Richard Roblin

Gene Therapy for Human Genetic Disease

In our view, gene therapy may ameliorate some human genetic diseases in the future. For this reason, we believe that research directed at the development of techniques for gene therapy should continue. For the foreseeable future, however, we oppose any further attempts at gene therapy in human patients because (i) our understanding of such basic processes as gene regulation and genetic recombination in human cells is inadequate; (ii) our understanding of the details of the relation between the molecular defect and the disease state is rudimentary for essentially all genetic diseases; and (iii) we have no information on the short-range and long-term side effects of gene therapy. We therefore propose that a sustained effort be made to formulate a complete set of ethicoscientific criteria to guide the development and clinical application of gene therapy techniques. Such an endeavor could go a long way toward ensuring that gene therapy is used in humans only in those instances where it will prove beneficial, and toward preventing its misuse through premature application. Two recent papers have provided new demonstrations of directed genetic modification of mammalian cells. Munyon et al. (44) restored the ability to synthesize the enzyme thymidine kinase to thymidine kinase-deficient mouse cells by infection with ultraviolet-irradiated herpes simplex virus. In their experiments the DNA from herpes simplex virus, which contains a gene coding for thymidine kinase, may have formed a hereditable association with the mouse cells. Merril et al. (45) reported that treatment of fibroblasts from patients with galactosemia with exogenous DNA caused increased activity of a missing enzyme, α-D-galactose-l-phosphate uridyltransferase. They also provided some evidence that the change persisted after subculturing the treated cells. If this latter report can be confirmed, the feasibility of directed genetic modification of human cells would be clearly demonstrated, considerably enhancing the technical prospects for gene therapy.

The 5′-terminus of bacteriophage R17 RNA: pppGp- - -☆

Abstract Alkaline hydrolysis of radioactive R17 RNA made in vivo yields pppGp from the 5′-terminus of the RNA. 0.75 mole pppGp/mole of RNA has been recovered by chromatography of the alkaline hydrolysate on DEAE-Sephadex columns using buffers containing 7 m -urea and NaCl gradient elution. Various degradative experiments have been carried out which prove the structure of pppGp. This nucleoside 5′-triphosphate group can be used as a natural label of the 5′-terminus for studies of the 5′-terminal nucleotide sequence of R17 RNA.

Proteolytic enzymes, cell surface changes, and viral transformation.

Publisher Summary This chapter reviews the evidence for and against the view that proteolysis plays a role in determining some of the phenotypic characteristics of virus-transformed cells. It discusses some aspects of the structure of the cellular membrane surface because one possible mechanism by which proteases might alter cell properties is via the selective proteolysis of cellular membrane surface components. It also reviews the current knowledge of some cellular phenotypic characteristics that are known to be altered by viral transformation and for which there exists at least suggestive evidence for the involvement of a proteolytic enzyme. The research into the involvement of the limited proteolytic enzymes of serum, in particular plasmin and thrombin, in virus-induced cell transformation has only just begun. It is possible to outline several mechanisms by which the plasmin or the thrombin system, or both, could, by limited proteolysis of the cellular membrane surface, produce several of the phenotypic changes characteristic of virus-transformed cells. A large body of circumstantial evidence implicating these enzyme systems in causing the altered phenotypic characteristics of virus-transformed cells has recently been obtained. However, critical experiments to prove that plasmin, thrombin, or other serum proteases modify the cellular membrane surface under the usual conditions of cell culture, in the presence of serum protease inhibitors, remain to be performed.

Nucleotides adjacent to the 5'-terminus of bacteriophage R17 RNA.

Abstract Information about the 5′-terminal nucleotide triplet of bacteriophage R17 RNA was obtained in order to test the hypothesis that it might be an N -formylmethionine initiator codon. Utilizing the fact that most R17 RNA chains made in vivo contain pppGp--- 5′-termini, a method for determination of the chain length of the 5′-terminal oligonucleotide resulting from pancreatic RNase hydrolysis of R17 RNA was developed. The results suggest that R17 RNA chains made in vivo have the 5′-terminal sequence pppGpXpYp--- (X = purine, Y = pyrimidine). Therefore, an N -formylmethionine initiator codon cannot be present directly at the 5′-terminus of this messenger RNA.

REFLECTIONS ON ISSUES POSED BY RECOMBINANT DNA MOLECULE TECHNOLOGY. I

Powerful new molecular techniques, which permit formation of new genetic combinations in vifro and cloning and amplification of the resulting recombinant DNA molecules in the bacterium f!’. coli, have recently been developed.’-4 These techniques make it possible t o combine restriction endonuclease fra ments of DNA from any source with a DNA vector or carrier (e.g., plasmids‘? or bacterial viruses3 9 ‘ ) and t o introduce the resulting recombinant DNA molecules into bacterial cells, where the newly created DNA molecules can replicate. Thus, specific fragments of DNA from any organism can now be isolated, purified, and prepared in large quantities using these techniques. Recombinant DNA molecule techniques have already been used in several different types of scientific investigations. In a study of the genetic control of plasmid replication’ the techniques were used to construct a recombinant plasmid DNA containing the genes for two distinct modes of plasmid DNA replication. In another investigation6 fragments of the region of Xenopus faevis DNA, which direct the cellular synthesis of ribosomal RNA, were cloned and their transcription into RNA in bacterial cells carrying a recombinant plasmid was demonstrated. The utility of recombinant DNA molecule techniques for genetic mapping of the genomes of higher organisms such as Drosophilu has also been d e m ~ n s t r a t e d . ~ y 7 These examples illustrate the usefulness of recombinant DNA molecule techniques in basic research in molecular genetics and some of the ways in which they are opening new molecular approaches t o previously intractable problems. The power and elegance of these new techniques practically guarantees that moderately large numbers of scientists will want to apply them in research investigations in the near future. As information about these new techniques spread and more scientists started t o use them, certain issues or questions regarding the use of the techniques began t o emerge. An early concern, voiced by scientists attending the 1973 Gordon Research Conference on Nucleic Acids,8 was that the biological activity of newly created recombinant DNA molecules might be unpredictable, and that “certain such hybrid molecules may prove hazardous t o laboratory workers and t o the public.” This statement raised the possibility that the conduct of basic scientific research (as opposed t o applications of such research) could prove harmful t o the public and thereby created a somewhat novel problem. The problem for the basic scientist was that if there were in fact biohazards inherent in the use of recombinant DNA molecule techniques, it could no longer be maintained that such scientific research was harmless, and that only misapplications of scientific research by social forces were detrimental. Using a 5-