Characterization of nuclease-resistant ribozymes directed against hepatitis B virus RNA.

Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.

Nucleic acid hybridization assays employing dA-tailed capture probes. I. Multiple capture methods.

A quantitative hybridization assay termed "reversible target capture" is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe "ternary complex" is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol (100 fg) of target with 32P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as few as six Listeria cells have been detected in the presence of 1.25 x 10(7) control cells.

Nucleic acid hybridization assays employing dA-tailed capture probes. Single capture methods.

Several novel hybridization techniques are described. Cells or specimens are treated to release nucleic acids and a liquid phase hybridization is carried out with a dA-tailed capture probe and a reporter probe in chaotropic salts or in salts containing SDS/proteinase K. In another format the tailed capture probe is preimmobilized on polystyrene and used to capture target nucleic acids from the solution. No phenol extraction or centrifugation is required to prepare the nucleic acids. Capture of the target on the poly (dT)-solid supports is used to remove excess labelled probe and sample impurities prior to non-radioisotopic or radioisotopic detection. This paper shows the advantage of a single round of capture on polystyrene, including the ability to assay large numbers of samples manually, the ability to analyse each sample for many analytes simultaneously, the use of rapid non-radioisotopic detection, and the ability to readily adapt the assay for automation.

A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen.

Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.