005  |  Pubmed ID: 15574516

Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.

Synthetic DsRNA Dicer Substrates Enhance RNAi Potency and Efficacy

Nature Biotechnology. Feb, 2005  |  Pubmed ID: 15619617

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25-30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

Lentiviral Vector Delivery of Recombinant Small Interfering RNA Expression Cassettes

Methods in Enzymology. 2005  |  Pubmed ID: 15644184

Lentiviral vectors are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. Many cells types, including brain, liver, muscle and hematopoietic stem cells, have been successfully transduced with lentiviral vectors carrying a variety of genes. These properties make lentiviral vectors attractive vehicles for delivering small interfering RNA (siRNA) genes into mammalian cells. RNA polymerase III (pol III) promoters are most commonly used for expressing siRNAs from lentiviral vectors. Pol III promoters are relatively small, have high activity, and use simple termination signals of short stretches of Us. It is possible to include several pol III expression cassettes in a single lentiviral vector backbone to express different siRNAs or to combine siRNAs with other transgenes. This chapter describes the delivery of pol III-promoted siRNAs by human immunodeficiency virus (HIV)-based lentiviral vectors and covers vector design, production, and verification of siRNA expression and function. This chapter should be useful for establishing a lentiviral vector-based delivery of siRNAs in experiments that require long-term gene knockdown or developing siRNA-based approaches for gene therapy applications.

A Potential Role for RNA Interference in Controlling the Activity of the Human LINE-1 Retrotransposon

Nucleic Acids Research. 2005  |  Pubmed ID: 15701756

Long interspersed nuclear elements (LINE-1 or L1) comprise 17% of the human genome, although only 80-100 L1s are considered retrotransposition-competent (RC-L1). Despite their small number, RC-L1s are still potential hazards to genome integrity through insertional mutagenesis, unequal recombination and chromosome rearrangements. In this study, we provide several lines of evidence that the LINE-1 retrotransposon is susceptible to RNA interference (RNAi). First, double-stranded RNA (dsRNA) generated in vitro from an L1 template is converted into functional short interfering RNA (siRNA) by DICER, the RNase III enzyme that initiates RNAi in human cells. Second, pooled siRNA from in vitro cleavage of L1 dsRNA, as well as synthetic L1 siRNA, targeting the 5'-UTR leads to sequence-specific mRNA degradation of an L1 fusion transcript. Finally, both synthetic and pooled siRNA suppressed retrotransposition from a highly active RC-L1 clone in cell culture assay. Our report is the first to demonstrate that a human transposable element is subjected to RNAi.

Importance of Public Engagement Found Through Involvement in Horse Slaughter Bill

Journal of the American Veterinary Medical Association. Feb, 2005  |  Pubmed ID: 15702679

Cytoplasmic and Nuclear Retained DMPK MRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells

The Journal of Biological Chemistry. Apr, 2005  |  Pubmed ID: 15722335

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( approximately 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.

SiRNA Target Site Secondary Structure Predictions Using Local Stable Substructures

Nucleic Acids Research. 2005  |  Pubmed ID: 15722476

The crystal structure based model of the catalytic center of Ago2 revealed that the siRNA and the mRNA must be able to form an A-helix for correct positing of the scissile phosphate bond for cleavage in RNAi. This suggests that base pairing of the target mRNA with itself, i.e. secondary structure, must be removed before cleavage. Early on in the siRNA design, GC-rich target sites were avoided because of their potential to be involved in strong secondary structure. It is still unclear how important a factor mRNA secondary structure is in RNAi. However, it has been established that a difference in the thermostability of the ends of an siRNA duplex dictate which strand is loaded into the RNA-induced silencing complex. Here, we use a novel secondary structure prediction method and duplex-end differential calculations to investigate the importance of a secondary structure in the siRNA design. We found that the differential duplex-end stabilities alone account for functional prediction of 60% of the 80 siRNA sites examined, and that secondary structure predictions improve the prediction of site efficacy. A total of 80% of the non-functional sites can be eliminated using secondary structure predictions and duplex-end differential.

Sensing the Danger in RNA

Nature Medicine. Mar, 2005  |  Pubmed ID: 15746933

Comparative Single-turnover Kinetic Analyses of Trans-cleaving Hammerhead Ribozymes with Naturally Derived Non-conserved Sequence Motifs

FEBS Letters. Mar, 2005  |  Pubmed ID: 15757651

trans-Cleaving hammerhead ribozyme variants were generated with mimicked non-conserved internal loop motifs derived from five structurally diverse natural cis-cleaving ribozymes. Most modified trans-cleaving variants showed enhanced single-turnover cleavage rates relative to minimal counterparts that lack tertiary interactions between internal loop motifs I and II, and relative to controls with sequence changes in loop I. The trans-cleaving ribozyme derived from the positive strand of peach latent mosaic viroid had the highest observed cleavage rate, suggesting a structurally optimized motif that facilitates rapid formation of the ribozyme catalytic center in a trans-reaction.

Ribozyme Cleavage Leads to Decreased Expression of Fibroblast Growth Factor Receptor 3 in Human Multiple Myeloma Cells, Which is Associated with Apoptosis and Downregulation of Vascular Endothelial Growth Factor

Oligonucleotides. 2005  |  Pubmed ID: 15788896

The aim of this study was to investigate the fibroblast growth factor receptor 3 (FGFR3) mRNA cleavage by ribozymes targeting FGFR3, effect of growth inhibition and associated with mechanism on multiple myeloma (MM). We designated two ribozyme-expressing plasmids that target the FGFR3 genes, Rz52 and Rz32. In vitro catalytic activity of Rz52 and Rz32 in KMS11 cells decreased FGFR3 mRNA expression to 45% (p < 0.05) and 80% (p < 0.5), respectively, of that of the control. In vivo examination of the Rz52-transfected KMS11 clone showed that FGFR3 mRNA expression decreased to 20% (p < 0.05) of the control. In the Rz52-transfected H929 clone, FGFR3 mRNA decreased to 50% of the control. Protein expression of FGFR3 decreased to 70% of the parental KMS11 and H929 clones. DNA synthesis in the Rz52-transfected KMS11 clone decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p < 0.01) of that of the control. Ribozyme cleavage-associated increase in apoptosis of Rz52 KMS11 transfectants was twice that of the control. The inhibition of FGFR3 expression by ribozymes was associated with decreased vascular endothelial growth factor (VEGF) expression and upregulation of Flt-1 but not of the KDR receptor. Our data indicate that FGFR3 is an important cell survival and antiapoptotic factor for MM cells and that ribozyme-targeted downregulation of FGFR3 might be useful as a novel therapeutic intervention in MM characterized by t(4;14).

The Antisense Strand of Small Interfering RNAs Directs Histone Methylation and Transcriptional Gene Silencing in Human Cells

RNA (New York, N.Y.). Feb, 2006  |  Pubmed ID: 16373483

To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to alpha-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.

Novel Pol II Fusion Promoter Directs Human Immunodeficiency Virus Type 1-inducible Coexpression of a Short Hairpin RNA and Protein

Journal of Virology. Feb, 2006  |  Pubmed ID: 16439542

We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

Antiviral Applications of RNAi

Current Opinion in Molecular Therapeutics. Apr, 2006  |  Pubmed ID: 16610763

RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) operates to specifically and potently downregulate the expression of a target gene. This downregulation has been thought to predominantly function at the level of mRNA, as post-transcriptional gene silencing. The discovery that siRNAs can suppress gene expression at the level of transcription, that is, transcriptional gene silencing, has created a major paradigm shift in mammalian RNAi. These findings significantly broaden the role that RNA, specifically siRNA and potentially microRNA, plays in the regulation of gene expression, as well as the breadth of potential siRNA target sites. Indeed, the specificity and simplicity of design makes the use of siRNAs to target and suppress virtually any gene of interest a realized technology. Furthermore, since siRNAs are small nucleic acid reagents, they are unlikely to elicit an immune response, theoretically making them good therapeutics. The development, delivery and potential therapeutic use of antiviral siRNAs in treating viral infections and emerging viral threats are reviewed.

The Diversity of RNAi and Its Applications

BioTechniques. Apr, 2006  |  Pubmed ID: 16629381

RNAi As a Treatment for HIV-1 Infection

BioTechniques. Apr, 2006  |  Pubmed ID: 16629384

Human immunodeficiency virus type 1 (HIV-1) was the first primate virus shown to be inhibited by RNA interference (RNAi). Early studies used both synthetic and promoter expressed small interfering RNAs (siRNAs) or expressed short hairpin RNAs (shRNAs) to demonstrate that this virus was susceptible to RNAi. In addition to targeting the virus itself RNAi-mediated down-regulation of cellular targets that encode receptors required for viral entry also proved to be effective. The power of RNAi as an anti-HIV agent has propelled development of RNAi-based gene therapy approaches for the treatment of HIV infection in humans. Nevertheless, extensive in vitro experimentation has revealed potential problems of viral escape mutants and other toxicities caused by the si/shRNAs. This review covers the progress and problems in the development of RNAi for the treatment of HIV infection. Potential modalities for clinical application of RNAi in the treatment of HIV-1 infection are also described.

Stable Expression of ShRNAs in Human CD34+ Progenitor Cells Can Avoid Induction of Interferon Responses to SiRNAs in Vitro

Nature Biotechnology. May, 2006  |  Pubmed ID: 16648841

RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs). In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter-expressed shRNAs in primary CD34+ progenitor-derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNalpha and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.

A Nucleolar Localizing Rev Binding Element Inhibits HIV Replication

AIDS Research and Therapy. 2006  |  Pubmed ID: 16712721

The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment.

Lentivirus-mediated RNA Interference Therapy for Human Immunodeficiency Virus Type 1 Infection

Human Gene Therapy. May, 2006  |  Pubmed ID: 16716105

RNA interference (RNAi) is a natural mechanism by which small interfering RNAs (siRNAs) operate to specifically and potently downregulate the expression of a target gene. This downregulation has been demonstrated by targeting siRNAs to the mRNA (posttranscriptional gene silencing) as well as to the gene promoter, regulating gene expression epigenetically by transcriptional gene silencing. These observations significantly broaden the role RNA plays in the cell and suggest that siRNAs could prove to be a potent future therapeutic for the treatment of diseases such as human immunodeficiency virus type 1 (HIV-1) infection. The specificity and simplicity of design and the ability to express siRNAs from mammalian promoters make the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a soon-to-be-realized technology. However, the delivery and stable expression of siRNAs to target cells remain an enigma that could be surmounted, at least regarding the treatment of HIV-1 infection, by the application of lentiviral vectors to deliver and express anti-HIV-1 siRNAs in target cells. This review focuses on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating HIV-1.

