Antisense RNA Sequences Research Articles

Cryptosporidium parvum cyclic GMP-dependent protein kinase (PKG): An essential mediator of merozoite egress

Cryptosporidiosis is an obligate intracellular pathogen causing diarrhea. Merozoite egress is essential for infection to spread between host cells. However, the mechanisms of egress have yet to be defined. We hypothesized that Cyclic GMP-Dependent Protein Kinase G (PKG) may be involved in Cryptosporidium egress. In this study, Cryptosporidium parvum PKG was silenced by using antisense RNA sequences. PKG-silencing significantly inhibited egress of merozoites from infected HCT-8 cells into the supernatant and led to retention of intracellular forms within the host cells. This data identifies PKG as a key mediator of merozoite egress, a key step in the parasite lifecycle.

Development of genetically modified chrysanthemums resistant to Chrysanthemum stunt viroid using sense and antisense RNAs

Chrysanthemums are one of the most popular flowers. Of known several pathogens causing serious diseases in chrysanthemums, Chrysanthemum stunt viroid (CSVd) is regarded as an important pathogen for chrysanthemums. To control CSVd, it is desirable to generate genetically modified (GM) chrysanthemums resistant to CSVd. In this study, we developed GM chrysanthemum by introducing sense and antisense RNA sequences of CSVd into a commercial chrysanthemum cultivar. A total of 39 GM chrysanthemum lines using four different constructs carrying sense or antisense RNAs were generated by Agrobacterium-mediated transformation. RT-PCR confirmed the expression of introduced RNA sequences in GM and was performed to identify GM lines resistant to CSVd. As a result, nine lines out of 16 tested GM chrysanthemum lines showed very strong resistance against CSVd infection. Phenotype analyses showed that GM plants have grown well as compared to WT plants after CSVd infection. Taken together, this is the first study which develops GM chrysanthemums resistant to CSVd using sense and antisense RNAs of CSVd.

A 1.8-kb insertion in the 3'-UTR of RXFP2 is associated with polledness in sheep.

SummarySheep breeds show a broad spectrum of different horn phenotypes. In most modern production breeds, sheep are polled (absence of horns), whereas horns occur mainly in indigenous breeds. Previous studies mapped the responsible locus to the region of the RXFP2 gene on ovine chromosome 10. A 4‐kb region of the 3′‐end of RXFP2 was amplified in horned and polled animals from seven Swiss sheep breeds. Sequence analysis identified a 1833‐bp genomic insertion located in the 3′‐UTR region of RXFP2 present in polled animals only. An efficient PCR‐based genotyping method to determine the polled genotype of individual sheep is presented. Comparative sequence analyses revealed evidence that the polled‐associated insertion adds a potential antisense RNA sequence of EEF1A1 to the 3′‐end of RXFP2 transcripts.

Efficient inhibition of HIV-1 replication by an artificial polycistronic miRNA construct.

BackgroundRNA interference (RNAi) has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs) against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study.ResultsAn artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed.ConclusionsThe strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.

Abstract 2951: Potent antisense pharmacology of highly optimized antisense oligonucleotides in multiple transgenic, spontaneous and patient derived xenograft models of cancer reveals antitumor activity for the non-coding RNA MALAT-1.

