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1. |
Toward a Broad-Based Antisense Technology |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 113-114
Richard W. Wagner,
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ISSN:1050-5261
DOI:10.1089/ard.1995.5.113
年代:1995
数据来源: MAL
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2. |
Stability and Pharmacokinetic Characteristics of Oligonucleotides Modified at Terminal Linkages in Mice |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 115-121
TAKENORI MIYAO,
YOSHINOBU TAKAKURA,
TAISHIN AKIYAMA,
FUMIO YONEDA,
HITOSHI SEZAKI,
MITSURU HASHIDA,
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摘要:
To construct the strategy for delivery systems that can controlin vivodisposition of antisense oligonucleotides, we studied the stability and basic pharmacokinetic characteristics of oligonucleotides. Decathymidylic acid (T10), a model oligodeoxynucleotide, and its derivatives, 5′-biotin-T10(5′B-T10) and 3′-methoxyethylamine 5′-biotin-T10(3′M5′B-T10), containing phosphoroamidate modification at 3′- and/or 5′-terminal internucleoside linkages, were synthesized. In phosphate-buffered saline (PBS,pH 7.4) containing 10% mouse serum, unmodified T10was degraded with a half-life of 45 minutes; the degradation half-lives of 5′B-T10and 3′M5′B-T10were 11 and 30 h, respectively. In mouse whole blood, 3′M5′B-T10was relatively stable, and 45% remained intact after 1 h incubation. After intravenous injection of [3H]3′M5′B-T10into mice at a dose of 1 mg/kg, the radioactivity was rapidly cleared from plasma with a half-life of 2 minutes and accumulated in the kidney, liver, and gallbladder. About 30% of the dose was excreted in the urine within 60 minutes. A much more rapid degradation of [3H]3′M5′B-T10was observedin vivothan expected fromin vitroexperiments: more than 90% of the radioactivity in plasma was degradation product at 2 minutes after injection. These results suggested that enzymatic degradation occurred in some compartments in addition to the blood pool. The apparent urinary excretion clearance of [3H]3′M5′B-T10was close to that of inulin, whereas the apparent hepatic uptake clearance was much greater than that of inulin and comparable to that of dextran sulfate, which is taken up by the liver by scavenger receptors for polyanions. Thus, the present study demonstrated that the disposition processes to be controlled involve stability, urinary excretion, and hepatic uptake for the developmen
ISSN:1050-5261
DOI:10.1089/ard.1995.5.115
年代:1995
数据来源: MAL
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3. |
Uptake, Intracellular Distribution, and Stability of Oligodeoxynucleotide Phosphorothioate bySchistosoma mansoni |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 123-129
LIANG-FENG TAO,
KENNETH A. MARX,
WARANYA WONGWIT,
ZHIWEI JIANG,
SUDHIR AGRAWAL,
ROBERT M. COLEMAN,
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摘要:
Thein vitrouptake, cellular distribution, efflux, stability, and toxicity levels of an oligodeoxynucleotide phosphorothioate (PS-oligonucleotide) have been studied in matureSchistosoma mansoniworms. The intracellular accumulation of35S-labeled PS-oligonucleotide occurred roughly in proportion to the worm body mass over a wide concentration range, whether the worms were exposed singly or in mating pairs. Cellular uptake was dependent on the extracellular concentration. A minor fraction (13%) of the PS-oligonucleotide taken up by the worm accumulated in the surface tegumental coat. Most of the PS-oligonucleotide taken up localized in the cytosol (54%) and the nuclei-enriched (33%) fractions. In a time course study on adult worms in culture, oligonucleotide uptake was observed within the first 2 h and peaked at about 36 h. A decrease in the intracellular concentration of the PS-oligonucleotide was observed by 42 h. Analysis of the extracted oligonucleotides showed that PS-oligonucleotide was digested slowly. Efflux of the oligonucleotide was time and temperature dependent. Significant toxicity to the cultured worms did not occur until the PS-oligonucleotide concentration was over 8 mg/ml (1 mM).
