Construction of adenoviral vectors.

Recombinant adenovirus vectors have proven to be useful tools in facilitating gene transfer. Construction of such vectors requires a knowledge of the adenovirus genome structure and its life cycle. A commonly used recombinant adenovirus involves deletion of the E1 region; such a recombinant is traditionally produced by overlap recombination after cotransfection of 293 cells with a plasmid shuttle vector and a large right-end restriction fragment of viral DNA. The shuttle vector contains a cassette for a transgene placed in region E1 and flanking sequences from adenovirus for recombination. Normally, a high background of parental virus results because of the difficulty in separating right-end restriction fragment length DNA from uncut DNA. This paper describes a negative selection based on the traditional cotransfection method using viral DNA from an E1-deleted adenoviral recombinant that expresses green fluorescent protein (GFP). In situ fluorescent microscopy is used to distinguish the recombinant plaques (white or nonfluorescent) from the parental virus plaques (green or fluorescent). In addition, this system allows for the detection of contaminating parental virus at later stages when production lots of the recombinant vector are being made.

Clinical infection control in gene therapy: a multidisciplinary conference.

Gene therapy is being studied for the treatment of a variety of acquired and inherited disorders. Retroviruses, adenoviruses, poxviruses, adeno-associated viruses, herpesviruses, and others are being engineered to transfer genes into humans. Treatment protocols using recombinant viruses are being introduced into clinical settings. Infection control professionals will be involved in reviewing the safety of these agents in their clinics and hospitals. To date, only a limited number of articles have been written on infection control in gene therapy, and no widely available recommendations exist from federal or private organizations to guide infection control professionals. The goals of the conference were to provide a forum where gene therapy experts could share their perspectives and experience with infection control in gene therapy and to provide an opportunity for newcomers to the field to learn about issues specific to infection control in gene therapy. Recommendations for infection control in gene therapy were proposed.

Production of recombinant adeno-associated virus.

Currently, rAAV appears to be one of the most promising vectors for gene therapy applications. Attractive features of the vector include nonpathogenicity, the ability to infect nondividing cells, escape from host immune responses, and integration into the host genome. Tremendous progress has been made in the production of this vector, which makes it possible to start to examine the vector performance in large animals and to implement the transition to phase I human clinical trials with a variety of target tissues and therapeutic genes. However, some major challenges remain to be addressed by more extensive studies. These include the current inability to provide rAAV vectors in sufficient quantity and purity for large-scale clinical human applications, lack of site-specific integration, and lack of efficient transduction in some tissues such as airway epithelial cells. There is a limited transgene capacity in recombinant virus particles, and repeated administration of the vectors may be necessary to treat patients with chronic forms of genetic disease. Nevertheless, it is reasonable to assume that significant refinements will be made in all these areas in the relatively near future. This will promote the potential for successful therapeutic applications in humans, using rAAV-mediated gene transfer for a variety of different diseases.

Methods of gene delivery.

Human gene therapy continues to be an exciting concept for the treatment of disease. This field of research remains in its early stages, but already a number of studies have provided "proof-of-principle." Although there is no unequivocal evidence of efficacy, there have been demonstrated physiologic changes that are relevant to the disease process. One of the major challenges still confronting the field is the design of more efficient vectors. The gene delivery systems being used today will undoubtedly be seen as crude when compared with future developments. It is unlikely that there will ever be a universal vector, but rather there will be multiple vectors specifically designed for certain organ sites and certain disease. It is reasonable to assume that, in the future, there will be synthetic vectors that co-opt the needed properties from both cells and viruses. It will be necessary to do much more fundamental research in cell biology, virology, immunology, and pathophysiology before vectors can be significantly improved. Many of the tools for this research are in place, and the driving force will be provided by the imaginativeness of the committed investigators.

Gene therapy. Molecular medicine of the 1990s.

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.

Germ-line gene modification and disease prevention: some medical and ethical perspectives.

There has been considerable debate about the ethics of human germ-line gene modification. As a result of recent advances in the micromanipulation of embryos and the laboratory development of transgenic mice, a lively discussion has begun concerning both the technical feasibility and the ethical acceptability of human germ-line modification for the prevention of serious disease. This article summarizes some of the recent research on germ-line gene modification in animal models. Certain monogenic deficiency diseases that ultimately might be candidates for correction by germ-line intervention are identified. Several of the most frequently considered ethical issues relative to human germ-line gene modification are considered in the context of professional ethics, parental responsibility, and public policy. Finally, it is suggested that there is merit in continuing the discussion about human germ-line intervention, so that this technique can be carefully compared with alternative strategies for preventing genetic disease.

