HIV Gene Therapy Using Hairpin Ribozymes in Hematopoietic Stem/Progenitor Cells

Abstract

Ribozymes are a class of small metalloenzymes composed entirely of RNA which cleave specific RNA sequences (Christoffersen and Marr, 1995; Kiehntopf et al., 1995; Pyle, 1993). Different natural ribozymal structures have been isolated from such diverse sources as tetrahymena, tobacco ringspot virus, plant viroids, virusoids, and satellite viruses; and hepatitis delta virus (Michel et al., 1989; Pyle, 1993). According to their molecular structures, the natural ribozymes are classified into at least six different groups: Group I introns (Cech, 1987), Group II introns (Michel et al., 1989), the Ml RNA subunit of the ribonucleoprotein enzyme RNase P (Guerrier-Takada, et al., 1983), hammerhead (Uhlenbeck, 1987), hairpin (Hampel and Tritz, 1989), and hepatitis δ RNA (Sharmeen et al., 1988; Wu et al., 1989). Members of the first three classes are typically larger (>200 nucleotides) and cleave RNA to produce a 3′ hydroxyl group (Cech, 1992; Michel et al., 1989). The latter three classes are typically smaller (30–80 nucleotides), cleave RNA by transesterification, and produce a 2′-, 3′-cyclic phosphate and a 5′ hydroxyl terminus (Cech, 1992). Definitive triplet RNA sequences are required to bind and cleave hammerhead and hairpin ribozymes. The hammerhead ribozyme recognizes an NUX-triplet, where N is any nucleotide and X represents A, C, or U, and cleaves at 3′ to the N residue (Ruffner et al., 1990). The hairpin ribozyme recognizes a GNZ-triplet, where Z is either U or C, and cleaves 5′ to the G residue (Chowrira el al., 1991). A GUC triplet is required for efficient trans cleavage of the hairpin ribozyme (Ojwang et al., 1992). Because of their relatively small sizes and minimal substrate sequence requirements, both hairpin and hammerhead ribozymes can be engineered to bind to specific RNA molecules by attaching sequences complementary to the nucleotides near the cleavage site of the target.