Bcl-2 Protein in 518A2 Melanoma Cells in Vivo and in Vitro

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Aug, 2006  |  Pubmed ID: 16914583

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.

Partnering Aptamer and RNAi Technologies

Molecular Therapy : the Journal of the American Society of Gene Therapy. Oct, 2006  |  Pubmed ID: 16920031

Expressing Short Hairpin RNAs in Vivo

Nature Methods. Sep, 2006  |  Pubmed ID: 16929313

Promoter-based expression of short hairpin RNAs (shRNAs) may in principle provide stable silencing of genes in any tissue. As for all approaches that require transgene expression, safe delivery is the biggest obstacle, but toxicity can also occur via expression of the sequence itself. Innate immunity mechanisms can be triggered by expressed hairpin RNAs, critical cellular factors can be saturated, and genes other than the intended target can be silenced. Nevertheless, shRNAs constitute a valuable tool for in vivo research and have great therapeutic potential if the challenges with delivery and side effects are appropriately addressed.

Argonaute-1 Directs SiRNA-mediated Transcriptional Gene Silencing in Human Cells

Nature Structural & Molecular Biology. Sep, 2006  |  Pubmed ID: 16936726

Argonaute proteins are the core components of effector complexes that facilitate RNA interference (RNAi). Small interfering RNAs (siRNAs) targeted to promoter regions mediate transcriptional gene silencing (TGS) in human cells through heterochromatin formation. RNAi effector complexes have yet to be implicated in the mechanism of mammalian TGS. Here we describe the role of the human Argonaute-1 homolog (AGO1) in directing TGS at the promoters for human immunodeficiency virus-1 coreceptor CCR5 and tumor suppressor RASSF1A. AGO1 associates with RNA polymerase II (RNAPII) and is required for histone H3 Lys9 dimethylation and TGS. AGO1, TAR RNA-binding protein-2 (7TRBP2) and Polycomb protein EZH2 colocalize to the siRNA-targeted RASSF1A promoter, implicating Polycomb silencing in the mechanism of mammalian TGS. These results establish a connection between RNAi components AGO1 and TRBP2, RNAPII transcription and Polycomb-regulated control of gene expression.

Toxicity in Mice Expressing Short Hairpin RNAs Gives New Insight into RNAi

Genome Biology. 2006  |  Pubmed ID: 16942630

Short hairpin RNAs can provide stable gene silencing via RNA interference. Recent studies have shown toxicity in vivo that appears to be related to saturation of the endogenous microRNA pathway. Will these findings limit the therapeutic use of such hairpins?

Interaction of Muscleblind, CUG-BP1 and HnRNP H Proteins in DM1-associated Aberrant IR Splicing

The EMBO Journal. Sep, 2006  |  Pubmed ID: 16946708

In myotonic dystrophy (DM1), both inactivation of muscleblind proteins and increased levels of CUG-BP1 are reported. These events have been shown to contribute independently to aberrant splicing of a subset RNAs. We demonstrate that steady-state levels of the splice regulator, hnRNP H, are elevated in DM1 myoblasts and that increased hnRNP H levels in normal myoblasts results in the inhibition of insulin receptor (IR) exon 11 splicing in a manner similar to that observed in DM1. In normal myoblasts, overexpression of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1, which is required to maximally inhibit IR exon 11 inclusion. Elevated levels of MBNL1 show RNA-independent interaction with hnRNP H and dampen the inhibitory activity of increased hnRNP H levels on IR splicing in normal myoblasts. In DM1 myoblasts, overexpression of MBNL1 in conjunction with si-RNA mediated depletion of hnRNP H contributes to partial rescue of the IR splicing defect. These data demonstrate that coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 dictate IR splicing in normal and DM1 myoblasts.

Small Interfering RNAs to the Rescue: Blocking L1 Retrotransposition

Nature Structural & Molecular Biology. Sep, 2006  |  Pubmed ID: 16955095

RNAi in Combination with a Ribozyme and TAR Decoy for Treatment of HIV Infection in Hematopoietic Cell Gene Therapy

Annals of the New York Academy of Sciences. Oct, 2006  |  Pubmed ID: 17145937

Combinatorial therapies for the treatment of HIV infection have changed the course of the AIDS epidemic in developed nations where the antiviral drug combinations are readily available. Despite this progress, there are many problems associated with chemotherapy for AIDS including toxicities and emergence of viral mutants resistant to the drugs. Our goal has been the development of a hematopoietic gene therapy treatment for HIV infection. Like chemotherapy, gene therapy for treatment of HIV infection should be used combinatorially. We have thus combined three different inhibitory genes for treatment of HIV infection into a single lentiviral vector backbone. The inhibitory agents engage RNAi via a short hairpin RNA targeting HIV tat/rev mRNAs, a nucleolar localizing decoy that binds and sequesters the HIV Tat protein, and a ribozyme that cleaves and downregulates the CCR5 chemokine receptor used by HIV for cellular entry. This triple combination has proven to be highly effective for inhibiting HIV replication in primary hematopoietic cells, and is currently on track for human clinical application.

Inhibition of Infectious Human Immunodeficiency Virus Type 1 Virions Via Lentiviral Vector Encoded Short Antisense RNAs

Oligonucleotides. 2006  |  Pubmed ID: 17155905

During the life cycles of most retroviruses and lentiviruses, dimerization and packaging of two copies of viral genomic RNA is required for the subsequent conversion of RNA into double stranded DNA by reverse transcriptase. For human immunodeficiency virus type 1 (HIV-1), dimerization is mediated by interactions of the stem-loop structures in the dimerization-packaging, or psi (Psi) domain. We have tethered anti-HIV gag ribozymes and small antisense RNAs to the HIV Psi domain in an HIV-1 lentiviral vector to facilitate copackaging of these replication inhibitors with HIV genomic RNAs during HIV infectious challenge. In order to maximize the base pairing of the ribozymes or antisense segments to the HIV-1 genomic target, sequences in HIV-1 were identified that are highly accessible to antisense pairing. Ribozymes or antisense RNAs designed to target these sequences were inserted in the lentiviral vector at the same relative distance to the Psi element as the HIV-1 target sites. Packaged vectors were transduced into CEM cells followed by challenges with HIV-1. Only the constructs that harbored short antisense segments complementary to HIV-1 gag produced replication incompetent HIV-1. These results demonstrate that a short stretch of antisense pairing downstream of the dimerization domain in an HIV-based vector can drive dimerization and provide a powerful approach for inhibition of HIV-1.

Chemical Modifications Rescue Off-target Effects of RNAi

ACS Chemical Biology. Jun, 2006  |  Pubmed ID: 17163754

RNAi's specificity has been questioned for some time. Three recent papers show that off-target effects should be considered normal, but one paper also provides insight on how chemical modifications of siRNAs may overcome the problem.

Endogenous Expression of an Anti-TAR Aptamer Reduces HIV-1 Replication

RNA Biology. Oct, 2006  |  Pubmed ID: 17299271

An anti-TAR RNA aptamer called R06, which binds tightly and specifically to the trans-activation responsive (TAR) element of the human immunodeficiency virus type 1 (HIV-1) through loop-loop interactions has been previously selected.(1) We used HIV-based retroviral vectors to express the R06 aptamer. Its synthesis was driven by the U16 snoRNA. We investigated the ability of this cassette to interfere with TAR-mediated transcription using HeLa P4 cells stably expressing the beta-galactosidase gene under the control of the HIV-1 5'LTR. We demonstrated that, upon HIV-1 infection, the beta-galactosidase activity was reduced in cells expressing the nucleolar U16-R06 transcript. The replication of HIV-1 in these cells was also reduced as shown by quantification of the HIV-1 protease gene 24 h post-infection. This effect was specific and related to the formation of R06 TAR complex as an aptamer with a mutated loop, which was no longer able to bind to TAR, did not show any effect. The nucleolus is likely a compartment of interest for targeting the TAR-protein complex responsible for the trans-activation of transcription of the HIV-1 genome.

Rational Design and in Vitro and in Vivo Delivery of Dicer Substrate SiRNA

Nature Protocols. 2006  |  Pubmed ID: 17406276

RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21-25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.

Safety and Efficacy of a Lentiviral Vector Containing Three Anti-HIV Genes--CCR5 Ribozyme, Tat-rev SiRNA, and TAR Decoy--in SCID-hu Mouse-derived T Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2007  |  Pubmed ID: 17406343

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes--namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme--have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

Epigenetics and MicroRNAs

Pediatric Research. May, 2007  |  Pubmed ID: 17413846

MicroRNAs (miRNAs) regulate protein-coding genes post transcriptionally in higher eukaryotes. Argonaute proteins are important in miRNA regulation and are also implicated in epigenetic mechanisms such as histone modifications and DNA methylation. Here, we review miRNA regulation and outline its connections to epigenetics.

Optimization and Characterization of TRNA-shRNA Expression Constructs

Nucleic Acids Research. 2007  |  Pubmed ID: 17426139

Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNA(Lys3)-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3' tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.

RNAi Therapeutics: Principles, Prospects and Challenges

Advanced Drug Delivery Reviews. Mar, 2007  |  Pubmed ID: 17449137

RNA interference (RNAi) was discovered less than a decade ago and already there are human clinical trials in progress or planned. A major advantage of RNAi versus other antisense based approaches for therapeutic applications is that it utilizes cellular machinery that efficiently allows targeting of complementary transcripts, often resulting in highly potent down-regulation of gene expression. Despite the excitement about this remarkable biological process for sequence specific gene regulation, there are a number of hurdles and concerns that must be overcome prior to making RNAi a real therapeutic modality, which include off-target effects, triggering of type I interferon responses, and effective delivery in vivo. This review discusses mechanistic aspects of RNAi, the potential problem areas and solutions and therapeutic applications. It is anticipated that RNAi will be a major therapeutic modality within the next several years, and clearly warrants intense investigation to fully understand the mechanisms involved.

