It follows that effective treatment for HIV infection might engage the virus prior to 
integration, or in already infected cells, at the level of the integrated DNA provirus. No 
practicable methods now exist for selectively eliminating a particular chromosomally 
integrated DNA sequence from a population of human cells. As an alternative, therapies 
aimed at the RNA level (ribozymes, anti-sense RNA) could target and destroy incoming 
viral RNA genomes prior to their reverse transcription or destroy new viral RNA 
transcripts. Our previous data (see below and ref. 7,8,9 which are attached to this 
document) indicate that in transduced, G41 8-selected T cells in vitro, an HIV-1 leader 
sequence hairpin ribozyme does in fact inhibit HIV-1 infection at two steps in the viral 
lifecycle: by cleaving both incoming viral RNA genomes and viral RNA expressed from 
integrated provirus. 
Most current gene therapy protocols use, as in this proposal, murine retroviral vectors 
to deliver therapeutic genes into target cells; this process (transduction) mimics the 
early events of retroviral infection (2,6,10,55). The crucial difference is that, unlike 
replication-competent retroviruses, the vector genome packaged within the viral coat 
contains no genes for viral proteins. The vector, LNL6, used in this study has been used 
in previous FDA-approved trials and is designed to have only long-terminal repeat 
sequences necessary for integration of the DNA intermediate into host cell chromosome 
and a packaging signal that allows packaging into viral structural proteins supplied by 
the packaging line in trans (2,10,37). We have inserted only a cassette containing a 
tRNA val promoter element to drive ribozyme expression and the ribozyme gene itself. 
Hence, genomic integration is the terminal step for the vector; it has no potential to 
make viral proteins in cells transduced with the packaged vector and therefore no 
opportunity to produce progeny virus. 
Ribozymes are RNA molecules that contain anti-sense sequences for specific recognition, 
and RNA-cleaving enzymatic activity (11-20, 48). We have reported that a hairpin 
ribozyme derived from the negative strand satellite RNA of the tobacco ringspot virus [- 
sTRSV] and designed to cleave HIV-1 RNA in the 5' leader sequence (positions 
+111/112 from the cap site in HXB2) suppressed virus expression in HeLa cells co- 
transfected with proviral DNA from diverse HIV-1 strains, suggesting that the ribozyme 
was effective in blocking virus expression from pre-existing viral DNA (7). 
Subsequently, in experiments more directly analogous to gene therapy, human T-cell 
lines and normal primary peripheral blood T cells transduced with ribozyme-bearing 
retroviral vectors resisted challenge with diverse strains of HIV-1 (9). No reverse 
[384] 
Recombinant DNA Research, Volume 18 
