Ribozyme On The Replication Of Hiv-1 In Human T Lymphocytes, The Inhibitory Effect And Its Influencing Factors

Gene therapy for AIDS will require the identification of genes which effectively inhibit HIV-1 replication coupled to an efficient vector system for gene delivery and expression. Hammerhead ribozymes are RNA moleculescapable of catalytic cleavage of complementary RNA molecules.Ribozymes targeted against two portions of the HIV-1 genome were designed to cleave in the tat gene (TAT) or in a common exon for tat and rev (TR). these ribozymes cleave the target HIV-1 RNA sequences at physiologic temperature when produced by in v-itro transcription. The genes of the ribozymes were cloned into the LN retr-oviral vector plasmids so that they would be expressed as part of the vecfor transcripts encoding the bacterial neomycin gene(neo).The vectors were packaged as amphotropic virions and used to transduce human CEM T lymphocytes. Expression of the vector transcripts containing the ribozyme sequences was readily detected by Northern blot analysis of the transduced CEM cells. The CEM cells expressing the anti-HIV-1 ribozymes showed resistance to HIV-1 replication. In contrast, celts expressing mutant ribozymes, containing substitutions of a key mucleotide in the catalytic domain which eliminate in vitro cleavage activity, supported replication of HIV-1To compare the strength of different promoters controlling the expression of ribozymes in vivo, to compare the effects of unrelated flanking sequences from the retroviral vectors on ribozyme activity in vitro and in vivo, and to investigate the relationship between the ribozyme expression and inhibition of targeted virus replication in vivo, three different retroviral vectors have been constructed, in which the tandem dimer of ribozymes (TR-TA T) was driven by MoMuLV LTR, CMV promoter or tRNA promoter for expression.Using an in vitro transcription system ribozymes with different sizes of flanking sequences were synthesized. In vitro cleavage assay was performed to measure the catalytic activity. the ribozymes with different sizes of flanking sequences show nearly the same degree of cleavage activity, suggesting that the flanking sequences from the vectors (up ot 2700 bases) have no significant effects on ribozyme cleavage in vitro.