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, alpha-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.

MIF-like activity in simian virus 40-transformed 3T3 fibroblast cultures.

Simian virus 40-transformed fibroblasts (SV3T3), as compared with their untransformed counterparts (3T3), elaborate a macromolecular product that inhibits macrophage migration and causes macrophages to aggregate and lose one type of cell coat material. I'he SV3T3 cells also lack this surface material relative to 3T3 cells. There may be a relationz between migration inhibition factor (MIF), the cell coat, and cell migration.

Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen.

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radioimmunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human and rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply.

Survey of cell lines in the American Type Culture Collection for bovine viral diarrhea virus.

Cell lines originating from cattle, sheep, goat, deer, bison, swine, rabbit, hamster, cat, dog, monkey, human, and mosquito were obtained from the American Type Culture Collection and tested for contamination with bovine viral diarrhea virus (BVDV). Immunocytochemical procedures and polymerase chain reaction (PCR) amplification were used to detect viral antigen or viral RNA in 13 of 41 cell lines. The results of these procedures correlated exactly. Cell lines derived from cattle, sheep, goat, deer, bison, rabbit, and domestic cat were found contaminated with BVDV. Attempts were made to experimentally infect 14 swine, rabbit, hamster, cat, dog, monkey, and human cell lines that had been found free of virus. All swine cell lines, and most rabbit and cat cell lines, became infected with BVDV. Hamster, human, dog, and certain rabbit and cat cells were refractory to BVDV infection. Experimental infection of monkey cells produced variable results.

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture.

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.

Detection of a cell line contaminated with hog cholera virus.

Cell lines from the repository of the American Type Culture Collection were examined for possible contamination with bovine viral diarrhea virus. During testing, hog cholera virus (HCV) was detected in the IB-RS-2 D10 porcine kidney cell line. This variant of HCV was avirulent for pigs and seldom induced detectable concentrations of antibody against reference viruses (HCV-Ames or bovine viral diarrhea virus-NY1) in serum of inoculated pigs. Additionally, this variant of HCV did not confer protection to pigs against virulent HCV. The contaminated cell line had been distributed to > 20 laboratories in the United States. The cell line was not used in field studies and has been destroyed.

Ethical and social aspects of experimental gene manipulation.

New genetic techniques for the isolation and rejoining of segments of DNA now permit construction of biologically active recombinant DNA molecules. Such recombinant DNA molecules can be multiplied many times by inserting them into the bacterium Escherichia coli. Thus, we now possess a powerful technology for cloning segments of DNA from any source and for adding new genetic capabilities to bacteria. Application of this technology promises to facilitate the solution of a variety of theoretical and practical biological questions. However, use of this technology will also result in the creation of novel organisms whose biological properties may not be completely predictable in advance. Some of these organisms may be hazardous to man or to the environment. This paper discusses some ethical and social control of the new genetic technologies.

Differential inhibition of two molecular forms of melanoma cell plasminogen activator by a placental inhibitor.

The inhibitory effect of a placental urokinase inhibitor on the one- and two-chain form of melanoma cell derived plasminogen activator (PA) was investigated. The melanoma cell PA has been shown to be similar to or identical with the PA found in normal tissue. With constant concentration of placental inhibitor the rate of inhibition of the two-chain form was fast in contrast to that of the one-chain form. With constant incubation time but increasing concentrations of the placental inhibitor the two-chain form was inactivated to a greater extent at lower concentrations of placental inhibitor than was the one-chain form. The increased reactivity of the two-chain form of melanoma PA compared to the single-chain form may explain the role of tissue PA in achieving high local concentrations of PA activity which facilitate selective fibrin clot lysis.

Protein synthesis in Simian virus 40-infected monkey cells.