Short Hairpin RNA Causes the Methylation of Transforming Growth Factor-beta Receptor II Promoter and Silencing of the Target Gene in Rat Hepatic Stellate Cells

Biochemical and Biophysical Research Communications. Jul, 2007  |  Pubmed ID: 17533113

Small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in plant and animal cells. RNA dependent DNA methylation (RdDM) accounts for TGS in plants, but it is unclear whether siRNA induces RdDM in mammalian cells. To determine whether stable expression of short hairpin siRNA (shRNA) induces DNA methylation in mammalian cells, we transduced rat hepatic stellate SBC10 cells with lentiviral vectors which encode an U6 promoter-driven shRNA expression cassette homologous to the transforming growth factor-beta receptor (TGFbetaRII) promoter region. Sequencing analysis of bisulfite-modified genomic DNA showed the methylation of cytosine residues both in CpG dinucleotides and non-CpG sites around the target region of the TGFbetaRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFbetaRII mRNA levels which were reversed by treatment with 5-aza-2-deoxycytidine. Our results demonstrate that recombinant lentivirus-mediated shRNA delivery resulted in the methylation of the homologous promoter area in mammalian cells, and this approach may be used as a tool for transcriptional gene silencing by epigenetic modification of mammalian cell promoters.

Molecular Medicine: Entry Granted

Nature. Jul, 2007  |  Pubmed ID: 17611531

Combinatorial Delivery of Small Interfering RNAs Reduces RNAi Efficacy by Selective Incorporation into RISC

Nucleic Acids Research. 2007  |  Pubmed ID: 17660190

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.

Cloning and Detecting Signature MicroRNAs from Mammalian Cells

Methods in Enzymology. 2007  |  Pubmed ID: 17720482

MicroRNAs (miRNAs) are about 19- to 24-nucleotides long noncoding regulatory small RNAs that could silence target gene expression through base pairing to the complementary sequences in the 3' untranslated region (3'UTR) of targeted genes. They are evolutionally conserved and play an important regulatory role in embryogenesis, cell differentiation, and proliferation. They are also involved in pathogenesis and progression of some human diseases. There are about 1000 human miRNAs predicted today, and it is estimated that they could target about 30% of all human transcripts. Profiling the miRNAs that are expressed in the experimental cells became an important issue as different cells express different signature miRNAs or express the same miRNAs at different level. Small RNA cloning is a reliable way to characterize those tissue- or cell-specific signature miRNAs. This chapter describes a relatively nonlaborious polyadenylation-mediated complementary DNA (cDNA) cloning method that will identify most of the small RNAs expressed in the cells of interest. This procedure can also be used to verify bioinformatic predictions of miRNAs/small interfering RNAs (siRNAs) as well as to identify new miRNAs/siRNAs.

Transcriptional Activation by Small RNA Duplexes

Nature Chemical Biology. Mar, 2007  |  Pubmed ID: 17301798

Strategies for Silencing Human Disease Using RNA Interference

Nature Reviews. Genetics. Mar, 2007  |  Pubmed ID: 17304245

Since the first description of RNA interference (RNAi) in animals less than a decade ago, there has been rapid progress towards its use as a therapeutic modality against human diseases. Advances in our understanding of the mechanisms of RNAi and studies of RNAi in vivo indicate that RNAi-based therapies might soon provide a powerful new arsenal against pathogens and diseases for which treatment options are currently limited. Recent findings have highlighted both promise and challenges in using RNAi for therapeutic applications. Design and delivery strategies for RNAi effector molecules must be carefully considered to address safety concerns and to ensure effective, successful treatment of human diseases.

A Facile Lentiviral Vector System for Expression of Doxycycline-inducible ShRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2007  |  Pubmed ID: 17311008

RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. During the past 3 years a number of systems for conditional RNAi have been developed that allow controlled expression of short hairpin RNA (shRNA) triggers of RNAi. The simplest strategy relies on tet-operable polymerase III-promoted shRNAs and co-expression of the tetracycline regulatory protein, TetR. In this study we have combined these features into a single lentiviral vector that upon delivery to target cells allows robust induction of shRNAs, even with low levels of doxycycline; importantly, we show minimal leakiness in the absence of inducer. We have exploited the regulatory properties of our system by targeting an essential cellular gene, the nuclear RNaseIII endonuclease Drosha. Drosha is the core catalytic component of the "microprocessor complex" and cleaves the primary microRNA (miRNA) transcripts into their pre-miRNA hairpin intermediates. We anticipate that our vector will facilitate functional studies of miRNA biogenesis.

Cross-clade Inhibition of Recombinant Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus SIVcpz Reverse Transcriptases by RNA Pseudoknot Aptamers

Journal of Virology. May, 2007  |  Pubmed ID: 17329328

Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.

MicroRNA-192 in Diabetic Kidney Glomeruli and Its Function in TGF-beta-induced Collagen Expression Via Inhibition of E-box Repressors

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2007  |  Pubmed ID: 17360662

Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-alpha 1 and -2 (Col1a1 and -2). Transforming growth factor beta1 (TGF-beta), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-beta increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, deltaEF1. TGF-beta treatment or short hairpin RNAs targeting deltaEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-beta also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to deltaEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-beta in MMC. TGF-beta treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3' UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with deltaEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-beta and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-beta-induced Col1a2 expression by down-regulating E-box repressors.

Distance Constraints Between MicroRNA Target Sites Dictate Efficacy and Cooperativity

Nucleic Acids Research. 2007  |  Pubmed ID: 17389647

MicroRNAs (miRNAs) have the potential to regulate the expression of thousands of genes, but the mechanisms that determine whether a gene is targeted or not are poorly understood. We studied the genomic distribution of distances between pairs of identical miRNA seeds and found a propensity for moderate distances greater than about 13 nt between seed starts. Experimental data show that optimal down-regulation is obtained when two seed sites are separated by between 13 and 35 nt. By analyzing the distance between seed sites of endogenous miRNAs and transfected small interfering RNAs (siRNAs), we also find that cooperative targeting of sites with a separation in the optimal range can explain some of the siRNA off-target effects that have been reported in the literature.

Potent SiRNA Inhibitors of Ribonucleotide Reductase Subunit RRM2 Reduce Cell Proliferation in Vitro and in Vivo

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Apr, 2007  |  Pubmed ID: 17404105

Ribonucleotide reductase (RR) is a therapeutic target for DNA replication-dependent diseases such as cancer. Here, a potent small interfering RNA (siRNA) duplex against the M2 subunit of RR (RRM2) is developed and shown to reduce the growth potential of cancer cells both in vitro and in vivo.

Targeted Cleavage: Tuneable Cis-cleaving Ribozymes

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2007  |  Pubmed ID: 17855561

MicroRNAs in Disease and Potential Therapeutic Applications

Molecular Therapy : the Journal of the American Society of Gene Therapy. Dec, 2007  |  Pubmed ID: 17878899

MicroRNAs (miRNAs) are 21-24 nucleotide (nt) duplex RNAs that are created from precursor transcripts by subsequent processing steps mediated by members of the RNAseIII family, Drosha and Dicer. One of the two strands is incorporated into the active sites of the Argonaute family of proteins, where it serves as a guide for Watson-Crick base pairing with complementary sequences in target messenger RNAs (mRNAs). In mammals, the majority of miRNAs guide the RNA-induced silencing complex (RISC) to the 3' untranslated regions (UTRs) of mRNA targets, with the consequence that translation of the target mRNAs is inhibited. The importance of miRNAs in normal cellular development and metabolism is only now being realized. miRNA deficiencies or excesses have been correlated with a number of clinically important diseases ranging from myocardial infarction to cancers. The loss or gain of miRNA function can be caused by a single point mutation in either the miRNA or its target or by epigenetic silencing of primary miRNA transcription units. This review summarizes miRNA biogenesis and biology, explores the potential roles miRNAs can play in a variety of diseases, and suggests some therapeutic applications for restoring or inhibiting miRNA function.

Genetic Therapies Against HIV

Nature Biotechnology. Dec, 2007  |  Pubmed ID: 18066041

Highly active antiretroviral therapy prolongs the life of HIV-infected individuals, but it requires lifelong treatment and results in cumulative toxicities and viral-escape mutants. Gene therapy offers the promise of preventing progressive HIV infection by sustained interference with viral replication in the absence of chronic chemotherapy. Gene-targeting strategies are being developed with RNA-based agents, such as ribozymes, antisense, RNA aptamers and small interfering RNA, and protein-based agents, such as the mutant HIV Rev protein M10, fusion inhibitors and zinc-finger nucleases. Recent advances in T-cell-based strategies include gene-modified HIV-resistant T cells, lentiviral gene delivery, CD8(+) T cells, T bodies and engineered T-cell receptors. HIV-resistant hematopoietic stem cells have the potential to protect all cell types susceptible to HIV infection. The emergence of viral resistance can be addressed by therapies that use combinations of genetic agents and that inhibit both viral and host targets. Many of these strategies are being tested in ongoing and planned clinical trials.

Lentivirus Transduction of Hematopoietic Cells

CSH Protocols. 2007  |  Pubmed ID: 21357086

INTRODUCTIONEfficient transfer and sustained expression of transgenes are among the most important issues in gene delivery. The majority of hematopoietic cells are nondividing or slowly self-renewing. Thus, they are refractory to most nonviral or retroviral delivery methods. Lentiviral vectors are capable of transducing nondividing cells and maintaining long-term and sustained expression of the transgenes. They are becoming useful for many delivery protocols, such as long-term expression of short hairpin RNA (shRNA) and functional genetics. They may also have great potential in gene therapy. This protocol describes lentivirus-vector-based delivery of foreign genes to hematopoietic cells. The method is applicable to various cell types in experiments that require long-term transgene expression.

Metalloproteases Regulate T-cell Proliferation and Effector Function Via LAG-3

The EMBO Journal. Jan, 2007  |  Pubmed ID: 17245433

Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases approximately 12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCtheta-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3(-/-) T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target.

