Showing papers on "Small hairpin RNA published in 2003"

Lentivirus-delivered stable gene silencing by RNAi in primary cells.

Abstract: Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Induction of an interferon response by RNAi vectors in mammalian cells

Abstract: DNA vectors that express short hairpin RNAs (shRNAs) from RNA polymerase III (Pol III) promoters are a promising new tool to reduce gene expression in mammalian cells. shRNAs are processed to small interfering RNAs (siRNAs) of 21 nucleotides (nt) that guide the cleavage of the cognate mRNA by the RNA-induced silencing complex. Although siRNAs are thought to be too short to induce interferon expression, we report here that a substantial number of shRNA vectors can trigger an interferon response.

Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference

Abstract: RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

RNA interference in mammalian cells by chemically-modified RNA

Abstract: RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing.

Abstract: Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.

Allele-specific silencing of dominant disease genes

Abstract: Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.

Human Immunodeficiency Virus Type 1 Escape from RNA Interference

Abstract: Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo

Abstract: The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease1,2. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from Eµ-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53‐/‐ lymphomagenesis in the Eµ-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an ‘epi-allelic series’ for dissecting gene function in vivo. RNAi is a powerful tool for manipulating gene expression in model organisms and cultured mammalian cells 3 . The technology arose from the observation that exogenous double-stranded RNAs induce gene silencing in plants and Caenorhabditis elegans. These double-stranded RNAs are processed into small interfering RNAs (siRNAs), which are incorporated into a conserved cellular machinery that mediates the suppression of homologous genes. Recently, small non-coding RNAs have been identified that can act as endogenous regulators of gene expression. These microRNAs typically form stem-loop structures, essentially short double-stranded RNAs, that enter the RNAi pathway 4‐7 . shRNAs, modeled after microRNAs, can be expressed from viral vectors to induce stable suppression of gene expression in cultured mammalian cells 8 . We reasoned that stable suppression of gene expression by RNAi should recapitulate the phenotype of mice harboring deletions of the targeted gene. To test this, we targeted the Tr p53 tumor suppressor, a gene with extensively characterized loss-of-function

Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing.

Abstract: The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation. This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants. We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin. This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation. A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs). These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells

Abstract: RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.

Transgenic RNA interference in ES cell–derived embryos recapitulates a genetic null phenotype

Abstract: Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.

RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)

Abstract: This invention relates to compounds, compositions, and methods useful for modulating gene expression using short interfering nucleic acid (siNA) molecules In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of genes, such as expressed pseudogenes associated with the maintenance or development of diseases, disorders, traits, and conditions in a subject or organism

RNA interference mediated treatment of Alzheimer's disease using short interfering nucleic acid (siNA)

Abstract: The present invention concerns methods and reagents useful in modulating BACE gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against beta-secretase (BACE), amyloid precurson protein (APP), pin-1, presenillin 1 (PS-1) and/or presenillin 2 (PS-2) gene expression and/or activity. The small nucleic acid molecules are useful in the treatment of Alzheimer's disease and any other condition that responds to modulation of BACE, APP, pin-1, PS-1 and/or PS-2 expression or activity.

Use of adeno-associated viral vector for delivery of small interfering RNA

Abstract: Post-transcriptional gene silencing by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. Delivery of siRNA into mammalian cells is usually achieved via the transfection of double-stranded oligonucleotides or plasmids encoding RNA polymerase III promoter-driven small hairpin RNA. Recently, retroviral vectors have been used for siRNA delivery, which overcome the problem of poor transfection efficiency seen with the plasmid-based systems. However, retroviral vectors have several limitations, such as the need for active cell division for gene transduction, oncogenic potential, low titers and gene silencing. In this report, we have adapted a commercially available adenoassociated virus (AAV) vector for siRNA delivery into mammalian cells. We demonstrate the ability of this modified vector to deliver efficiently siRNA into HeLa S3 cells and downregulate p53 and caspase 8 expression. Our results suggest that AAV-based vectors are efficient vectors for the delivery of siRNA into mammalian cells. Based on the known ability of these vectors to infect both dividing and nondividing cells, their use as a therapeutic tool for the delivery of siRNA deserves further study.

Inducible shRNA expression for application in a prostate cancer mouse model

Abstract: RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110α and p110β, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.

Establishment of conditional vectors for hairpin siRNA knockdowns

Abstract: Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown.

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.

Abstract: Summary RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase ( SOD1 ) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires singlenucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo , we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused

RNA silencing in the phytopathogenic fungus Magnaporthe oryzae.

Abstract: Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi Overall, these results indicate that RNA silencing operates in M oryzae, which gives us a new tool for genome-wide gene analysis in this fungus

Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules.

Abstract: We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express β-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.

RNA INTERFERENCE MEDIATED INHIBITION OF EPIDERMAL GROWTH FACTOR RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

Abstract: The present invention concerns methods and reagents useful in modulating EGFR gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against HER1, HER2, HER3 and/or HER4. The small nucleic acid molecules are useful in the treatment and diagnosis of cancer.

Nucleic Acid-Based Immune System: the Antiviral Potential of Mammalian RNA Silencing

Abstract: The discovery of a highly conserved cellular machinery that can regulate gene expression in response to double-stranded RNA may revolutionize mammalian virology. This revolution promises not only a deeper understanding of host-pathogen interactions and a novel set of experimental tools to explore the mechanism of viral replication but may also yield new therapeutic approaches. Even though the field of RNA silencing (or RNA interference [RNAi]) as applied to mammalian viruses is barely a year old, there is already enough material to appreciate its importance and to discuss its implications. Since an impressive number of excellent reviews on RNAi have been published recently (25, 27, 62), here we emphasize mammalian RNA silencing as it concerns one of its (presumably) natural targets, viruses.

