ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT 6:111-118 (1996) Mary Ann Liebert, Inc. Correlation of Activity with Stability of Chemically Modified Ribozymes in Nuclei Suspension OLAF HEIDENREICH,1 XIAO XU,1 PIOTR SWIDERSKI,2 JOHN J. ROSSI,2 and MICHAEL NERENBERG' ABSTRACT To examine hammerhead ribozyme activity in the nuclear environment, we have used nuclei isolated from HTLV-I tax transformed fibroblasts to evaluate ribozymes targeted against HTLV-I tax RNA. The ribozyme activity in nuclei suspension was strongly dependent on the resistance of the particular ribozyme to endoge¬ nous nucleases. A ribozyme containing exclusively 2'-deoxynucleotides in its stems cleaved target RNA by its catalytic activity in the absence of proteins and caused degradation in their presence by induction of nuclear RNase H activity. A ribozyme containing 2-amino- and 2'-fluoropyrimidine nucleosides in combination with terminal phosphorothioate linkages was significantly more stable in nuclei suspension and also exhibited a more than threefold higher cleavage efficacy than its unmodified counterpart. The increased resistance against nuclease degradation is mainly due to terminal phosphorothioate linkages, suggesting that both 5' and 3'-ex¬ onucleases are primarily responsible for the nuclear degradation of oligonucleotides. INTRODUCTION The development of rrans-cleaving hammerhead ri¬ bozymes offers a promising alternative to more conven¬ tional antisense approaches (Marschall et al., 1994). However, a major problem for the exogenous delivery of such oligori¬ bonucleotides is their susceptibility to ribonucleases, which are abundant in body fluids, such as blood serum. Unlike oligodeoxynucleotides, ribozymes cannot be fully internally modified to enhance their nuclease stability, as several posi¬ tions within the central core region of the ribozyme are very sensitive to chemical modifications (Heidenreich et al., 1993, and citations therein). Thus, extensive modification results in a severe loss of RNA cleavage activity of the ribozyme. One possibility to circumvent this problem is to modify only the stem but not the central core region of the ribozyme. Ribozymes with stems consisting exclusively of 2'-deoxynu- cleotides have been shown to cleave their target RNA very ef¬ fectively (Hendry et al., 1992). Whereas the stability of such chimeric ribozymes is still very low in serum, their intracellular activity is at least twofold enhanced (Taylor et al., 1992). The incorporation of phosphorothioate linkages into the stems and into selected positions within the core region results in a further increase in stability (Shimayama et al., 1993). Alternatively, se¬ lective introduction of 2'-modified analogs, such as 2'-alkoxy- (Paolella et al., 1992), 2'-amino-, or 2'-fluoronucleosides (Pieken et al., 1991), into the ribozyme sequence in combina¬ tion with terminal phosphorothioate (Heidenreich and Eckstein, 1992) yields ribozymes that are highly resistant to serum nucle¬ ases and are still as active as their unmodified counterparts. However, the uridines at positions 4 and 7 in the central core are particularly crucial and tolerate 2'-amino but not 2'-fluoro substitution without loss of activity (Heidenreich et al., 1994). Such modified ribozymes are amenable to the exogenous deliv¬ ery to cells (Kiehntopf et al., 1994, 1995). However, there is very little information available about the stability and activity of ribozymes in an intracellular environ¬ ment, and many questions are raised. Is a chemically modified ribozyme more effective inside the cell than an unmodified one? Which modifications are necessary to protect a ribozyme against, for example, nucleases located in the cell nucleus? When chimeric ribozymes bind to their RNA target sequence, an RNA-DNA heteroduplex is formed. Does such a ribozyme cleave RNA intracellularly by its catalytic activity, or does it trigger RNase -mediated RNA degradation? These questions prompted us to compare the cleavage activity and stability of several modified and unmodified ribozymes in a system simu¬ lating an intracellular compartment. It is shown that chemical modifications increase the efficacy of ribozymes in cell nuclei suspension by improving their stability. In addition, the type of 'The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA 92037. department of Molecular Biology and Gene Therapy, Loma Linda Medical Center, Loma Linda, CA 92350. Ill