Abstract Antisense technology holds great promise as a novel drug discovery platform that can rapidly translate discoveries from cancer genomics into highly selective therapeutics. Antisense oligonucleotices (ASOs) are particularly attractive, as they can be applied to difficult to drug target classes currently not tractable by other therapeutic modalities. Recent clinical demonstration of activity for ASOs in cancer patients supports the potential of this drug class, however, challenges in demonstrating robust antisense pharmacology in preclinical cancer models has slowed the progress of this technology as cancer therapeutics. Here we have employed a high resolution in-situ hybridization-based methodology (QuantigeneTM) among other detection methods, to demonstrate visually and quantitatively the activity of systemically administered, high potency next generation antisense oligonucleotides, in multiple preclinical cancer models. Cancer models evaluated include, transgenic models, chemically induced tumor, genetically predisposed mouse strains, cell line derived xenograft and patient derived xenograft models. As a test antisense target RNA sequence we chose the non-coding RNA MALAT1 (also called NEAT2) because it is ubiquitously expressed at high levels in most cell types and thus RNA levels could be readily visualize at the cellular level by Quantigene method. In addition, as MALAT-1 is overexpressed in many human tumors it also had the potential to be a therapeutically relevant target. We screened >1000 ASO sequences in vitro and identified highly potent mouse and mouse/human cross reactive MALAT-1 ASOs that reduced target RNA in cells in culture with IC50 values in the low nanomalor range (10-50 nM), without any lipid mediated delivery vehicles (ASO free uptake). Systemic delivery (s.c. administration of ASOs formulated in saline) of MALAT1 ASOs in vivo were well tolerated in all animals tested, and reduced target RNA by 70–>90% in the tumor cells of APC/min- mice, prostate tumor cells of the TRAMP model, DEN-induced HCC tumors as well as in the tumor cells of several human tumor xenograft models and in a patient derived NSCLC primary tumor explants model. Interestingly, MALAT-1 inhibition by ASOs was also associated with significant antitumor effects including inhibition of tumor formation and decreased BrdU positive cells in the polyps of APC/min- mice, decrease tumor growth in TRAMP prostate tumors and DEN HCC tumors and significant tumor growth delays in several xenograft and human tumor explant models. These data demonstrate unequivocal, potent, ASO mediated antisense activity of highly optimized next generation ASOs targeting MALAT-1 by systemic administration and highlight a previously uncharacterized role of the ncRNA MALAT1 as regulator of tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2951. doi:1538-7445.AM2012-2951

Obesity/hyperleptinemic phenotype adversely affects hippocampal plasticity: Effects of dietary restriction

Epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese and there is a growing appreciation that obesity affects the functional integrity of the central nervous system (CNS). We recently developed a lentivirus (LV) vector that produces an insulin receptor (IR) antisense RNA sequence (IRAS) that when injected into the hypothalamus selectively decreases IR signaling in hypothalamus, resulting in increased body weight, peripheral adiposity and plasma leptin levels. To test the hypothesis that this obesity/hyperleptinemic phenotype would impair hippocampal synaptic transmission, we examined short term potentiation (STP) and long term potentiation (LTP) in the hippocampus of rats that received the LV-IRAS construct or the LV-Control construct in the hypothalamus (hypo-IRAS and hypo-Con, respectively). Stimulation of the Schaffer collaterals elicits STP that develops into LTP in the CA1 region of hypo-Con rats; conversely, hypo-IRAS rats exhibit STP that fails to develop into LTP. To more closely examine the potential role of hyperleptinemia in these electrophysiological deficits, hypo-IRAS were subjected to mild food restriction paradigms that would either: 1) prevent the development of the obesity phenotype; or 2) reverse an established obesity phenotype in hypo-IRAS rats. Both of these paradigms restored LTP in the CA1 region and reversed the decreases in the phosphorylated/total ratio of GluA1 Ser845 AMPA receptor subunit expression observed in the hippocampus of hypo-IRAS rats. Collectively, these data support the hypothesis that obesity impairs hippocampal synaptic transmission and support the hypothesis that these deficits are mediated through the impairment of hippocampal leptin activity.

A Novel Targeted Therapeutic Approach to Multiple Myeloma Using Tumor Specific Anti-Sense RNA Sequences