Rna interference mediated inhibition of hepatitis c virus (hcv) gene expression using short interfering nucleic acid (sina)

Abstract: The present invention concerns methods and reagents useful in modulating hepatitis C virus (HCV) gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against hepatitis C virus (HCV) gene expression and/or activity. The small nucleic acid molecules are useful in the treatment and diagnosis of HCV infection, liver failure, hepatocellular carcinoma, cirrhosis and any other disease or condition that responds to modulation of HCV expression or activity.

RNA interference mediated inhibition of TGF-beta and TGF-beta receptor gene expression using short interfering nucleic acid (siNA)

Abstract: This invention relates to compounds, compositions, and methods useful for modulating tumor necrosis factor and/or tumor necrosis factor receptor gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of tumor necrosis factor and/or tumor necrosis factor receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of tumor necrosis factor and/or tumor necrosis factor receptor genes, (TNF and/or TNF receptor).

In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats

Abstract: Background Gene silencing in mammalian cells is possible with RNA interference (RNAi) with expression plasmids that encode for short hairpin RNAs (shRNA) that hybridize to a specific sequence within a target mRNA. The limiting factor in developing RNAi therapeutics in mammals is the gene delivery system. Methods The present studies describe the production of anti-luciferase shRNA expression plasmids, which are encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs). C6 rat glioma cells, permanently transfected with the luciferase gene, are implanted in the caudate-putamen nucleus of adult rats, which produces luciferase-expressing intracranial brain cancer. The PILs are targeted across the blood-brain barrier and across the tumor cell membrane in vivo with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). The TfRMAb is tethered to the tips of 1–2% of the poly(ethylene glycol) strands conjugated to the surface of the liposome. Results The TfRMAb-targeted PILs inhibit luciferase gene expression in the brain cancer by 90%, and this effect persists for at least 5 days after a single intravenous injection of 10 µg/rat of plasmid DNA. RNAi therapy directed against the luciferase gene caused no change in expression of tumor γ-glutamyl transpeptidase. Targeting the empty expression plasmid with the TfRMAb-PIL resulted in no change in luciferase activity in the brain cancer in vivo. Conclusions In vivo RNAi is enabled with a new form of gene delivery system that encapsulates expression plasmids in PILs, which are targeted to distant sites based on the specificity of a receptor-specific monoclonal antibody. The combined application of the PIL gene delivery system and RNAi expression plasmids enables gene silencing in remote sites such as brain cancer in mammals after intravenous administration. Copyright © 2003 John Wiley & Sons, Ltd.

Lentiviral Vector-Mediated Delivery of Short Hairpin RNA Results in Persistent Knockdown of Gene Expression in Mouse Brain

Abstract: RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene-specific silencing. For in vivo applications, RNAi has been hampered until recently by inefficient delivery methods and by the transient nature of the gene suppression. Lentiviral vectors (LVs) hold great promise for gene therapeutic applications, pharmaceutical target validation, and functional genomics because stable gene transfer is mediated both in dividing and nondividing cells. We have used a lentiviral vector-based system for RNAi. We produced human immunodeficiency virus type 1-derived LVs encoding a short hairpin RNA specific for enhanced green fluorescent protein (EGFP) mRNA that were capable of inhibiting EGFP expression in mammalian cells. EGFP knockdown persisted after multiple passages of the cells. Of particular interest, our RNAi LVs were equally effective in suppression and prevention of EGFP expression after stereotactic injection in adult mouse brain. Therefore, we believe that the use of LVs for stable RNAi in brain will become a powerful aid to probe gene function in vivo and for gene therapy of diseases of the central nervous system.

General plasmids for producing RNA in vitro transcripts with homogeneous ends.

Abstract: In vitro transcripts of bacteriophage RNA polymerases (RNAPs), such as T7 RNAP, often suffer from a considerable degree of 3'-end heterogeneity and, with certain promoter sequences, 5'-end heterogeneity. For some applications, this transcript heterogeneity poses a significant problem. A potential solution is to incorporate ribozymes into the transcripts at the 5'- and/or 3'-end of the target RNA sequence. This approach has been used quite widely but has required the generation of new transcription vectors or PCR-derived templates for each new RNA to be studied. To overcome this limitation, we have created two general plasmids for producing homogeneous RNA transcripts: one encodes a 3'- hepatitis delta virus (HDV) ribozyme and the other, used in combination with a two-step PCR, allows the production of double [5'-hammerhead (HH) and 3'-HDV] ribozyme constructs. A choice of cloning and run-off transcription linearisation restriction enzyme sites ensures that virtually any RNA sequence can be cloned and transcribed from these plasmids. For all the RNA sequences tested, good yields of transcript were obtained. These plasmids provide the tools for the simple, rapid creation of new RNA-coding plasmids to produce milligram quantities of homogeneous in vitro transcripts for all applications.

Promoter choice affects the potency of HIV‐1 specific RNA interference

Abstract: RNA interference (RNAi) is mediated by small interfering (si) RNAs that target and degrade mRNA in a sequence-specific manner. Cellular expression of siRNA can be achieved by the use of expression cassettes driven by RNA polymerase III (pol III) promoters. Here, we demonstrate that a modified tRNA(met)-derived (MTD) promoter effectively drives the cellular expression of HIV-1-specific siRNA. We observed up to 56% greater inhibition of virus production when the MTD promoter was used to drive the expression of short hairpin (sh) RNA targeting the HIV-1 transactivator protein tat compared to cassettes containing other pol III promoters such as H1, U6+1 and U6+27. We conclude that the MTD promoter is ideally suited to drive intracellular expression of HIV-1 specific siRNA and may serve as an important component of future RNAi vector delivery systems.