Expression Strategies for Short Hairpin RNA Interference Triggers

Human Gene Therapy. Apr, 2008  |  Pubmed ID: 18363506

Since the discovery that the triggers for RNA interference (RNAi), small interfering RNAs, could mediate silencing in mammalian cells without triggering a toxic response, RNAi has become the standard tool for sequence-specific knockdown of gene expression in molecular biology. This is due in part to the development of methods for promoter-based expression of RNAi triggers that can mediate stable silencing in mammalian cells. Numerous systems with slightly different characteristics exist, but despite incredible progress in a field that moves very rapidly, challenges still remain. The biggest challenge is to successfully and safely apply RNAi in vivo. Aside from potential issues of delivery, which is one of the most important considerations, successful application of short hairpin RNAs (shRNAs) in vivo requires expression systems that yield potent and specific knockdown of the target in the absence of toxicity. With a couple of exceptions, the current systems available for shRNA expression have not generally resulted in unexpected toxicities, while still providing strong knockdown of the intended targets; however, we do not know enough about how sequence-specific off-target effects will affect various cell and tissue types, or to what extent ectopic expression of RNAi triggers will perturb the endogenous RNAi mechanisms.

Principles of Dicer Substrate (D-siRNA) Design and Function

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 18369774

An efficient RNAi largely depends on optimal design of the siRNA. In recent studies, Dicer substrates were found to be more potent than classical synthetic 21-mer siRNAs, suggesting a coupling of the Dicer-mediated processing step to the efficient assembly of the silencing complex, RISC. We describe the fundamental principles and experimental results leading to optimal Dicer substrates.

Use of a U16 SnoRNA-containing Ribozyme Library to Identify Ribozyme Targets in HIV-1

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2008  |  Pubmed ID: 18388915

Hammerhead ribozymes have been shown to silence human immunodeficiency virus-1 (HIV-1) gene expression by site-specific cleavage of viral mRNA. The two major factors that determine whether ribozymes will be effective for post-transcriptional gene silencing are colocalization of the ribozyme and the target RNAs, and the choice of an appropriate target site on the mRNA. An effective screening strategy for potential targets on the viral genome is the use of ribozyme libraries in cell culture. Capitalizing on previous findings that HIV-1 and ribozymes can be colocalized in the nucleolus, we created a novel hammerhead ribozyme library by inserting hammerhead ribozymes with fully randomized stems 1 and 2 into the body of the U16 small nucleolar RNA (snoRNA). Following three rounds of cotransfection with an HIV-1 proviral DNA harboring the herpes simplex virus thymidine kinase (HSV-TK) gene, we selected for gancyclovir-resistant cells and identified a ribozyme sequence that could potentially target both the U5 and gag genes of HIV-1 regions on the HIV-1 genome through partial homologies with these targets. When the ribozymes were converted to full complementarity with the targets, they provided potent inhibition of HIV-1 replication in cell culture. These results provide a novel approach for identifying ribozyme targets in HIV-1.

Tat-regulated Expression of RNA Interference: Triggers for the Treatment of HIV Infection

Current HIV/AIDS Reports. Feb, 2008  |  Pubmed ID: 18417034

HIV infection is a lifelong problem requiring continual medication for suppressing viral replication. Current strategies of antiretroviral drug combinations have proven effective in prolonging the time from infection to the symptoms of AIDS. Nevertheless, chemotherapy is not without its problems, which include toxicities and eventual emergence of virus mutants that are resistant to current antiretrovirals. Gene therapy refers to the introduction of effector oligonucleotides to transiently or stably alter gene expression or the delivery and expression of an exogenous gene within a specific target cell. A number of studies have demonstrated effective silencing/inhibition of HIV-1 replication by using RNA-based effector oligonucleotides for RNA interference. In this study, we have taken advantage of lentiviral vector-mediated delivery of anti-HIV short hairpin RNA for the treatment of HIV infection in hematopoietic cells.

Realizing the Promise of RNAi

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2008  |  Pubmed ID: 18432272

Novel Dual Inhibitory Function Aptamer-siRNA Delivery System for HIV-1 Therapy

Molecular Therapy : the Journal of the American Society of Gene Therapy. Aug, 2008  |  Pubmed ID: 18461053

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. In this study, we demonstrate cell type-specific delivery of anti-human immunodeficiency virus (anti-HIV) siRNAs through fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1-infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the appended siRNA is processed by Dicer; this releases an anti-tat/rev siRNA which, in turn, inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also show that gp120 expressed on the surface of HIV-infected cells can be used for aptamer-mediated delivery of anti-HIV siRNAs.

RNAi Mechanisms and Applications

BioTechniques. Apr, 2008  |  Pubmed ID: 18474035

Within the past two decades we have become increasingly aware of the roles that RNAs play in regulation of gene expression. The RNA world was given a booster shot with the discovery of RNA interference (RNAi), a compendium of mechanisms involving small RNAs (less than 30 bases long) that regulate the expression of genes in a variety of eukaryotic organisms. Rapid progress in our understanding of RNAi-based mechanisms has led to applications of this powerful process in studies of gene function as well as in therapeutic applications for the treatment of disease. RNAi-based therapies involve two-dimensional drug designs using only identification of good Watson-Crick base pairing between the RNAi guide strand and the target, thereby resulting in rapid design and testing of RNAi triggers. To date there are several clinical trials using RNAi, and we should expect the list of new applications to grow at a phenomenal rate. This article summarizes our current knowledge about the mechanisms and applications of RNAi.

Wandering Eye for RNAi

Nature Medicine. Jun, 2008  |  Pubmed ID: 18535575

Bivalent Aptamers Deliver the Punch

Chemistry & Biology. Jul, 2008  |  Pubmed ID: 18635000

Aptamers, sometimes termed "chemical antibodies," have been engineered into multimerized versions for therapeutic application. The groups of Gilboa and Sullenger now report the development of a bivalent aptamer-molecular device as a receptor agonist that has the same functional properties, but stronger avidity than a corresponding antibody.

Chemical Modification Patterns Compatible with High Potency Dicer-substrate Small Interfering RNAs

Oligonucleotides. Jun, 2008  |  Pubmed ID: 18637735

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

Lentiviral Vector Delivery of SiRNA and ShRNA Encoding Genes into Cultured and Primary Hematopoietic Cells

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 18679631

Lentiviral vectors are able to transduce non-dividing cells and maintain sustained long-term expression of the transgenes. Many cell types including brain, liver, muscle, and hematopoietic stem cells have been successfully transduced with lentiviral vectors carrying a variety of genes. These properties make lentiviral vectors attractive vehicles for delivering small interfering RNA (siRNA) genes into mammalian cells. RNA polymerase III (Pol III) promoters are most commonly used for expressing siRNAs from lentiviral vectors. Pol III promoters are relatively small, have high activity, and use simple termination signals of short stretches of U. It is possible to include several Pol III expression cassettes in a single lentiviral vector backbone to express different siRNAs or to combine siRNAs with other transgenes. This chapter describes the delivery of Pol III promoted siRNAs by HIV-based lentiviral vectors and covers vector design, production, and verification of siRNA expression and function. This chapter should be useful for establishing a lentiviral vector-based delivery of siRNAs in experiments that require long-term gene knockdown or developing siRNA-based approaches for gene therapy applications.

Functional and Intracellular Localization Properties of U6 Promoter-expressed SiRNAs, ShRNAs, and Chimeric VA1 ShRNAs in Mammalian Cells

RNA (New York, N.Y.). Sep, 2008  |  Pubmed ID: 18697923

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.

Toward a Zoocentric Animal Ethics

The American Journal of Bioethics : AJOB. Jun, 2008  |  Pubmed ID: 18726783

MicroRNA-directed Transcriptional Gene Silencing in Mammalian Cells

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2008  |  Pubmed ID: 18852463

MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level in the cytoplasm, but recent findings suggest additional roles for miRNAs in the nucleus. To address whether miRNAs might transcriptionally silence gene expression, we searched for miRNA target sites proximal to known gene transcription start sites in the human genome. One conserved miRNA, miR-320, is encoded within the promoter region of the cell cycle gene POLR3D in the antisense orientation. We provide evidence of a cis-regulatory role for miR-320 in transcriptional silencing of POLR3D expression. miR-320 directs the association of RNA interference (RNAi) protein Argonaute-1 (AGO1), Polycomb group (PcG) component EZH2, and tri-methyl histone H3 lysine 27 (H3K27me3) with the POLR3D promoter. Our results suggest the existence of an epigenetic mechanism of miRNA-directed transcriptional gene silencing (TGS) in mammalian cells.

A Role for the Dicer Helicase Domain in the Processing of Thermodynamically Unstable Hairpin RNAs

Nucleic Acids Research. Nov, 2008  |  Pubmed ID: 18927112

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.

Designing and Utilization of SiRNAs Targeting RNA Binding Proteins

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 18982303

Small interfering RNA (siRNA)-mediated RNA interference (RNAi) is a very powerful tool for triggering posttranscriptional gene silencing in several organisms. We discuss the improvement of two different sources of siRNAs synthesized either chemically or by an enzymatic method. When the siRNAs are synthesized by in vitro transcription using a phage polymerase, the initiating triphosphates trigger a potent interferon induction that can lead to misinterpretation of the data. A novel method is presented to minimize the nonspecific effect of enzymatic siRNAs while maintaining the advantages of lower cost and less turnaround time. When chemical siRNAs are used, the expense and long turnaround time can be a problem, especially if the selected siR-NAs are not highly functional in triggering RNAi. The new format for making double-stranded RNAs (dsRNAs) is described to achieve more efficient suppression. The format has been tested by creating siRNAs targeting two RNA binding proteins, La and hnRNP (heterogeneous nuclear ribonucleoprotein) H, and has shown better potency at lower concentrations than the conventional 21-mer siRNA.

Expression of Long Anti-HIV-1 Hairpin RNAs for the Generation of Multiple SiRNAs: Advantages and Limitations

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2008  |  Pubmed ID: 17726454

Promoter expressed long-hairpin RNAs (lhRNAs) that can be processed into multiple small interfering RNA (siRNAs) are being considered as effective agents for treating rapidly mutating viruses such as human immunodeficiency virus (HIV). In the present study, we have generated human U6 promoter-driven lhRNAs of 50, 53, and 80 base pairs (bp) targeting contiguous sequences within the tat and rev genes of HIV-1 and evaluated the efficacy of these lhRNAs as well as their processing in cells. By using multiple G:U mismatches in the stems, we have been able to readily incorporate the long-hairpin structures into a lentiviral vector transduction system. Here we show that such long hairpins can be stably and functionally expressed for a long term in HIV-1 susceptible T cells, where they provide potent inhibition of HIV replication against both non-mutant and mutant variants of HIV-1. Our studies provide strong support for the use of the G:U wobble pair containing lhRNAs to generate multiple siRNAs from a single transcript, but we also show that lhRNAs of 80 bp may be the upper size limit for effectively producing multiple, functional siRNAs.

