Development of ribozymes that target stathmin, a major regulator of the mitotic spindle

Stathmin is a major cytosolic phosphoprotein that plays an important role i n the control of cellular proliferation by regulating the dynamics of the m icrotubules that make up the mitotic spindle. Because stathmin is expressed at high levels in ail human cancers, it is an attractive molecular target for anticancer interventions. We had shown previously that antisense stathm in inhibition results in marked abrogation of the transformed phenotype of leukemic cells in vitro and in vivo. Unlike the antisense approach, ribozym es can catalytically cleave several molecules of target RNA. This may provi de a more efficient strategy for downregulating genes, such as stathmin, th at are expressed at very high levels in cancer cells, We designed several a ntistathmin hammerhead ribozymes and tested their cleavage activity against short synthetic stathmin RNA substrates, In vitro cleavage studies demonst rated site-specific cleavage of stathmin RNA that was dependent on ribozyme concentration and duration of exposure to ribozyme, The most active antist athmin ribozyme was capable of cleaving >90% stathmin RNA in a catalytic ma nner, cleaving multiple substrate molecules per ribozyme molecule, We also demonstrated that the designed antistathmin ribozymes are capable of select ively cleaving native stathmin RNA in a mixture of total RNA isolated from leukemic cells, These antistathmin ribozymes may provide a novel and effect ive form of gene therapy that may be applicable to a wide variety of human cancers.