Transfer of an Anti-HIV-1 Ribozyme Gene into Primary Human Lymphocytes

Abstract

We reported previously that human CD4+ T cell lines stably expressing a hairpin ribozyme targeted to the human immunodeficiency virus type 1 (HIV-1) U5 leader sequence were resistant to challenge with diverse HIV-1 viral clones and clinical isolates (Yamada et al., 1994). To simulate more closely the in vivo infection process for investigations of anti-HIV-1 ribozyme gene therapy, we developed a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes (PBLs) using a murine retrovirus vector. Following transduction and G418 selection, human PBLs from multiple donors expressed the ribozyme and resisted challenge by HIV-1 viral clones and clinical isolates, while control vector-transduced PBLs remained fully permissive for HIV-1 infection. No inhibition of an HIV-2 clone lacking the target was seen in ribozyme-expressing PBLs. Ribozyme expression had no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T cells of resistance to HIV-1 infection conferred by gene transfer. A human clinical trial is in development to test further the safety and efficacy of this ribozyme in PBLs of HIV-1-infected patients in vivo.

Long-term RNase P-mediated inhibition of HIV-1 replication and pathogenesis

“…This region is highly conserved across HIV-1 clades and is present on all forms of HIV RNA including incoming RNA strands and mRNA transcripts expressed following infection. [21][22][23][24] Successful cleavage at this region of the viral RNA results in degradation of the genome due to removal of the pro- tective 5'cap. A disabled EGS devoid of the T-stem and loop required for RNase P enzyme recognition and cleavage was constructed to control for antisense inhibition (Figure 1b).…”

Ribozymes in gene therapy of HIV-1

“…These include ribozymes targeting other sequences within the tat gene (54), the HIV-1 5'-leader sequence (55), env (58) and gag (59). Hairpin ribozyme constructs targeted to tat and env and expressed from a tRNA Val transcriptional cassette within various expression vectors have also been shown to confer resistance to several HIV-1 isolates (52,(60)(61)(62).…”

Inhibition of HIV-1 replication by retroviral vectors expressing monomeric and multimeric hammerhead ribozymes

“…[1][2][3] HIV-1 replication for 10-25 days after infection. 23 This HIV-1 RNA-specific hammerhead ribozymes targeting hairpin ribozyme was also shown to inhibit HIV-1 replithe 5′ untranslated region, 4,5 trans-activation responsive cation by up to 90% at day 6-10 after infection in (TAR) region, 6 regulator of expression of virion protein macrophage-like cells derived from stably transduced (Rev) response element and repeat regions, 7 packaging CD34 + hematopoietic stem cells from human cord signal region, 8 as well as group antigen, 9,10 polymerase, 7,9 blood. 24 Env, 9,11 trans-activator of transcription (Tat), 9,[12][13][14][15][16] The above studies suggest that although effective, Tat/Rev, 14 and Nef 17 coding regions have been shown monomeric ribozymes are unable to inhibit HIV-1 replito inhibit HIV-1 replication following transient or stable cation completely.…”