Development and testing of retroviral vectors expressing multimeric hammerhead ribozymes targeted against all major clades of HIV-1.

Rz1-9 is a multimeric hammerhead ribozyme targeted against nine highly conserved sites within the env coding region of human immunodeficiency virus type-1 (HIV-1) RNA. This ribozyme was shown to inhibit HIV-1 replication (1, 2). However, Rz1-9 target sites are only conserved within the clade B of HIV-1. In this study, we have designed another multimeric ribozyme, Rz10-14. This multimeric ribozyme is targeted against five sites that are highly conserved amongst all major clades of HIV-1. A third multimeric ribozyme, Rz1-14, was obtained by combining both Rz1-9 and Rz10-14. A mouse stem cell virus-based MGIN vector (3) was used to express these ribozymes in a human CD4+ T lymphoid cell line. Stable transductants expressed vector RNA containing ribozymes which were shown to be active. In HIV-1 challenge experiments, very little or no virus production could be detected in the pools of stable MT4 transductants expressing Rz1-14 for 60 days tested. Inhibition of virus replication was most prominent with Rz1-14, followed by Rz1-9, and then Rz10-14. Thus, the combined multimeric ribozyme, Rz1-14, is more effective than Rz1-9 or Rz10-14. As Rz1-14 is targeted against all major clades of HIV-1, it will be further pursued for use in HIV-1 gene therapy.