Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments.

3T3 Cells↗

PubMed1992

PCR and other test systems in human gene therapy.

Three protocols using retroviral-mediated gene transfer are currently undergoing clinical trials. The uniqueness of these protocols, which were the first approved experiments in which genetically engineered cells were administered to human beings, necessitated the development of novel biological and physical safety testing procedures. In addition to standard testing for contamination with exogenous pathogens and toxins, specific tests were developed to assay for the presence of recombinant helper virus in both retroviral vector preparations and in the engineered human cells. Two new assays were developed, an enhanced S+/L-virus assay called 3T3 amplification, and a specific polymerase chain reaction protocol of great sensitivity. The results of these safety tests, along with pre-clinical primate safety studies, and the accumulating clinical data, suggest that retroviral-mediated gene transfer does not pose any serious short-term safety problems for patients enrolled in gene therapy protocols.

Animals↗

PubMed1992

Prospects for human gene therapy.

Retroviral vectors containing marker genes and the sequence for human proteins have been used to transduce cultured lymphocytes, which have then been reinfused into patients. Circulating hematopoietic progenitor cells from human fetal cord blood obtained at the time of term and premature deliveries as early as 19 weeks of gestation have been shown to express such transduced genes in vitro. Cord blood cells from fetal sheep sampled and transduced ex vivo and transfused back in utero expressed marker genes up to two years after birth. Although the efficiency of gene transfer into cells and their long-term expression need to be improved, the potential exists for treating some genetic diseases after prenatal diagnosis either in utero or shortly after birth.

Animals↗

PubMed1991

Different interactions of Cro repressor dimer with the left and right halves of OR3 operator DNA.

lambda Cro repressor protein is titrated with two half-operator DNA duplexes comprising the right and left halves of the major binding site on phage lambda DNA, the OR3 operator. The comparison of binding strengths and the conformation of Cro repressor in the two protein-DNA complexes shows that base pair differences between the two halves of the OR3 operator affect the binding of Cro repressor protein. Some 1H NMR resonances are assigned for both protein and DNA in the Cro-operator DNA complexes which are then used to highlight differences between Cro right half and Cro left half protein-operator DNA interactions. These differences are compared to the asymmetry found in the lambda C1 repressor-operator DNA complex. Mechanisms for the recognition of the Cro transcriptional regulatory protein have considered only interactions between a single Cro monomer and a consensus half-operator site with the assumption that the interactions in the remaining half-site are related by the 2-fold symmetry of the complex. A revised model is suggested that allows asymmetry in the two halves of the protein-DNA complex. Methods are proposed to avoid problems in the general use of 1H NMR spectroscopy to study protein-DNA interaction such as intermediate exchange behavior and sample aggregation.

Amino Acid Sequence↗

PubMed1991

Expression of a swine class II gene in murine bone marrow hematopoietic cells by retroviral-mediated gene transfer.

As a first step in assessing the efficacy of a gene transfer approach to the induction of transplantation tolerance in our miniature swine model, double-copy retroviral vectors engineered to express a drug-resistance marker (neomycin) and a swine class II DRB cDNA were constructed. Infectious particles containing these vectors were produced at a titer of greater than 1 x 10(6) G418-resistant colony-forming units/ml using both ecotropic and amphotropic packaging cell lines. Flow cytometric analysis of DRA-transfected murine fibroblasts subsequently transduced with virus-containing supernatants demonstrated that the transferred sequences were sufficient to produce DR surface expression. Cocultivation of murine bone marrow with high-titer producer lines leads to the transduction of 40% of granulocyte/macrophage colony-forming units (CFU-GM) as determined by the frequency of colony formation under G418 selection. After nearly 5 weeks in long-term bone marrow culture, virus-exposed marrow still contained G418-resistant CFU-GM at a frequency of 25%. In addition, virtually all of the transduced and selected colonies contained DRB-specific transcripts. These results suggest that a significant proportion of very primitive myelopoietic precursor cells can be transduced with the DRB recombinant vector and that vector sequences are expressed in the differentiated progeny of these cells.

Animals↗

PubMed1991

Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow.