The Promises and Pitfalls of RNA-interference-based Therapeutics

Nature. Jan, 2009  |  Pubmed ID: 19158789

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.

Selection, Characterization and Application of New RNA HIV Gp 120 Aptamers for Facile Delivery of Dicer Substrate SiRNAs into HIV Infected Cells

Nucleic Acids Research. May, 2009  |  Pubmed ID: 19304999

The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2'-F substituted RNA aptamers that bind to the HIV-1(BaL) gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer-siRNA chimeras. The aptamers and aptamer-siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a 'sticky' sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.

Overview of Gene Silencing by RNA Interference

Current Protocols in Nucleic Acid Chemistry / Edited by Serge L. Beaucage ... [et Al.]. Mar, 2009  |  Pubmed ID: 19319858

The potential of harnessing RNA interference (RNAi) for sequence-specific gene silencing has generated much excitement and progress in the field. Recent advances in RNAi technology suggest that RNAi-based approaches may soon become an effective therapeutic strategy against a myriad of diseases. This overview provides a brief description of important considerations when designing an RNAi-based method for gene silencing and therapeutic development: (a) mechanistic aspects of RNAi-mediated gene silencing in mammalian cells; (b) structural requirements for potent siRNA duplexes; (c) off-target effects and interferon responses; and (d) effective delivery of RNAi-inducing agents. Promising therapeutic applications of RNAi that are currently in the developmental pipeline are also described.

Nonhuman Primate Research: the Wrong Way to Understand Needs and Necessity

The American Journal of Bioethics : AJOB. May, 2009  |  Pubmed ID: 19396676

Dotting the I's and Crossing the T's: Integration Analyses in Transduced Patient T Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2009  |  Pubmed ID: 19404325

Transcriptional Gene Silencing Using Small RNAs

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19495692

RNA interference is a potent gene silencing pathway initiated by short molecules of double-stranded RNA. Small interfering RNAs (siRNAs) with full sequence complementarity to mRNAs induce cleavage of their target transcripts in the cytoplasm. Recent evidence has shown, however, that siRNAs can also function in the nucleus of mammalian cells to affect changes in chromatin structure. When targeted to promoter regions, siRNAs load into the effector protein Argonaute-1 (AGO1) and direct the formation of silent chromatin domains. This mechanism is known as transcriptional gene silencing (TGS), and the development of TGS as a novel therapeutic modality would be applicable to chronic diseases where long-term, heritable silencing of target genes is warranted. Here we discuss how small RNAs can be used to direct TGS in mammalian cells.

New Hope for a MicroRNA Therapy for Liver Cancer

Cell. Jun, 2009  |  Pubmed ID: 19524500

The loss of expression of particular microRNAs can contribute to tumorigenesis. Kota et al. (2009) now explore in a mouse model a promising new approach for the treatment of liver cancer-re-establishing the expression of an miRNA using a viral vector.

TGF-beta Activates Akt Kinase Through a MicroRNA-dependent Amplifying Circuit Targeting PTEN

Nature Cell Biology. Jul, 2009  |  Pubmed ID: 19543271

Akt kinase is activated by transforming growth factor-beta1 (TGF-beta) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells. However, the mechanisms of Akt activation by TGF-beta are not fully understood. Here we show that TGF-beta activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-beta and miR-192 through E-box-regulated mechanisms, as shown previously. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-beta. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-beta. Due to the diversity of PTEN function, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Evidence for X-chromosomal Schizophrenia Associated with MicroRNA Alterations

PloS One. 2009  |  Pubmed ID: 19568434

Schizophrenia is a severe disabling brain disease affecting about 1% of the population. Individual microRNAs (miRNAs) affect moderate downregulation of gene expression. In addition, components required for miRNA processing and/or function have also been implicated in X-linked mental retardation, neurological and neoplastic diseases, pointing to the wide ranging involvement of miRNAs in disease.

SNPs in Human MiRNA Genes Affect Biogenesis and Function

RNA (New York, N.Y.). Sep, 2009  |  Pubmed ID: 19617315

MicroRNAs (miRNAs) are 21-25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.

Problems Associated with Reporter Assays in RNAi Studies

RNA Biology. Sep-Oct, 2009  |  Pubmed ID: 19617709

Reporter assays represent a facile tool for studies of RNA interference and are routinely used in functional tests or mechanistic studies of siRNAs and miRNAs. Unfortunately some reporter assays were developed without careful consideration when they were adopted from studies of transcriptional regulation using promoter-reporter gene fusions. Here we report on some problems in the use of reporter systems for studies of RNA interference and make some suggestions for avoiding such problems in future studies.

A Risk Variant in an MiR-125b Binding Site in BMPR1B is Associated with Breast Cancer Pathogenesis

Cancer Research. Sep, 2009  |  Pubmed ID: 19738052

MicroRNAs regulate diverse cellular processes and play an integral role in cancer pathogenesis. Genomic variation within miRNA target sites may therefore be important sources for genetic differences in cancer risk. To investigate this possibility, we mapped HapMap single nucleotide polymorphisms (SNP) to putative miRNA recognition sites within genes dysregulated in estrogen receptor-stratified breast tumors and used local linkage disequilibrium patterns to identify high-ranking SNPs in the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer genome-wide association study for further testing. Two SNPs, rs1970801 and rs11097457, scoring in the top 100 from the CGEMS study, were in strong linkage disequilibrium with rs1434536, an SNP that resides within a miR-125b target site in the 3' untranslated region of the bone morphogenic receptor type 1B (BMPR1B) gene encoding a transmembrane serine/threonine kinase. We validated the CGEMS association findings for rs1970801 in an independent cohort of admixture-corrected cases identified from families with multiple case histories. Subsequent association testing of rs1434536 for these cases and CGEMS controls with imputed genotypes supported the association. Furthermore, luciferase reporter assays and overexpression of miR-125b-mimics combined with quantitative reverse transcription-PCR showed that BMPR1B transcript is a direct target of miR-125b and that miR-125b differentially regulates the C and T alleles of rs1434536. These results suggest that allele-specific regulation of BMPR1B by miR-125b explains the observed disease risk. Our approach is general and can help identify and explain the mechanisms behind disease association for alleles that affect miRNA regulation.

In Vivo Delivery of SiRNA to Immune Cells by Conjugation to a TLR9 Agonist Enhances Antitumor Immune Responses

Nature Biotechnology. Oct, 2009  |  Pubmed ID: 19749770

Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9(+) myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

Effect of Saphenous Vein Diameter on Closure Rate with ClosureFAST Radiofrequency Catheter

Vascular and Endovascular Surgery. Dec, 2009  |  Pubmed ID: 19828586

Radiofrequency ablation (RFA) of veins >12 mm in diameter has been a controversial subject since the first-generation device was submitted for Food and Drug Administration (FDA) approval. Veins >12 mm were excluded in the initial study. Many insurance carriers used >12 mm size as reason to not approve the procedure. As the concept of tumescent anesthesia was better communicated, RFA was used for large veins. The 12-mm size limit was not used in the studies for the newer ClosureFAST catheter approval, yet remains in force with some insurance companies. Our objective was to determine whether vein diameter >12 mm had effect on closure rates with the Closure-FAST catheter.

Protection of Stem Cell-derived Lymphocytes in a Primate AIDS Gene Therapy Model After in Vivo Selection

PloS One. 2009  |  Pubmed ID: 19888329

There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Delta32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation.

CRM1 Mediates Nuclear-cytoplasmic Shuttling of Mature MicroRNAs

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2009  |  Pubmed ID: 19955415

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, Topo2alpha, EzH2, and Mta, consistent with a role of Argonautes and small RNAs in chromatin remodeling.

RNAi-based Therapeutics-current Status, Challenges and Prospects

EMBO Molecular Medicine. Jun, 2009  |  Pubmed ID: 20049714

RNA interference (RNAi) is a collection of small RNA directed mechanisms that result in sequence specific inhibition of gene expression. The notion that RNAi could lead to a new class of therapeutics caught the attention of many investigators soon after its discovery. The field of applied RNAi therapeutics has moved very quickly from lab to bedside. The RNAi approach has been widely used for drug development and several phase I and II clinical trials are under way. However, there are still some concerns and challenges to overcome for therapeutic applications. These include the potential for off-target effects, triggering innate immune responses and most importantly obtaining specific delivery into the cytoplasm of target cells. This review focuses on the current status of RNAi-based therapeutics, the challenges it faces and how to overcome them.

The Therapeutic Potential of Cell-internalizing Aptamers

Current Topics in Medicinal Chemistry. 2009  |  Pubmed ID: 19860714

Aptamers that are evolved by the SELEX procedure (Systematic Evolution of Ligands by Exponential enrichment) can specifically recognize and tightly bind their cognate targets by means of well-defined secondary and three-dimensional structures. In comparison to antibodies, nucleic acid-based aptamers offer some exciting advantages, including the potential for chemical synthesis, convenient modification, chemical versatility, stability and lack of immunogenicity. During the past 20 years, aptamers have been developed for various applications such as diagnostics, drug development, target validation and therapeutics. Aptamers targeting cell surface proteins are being explored as promising delivery vehicles to target a distinct disease or tissue in a cell-type-specific manner. In this review, we summarize the recent developments in creatively using cell-internalizing aptamers as drug delivery escorts to deliver, enhance and modulate the activity of other therapeutic agents, including chemical drugs, toxins, small interfering RNAs and nanoparticle-encapsulated drugs. Specifically, several attractive aptamer-mediated cell-type specific siRNA delivery systems are highlighted, and their promise in clinical development is also discussed.

