TRENDS in Molecular Medicine Vol.7 No.5 May 2001 http://tmm.trends.com 221 Review Review Review Alfred S. Lewin* Dept of Molecular Genetics and Microbiology and the Powell Gene Therapy Center. *e-mail: lewin@mgm.ufl.edu William W. Hauswirth Dept of Ophthalmology and the Powell Gene Therapy Center, Box 100266 University of Florida, Gainesville, FL 32610-0266, USA. There are two basic modes for therapy that targets the genetic basis of disease: replace or resect. For diseases caused by recessive mutations, gene therapists try to complement the defective gene. For dominant disease mutations, however, introducing a normal gene will not work. Many of these diseases are associated with hyperactivation (in the case of oncogenes), or aggregation of a mutant protein (in the case of neurodegenerative diseases), with a normal copy of the same gene being present on the partner chromosome. In these cases, expression of the defective gene must be silenced or at least limited. For such autosomal dominant genetic diseases, ribozymes are a particularly appealing tool: they can be used to reduce expression of a pathogenic gene by digesting the mRNA it encodes. RNA catalysts Ribozymes are RNA enzymes that catalyze a variety of reactions in cells. The most complex ribozyme is undoubtedly the ribosome, which synthesizes sequence-specific peptide bonds in an RNA-dependent reaction. Structural analogies with the self-splicing group II introns indicate that the spliceosome, which comprises five RNA molecules and over 50 proteins, might also be a ribozyme. The catalysts under development for therapy, however, are much simpler than these organelle-sized ribozymes (Fig. 1). Such catalysts include small nucleolytic activities, such as the hammerhead and the hairpin, derived from plant virus satellite RNA, the tRNA processing activity ribonuclease P (RNase P), and group I and group II ribozymes, which occur as introns in organelles and bacteria but can be engineered to act in trans on RNA or DNA (Table 1). In addition to these naturally occurring ribozymes, novel ribozymes have been developed by random sequence in vitro selection 1 . This technique relies on the prospect that a large population of molecules will include some that can perform a given task, for example, cutting a specific sequence of RNA. The challenge is to isolate these molecules from the remainder of the pool and subsequently amplify them for additional rounds of selection. Fortunately, several research groups have developed strategies for isolation of new ribozymes from pools of random sequences 2 . This method is not limited to RNA catalysts, and in fact, some of the best site-specific ribonucleases are made of DNA (Ref. 3). Despite their structural diversity, ribozymes and DNA enzymes catalyze only a few reactions: primarily, site-specific cleavage or ligation of RNA enzymes – ribozymes – are being developed as treatments for a variety of diseases ranging from inborn metabolic disorders to viral infections and acquired diseases such as cancer.Ribozymes can be used both to downregulate and to repair pathogenic genes. In some instances, short-term exogenous delivery of stabilized RNA is desirable,but many treatments will require viral-mediated delivery to provide long-term expression of the therapeutic catalyst.Current gene therapy applications employ variations on naturally occurring ribozymes,but in vitro selection has provided new RNA and DNA catalysts, and research on trans-splicing and RNase P has suggested ways to harness the endogenous ribozymes of the cell for therapeutic purposes. Ribozyme gene therapy: applications for molecular medicine Alfred S. Lewin and William W. Hauswirth 67 Knudson, W. et al. (1993) Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes. Proc. Natl. Acad. Sci. U. S. A. 90, 4003–4007 68 Hart, S.P. et al. (1997) CD44 regulates phagocytosis of apoptotic neutrophil granulocytes, but not apoptotic lymphocytes, by human macrophages. J. Immunol. 159, 919–925 69 Fraser, J.R. et al. 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