Nelson A. Wivel

Transfer of murine intracisternal A particle phenotype in chloramphenicol-resistant cytoplasts

Murine intracisternal A particles have a number of properties which are common to known RNA tumor viruses, but horizontal transmission has not been previously demonstrated. The apparent absence of infectivity may be related to the failure of these particles to be released from cisternae of endo-plasmic reticulum. Previous biological studies using isolated, purified A particles have been compromised by the fact that the isolation procedure requires small amounts of nonionic detergent. Using some techniques of somatic cell hybridization, we have assessed the capacity for A particle genome transfer from positive to negative cells. Since it has been previously shown that some chloramphenicol-resistant cell lines can transfer this resistance in the cytoplasm, we have used this characteristic as a marker for cytoplasmic fragments. Mouse cells containing A particles were mutagenized, and clones resistant to chloramphenicol were selected; by enucleating these cells and fusing the resultant cytoplasts to each of two recipient mouse cell lines negative for A particles, it is possible to identify clones of cells known to be the product of a fusion event between a cytoplast and a whole cell (cybrids). Under these conditions, intracisternal A particles appear in the cybrid clones as a phenotypic trait that has not been segregated over at least 60-80 cell generations.

Some characteristics of an isolated group antigen common to most strains of murine leukemia virus.

Abstract Various density gradient isolates were prepared from intact murine leukemia virus (Rauscher) and from the aqueous phase after virus disruption with Tween-ether. Each isolate was examined for the presence of viral antigens by three serological techniques—immunodiffusion, passive hemagglutination, and complement fixation—and by electron microscopy. The 1.24–1.26 density isolate derived from the nucleoid of the virion contained an antigen with the properties of the classical myxovirus “g” antigen. By the use of antisera prepared against 7 strains, and antigens of 12 strains of murine leukemia virus, the “g” antigen was shown to be identical in 7 strains, and strongly cross reactive, if not identical, in several others. Only one strain showed a reaction of partial identity in immunodiffusion.

Regulatory considerations for gene-therapy strategies and products

through the R & D companies, rather than through in-house research. More ideas originate in R&D companies, and if a particular research direction appears to be a cul-de-sac, it can be easily dropped by not renewing the agreement. If, on the other hand, the research looks promising, further agreements would be drawn up, or even purchase of the company would be considered. Sandoz, Merck, Parke-Davis (a division o f Warner-Lambert) and Green Cross Corporation have invested millions of dollars in GTI, Vical and Viagene, respectively, to develop clinical applications of gene therapy. Other drug-company giants are scouting for deals. It is hard to tell, at present, which company will be first to bring gene therapy to the market, and whether it will be a commercial success. GTI, the pioneer in the field, has been involved in more than a dozen clinical trials and has acquired much experience. However, the other companies that also have a strong scientific environment could become important competitors. Firms such as TargeTech or Vical, which are developing non-viral vectors, might be first to commercialize gene-therapy products, since gaining regulatory approval is likely to be easier. However, even when a gene-therapy protocol has been shown to be safe and effective, and has regulatory approval, commercial success will depend on the market size, and on the willingness of the company to meet the costs. Many governments are currently re-evaluating the cost of health-care systems and the problems of financing expensive new treatments. The costs o f gene therapy must, however, be compared with those for current treatments. A costly, but definitive cure for a disabling genetic disease may, in the long-run, be cheaper than lifelong palliative treatment the cost of current treatment for Gauchers disease is estimated at US

Properties of murine intracisternal A particles electron microscopic appearance after critical point drying and platinum shadowing

450 000 annually. For some, even if not all, the diseases which are currently being targeted, gene therapy is likely to prove commercially as well as medically beneficial.

Gene Therapy: Molecular Medicine of the 1990s

Abstract Intracisternal A particles (IAP) are present in mouse neoplasms; morphologically they resemble certain immature forms of oncogenic RNA viruses, and they have certain biochemical markers found in known RNA tumor viruses. By combining critical point drying with uranyl acetate positive staining and shadowing, it is possible to observe whole unsectioned particles while simultaneously demonstrating internal detail. IAP exist in the isolated state as budding and intact forms, and consist of two concentric spherical shells surrounding a roughly spherical electron lucent core. The outer shell is sensitive to the detergent Na deoxycholate, but the inner shell is resistant to this agent and retains its spherical construct following treatment with it. The inner shell is not solublized by 1% Na dodecyl sulfate but it does lose its structural integrity and collapses. Exposure to 0.15% trypsin for limited time periods reveals concentric linear densities in the inner shell; such structures are faintly visible in untreated particles.

Reversible suppression of alkaline phosphatase in human thyroid medullary carcinoma cells transformed by SV40.

In less than four years, the techniques of gene transfer have been taken from the laboratory and translated into numerous clinical trials. Although gene therapy was initially designed for the molecular repair of monogenic deficiency diseases, most of the current studies of gene therapy are targeting cancer.

Long-term in vitro cultivation of Rauscher Leukemia virus in rat embryo cells.

Summary Two tumors, medullary carcinomas from human thyroid glands, were used to establish tissue culture lines. These lines were assayed for alkaline phosphatase activity and were found to be consistently positive. Following transformation of the cell lines with SV40, the alkaline phosphatase levels were markedly depressed or undetectable. When hydrocortisone was added to the in vitro system, the enzyme activity returned to preinfection levels. This increase in alkaline phosphatase was dependent on the continuous presence of steroid; when steroid was withdrawn there was a loss of activity to pretreatment levels. No increase in enzyme levels was detected in uninfected tumor cells treated with hydrocortisone.

Distribution of intracisternal a-particles in a variety of normal and neoplastic mouse tissues

Summary The long-term in vitro cultivation of Rauscher leukemia virus (RLV) in rat embryo (RE) cells is reported. RE cells infected initially with RLV have replicated infectious virus and produced complement-fixing (CF) antigen characteristic of the murine leukemia-sarcoma virus complex for more than 12 months. Production of complete virus has been demonstrated by means of electron microscopy; and of infectious virus by the in vitro assay technic (COMuL test) based on detection of the group-specific CF antigen of murine leukemia viruses. Interferon production was not demonstrated in infected cultures. The data indicate that RE cells could be used for cultivation of RLV in a carrier state.