Despite recent advances in the therapy of multiple myeloma (MM), the disease remains incurable. Current drugs for MM target not only MM but also normal cells. This limits the efficacy of these agents and may lead to significant morbidities for patients. Therefore, novel strategies are needed that allow targeting of tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells of these patients. We have explored the possibility of using a unique sequence from this transcript, the complementarity determining region (CDR) gene sequence, to direct drug release into MM cells. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe, our results have demonstrated that quenched fluorescent signals of this RPMI8226-specific oligonucleotide were released upon binding to the CDR3 gene sequence of RPMI8226, but not to oligo(dT), random sequences or the CDR3 gene sequence from another MM cell line, U266. Similarly, using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from U266 as a probe, our results have also shown that quenched fluorescent signals of the U266-specific oligonucleotide were released upon binding to the CDR3 gene sequence of U266, but not to oligo(dT), random sequences or the CDR3 gene sequence from RPMI8226. In addition, transfection of 8226- or U266-specific oligonucleotides into 8226 and U266 cells indicated that the oligonucleotides specifically discriminated the two cell lines. Furthermore, these oligonucleotides are stable in PBS or in transfected cells for at least 48hrs at 37°C, long enough for killing of MM cells with most currently available anti-MM drugs. Currently, we are evaluating the efficacy of this targeted approach to specifically identify tumor cells, and developing therapeutic modification of the oligonucleotide to allow drug delivery specifically to the tumor cells. Our studies validate a new strategy for MM treatment that may introduce a new targeted therapeutic approach in MM as well as other B cell malignancy, without impacting nonmalignant cells, which should allow much higher concentrations of drugs to be delivered into the tumor cells. Updated results using this novel targeted treatment approach will be reported at the meeting.

A Negatively Acting Bifunctional RNA Increases Survival Motor Neuron Both In Vitro and In Vivo

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.

A Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron in vitro and in vivo

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.

Stimulating Full-Length SMN2 Expression by Delivering Bifunctional RNAs via a Viral Vector

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present, called SMN2. SMN2 is retained in all SMA patients and encodes an identical protein compared to SMN1. However, a single silent nucleotide difference in SMN2 exon 7 results in the production of a spliced isoform (called SMNΔ7) that encodes a nonfunctional protein. The presence of SMN2 represents a unique therapeutic target since SMN2 has the capacity to encode a fully functional protein. Here we describe an in vivo delivery system for short bifunctional RNAs that modulate SMN2 splicing. Bifunctional RNAs derive their name from the presence of two domains: an antisense RNA sequence specific to a target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. Plasmid-based and recombinant adeno-associated virus vectors were developed that expressed bifunctional RNAs that stimulated SMN2 exon 7 inclusion and full-length SMN protein in patient fibroblasts. These experiments provide a mechanism to modulate splicing from a variety of genetic contexts and demonstrate directly a novel therapeutic approach for SMA.

Effect of p53 Silencing on Asenic Tioxide-Induced Apoptotic Pathways in a Myeloma Cell Line Expressing w.t.p53.

Mutations in the p53 gene are the most common genetic abnormality in human cancers. P53 is a tumor suppressor gene, which regulates a wide variety of target genes controlling cell cycle and apoptosis. Arsenic Trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. Our finding in this respect were further supported by our recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al., AACR ; Abstract #5344, 2005). We hypothesized that knocking out of p53 or p21 mRNA will result in modifying ATO-induced apoptotic pathways as observed using the p53-Ts conditional mutant of BRK cells. Thus, IM9 cells (expressing w.t.p53) were transfected [AMAXA Nucleoporator, ~60% transfection by green fluorescence protein (GFP) tag] with a GFP-SiRNA expressing plasmid co-expressing anti-sense RNA sequences for p53 (GFP/Sip53) and p21 (GFP/Sip21). As a control, GFP expressing plasmid not containing SiRNA (GFP/cont) was used. We observed in GFP/cont-transfected cells a G1 arrest through activation of p21 and moderate apoptosis, primarily through activation of procaspase 9, with no activation of BID and procaspase 8. In cells transfected with GFP/Sip53 or GFP/Sip21 plasmids we observed rapid mitochondrial membrane depolarization and apoptosis primarily through activation of the extrinsic apoptotic pathway with downregulation of FLIP, activation of procaspase 8 and BID, enhanced release of AIF from mitochondria and rapid apoptosis. Depletion of glutathione was observed with or without silencing of p21 or p53. Furthermore, silencing of p53 or p21 resulted with a decrease in ATO-induced activation of c-jun and Erk and in a decrease in the activation of Akt. These results further substantiate the model we put forward for the role of p53 in ATO-induced apoptosis in myeloma and other cancer cell lines.

Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells.

RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber viroid, hop latent viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNA(Tyr) gene or near the 3' end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub-cellular compartments.

Inhibiting expression of a tomato ripening-associated membrane protein increases organic acids and reduces sugar levels of fruit.

Tomato (Lycopersicon esculentum Mill.) ripening-associated membrane protein (TRAMP) is a channel protein of the membrane intrinsic protein (MIP) class encoded by the cDNA clone pNY507 [R.G. Fray et al. (1994) Plant Mol Biol 24: 539-543]. It has been suggested that these proteins encode water channels or aquaporins. TRAMP mRNA accumulated in all tomato tissues tested and was elevated in fruit during post-anthesis development and again during ripening. Transgenic plants that constitutively expressed a TRAMP antisense RNA sequence were generated with a 94% reduction of endogenous TRAMP mRNA in fruit. They showed no obvious phenotype that could be associated with gross perturbation of water relations, but ripening fruit of these plants showed marked alterations in the normal pattern of accumulation of both organic acids and sugars. At the onset and during ripening, levels of the organic acids L-malate and citrate were significantly elevated while levels of D[+]-glucose and D[+]-fructose were reduced. Additional transgenic lines were generated with reduced TRAMP mRNA, and the phenotype of increased acids and reduced sugars during fruit maturation and ripening was shown to be reproducible and stably inherited. Fruit of plants that over-expressed TRAMP mRNA showed no significant alteration in the sugars or acids investigated. These results suggest a role for TRAMP in the movement of solutes between cell compartments.

Accelerated assessing of antisense RNA efficacy using a chimeric enhanced green fluorescent protein-antisense RNA-producing vector.

The selection of suitable parts of a gene as antisense RNA sequences is largely a matter of trial and error and, as a consequence, a rather time-consuming process. In this study, we present a rapid and reproducible method to bypass this protracted procedure by using a chimeric enhanced green fluorescent protein (EGFP)-antisense RNA-producing vector. The combination of a reporter gene and antisense RNA allows easy measurement by flow cytometry of antisense RNA efficacy in successfully transfected cells shortly after transfection. Four chimeric EGFP-p185c-erbB-2-antisense RNA vectors were constructed and transfected into the p185-c-erbB-2-overexpressing cell line SKBR3. Within 1 week, we were able to estimate the inhibitory capacities of the different antisense RNA sequences used in this study. Our results strongly suggest that a chimeric EGFP-antisense RNA vector is an appropriate tool to expedite the laboratory work and time in screening the efficacy of antisense RNA strategies.

Antisense RNA sequences targeting the 5' leader packaging signal region of human immunodeficiency virus type-1 inhibits viral replication at post-transcriptional stages of the life cycle.

Antisense RNA has proven a potent inhibitor of gene expression and has the potential to inhibit retroviral replication at a number of stages in the virus life cycle by targeting both viral and cellular RNA sequences. Antisense RNA complementary to three target regions in the 5' leader/LTR of human immunodeficiency virus type-1 (HIV-1), the TAR region, the primer binding site and the splice donor (SD)-packaging signal (psi) region were stably expressed from the CMV IE promoter in Jurkat cells, and expression confirmed by RT-PCR. When challenged with HIV-1, cell lines expressing antisense RNA targeting the SD/psi region showed significant inhibition of replication (at up to 10(6) TCID 50/ml). These sequences were also expressed in lymphocytes after transduction using recombinant retroviruses and one sequence complementary to the SD/psi region inhibited replication of HIV-1. A co-transfection assay using COS-1 cells was also developed both to confirm the antiviral potential of these sequences, and to determine the predominant site of action of these molecules. Antisense RNAs targeting the psi region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore, analyses of relative levels of cellular and virion RNA from these assays suggest each of these antisense molecules exerts its effect at an early stage in the transcription-translation pathway, while the longer of the sequences also inhibited packaging of virion RNA. These results suggest that the packaging signal (psi) of HIV-1 represents an attractive target for antisense RNA-based gene therapy, although the main mode of action of such molecules may well be through antisense effects at an earlier stage of replication than packaging.

