ABSTRACT
Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5'-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2'-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.
Subject(s)
Proto-Oncogene Proteins/metabolism , RNA, Catalytic/metabolism , Trans-Activators/metabolism , Animals , Carbohydrates/chemistry , Cell Division , Cells, Cultured , Female , Humans , Mice , Molecular Sequence Data , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myb , RNA, Catalytic/chemistry , Rats , Rats, Sprague-Dawley , Substrate SpecificityABSTRACT
Proliferation of injured smooth muscle cells contributes to the reocclusion or restenosis of coronary arteries that often occurs following angioplasty procedures. We have identified and optimized nuclease-resistant ribozymes that efficiently cleave c-myb RNA. Three ribozymes targeting different sites in the c-myb mRNA were synthesized chemically and delivered to rat aortic smooth muscle cells with cationic lipids; all three inhibited serum-stimulated cell proliferation significantly. RNA molecules with two base substitutions in the catalytic core that render the ribozyme catalytically inactive had little effect on smooth muscle cell proliferation. Ribozymes with scrambled binding arm sequences also failed to affect cell cycle progression of vascular smooth muscle cells. Furthermore, inhibition of rat smooth muscle cell proliferation correlated with a reduction in intact c-myb mRNA. Efficacy of the chemically-modified ribozyme was compared directly to phosphorothioate antisense oligodeoxynucleotides targeting the same site in the c-myb RNA; the ribozyme had superior efficacy and showed greater specificity than the antisense molecules. Exogenously delivered ribozymes also inhibited porcine and human smooth muscle cell proliferation effectively. Ribozymes targeting c-myb or other regulators of smooth muscle cell proliferation may represent novel therapeutics for the treatment of restenosis after coronary angioplasty.
Subject(s)
Muscle, Smooth, Vascular/cytology , Proto-Oncogene Proteins/genetics , RNA, Catalytic/metabolism , Trans-Activators/genetics , Animals , Aorta , Base Sequence , Cell Division , Cells, Cultured , DNA Primers , Female , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Proto-Oncogene Proteins c-myb , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , SwineABSTRACT
An outbreak of histomonad infection of an apparent atypical nature was diagnosed in a flock of about 850 Bobwhite quail. Mortality was 95% over a 3-week period. The most prominent gross pathologic lesions were in the livers: disseminated white foci of necrosis, 1 to 2 mm in diameter, and subcapsular multifocal splenic necrosis was seen occasionally; lower intestinal lesions were infrequent. Histologic examination of liver and spleen sections revealed focal necrosis associated with variable numbers of protozoal organisms identified as a Histomonad spp. Identification of the protoza was ascertained by electron microscopy. Histmonads were isolated from affected quail livers and propagated in specific-pathogen-free chicken embryos. Lesions produced in embryos were evaluated. Isolates of the organism were used to reproduce the disease in young Bobwhite quail.
Subject(s)
Colinus , Poultry Diseases/epidemiology , Protozoan Infections/epidemiology , Quail , Animals , Chickens , Liver/pathology , Poultry Diseases/pathology , Protozoan Infections/pathology , Spleen/pathologyABSTRACT
Chickens were vaccinated and revaccinated with inactivated Newcastle disease (ND) vaccines from 2 different sources and also with LaSota strain, live Newcastle disease virus (NDV). Both inactivated vaccines induced higher virus neutralizing (VN) and hemagglutination-inhibition (HI) titers than the LaSota virus. One of the inactivated preparations was found superior to the other by both the VN and HI tests. However, poor protection from apparent virus replication, virus shed, and transmission occurred after challenge with a velogenic NDV strain in those vaccinated with each of the inactivated vaccines. In contrast, LaSota virus, given by the eye drop route, produced excellent protection by the same criteria. In one of the groups of chickens, gross lesions of airsacculitis were seen after vaccination with an inactivated vaccine and subsequent challenge. Revaccination with inactivated vaccines did not enhance the protection of the respiratory tract but did result in an anamnestic serological response (VN and HI). In the post-challenge period, the use of tracheal swabs proved more sensitive as an indicator of virus shed than did cloacal swabs with the velogenic NDV strain used. The practical implications of observations made from the trials are discussed.