Induction of B-cell lymphomas in athymic NIH Swiss nu/nu mice.

A B-cell lymphoma was induced in athymic NIH Swiss nu/nu mice by challenging the animals with NIH3T3 cells, previously transfected with a recombinant DNA carrying a human oncogene hhcM, ligated to an SV40 promoter with a neomycin-resistance marker. The gross pathology of the tumor-bearing animal revealed generalized lymphadenopathy and the histopathology indicated widespread infiltration of lymphocytes into organs, such as brain, liver, kidney and lung, in addition to the lymphoid tissues and spleen; the appearance was consistent with diffuse histiocytic lymphoma. Direct immunofluorescence assays with specific typing anti-sera on live cells prepared from spleen and various lymph nodes in short-term culture, suggested the cells were B-cells. This B-cell lymphoma provides an experimental model, not only for studying possible oncogene activation but also for studying the various interactions involved in signal transduction essential for the activation of B-cell proliferation and differentiation.

Murine type C retroviruses and intracisternal A-particles in human tumors serially passaged in nude mice.

An ultrastructural survey of 11 human tumors passaged in N:NIH(S) (nu/nu) mice showed two instances of type C virus production. In one instance type C virus particles were observed in the endothelial murine stromal cell component of an embryonal carcinoma but not in the human tumor cells. In another instance type C virus particles were seen replicating in the chondroblastic human cells of a xenografted osteosarcoma. The type C virus produced in the human cells failed to transform NIH/3T3 cells, the C-127 rat cell line, or mink cells. Nucleic acid hybridization studies in which a human endogenous retroviral probe and a xenotropic murine leukemia virus envelope probe were used suggested that the retrovirus present in the human osteosarcoma cells is related to murine leukemia viruses. Intracisternal A-particles (IAP) were also detected in the human osteosarcoma cells. Their presence in the human cells was demonstrated by simultaneous visualization of IAP and human HLA determinants at the cell surface. The literature on type C virus infection of human cells and tumors grafted in nude mice is reviewed.

Interferon treatment of murine Meth-A sarcoma cells: effects on the malignant phenotype and expression of tumor-specific and H-2 antigens.

A virus-free methycholanthrene-induced sarcoma (Meth-A) in BALB/c mice was grown in culture and treated with purified mouse interferon (alpha and beta mixture) prior to testing for oncogenicity in the host animal. Use of interferon in vitro caused growth inhibition, but not cytotoxic effects; such effects were fully reversible upon interferon removal from the system. There was a significant decrease in tumor incidence in mice challenged with interferon-treated cells, but this could be overcome by sufficiently increasing the number of cells in the inoculating dose. By transplanting these BALB/c sarcoma cells into Sprague-Dawley nude mice, the effects of interferon could be negated. Since these mice have an unaltered activity of NK cells, the results suggest that NK cells do not play a major role in rejection of the Meth-A tumor, but that the reduction in tumor incidence is dependent on the presence of functional T cells. Interferon caused a detectable reduction in the expression of the tumor-specific transplantation antigen (TSTA) associated with Meth-A cells, but increased the expression of H-2 antigens. It has recently been shown that H-2 antigens play a role in host recognition of the TSTA of methylcholanthrene-induced sarcomas. It is suggested that the increased expression of H-2 antigens on interferon-treated Meth-A cells could lead to an increased frequency of recognition by T-cells.

Effect of interferon on the replication of mink cell focus-inducing virus in murine cells: synthesis, processing, assembly, and release of viral proteins.