ISSN:1050-5261
DOI:10.1089/ard.1995.5.123
年代:1995
数据来源: MAL
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4. |
Characterization of Binding Sites, Extent of Binding, and Drug Interactions of Oligonucleotides with Albumin |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 131-139
SHASHI KUMAR SRINIVASAN,
HEMANT K. TEWARY,
PATRICK L. IVERSEN,
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摘要:
Phosphorothioate oligonucleotides (S-ODNs) have the ability to modulate gene expression selectively and thus have potential therapeutic capabilities. This potential led us to investigate the protein binding characteristics of selected S-ODNs. We evaluated S-ODN interactions with bovine serum albumin (BSA) and human serum albumin (HSA)in vitro. The equilibrium dissociation constantsKmfor the binding of a 20 mer S-ODN with BSA and HSA range between 1.1-5.2 × 10-5and 2.4-3.1 × 10-4M, respectively. TheKmfor an unrelated 15 mer S-ODN binding with HSA ranges between 3.7 and 4.8 × 10-5M. Studies with a fluorescently labeled 27 mer S-ODN suggest cooperative binding (Hill slope = 1.67) and/or the presence of secondary binding sites on the S-ODN. HSA or BSA linked to Sepharose was incubated with a 15,20, or 24 mer S-ODN followed by the addition of selected drugs known to be highly protein bound (nifedipine, warfarin, midazolam, probenecid, indomethacin, and mitoxantrone). Up to 30% of S-ODN was displaced by warfarin in competition binding assays. Conversely, HSA-bound warfarin was incubated with a variety of oligonucleotides, including RNA and genomic dsDNA. Maximum displacement of warfarin-bound HSA was observed following incubation with 5′-cholesterol-conjugated 20 mer S-ODN. In summary, S-ODNs are likely to interact and displace other therapeutic agents that bind to albumin, particularly those binding at si
ISSN:1050-5261
DOI:10.1089/ard.1995.5.131
年代:1995
数据来源: MAL
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5. |
Transient Expression Assay for Antisense RNAs Using Episomal Replication of Plasmids: Effective Reduction of Retinoblastoma Gene (Rb-1) Product by Its Antisense RNA Complementary to 3′-Untranslated Region |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 141-148
MASAYUKI KOBAYASHI,
YUKIKA YAMAUCHI,
KAZUNORI YAMAGUCHI,
AKIKO TANAKA,
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摘要:
We have developed a transient expression assay for selection of effective antisense RNAs using episomal replication of plasmids in COS-7 cells, an African green monkey kidney-derived cell line expressing SV40 large T antigen. The transient expression assay was enabled by a liposome-mediated DNA transfection method, by which about 70% of the cells were reproducibly transfected with exogenous DNAs. Plasmids expressing antisense RNAs for the retinoblastoma gene (Rb-1) mRNA and harboring SV40oriwere constructed and introduced into COS-7 cells to examine their inhibitory effect on the accumulation of endogenous Rb protein (pRb). Only the antisense RNA complementary to the 3′-untranslated region (UTR) inRb-1mRNA was expressed stably at high levels for 3 days after the transfection. This antisense RNA reduced by 73% the content of endogenous pRb 70 h after transfection. A similar inhibition was detected in mouse mammary carcinoma cells (FM3A) that were stably transfected with the antisense RNA expressing vector directed to 3′UTR. In contrast, no obvious change in pRb was observed with antisense RNAs complementary to the coding region ofRb-1mRNA. The cellular content of these antisense RNAs was lowered by degradation; thus these RNAs did not affect the levels of pRb in COS-7 and FM3A cells. These results, taken together, suggest that the expression levels and the stability of antisense RNAs are involved in their repressive activity, and our transient expression assay provides a rapid and easy system for evaluation of ectopic antisense RNA activity in COS-7 ce
ISSN:1050-5261
DOI:10.1089/ard.1995.5.141
年代:1995
数据来源: MAL
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6. |
Helix-Stabilizing Agent, CC-1065, Enhances Suppression of Translation by an Antisense Oligodeoxynucleotide |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 149-154
DAE-YONG KIM,
DAVID H. SWENSON,
D.-Y. CHO,
H. WAYNE TAYLOR,
DING S. SHIH,
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摘要:
The antitumor antibiotic CC-1065 is known to bind at selected sequences in the minor groove of duplex nucleic acids and to hyperstabilize the duplexes against thermal melting. These properties suggested that CC-1065 may enhance translation inhibition by antisense oligonucleotides directed against a specific mRNA. A 585 bp mRNA transcript containing the equine infectious anemia virus (EIAV)S2gene and a portion of theenvgene was prepared. Also, a complementary 20 mer antisense oligodeoxynucleotide (5′-TGTTGGGTAATAGGGGTTGA-3′) was prepared against a target sequence in the mRNA located near the translational initiation sites of the overlappingS2andenvgenes. The center of the target sequence had an expected CC-1065 recognition sequence (5′-UAUUA-3′). Translation in the presence of CC-1065 and antisense was markedly suppressed compared with that of antisense alone. Addition of a sense 20 mer strand, with or without CC-1065, had little or no effect on translation. CC-1065 and related compounds may be useful as ligands for enhancing the stability of sense-antisense duplexes and for promoting the inhibition of translation by antisense oligonucl
ISSN:1050-5261
DOI:10.1089/ard.1995.5.149
年代:1995
数据来源: MAL
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7. |
Influence of Chromosomal Position and Copy Number of awhite-Directed Ribozyme Gene on the Suppression of Eye Pigmentation inDrosophila melanogaster |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 155-160
JÖRG-CHRISTIAN HEINRICH,
MARTIN TABLER,
CHRISTOS LOUIS,
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摘要:
Different strains of transgenicDrosophila melanogastercarrying one, two, or three copies of a heat-shock promoter 70 (hsp70)-driven catalytic antisense RNA gene, directed against thewhitegene, were investigated for the expression level of ribozyme RNA. It was found that the steady-state concentrations of the hammerhead ribozyme were proportional to the copy number of the genes and that the suppressive effect on eye pigment accumulation was dosage dependent. In a further experiment, aD. melanogasterstrain, deficient in eye pigmentation caused by a deletion of thewhitegene, was used for P element-mediated germline transformation: the transposon used contained thehsp70-driven.white-directed ribozyme gene and, on the same DNA, themini-whitegene under its own promoter. The spatial coupling of the transcription of ribozyme and target RNA resulted in more effective ribozyme-mediated inhibition of eye pigmentation under heat-shock conditions. These effects were dependent on the chromosomal integration site of the transposon.
ISSN:1050-5261
DOI:10.1089/ard.1995.5.155
年代:1995
数据来源: MAL
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8. |
Selected Abstracts from the 2nd International Conference on Antisense Nucleic Acids |
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Antisense Research and Development,
Volume 5,
Issue 2,
1995,
Page 161-166
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ISSN:1050-5261
DOI:10.1089/ard.1995.5.161
年代:1995
数据来源: MAL
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