Interference with virus and bacteria replication by the tissue specific expression of antibodies and interfering molecules.

Historically, protection against virus infections has relied on the use of vaccines, but the induction of an immune response requires several days and in certain situations, like in newborn animals that may be infected at birth and die in a few days, there is not sufficient time to elicit a protective immune response. Immediate protection in new born could be provided either by vectors that express virus-interfering molecules in a tissue specific form, or by the production of animals expressing resistance to virus replication. The mucosal surface is the largest body surface susceptible to virus infection that can serve for virus entry. Then, it is of high interest to develop strategies to prevent infections of these areas. Virus growth can be interfered intracellularly, extracellularly or both. The antibodies neutralize virus intra- and extracellularly and their molecular biology is well known. In addition, antibodies efficiently neutralize viruses in the mucosal areas. The autonomy of antibody molecules in virus neutralization makes them functional in cells different from those that produce the antibodies and in the extracellular medium. These properties have identified antibodies as very useful molecules to be expressed by vectors or in transgenic animals to provide resistance to virus infection. A similar role could be played by antimicrobial peptides in the case of bacteria. Intracellular interference with virus growth (intracellular immunity) can be mediated by molecules of very different nature: (i) full length or single chain antibodies; (ii) mutant viral proteins that strongly interfere with the replication of the wild type virus (dominant-negative mutants); (iii) antisense RNA and ribozyme sequences; and (iv) the product of antiviral genes such as the Mx proteins. All these molecules inhibiting virus replication may be used to obtain transgenic animals with resistance to viral infection built in their genomes. We have developed two strategies to target into mucosal areas either antibodies to provide immediate protection, or antigens to elicit immune responses in the enteric or respiratory surfaces in order to prevent virus infection. One strategy is based on the development of expression vectors using coronavirus derived defective RNA minigenomes, and the other relies on the development of transgenic animals providing virus neutralizing antibodies in the milk during lactation. Two types of expression vectors are being engineered based on transmissible gastroenteritis coronavirus (TGEV) defective minigenomes. The first one is a helper virus dependent expression system and the second is based on self-replicating RNAs including the information required to encode the TGEV replicase. The minigenomes expressing the heterologous gene have been improved by using a two-step amplification system based on cytomegalovirus (CMV) and viral promoters. Expression levels around 5 micrograms per 10(6) cells were obtained. The engineered minigenomes will be useful to understand the mechanism of coronavirus replication and for the tissue specific expression of antigen, antibody or virus interfering molecules. To protect from viral infections of the enteric tract, transgenic animals secreting virus neutralizing recombinant antibodies in the milk during lactation have been developed. Neutralizing antibodies with isotypes IgG1 or IgA were produced in the milk with titers of 10(6) in RIA that reduced virus infectivity by one million-fold. The recombinant antibodies recognized a conserved epitope apparently essential for virus replication. Antibody expression levels were transgene transgene copy number independent and were related to the transgene integration site. This strategy may be of general use since it could be applied to protect newborn animals against infections of the enteric tract by viruses or bacteria for which a protective MAb has been identified. Alternatively, the same strategy could be used to target the expression of antibio

Anti-angiogenin Activity of the Peptides Complementary to the Receptor-binding Site of Angiogenin

Angiogenesis promotes growth and metastasis of tumor cells. In this study, we have developed two peptide antagonists of human angiogenin by deducing the codes from the antisense RNA sequence corresponding to the receptor-binding site of angiogenin in either 5' --> 3' (chANG) or 3' --> 5' (chGNA) direction. chANG and chGNA peptides bind to angiogenin with specificity and high affinity (Kd approximately 44 nM) and inhibit the interaction of angiogenin with actin, which is regarded as the angiogenin-binding protein on the surface of endothelial cells. The peptides inhibit the neovascularization induced by angiogenin in the chick chorioallantoic membrane assay. The anti-angiogenic activity of the peptides is specific for angiogenin, and the peptides do not have any apparent effect on embryonic angiogenesis or the preexisting blood vessels. chANG and chGNA also inhibit the angiogenesis induced by the angiogenin-secreting PC 3 human prostate adenocarcinoma cells and have no direct effect on the proliferation as well as the adhesion of PC 3 cells to angiogenin. Therefore, the inhibition of the tumor-induced angiogenesis by the peptides is most likely caused by neutralization of the extracellular angiogenin secreted by PC 3 cells. Based on our results, chANG and chGNA peptides may be effective for treatment of various human tumors which secrete angiogenin.