The proteins of purified Simian virus 40 (SV40) were examined by sodium dodecyl sulfate-acrylamide gel electrophoresis and compared with the polypeptides synthesized in SV40-infected monkey cells. Purified virions contain two major components, with molecular weights of 44,000 and 31,000. Together they make up 83% of the total virion proteins. In addition, the virus contains 12 minor polypeptides, which are believed to be cellular proteins, or peptides derived from proteolytic degradation of the 44,000 molecular weight polypeptide. Pulse-label experiments show that about 90% of the polypeptides synthesized after SV40 infection are host-cell proteins; 10% represent the two major structural components of the virion. A small fraction (about 0.5%) consists of three polypeptides (molecular weights 70,000, 60,000, and 8,000) that are neither part of the virion nor detectable in uninfected cells. They are either virus-induced cellular proteins or, more likely, proteins coded for by the SV40 genome.

Lipids of whole cells and plasma membrane fractions from Balb/c3T3, SV3T3, and concanavalin A-selected revertant cells.

The lipid composition of Balb/c3T3, SV3T3, and the concanavalin A-selected SV3T3 revertant cells has been analyzed at the whole cell and plasma membrane levels. In comparison to untransformed 3T3 whole cells, SV3T3 cells showed an unchanged content of triacylglycerols, free fatty acids, and glycerylether diesters but a lower concentration of total phospholipids, while no significant difference was found in the phospholipid composition. Whole SV3T3 revertant cells exhibited a lipid composition similar to that in untransformed 3T3 cells with the exception of a higher proportion of sphingomyelin. Analysis of isolated plasma membranes did not reveal any significant differences in the cholesterol to phospholipid molar ratio between 3T3 and SV3T3 or SV3T3 revertant cells. The major changes in the acyl chain pattern SV3T3 compared with whole 3T3 cells consisted of an increase of oleic and palmitoleic acids coupled with a decrease of C20 and C22 polyunsaturated acids in phosphatidylethanolamine and phosphatidylcholine; an increase of oleic acid was also evident in SV3T3 phosphatidylinositol plus phosphatidylserine. An increase of palmitoleic and oleic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine of SV3T3 plasma membranes; the only change in SV3T3 plasma membrane phosphatidylcholine was an increase of oleic acid. An increase of monoenoic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine of SV3T3 revertant cells at the level of both whole cells and plasma membranes.

Non-selective inhibition of transformed cell growth by a protease inhibitor.

The protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) and N-tosyl-L-lysylchloromethyl ketone (TLCK) have previously been shown to selectively inhibit growth of simian virus 40-transformed cells, suggesting that proteolytic enzymes play a role in loss of cellular growth control following viral transformation. In contrast, this study shows that TPCK-mediated growth inhibition is non-selective, since the growth of both simian virus 40-transformed and untransformed 3T3 cells is similarly reduced by TPCK treatment. Under certain conditions, TPCK treatment of simian virus 40-transformed cells yields a reversible "growth plateau" condition which mimics, but is not equivalent to, contact inhibition of growth. The growth inhibitory effects of TPCK are due to inhibition of protein synthesis, since TPCK treatment resulted in a diminution of protein synthesis and since the "growth plateau" effect was also observed in cultures treated with cycloheximide.

Calcium stimulation of plasminogen activator secretion/production by swiss 3T3 cells.

Actively growing Swiss 3T3 cells secret high levels of plasminogen activator which decreases after the cells become confluent. In contrast, simian virus 40-transformed 3T3 cells secrete large amounts of plasminogen activator independent of cell density (Chou, I.-N., O'Donnel, S.P., Black, P.H., and Roblin, R.O. (1977) J. Cell. Physiol. 91, 31-38). These results suggest a correlation between active cell multiplication and plasminogen activator secretion in both 3T3 and simian virus-transformed 3T3 cells. The data reported herein indicate that treatment of both subconfluent and confluent Swiss 3T3 cells with high concentrations of Ca2+ (final 3.0 to 4.9 mM) increases the amounts of both secreted and cell-associated plasminogen activator in a dose-dependent manner. In addition, the ionophore A23187 (19 to 95 nM) in the presence of a normal level of Ca2+ (1.8 mM) stimulates both production and secretion of plasminogen activator from growing 3T3 cells. The Ca2+ stimulation of plasminogen activator production/secretion may be related to the mitogenic effect of Ca2+.