RETROVIRUS-MEDIATED TRANSDUCTION OF AN ENGINEERED INTRON-CONTAINING PURINE NUCLEOSIDE PHOSPHORYLASE GENE

We constructed and tested several retroviral vectors containing abbrev iated purine nucleoside phosphorylase (PNP) genes in the reverse orien tation, a strategy compatible with transduction of intron-containing g enes. We observed two types of deletions in these vectors after one ro und of replication: (i) Deletions flanked by direct repeats with one c opy of the repeat retained in the provirus, presumably resulting from reverse transcriptase slippage during (-) strand DNA synthesis. (ii) D eletions due to fortuitous splice sites in the PNP complementary stran d. Two splice donor sites and three splice acceptor sites were identif ied ina 3.0-kb PNP minigene. We found that the splice donor sites (but not the splice acceptor sites) could be predicted by sequence analysi s of the PNP complementary strand. To increase the frequency of intact PNP gene transduction, we introduced sequence modifications: The puta tive PNP polyadenylation signal and a truncated 117-bp 3' flank were r ecovered from a rearranged provirus and inserted in place of a 1.2-kb genomic 3' flank, Sequences associated with deletions were eliminated from the PNP 5' untranslated region, and two fortuitous splice donor s ignals in the complementary strand were inactivated. A retroviral vect or LN-PMG11, containing the engineered 2.9-kb PNP minigene in the reve rse orientation, was transduced intact in 23% (5/22) of clones after o ne round of replication and in 87% (20/23) of clones after a second ro und of replication from two primary virus producer clones. Directed mu tagenesis of sequences preventing intact retroviral transduction thus provided a 2.9-kb PNP gene that was transduced intact and expressed at a high level.