transcriptase activity or virus infectivity was detectable in the culture supernatants for 
up to 35 days (the duration of the experiments) which represents a more than 10 4 -fold 
reduction in recovered virus. There was no detection of escape or resistant virus after 
long term culture. Identical cells transduced with a control vector (identical except for 
lacking the ribozyme cassette) were not protected. Furthermore, the ribozyme is 
effective against a broad spectrum of HIV isolates, including an uncloned clinical isolate 
(8,9). The latter more directly simulates the in vivo milieu where heterogenous viral 
quasispecies or “swarms” predominate. 
Expression of ribozyme significantly decreased (50-100 fold) the efficiency of 
incoming virus to synthesize viral DNA. These results indicate that transfer and 
expression of the ribozyme gene interfered with both early and late events in the HIV 
replication cycle; this double-target “intracellular immunization" by a single agent 
makes the ribozyme a unique molecular genetic intervention for treatment of HIV 
infection. The transduced T cells persistently express the ribozyme (no diminution in 
expression has been seen in over 6 months) and, equally important, there are no 
apparent deleterious effects on cell proliferation (assessed by thymidine uptake) or 
long-term viability (9). The three publications (ref. 7,8,9) detailing these previous 
results are attached (following REFERENCES). 
We have generated a retroviral particle-producing cell line, PAMJT, by the following 
methods: the ecotropic packaging cell line y2 was transfected by the calcium-phosphate 
method with a retroviral vector carrying the ribozyme driven by the human tRNA va ' 
promoter cloned in a widely used (2,10,21-25,37) vector for gene therapy, pLNL6. 
The transfections were performed in DMEM supplemented with HAT (hypoxanthine, 
13|ig/ml, thymidine 3.9 pg/ml and aminopterin 18 ng/ml) and 10% fetal bovine serum 
(FBS). The culture supernatants were filtered through 0.45 |im pore size filter to 
remove cell debris and then used to infect the amphotropic packaging line PA317 in the 
presence of polybrene (final concentration 4 jig/ml). The infected PA317 cells were 
grown in HAT medium containing G418 (400 |ig/ml), changing the medium every three 
days. G418 resistant colonies were scored and picked and expanded. A high titer clone, 
PAMJT, (10 5 CFU/ml) was identified by the standard method and frozen. Higher titre 
producing clones are in preparation. To produce retroviral particles (MJT) for the 
transduction protocol described below, the frozen PAMJT will be thawed and selected in 
DMEM with HAT and G418 and the cells will be expanded in large quantity so that 500 ml 
of supernatant containing MJT retroviral particles will be obtained. The supernatant so 
Recombinant DNA Research, Volume 18 
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