Identification and location of human papillomavirus type 16 antisense early promoter and characterisation of antisense RNA.

Antisense RNA sequences of various regions of human papillomavirus type 16 (HPV 16) were previously found in a number of cervical lesions, but the viral or cellular promoter has not been identified. HPV 16 E7 oncogene antisense transcripts expressed from an antisense promoter in viral DNA were found in the present study by RNase protection assays for total and cytoplasmic RNA. The antisense promoter for these transcripts was located within HPV 16 nt 4030-4230 by deletion analyses. The results also suggested that most of the antisense RNA was relatively short. The antisense promoter of HPV 16 was functional for expression of antisense RNA of a heterologous gene. Antisense-sense double-stranded E7 RNA was detected, and the sense RNA of this duplex was apparently inefficient for splicing or cleavage/poly(A) addition. These results show that HPV 16 can produce early region antisense RNA, which is from a promoter within a defined region of the viral genome. The possible importance of these transcripts for the regulation of episomal HPV 16 gene expression in infected and premalignant lesions and the possible importance of their deregulation for expression in malignant lesions are discussed.

Protection of a T-Cell Line from Human Immunodeficiency Virus Replication by the Stable Expression of a Short Antisense RNA Sequence Carried by a Shuttle RNA Molecule

Adenovirus VA1 gene is efficiently transcribed by RNA polymerase III and gives rise to a small highly ordered RNA. To inhibit replication of human immunodeficiency virus (HIV), a chimeric VA1 RNA molecule was designed that contained a short antisense RNA sequence complementary to a conserved region of the HIV-1 rev encoding mRNA (28 nucleotides). This sequence, which was inserted into a projecting loop of the VA1 RNA central domain, was mainly single stranded and available for binding with its complementary sequence. The chimeric VA1 antisense was abundantly expressed in human cells constituting 3% of mRNA and promoted strong and specific inhibition of HIV-1 gene replication. The stable expression of antisense RNA in human T cells (CEM) protected these cells from HIV-1 multiplication for at least 3 months. No side effects were detected because of the lack of antisense effect upon replication of the closely related HIV-2. The VA1 gene may provide a suitably compact gene cassette for the intracellular expression of short antisense RNA directed against HIV.

Transient Gene Expression of Antisense RNA and Coat Protein-Encoding Sequences Reduced Accumulation of Cherry Leafroll Virus in Tobacco Protoplasts

Cherry leafroll nepovirus (CLRV) is the causative agent of blackline disease, which results in a fatal necrosis of the graft union of English walnut scion on certain rootstocks. Tobacco suspension cell protoplasts were electroporated with plasmid constructions, bearing or not bearing RNA-derived sequences, and, subsequently, were electroporated with CLRV virions or virion RNA. Replication of CLRV in protoplasts was demonstrated by accumulation of both positive- and negative-sense CLRV genomic RNAs 1 and 2, capsid antigen, and virions. Three plasmids were tested for antiviral action. These have inserts that were derived from the coat protein gene, inserted in both orientations, and from the 3′ terminal sequence that is nearly identical in RNA 1 and RNA 2, oriented for expression of antisense RNA. Plasmids were introduced into protoplasts 12 hr prior to introducing the virions or virion RNA. CLRV accumulation was reduced significantly by prior electropotation of plasmids intended to express coat protein or 3′ antisense RNA, but not by electropotation of plasmid without insert or plasmid with coat protein encoding sequences in the antisense orientation. These results demonstrate the utility of transient expression in a protoplast system for comparing the efficacy of a variety of virus-derived and other sequences for their potential application in virus control strategies.