Identification of a Sensitive Anti-erythropoietin Receptor Monoclonal Antibody Allows Detection of Low Levels of EpoR in Cells

Journal of Immunological Methods. Jan, 2010  |  Pubmed ID: 19887071

Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on non-erythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the EpoR protein on intact cells by flow cytometry and by western blot analysis with cell lysates. Both methods were optimized and using them, EpoR protein was detected by western immunoblotting with lysates from fewer than 200 EpoR positive control cells and the positive signals were proportional to EpoR protein expression level with a minimal signal in EpoR negative cells. The proteins detected by western blot analysis using A82 included full-length EpoR ( approximately 59kDa) as well as smaller EpoR fragments derived from the EPOR gene. These results indicate that A82 can be used to examine low level EpoR expression in cells by western and flow cytometry allowing an improved understanding of EpoR expression and metabolism.

Biogenesis and Function of Endogenous and Exogenous SiRNAs

Wiley Interdisciplinary Reviews. RNA. Jul, 2010  |  Pubmed ID: 21956909

RNA interference (RNAi) is a sequence-specific gene silencing, or 'knockdown', mechanism facilitated by short duplex strands of RNA with sequence complementarity to target mRNAs. RNAi has many different forms, including posttranscriptional gene silencing (PTGS), and transcriptional gene silencing (TGS). Here, we review the biogenesis and function of an endogenous set of small RNA gene regulators, called microRNAs, as well as the mechanism of exogenously delivered small interfering RNAs. The potential applications of RNAi-based therapeutics are also highlighted.

Post-transcriptional Up-regulation of Tsc-22 by Ybx1, a Target of MiR-216a, Mediates TGF-{beta}-induced Collagen Expression in Kidney Cells

The Journal of Biological Chemistry. Oct, 2010  |  Pubmed ID: 20713358

Increased accumulation of extracellular matrix proteins and hypertrophy induced by transforming growth factor-β1 (TGF-β) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy. Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-β increased microRNA-216a (miR-216a) levels in mouse MC, with parallel down-regulation of Ybx1, a miR-216a target and RNA-binding protein. TGF-β also enhanced protein levels of Tsc-22 (TGF-β-stimulated clone 22) and collagen type I α-2 (Col1a2) expression in MC through far upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-β, miR-216a mimics, or Ybx1 shRNA to increase Tsc-22 protein levels but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-β could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-immunoprecipitation assays revealed that TGF-β promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-β-induced Col1a2 in MC related to the pathogenesis of diabetic nephropathy.

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-bound Fusion Inhibitor Following HIV-1 Infection

PloS One. 2010  |  Pubmed ID: 20808813

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

RNAi and Small Interfering RNAs in Human Disease Therapeutic Applications

Trends in Biotechnology. Nov, 2010  |  Pubmed ID: 20833440

Small interfering RNAs (siRNAs) have been shown to effectively downregulate gene expression in human cells, giving them potential to eradicate disease. Prospects for clinical applications are discussed in this review, along with an overview of recent history and our current understanding of siRNAs used for therapeutic application in human diseases, such as cancer and viral infections. Over recent years, progress has been made in lipids, ligands, nanoparticles, polymers and viral vectors as delivery agents and for gene-based expression of siRNA to enhance the efficacy and specificity of these methods while at the same time reducing toxicity. It has become apparent that given the recent advances in chemistry and delivery, RNAi will soon prove to be an important and widely used therapeutic modality.

Is Equal Moral Consideration Really Compatible with Unequal Moral Status?

Kennedy Institute of Ethics Journal. Sep, 2010  |  Pubmed ID: 21133335

The issue of moral considerability, or how much moral importance a being's interests deserve, is one of the most important in animal ethics. Some leading theorists--most notably David DeGrazia--have argued that a principle of "equal moral consideration" is compatible with "unequal moral status." Such a position would reconcile the egalitarian force of equal consideration with more stringent obligations to humans than animals. The article presents arguments that equal consideration is not compatible with unequal moral status, thereby forcing those who would justify significantly different moral protections for humans and animals to argue for unequal consideration.

Rational Design of Micro-RNA-like Bifunctional SiRNAs Targeting HIV and the HIV Coreceptor CCR5

Molecular Therapy : the Journal of the American Society of Gene Therapy. Apr, 2010  |  Pubmed ID: 20104214

Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

Absence of Functional EpoR Expression in Human Tumor Cell Lines

Blood. May, 2010  |  Pubmed ID: 20124514

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies, those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here, we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast, 54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further, and, although one line, NCI-H661, bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo), none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5, AKT, ERK, or S6RP with rHuEpo. In addition, EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors.

A Dual Function TAR Decoy Serves As an Anti-HIV SiRNA Delivery Vehicle

Virology Journal. 2010  |  Pubmed ID: 20144240

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed.

Strategies in Designing Multigene Expression Units to Downregulate HIV-1

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 20217548

The treatment of viral diseases such as HIV and HCV is limited by the genetic diversity of the viruses, especially when they are under the selective pressure of drugs. This problem holds true for gene-based therapies using RNAi in which there is evolution of drug-resistant strains under the discriminating pressure of treatment (1, 2). In a gene therapy setting for treatment of HIV, the incorporation of multiple effector molecules, targeting different viral and cellular sequences, can improve viral inhibition by substantially delaying the emergence of escape mutants (3-8). However, for short hairpin RNA triggers of RNAi, high levels of expression by strong Pol III promoters has led to cell toxicity, and even death in experimental animals (9, 10). Here, we describe a new combinatorial anti-HIV gene expression system allowing simultaneous expression of multiple RNAi effector units from a single Pol II polycistronic transcript. Our platform is suitable for the inclusion of any shRNA sequence and can be combined with other types of small RNA antiviral inhibitors.

Aptamer-targeted Cell-specific RNA Interference

Silence. 2010  |  Pubmed ID: 20226078

This potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of siRNAs into specific cell populations is still the principal challenge in the clinical development of RNAi therapeutics. With the increasing enthusiasm for developing targeted delivery vehicles, nucleic acid-based aptamers targeting cell surface proteins are being explored as promising delivery vehicles to target a distinct disease or tissue in a cell-type-specific manner. The aptamer-based delivery of siRNAs can often enhance the therapeutic efficacy and reduce the unwanted off-target effects of siRNAs. In particular, for RNA interference-based therapeutics, aptamers represent an efficient agent for cell type-specific, systemic delivery of these oligonucleotides. In this review, we summarize recent attractive developments in creatively using cell-internalizing aptamers to deliver siRNAs to target cells. The optimization and improvement of aptamer-targeted siRNAs for clinical translation are further highlighted.

Combinatorial Latency Reactivation for HIV-1 Subtypes and Variants

Journal of Virology. Jun, 2010  |  Pubmed ID: 20357084

The eradication of HIV-1 will likely require novel clinical approaches to purge the reservoir of latently infected cells from a patient. We hypothesize that this therapy should target a wide range of latent integration sites, act effectively against viral variants that have acquired mutations in their promoter regions, and function across multiple HIV-1 subtypes. By using primary CD4(+) and Jurkat cell-based in vitro HIV-1 latency models, we observe that single-agent latency reactivation therapy is ineffective against most HIV-1 subtypes. However, we demonstrate that the combination of two clinically promising drugs-namely, prostratin and suberoylanilide hydroxamic acid (SAHA)-overcomes the limitations of single-agent approaches and can act synergistically for many HIV-1 subtypes, including A, B, C, D, and F. Finally, by identifying the proviral integration position of latent Jurkat cell clones, we demonstrate that this drug combination does not significantly enhance the expression of endogenous genes nearest to the proviral integration site, indicating that its effects may be selective.

Dual-targeting SiRNAs

RNA (New York, N.Y.). Jun, 2010  |  Pubmed ID: 20410240

We have developed an algorithm for the prediction of dual-targeting short interfering RNAs (siRNAs) in which both strands are deliberately designed to separately target different mRNA transcripts with complete complementarity. An advantage of this approach versus the use of two separate duplexes is that only two strands, as opposed to four, are competing for entry into the RNA-induced silencing complex. We chose to design our dual-targeting siRNAs as Dicer substrate 25/27mer siRNAs, since design features resembling pre-microRNAs (miRNAs) can be introduced for Dicer processing. Seven different dual-targeting siRNAs targeting genes that are potential targets in cancer therapy have been developed including Bcl2, Stat3, CCND1, BIRC5, and MYC. The dual-targeting siRNAs have been characterized for dual target knockdown in three different cell lines (HEK293, HCT116, and PC3), where they were as effective as their corresponding single-targeting siRNAs in target knockdown. The algorithm developed in this study should prove to be useful for predicting dual-targeting siRNAs in a variety of different targets and is available from http://demo1.interagon.com/DualTargeting/.

Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs

Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et Al.]. Jun, 2010  |  Pubmed ID: 20521232

The discovery of RNA interference (RNAi) and related small RNA-mediated regulatory pathways has significantly altered the understanding of gene regulation in eukaryotic cells. In the RNAi pathway, small interfering RNAs (siRNAs) approximately 21 to 23 nucleotides in length serve as the regulatory molecules that guide and induce sequence-specific gene silencing. The use of siRNA-mediated silencing as a tool for investigating gene function is well established in cultured mammalian cells. This unit provides basic approaches to explore the field of RNAi, and hopes to address the importance of optimizing transfection conditions after empirical determinations in order to understand various degrees of silencing efficiency.

International Research and Positive Obligations: Are They "transaction Specific"?

The American Journal of Bioethics : AJOB. Jun, 2010  |  Pubmed ID: 20526974

RNA-based Gene Therapy for HIV with Lentiviral Vector-modified CD34(+) Cells in Patients Undergoing Transplantation for AIDS-related Lymphoma

Science Translational Medicine. Jun, 2010  |  Pubmed ID: 20555022

AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell-based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood-derived (CD34(+)) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to approximately 1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.

