DIPHTHERIA TOXIN STRUCTURAL GENE FRAGMENT CLONING

Executive Summary

DIPHTHERIA TOXIN STRUCTURAL GENE FRAGMENT CLONING proposals were recommended for approval by an NIH working group on toxins Aug. 16. The working group endorsed the start up of a series of DNA experiments which could eventually lead to a treatment strategy against malignant melanoma. Boston University biomolecular medicine chief John Murphy, PhD, heads a research team which proposed the cloning experiments. Murphy's research team plans to clone fragments of the diphtheria toxin structural gene; modify the cloned gene with cysteine, an amino acid; and clone a fusion product composed of fragments of the genes for diphtheria toxin and for the polypeptide hormone which stimulates the growth of melanocytes, tan-producing cells. All three genetic constructs would be cloned in E. coli K-12. Murphy said that the research team has already shown that partially purified periplasmic extracts containing the fusion gene product are cytotoxic to human malignant melanoma cells in culture. He added that the extracts are not active against cells which do not have the receptor for the melanocyte-stimulating hormone, in animal species that are highly sensitive to diphtheria toxin. The researchers sought approval to conduct the cloning experiments under the lowest biosafety physical containment level (BL1), but the working group recommended that the cloning be done at "BL2 containment with BL3 practices." The working group recommendation would require the use of stricter bio-safety measures, such as extra containment equipment and special lab design, than would normally be required of most experiments conducted at BL2 physical containment. The NIH panel, which consists of consultants and members of the agency's Recombinant DNA Advisory Committee (RAC), endorsed approval of the diphtheria toxin proposals by a vote of 6-0. The recommendation for approval goes directly to NIH Director Wyngaarden, bypassing further review by RAC. The working group recommended approval of another Murphy proposal, which would involve the cloning of a diphtheria toxin-related interleukin-2 (IL-2) hybrid gene. Murphy intends to employ the same portion of the diphtheria toxin structural gene used in the melanocyte hormone fusion gene experiments, with a modification. The Boston University researcher proposed that the cloning be conducted at BL4 containment, but the working group recommended that the IL-2 experiment be done at BL3 containment, using "non-mobilizing plasmids." The proposal has to be reviewed by RAC. The working group also recommended approval of a request by two Defense Department researchers to expand their cloning of the Shiga-like toxin genes from bacterial species other than E. coli. NIH had previously approved a request from the investigators to clone the structural gene of the Shiga-like toxin of E. coli(END ITALICS) in E. coli K-12, under BL3 + EKI containment. The working group recommended approval of the additional bacterial species cloning under the previously approved conditions, except that physical containment may be lowered to BL2 if toxicity levels permit. RAC must review the working group recommendation.