Wayne Gerlach

RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy

We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline—seven enzymatic steps upstream of codeinone—accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

Suppression of Smooth Muscle Cell Proliferation by a c-myc RNA-cleaving Deoxyribozyme

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262–4266) and is known as the “10-23” general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3′-3′-internucleotide inversion at the 3′-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity.

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

An anti‐HIV‐1 tat ribozyme, termed Rz2, has been shown to inhibit HIV‐1 infection/replication and to decrease HIV‐1‐induced pathogenicity in T‐lymphocyte cell lines and normal peripheral blood T‐lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2.

Morphine-pathway block in top1 poppies

The opium poppy is a source of the pharmaceuticals codeine, morphine and their derived analgesics. Here we describe the initial characterization of the poppy mutant known as top1 (for ‘thebaine oripavine poppy 1’), which accumulates the morphine and codeine precursors thebaine and oripavine and does not complete their biosynthesis into morphine and codeine. The original discovery of top1 stimulated a re-engineering of the opioid industry in the island state of Tasmania, which grows over 40% of the worlds licit opiates, in order to produce thebaine and oripavine efficiently from morphine-free poppy crops to provide precursors for highly effective analgesics and for treatment of opioid addiction.

Pollination biology of oilseed poppy, Papaver somniferum L

Although poppies (Papaver somniferum L.) are one of the oldest cultivated plants relatively little is known of their pollination biology. We have investigated the relative importance of wind and insects in the pollination of poppies and identified potential insect pollinators. Wind pollination was found to be negligible, insect pollination was responsible for the majority of out-crossing, and self-pollination was the dominant mode of poppy fertilisation. Honeybees and flies were identified as the main potential cross-pollinators of Tasmanian poppies. Using a transgenic poppy field trial in which approximately 50% of the pollen grains produced were transgenic, we have determined the level of pollen-mediated gene flow by scoring over 50 000 seeds for the presence of a selectable marker gene. Gene flow was measured using a 10-m buffer area that surrounded the field trial. It was highest at 0.1 m with 3.26% of seeds found to be transgenic and declined over distance with 1.73% transgenic seeds at 0.5 m, 1.80% at 1 m, 0.86% at 2 m, 0.34% at 5 m, 0.12% at 9 m, and 0.18% at 10 m. These results demonstrate that under Tasmanian conditions, pollen-mediated gene flow occurs at modest levels in poppies that are in close proximity to each other and is most probably mediated by honeybees and flies.