Treatment of mink cell focus-inducing (MCF) virus (isolate AK-13) producing SC-1 cells with mouse fibroblast interferon (150 to 600 U/ml) led to a 100-fold decrease in the release of infectious virus, whereas there was a 2.5- to 10-fold decrease in various parameters of virus particle release. Analysis of labeled virion proteins indicated that a temporal change in virion protein composition occurred after interferon treatment. After a 24-h exposure of chronically infected cells to interferon, the virions produced contained a 85,000-dalton glycoprotein (apparently of nonviral origin) which was in excess of the virus envelope glycoprotein gp70. Particles produced from cells treated with interferon for 32 to 48 h were nearly devoid of gp70 and contained substantially lower quantities of p30. Intracellular processing of viral precursor polyproteins to the mature virion structural proteins was not altered in the presence of interferon. However, an accumulation of the viral p30 and p12E proteins was observed in interferon-treated cells, consistent with an increase in cell-associated virions. Immunoprecipitation analysis of the tissue culture fluids from [35S]methionine-labeled control and interferon-treated cells revealed marked decrease in p30 and p15E/p12E released after interferon treatment. In contrast, gp70 did not accumulate in interferon-treated cells, but was released into the culture medium in a form that was neither pelletable nor associated with p15E/p12E.

possible role of a retrovirus in the expression of tumor-specific antigens of the Meth A sarcoma.

The serologically defined tumor-specific surface antigen (TSSA) of the chemically-induced BALB/c Meth A sarcoma, highly restricted to one of 20 sarcomas of BALB/c origin, has been detected on a Moloney murine sarcoma virus (Mo-MuSV)-transformed BALB/c 3T3 cell lines, designated IIA(v). The immunogenicity of the IIA(v) cell in tumor-rejection assays was specific for the Meth A sarcoma, supporting the evidence for a close relationship between the TSSA and the tumor-associated transplantation antigen (TATA) of this tumor. Infection of SC-I cells with retroviruses present in cultured filtrates of IIA(v) cells resulted in Meth A antigen expression. The retroviruses associated with Meth A antigen expression have been tentatively identified as replication and/or transformation-defective XC- MuLV. The possible roles of Mo-MuSV and cellular genes of the BALB/c strain of mice in the expression of the Meth A antigen are discussed.

Effect of interferon on mouse leukaemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles ('interferon' virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but 'interferon' virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in 'interferon' virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted 'interferon' virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.

Effect of interferon on murine leukaemia virus infection. IV. Formation of non-infectious virus in chronically infected cells.

Interferon (150 units/ml) was used to treat SC-1 and AKR-2B cells which were chronically infected with murine leukaemia virus (MuLV). This led to a 100-fold decrease in the amount of infectious virus released into the medium and a 10-fold decrease in the number of virus particles measured by the virion-associated reverse transcriptase assay. However, there was little change in the amount of cell-associated infectious virus, though nearly twice as many cell-associated virions were counted in electron micrographs. With both types of cells, interferon blocked MuLV replication at the post-budding stage, but it did not change the morphology of the particles produced or their content of virion 70S RNA. Infectious virus assembled on the cell membranes of interferon-treated cells was less stable at 37 degrees C than that grown in the absence of interferon. Release of infectious virus from interferon-treated cells was not inhibited by actinomycin D or cycloheximide, though both agents inhibited virus production in controls. These results show that interferon inhibits MuLV replication through effects on virion assembly; these lead both to the formation of non-infectious particles and of fewer virions. Kinetic analysis further shows that interferon affects MuLV assembly rapidly and induction of an antiviral protein may not be required.

The errors in assembly of MuLV in interferon treated cells.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.

Immunoglobulin production by lymphosarcomas induced by Abelson virus in mice.

A brief review of the origin and tumor-inducing properties of Abelson murine leukemia virus is given. The most common neoplasm induced by this virus in vivo is a nonthymic lymphocytic tumor of bone marrow and lymph node origin. Two morphologic types of lymphosarcomas are the undifferentiated lymphosarcoma (LS) and the plasmacytic lymphosarcoma (PL). With the electron microscope, both tumor cell types may be mixed and contain undifferentiated cells or cells with a moderate amount of rough endoplasmic reticulum and polysomes. PL tumors are composed predominantly of the latter. In biosynthetic studies, PL tumors produce more immunoglobulin (Ig) than LS and more of the Ig-heavy chain, which is thought to be the murine counterpart of IgD. PL-cells sensitized with rabbit antisera to mouse kappa chains formed rosettes with formalinized protein-A producing Staphylococcus aureus Cowan I strain. The rabbit antisera were specific for kappa chains by absorption. The failure of lymphosarcoma cells to secrete Ig indicates their differentiation is blocked by the transformation process. Lymphosarcoma cells appear then to be derived from B-lymphocytes.