Sequence Context Outside the Target Region Influences the Effectiveness of MiR-223 Target Sites in the RhoB 3'UTR

Nucleic Acids Research. Jan, 2010  |  Pubmed ID: 19850724

MicroRNAs (miRNAs) are 21-22 nucleotide regulatory small RNAs that repress message translation via base-pairing with complementary sequences in the 3' untranslated region (3'UTR) of targeted transcripts. To date, it is still difficult to find a true miRNA target due to lack of a clear understanding of how miRNAs functionally interact with their targeted transcripts for efficient repression. Previous studies have shown that nucleotides 2 to 7 at the 5'-end of a mature miRNA, the 'seed sequence', can nucleate miRNA/target interactions. In the current study, we have validated that the RhoB mRNA is a bona fide miR-223 target. We have analyzed the functional activities of two miR223-binding sites within the RhoB 3'UTR. We find that the two miR-223 target sites in the RhoB 3'UTR contribute differentially to the total repression of RhoB translation. Moreover, we demonstrate that some AU-rich motifs located upstream of the distal miRNA-binding site enhance miRNA function, independent of the miRNA target sequences being tested. We also demonstrate that the AU-rich sequence elements are polar, and do not affect the activities of miRNAs whose sites lie upstream of these elements. These studies provide further support for the role of sequences outside of miRNA target region influencing miRNA function.

Population Pharmacokinetic/pharmacodynamic Model of Subcutaneous Adipose 11β-hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Activity After Oral Administration of AMG 221, a Selective 11β-HSD1 Inhibitor

Journal of Clinical Pharmacology. Jun, 2011  |  Pubmed ID: 20663992

Inhibition of 11β-HSD1 is hypothesized to improve measures of insulin sensitivity and hepatic glucose output in patients with type II diabetes. AMG 221 is a potent, small molecule inhibitor of 11β-HSD1. The objective of this analysis is to describe the pharmacokinetic/pharmacodynamic (PK/PD) relationship between AMG 221 and 11β-HSD1 inhibition in ex vivo adipose tissue samples. Healthy, obese subjects were administered a single dose of 3, 30, or 100 mg of oral AMG 221 (n = 44) or placebo (n = 11). Serial blood samples were collected over 24 hours. Subcutaneous adipose tissue samples were collected by open biopsy. Population PK/PD analysis was conducted using NONMEM. The inhibitory effects (mean ± standard error of the estimate) of AMG 221 on 11β-HSD1 activity were directly related to adipose concentrations with I(max) (the maximal inhibition of 11β-HSD1 activity) and IC₅₀ (the plasma AMG 221 concentration associated with 50% inhibition of enzyme activity) of 0.975 ± 0.003 and 1.19 ± 0.12 ng/mL, respectively. The estimated baseline 11β-HSD1 enzyme activity was 755 ± 61 pmol/mg. An equilibration rate constant (k(eo)) of 0.220 ± 0.021 h⁻¹ described the delay between plasma and adipose tissue AMG 221 concentrations. AMG 221 potently blocked 11β-HSD1 activity, producing sustained inhibition for the 24-hour study duration as measured in ex vivo adipose samples. Early characterization of concentration-response relationships can support rational selection of dose and regimen for future studies.

Inducible and Reversible Breaching of the Blood Brain Barrier by RNAi

EMBO Molecular Medicine. Apr, 2011  |  Pubmed ID: 21394916

RNA Nanoparticles Come of Age

Acta Biochimica Et Biophysica Sinica. Apr, 2011  |  Pubmed ID: 21430091

Progress in RNAi-based Antiviral Therapeutics

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21431679

RNA interference (RNAi) refers to the conserved sequence-specific degradation of message RNA mediated by small interfering (si)RNA duplexes 21-25 nucleotides in length. Given the ability to specifically silence any gene of interest, siRNAs offers several advantages over conventional drugs as potential therapeutic agents for the treatment of human maladies including cancers, genetic disorders, and infectious diseases. Antiviral RNAi strategies have received much attention and several compounds are currently being tested in clinical trials. In particular, the development of siRNA-based HIV (human immunodeficiency virus) therapeutics has progressed rapidly and many recent studies have shown that the use of RNAi could inhibit HIV-1 replication by targeting a number of viral or cellular genes. Therefore, the present chapter mainly focuses on the recent progress of RNAi-based anti-HIV gene therapeutics, with particular attention to molecular targets and delivery strategies of the siRNAs.

Aptamer-targeted RNAi for HIV-1 Therapy

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21431697

The highly specific mechanism of RNA (RNAi) that inhibits the expression of disease genes is increasingly being harnessed to develop a new class of therapeutics for a wide variety of human maladies. The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Herein, we demonstrate novel cell type-specific dual inhibitory function anti-gp120 aptamer-siRNA delivery systems for HIV-1 therapy, in which both the aptamer and the siRNA portions have potent anti-HIV activities. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. The Dicer substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells. Our results provide a set of novel aptamer-targeted RNAi therapeutics to combat HIV and further validate the use of anti-gp120 aptamers for delivery of Dicer substrate siRNAs.

Stopping RNA Interference at the Seed

Nature Genetics. Apr, 2011  |  Pubmed ID: 21445071

MicroRNAs (miRNAs) regulate expression of more than one half of the genes in the human genome. A study now reports a new method for selectively silencing whole families of miRNAs, thus providing a new paradigm for disease therapy.

Ex Vivo Gene Therapy for HIV-1 Treatment

Human Molecular Genetics. Apr, 2011  |  Pubmed ID: 21505069

Until recently, progress in ex vivo gene therapy (GT) for human immunodeficiency virus-1 (HIV-1) treatment has been incremental. Long-term HIV-1 remission in a patient who received a heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5(-/-) donor, even after discontinuation of conventional therapy, has energized the field. We review the status of current approaches as well as future directions in the areas of therapeutic targets, combinatorial strategies, vector design, introduction of therapeutics into stem cells and enrichment/expansion of gene-modified cells. Finally, we discuss recent advances towards clinical application of HIV-1 GT.

Thermodynamic Stability of Small Hairpin RNAs Highly Influences the Loading Process of Different Mammalian Argonautes

Proceedings of the National Academy of Sciences of the United States of America. May, 2011  |  Pubmed ID: 21576459

MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage- and non-cleavage-based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1-Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila. Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.

Human Brain EEG Indices of Emotions: Delineating Responses to Affective Vocalizations by Measuring Frontal Theta Event-related Synchronization

Neuroscience and Biobehavioral Reviews. Oct, 2011  |  Pubmed ID: 21596060

At present there is no direct brain measure of basic emotional dynamics from the human brain. EEG provides non-invasive approaches for monitoring brain electrical activity to emotional stimuli. Event-related desynchronization/synchronization (ERD/ERS) analysis, based on power shifts in specific frequency bands, has some potential as a method for differentiating responses to basic emotions as measured during brief presentations of affective stimuli. Although there appears to be fairly consistent theta ERS in frontal regions of the brain during the earliest phases of processing affective auditory stimuli, the patterns do not readily distinguish between specific emotions. To date it has not been possible to consistently differentiate brain responses to emotion-specific affective states or stimuli, and some evidence to suggests the theta ERS more likely measures general arousal processes rather than yielding veridical indices of specific emotional states. Perhaps cortical EEG patterns will never be able to be used to distinguish discrete emotional states from the surface of the brain. The implications and limitations of such approaches for understanding human emotions are discussed.

A Boost for the Emerging Field of RNA Nanotechnology

ACS Nano. May, 2011  |  Pubmed ID: 21604810

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23-25, 2010) ( http://www.eng.uc.edu/nanomedicine/RNA2010/ ), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCI. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries.

A Critical Role for SHP2 in STAT5 Activation and Growth Factor-mediated Proliferation, Survival, and Differentiation of Human CD34+ Cells

Blood. Aug, 2011  |  Pubmed ID: 21670473

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.

Phylogenetic Comparison of Small RNA-triggered Transcriptional Gene Silencing

The Journal of Biological Chemistry. Aug, 2011  |  Pubmed ID: 21730056

The discovery of RNA interference has revealed complex roles for small RNAs in regulating gene expression and cellular physiology. Small RNAs have been demonstrated to be involved in post-transcriptional suppression of translation, targeted degradation of messenger RNAs, and transcriptional suppression via epigenetic modifications of histones and DNA. In fission yeast, RNAi mediates suppression of centromeric transcripts, whereas in plants, transcriptional gene silencing appears to be primarily an antiviral mechanism. In mammals, the well annotated functional role of RNAi is primarily post-transcriptional, but there is increasing evidence that this mechanism can also work to suppress or modulate gene transcription, although it is not clear what primary function this serves. We overview, compare, and contrast the transcriptional silencing pathways in yeast, plants, and mammals in this article. This minireview is intended to provide the reader with a framework of how the RNAi machinery appears to be universally involved in various aspects of transcriptional regulation with discussions of similarities and differences in the components and mechanisms of achieving transcriptional silencing.

Current Progress of SiRNA/shRNA Therapeutics in Clinical Trials

Biotechnology Journal. Sep, 2011  |  Pubmed ID: 21744502

Through a mechanism known as RNA interference (RNAi), small interfering RNA (siRNA) molecules can target complementary mRNA strands for degradation, thus specifically inhibiting gene expression. The ability of siRNAs to inhibit gene expression offers a mechanism that can be exploited for novel therapeutics. Indeed, over the past decade, at least 21 siRNA therapeutics have been developed for more than a dozen diseases, including various cancers, viruses, and genetic disorders. Like other biological drugs, RNAi-based therapeutics often require a delivery vehicle to transport them to the targeted cells. Thus, the clinical advancement of numerous siRNA drugs has relied on the development of siRNA carriers, including biodegradable nanoparticles, lipids, bacteria, and attenuated viruses. Most therapies permit systemic delivery of the siRNA drug, while others use ex vivo delivery by autologous cell therapy. Advancements in bioengineering and nanotechnology have led to improved control of delivery and release of some siRNA therapeutics. Likewise, progress in molecular biology has allowed for improved design of the siRNA molecules. Here, we provide an overview of siRNA therapeutics in clinical trials, including their clinical progress, the challenges they have encountered, and the future they hold in the treatment of human diseases.

Small Interfering RNAs Targeting Cyclin D1 and Cyclin D2 Enhance the Cytotoxicity of Chemotherapeutic Agents in Mantle Cell Lymphoma Cell Lines

Leukemia & Lymphoma. Nov, 2011  |  Pubmed ID: 21745168

Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40-60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC(50) (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.

