摘要:

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

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.1

年代:1998

数据来源: MAL

摘要:

Thebcr-ablchimeric gene is found in 95% of chronic myeloid leukemia (CML) patients and is thought to be seminal to the etiology of the disease. The possibility of using ribozymes to suppressbcr-ablgene expression and subsequently alter the malignant phenotype of hematopoietic cells may provide an alternative therapeutic approach to current regimens. A series of hammerhead ribozymes targeted to a b3a2bcr-abltranscript has been developed and previously shown to be capable of cleaving the desired sequence with varying degrees of specificity. This study investigated theex vivoeffects of endogenous expression of these ribozymes in a CML cell line, K562. We demonstrated a 53% decrease inbcr-ablmRNA levels in a clone induced to express Rz8, compared with its uninduced control. Phenotypic analysis of this clone also revealed a 63% decrease in colonyforming ability and a 43% inhibition of cell proliferation following ribozyme expression. Morphologic analysis of cells showed there was a slight increase (2.5% to 15%) in the number of cells undergoing apoptosis. These results suggest that Rz8 was effective in suppressingbcr-ablgene expression within a cellular environment and altering the leukemic nature of a CML cell line.

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.15

年代:1998

数据来源: MAL

摘要:

By describing the behavior of myotonin mRNA levels, from the quiescent to the differentiated state in C2 mouse myoblasts, we produced evidence bearing on the role of myotonin gene product in the control of cell growth and differentiation. To study the role of myotonin in myotonic dystrophy (DM) pathogenesis, we developed a suitable cellular model where myotonin gene expression was modulated by phosphorothioate antisense oligonucleotides in C2 cultured cells. Furthermore, an isoform of the gene product, similar to that described in humans and not yet described in the mouse, was found.

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.25

年代:1998

数据来源: MAL

摘要:

For the enzymatic digestion of a 25-mer phosphorothioate (PS) oligonucleotide, the reaction kinetics was previously determined to be the sum of two parallel processes: a fast and a very slow phase of digestion suggesting a two-exponential model. A characteristic metabolite profile was observed bothin vitroandin vivo. This behavior is shown to be the result of the stereoselective cleavage of chiral R-configuration and S-configuration PS internucleotide linkages by 3′-exonucleases. The stereoselective nature of 3′-exonuclease action was analyzed by reverse-phase HPLC. The separation of eight diastereomers of the tetramer TTCT (5′-3′) was used to follow the stereoselective course of exonuclease hydrolysis of PS internucleotide linkages. Degradation of the 25-mer parent compound having a 3′ S-terminal internucleotide linkage was calculated to be more than 300 times slower than an analog with a 3′-terminal R-configuration. These results support an approach for protecting antisense oligonucleotides based on the chirality of only the 3′-end internucle

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.35

年代:1998

数据来源: MAL

摘要:

The pharmacokinetics and metabolism of an antisense oligonucleotide phosphorothioate (GEM91®) were studied in cynomolgus monkeys following intravenous infusion. [35S]-Labeled GEM91 was administered to 12 monkeys by means of a 2-hour intravenous infusion at a dose of 4 mg/kg. Plasma pharmacokinetic analysis revealed that the maximum plasma concentration was 41.7 μg equivalents/ml, which was achieved in 2.13 hours. The plasma elimination half-life was 55.8 hours based on radioactivity levels. Urinary excretion represented the major pathway of elimination, with 70% of the administered dose excreted in urine over 240 hours. The oligonucleotide was widely distributed to tissues. The highest concentrations were observed in the liver and kidney. Analysis of the extracted oligonucleotide following post-labeling with [32P] on polyacrylamide gel electrophoresis showed the presence of both intact and degraded oligonucleotide in plasma, kidney, liver, spleen, and lymph nodes. Based on the methods used for post-labeling (either 3′-end or 5′-end), different patterns of bands were observed on polyacrylamide gel electrophoresis, suggesting metabolic modification of the administered oligonucle

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.43

年代:1998

数据来源: MAL

摘要:

We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H. The enzyme has a molecular weight of 32 kDa, is Mg2+dependent, and is inhibited by Mn2+and N-ethylmaleimide. Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H. The human RNase H we have cloned is highly homologous toEscherichia coliRNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous toE. coliRNase HI. The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues.

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.53

年代:1998

数据来源: MAL

摘要:

Hammerhead ribozymes and stable stem loop structures function as inhibitors of 3‵-5‵-exonuclease degradation of external guide sequences (EGSs) when covalently linked to the 3‵-end of EGS RNAs. This observation may be of use in improving the efficiency of gene inactivation techniques that use single-stranded RNA (e.g., antisense RNA, EGS RNA)in

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.63

年代:1998

数据来源: MAL

摘要:

The objective of this study was to clarify the renal uptake characteristics of oligonucleotides at a cellular level using LLC-PK1renal epithelial cells derived from the proximal tubule. The association of [35S]-labeled 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides with the monolayers of polarized LLC-PK1cells cultured on polycarbonate filter was characterized after apical or basolateral application. The cellular association of PO and PS at both apical and basolateral membranes was time dependent and temperature dependent, and the apparent association amount of PS was larger than that of PO. The PO and PS association after apical application was saturable, with the apparentKmand Vmaxvalues determined to be 5.4 μM and 0.14 nmol/mg protein for PO and 0.22 μM and 0.11 nmol/mg protein for PS, respectively. In contrast, almost linear kinetics were observed after basolateral application within a tested concentration range. The association was inhibited significantly by sodium azide and chloroquine, suggesting that an energy-dependent endocytotic process was involved. Internalization and subsequent transport to endosome and lysosome compartments of FITC-labeled oligonucleotides were shown by confocal laser scanning microscopy. The present study has demonstrated that both types of oligonucleotides are taken up by LLC-PK1cells from both apical and basolateral surfaces probably via an endocytosis mechanis

ISSN:1087-2906    

DOI:10.1089/oli.1.1998.8.67

年代:1998

数据来源: MAL