MicroRNAs and Their Potential Involvement in HIV Infection

Trends in Pharmacological Sciences. Nov, 2011  |  Pubmed ID: 21862142

Treatment and cure of HIV-1 infection remain one of the greatest therapeutic challenges owing to its persistent infection, which often leads to AIDS. Although it has been 28 years since the discovery of the virus, the development of an effective vaccine is still years away. Relatively newly discovered miRNAs are a family of small noncoding RNAs that can regulate gene expression primarily by binding to the 3' untranslated region of targeted transcripts. An understanding of how HIV-1 infection affects the host miRNA pathway could generate new insights into the basic mechanisms underlying HIV-1-mediated pathologies and T-lymphocyte depletion. Here, we review literature on the biogenesis of HIV-1-encoded miRNAs, cellular miRNAs that can directly target HIV-1 or essential cellular factors required for HIV-1 replication. We also discuss the feasibility of using miRNAs for HIV-1 therapy.

Systemic Administration of Combinatorial DsiRNAs Via Nanoparticles Efficiently Suppresses HIV-1 Infection in Humanized Mice

Molecular Therapy : the Journal of the American Society of Gene Therapy. Dec, 2011  |  Pubmed ID: 21952167

We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2(-)/-γc-/- (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2-/-γc-/- mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4(+) T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.

A Novel Nuclear MiRNA Mediated Modulation of a Non-coding Antisense RNA and Its Cognate Sense Coding MRNA

The EMBO Journal. Nov, 2011  |  Pubmed ID: 22048334

The Development of RNA Interference Therapeutics

Nucleic Acid Therapeutics. Dec, 2011  |  Pubmed ID: 22196369

Cell-specific Aptamer-mediated Targeted Drug Delivery

Oligonucleotides. Feb, 2011  |  Pubmed ID: 21182455

Nucleic acid aptamers are in vitro-selected small, single-stranded DNA or RNA oligonucleotides that can specifically recognize their target on the basis of their unique 3-dimensional structures. Recent advances in the development of escort aptamers to deliver and enhance the efficacy of other therapeutic agents have drawn enthusiasm in exploiting cell-type-specific aptamers as drug delivery vehicles. This review mainly focuses on the recent developments of aptamer-mediated targeted delivery systems. We also place particular emphasis on aptamers evolved against cell membrane receptors and possibilities for translation to clinical applications.

OTS Highlights Advance in Basic and Translational Aspects of Oligonucleotide Research

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2011  |  Pubmed ID: 21200390

An Aptamer-siRNA Chimera Suppresses HIV-1 Viral Loads and Protects from Helper CD4(+) T Cell Decline in Humanized Mice

Science Translational Medicine. Jan, 2011  |  Pubmed ID: 21248316

Therapeutic strategies designed to treat HIV infection with combinations of antiviral drugs have proven to be the best approach for slowing the progression to AIDS. Despite this progress, there are problems with viral drug resistance and toxicity, necessitating new approaches to combating HIV-1 infection. We have therefore developed a different combination approach for the treatment of HIV infection in which an RNA aptamer, with high binding affinity to the HIV-1 envelope (gp120) protein and virus neutralization properties, is attached to and delivers a small interfering RNA (siRNA) that triggers sequence-specific degradation of HIV RNAs. We have tested the antiviral activities of these chimeric RNAs in a humanized Rag2(-/-)γc(-/-) (RAG-hu) mouse model with multilineage human hematopoiesis. In this animal model, HIV-1 replication and CD4(+) T cell depletion mimic the situation seen in human HIV-infected patients. Our results show that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4(+) T cell decline. In comparison to the aptamer alone, the aptamer-siRNA combination provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose. The aptamer thus acts as a broad-spectrum HIV-neutralizing agent and an siRNA delivery vehicle. The combined aptamer-siRNA agent provides an attractive, nontoxic therapeutic approach for treatment of HIV infection.

Dual Functional RNA Nanoparticles Containing Phi29 Motor PRNA and Anti-gp120 Aptamer for Cell-type Specific Delivery and HIV-1 Inhibition

Methods (San Diego, Calif.). Jun, 2011  |  Pubmed ID: 21256218

The potent ability of small interfering RNA (siRNA) to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for diseases including HIV. However, efficient delivery of siRNAs remains a key obstacle to successful application. A targeted intracellular delivery approach for siRNAs to specific cell types is highly desirable. HIV-1 infection is initiated by the interactions between viral glycoprotein gp120 and cell surface receptor CD4, leading to fusion of the viral membrane with the target cell membrane. Once HIV infects a cell it produces gp120 which is displayed at the cell surface. We previously described a novel dual inhibitory anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also demonstrated that gp120 can be used for aptamer mediated delivery of anti-HIV siRNAs. Here we report the design, construction and evaluation of chimerical RNA nanoparticles containing a HIV gp120-binding aptamer escorted by the pRNA of bacteriophage phi29 DNA-packaging motor. We demonstrate that pRNA-aptamer chimeras specifically bind to and are internalized into cells expressing HIV gp120. Moreover, the pRNA-aptamer chimeras alone also provide HIV inhibitory function by blocking viral infectivity. The Ab' pRNA-siRNA chimera with 2'-F modified pyrimidines in the sense strand not only improved the RNA stability in serum, but also was functionally processed by Dicer, resulting in specific target gene silencing. Therefore, this dual functional pRNA-aptamer not only represents a potential HIV-1 inhibitor, but also provides a cell-type specific siRNA delivery vehicle, showing promise for systemic anti-HIV therapy.

A Role for Human Dicer in Pre-RISC Loading of SiRNAs

Nucleic Acids Research. Mar, 2011  |  Pubmed ID: 20972213

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.

A Bias-reducing Strategy in Profiling Small RNAs Using Solexa

RNA (New York, N.Y.). Dec, 2011  |  Pubmed ID: 22016383

Small RNAs (smRNAs) encompass several different classes of short noncoding RNAs. Progress in smRNA research and applications has coincided with the advance of techniques to detect them. Next-generation sequencing technologies are becoming the preferred smRNA profiling method because of their high-throughput capacity and digitized results. In our small RNA profiling study using Solexa, we observed serious biases introduced by the 5' adaptors in small RNA species coverage and abundance; therefore, the results cannot reveal the accurate composition of the small RNAome. We found that the profiling results can be significantly optimized by using an index pool of 64 customized 5' adaptors. This pool of 64 adaptors can be further reduced to four smaller index pools, each containing 16 adaptors, to minimize profiling bias and facilitate multiplexing. It is plausible that this type of bias exists in other deep-sequencing technologies, and adaptor pooling could be an easy work-around solution to reveal the "true" small RNAome.

Interplay Between HIV-1 Infection and Host MicroRNAs

Nucleic Acids Research. Nov, 2011  |  Pubmed ID: 22080513

Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

Lack of Expression and Function of Erythropoietin Receptors in the Kidney

Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association. Dec, 2011  |  Pubmed ID: 22167585

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) stimulate formation of red blood cells by binding to and activating Epo receptors (EpoR) on erythroid progenitor cells. Beyond successful treatment of anemia, ESAs have been reported to reduce damage following insult to organs, including the kidney, possibly via direct activation of EpoR. However, data on ESA effects outside hematopoietic functions are conflicting. Furthermore, limited use of appropriate EpoR-positive and EpoR-negative controls and lack of specific anti-EpoR antibodies make interpretation of data difficult. Recently positive and negative control cell types were validated and a sensitive and specific anti-EpoR antibody (A82) that detects low levels of EpoR protein was described.METHODS: A82 was used to measure EpoR protein levels in tissues, human renal cells and human cell lines by western blot analysis. Surface EpoR was examined on renal cells by measuring binding of [(125)I]-rHuEpo or antibodies. Renal cells and cell lines were treated with rHuEpo to see if phosphorylation of signaling proteins or proliferation/survival could be induced. Small inhibitory RNA (siRNA) were used to determine if EpoR knockdown affected cell viability.RESULTS: Total EpoR protein was low to undetectable in tissues and renal cells with no detectable EpoR on cell surfaces. EpoR knockdown had no effect on viability of renal cell lines. RHuEpo had no detectable effect on intracellular signaling on renal cell lines with no growth-promoting or survival effect on primary human renal cells.CONCLUSIONS: These results suggest that functional EpoR protein is absent on renal cells and that non-EpoR mechanisms should be explored to explain non-hematopoietic effects of ESAs.

RNA-Based Therapeutics: Current Progress and Future Prospects

Chemistry & Biology. Jan, 2012  |  Pubmed ID: 22284355

Recent advances of biological drugs have broadened the scope of therapeutic targets for a variety of human diseases. This holds true for dozens of RNA-based therapeutics currently under clinical investigation for diseases ranging from genetic disorders to HIV infection to various cancers. These emerging drugs, which include therapeutic ribozymes, aptamers, and small interfering RNAs (siRNAs), demonstrate the unprecedented versatility of RNA. However, RNA is inherently unstable, potentially immunogenic, and typically requires a delivery vehicle for efficient transport to the targeted cells. These issues have hindered the clinical progress of some RNA-based drugs and have contributed to mixed results in clinical testing. Nevertheless, promising results from recent clinical trials suggest that these barriers may be overcome with improved synthetic delivery carriers and chemical modifications of the RNA therapeutics. This review focuses on the clinical results of siRNA, RNA aptamer, and ribozyme therapeutics and the prospects for future successes.

Screening Effective Target Sites on MRNA: A Ribozyme Library Approach

Methods in Molecular Biology (Clifton, N.J.). 2012  |  Pubmed ID: 22315078

Hammerhead ribozymes have been extensively used as RNA-inactivating agents for therapy as well as forward genomics. A ribozyme can be designed so as to specifically pair with virtually any target RNA, and cleave the phosphodiester backbone at a specified location, thereby functionally inactivating the RNA. Two major factors that determine whether ribozymes will be effective for posttranscriptional gene silencing are colocalization of the ribozyme and the target RNAs, and the choice of an appropriate target site on the mRNA. Complex secondary structures and the ability to bind to some of the cellular proteins mandate that some RNA sequences could stearically occlude binding of RNA-based antivirals like ribozymes to these sites. The use of ribozyme libraries in cell culture factors in these interactions to select for target sites on the RNA, which are more accessible to RNA-based antivirals like ribozymes or siRNA. This chapter provides a useful guide toward using ribozyme libraries to screen for effective target sites on mRNA.

Value-ladenness and Rationality in Health Communication

The American Journal of Bioethics : AJOB. Feb, 2012  |  Pubmed ID: 22304512