A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony-forming unit granulocyte-macrophage after more than 200 days. Two of the four cats unexpectedly developed diabetes mellitus 90 days posttransplantation. The expression of a foreign gene in cats, albeit at a low level, demonstrates that retroviral vectors can be used for gene transfer in noninbred large animal species and may be useful for gene therapy of humans. The development of diabetes mellitus in two of the subjects emphasizes the value of animal models for the study of possible deleterious effects of retroviral vector-mediated gene transfer.

Animals↗

PubMed1991

Lymphocytes as cellular vehicles for gene therapy in mouse and man.

The application of bone marrow gene therapy has been stalled by the inability to achieve stable high-level gene transfer and expression in the totipotent stem cells. We show that retroviral vectors can stably introduce genes into antigen-specific murine and human T lymphocytes in culture. Murine helper T cells were transduced with the retroviral vector SAX to express both neomycin-resistance and human adenosine deaminase genes. These cells were expanded in culture and selected for expression of neomycin resistance with G418. The gene insertion, selection, and culture expansion did not alter antigen specificity or growth characteristics of the T cells in vitro. To determine if cultured T cells might be used for gene therapy, their persistence and continued expression of the introduced genes was evaluated in nude mice transplanted with the SAX-transduced T cells. G418-resistant cells could be readily recovered from the spleens of recipients of transduced T cells for several months. In addition, recovered cells continued to produce human adenosine deaminase. Based on these observations, we studied cultured human tumor-infiltrating lymphocytes as a candidate cell for a trial of gene transfer in man. Exponential cultures of interleukin-2-stimulated tumor-infiltrating lymphocytes were efficiently transduced with the neomycin-resistance gene using the retroviral vector N2. Gene insertion and subsequent G418 selection did not substantially alter the growth characteristics, interleukin 2 dependence, membrane phenotype, or cytotoxicity profile of the transduced T cells. These studies provided a portion of the experimental evidence supporting the feasibility of the presently ongoing clinical trials of lymphocyte gene therapy in cancer as well as in patients with adenosine deaminase deficiency.

Animals↗

PubMed1991

Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding.

Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects. The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.

Amino Acids↗

PubMed1991

Increased efficiency of retroviral-mediated gene transfer and expression in primate bone marrow progenitors after 5-fluorouracil-induced hematopoietic suppression and recovery.

To define conditions for improved efficiency of retroviral-mediated gene transfer and expression in primate progenitor cells, four rhesus monkeys were treated with a 200 mg/kg intravenous bolus of 5-fluorouracil (5-FU). The kinetics of hematopoietic suppression and recovery were assessed in peripheral blood, bone marrow mononuclear cells, and bone marrow cells fractionated in an albumin density gradient. Bone marrow mononuclear cells were transduced with N2, a retroviral vector carrying the bacterial neomycin phosphotransferase gene (NPT), which confers resistance to the otherwise toxic neomycin analogue, G418. Circulating colony-forming units-granulocyte-macrophage (CFU-GM) disappeared at 2 days. CFU-GM, transducible CFU-GM, CD34+ cells, and the percent of cells in cycle decreased at 3 days in unfractionated bone marrow cells and in a light density population known to be enriched for these progenitors and for stem cells. NPT activity in the light-density fraction, marginally detectable before treatment, disappeared at 3 days as well. At day 7 the CFU-GM plating efficiency, the CD34+ cell content, and the percentage of cells in cell cycle began to increase in the light-density fraction. The NPT assay became faintly positive again but the CFU-GM were not yet transducible, implying that it was an earlier progenitor population that was dividing and differentiating. By day 15, there was a marked rebound in all of the progenitors measured, and transduction efficiency assessed by G418R CFU-GM and NPT assay rebounded to several times pretreatment levels. The data suggest that CFU-GM are optimally transduced at 15 days but that earlier progenitors are more likely cycling and transducible before 5 days, a time when a gene transfer experiment would probably have the best chance to succeed.

Animals↗

PubMed1991

Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer.

In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t-PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

Animals↗

PubMed1991

Retroviral vector-mediated in vivo expression of low-density-lipoprotein receptors in the Watanabe heritable hyperlipidemic rabbit.

We have achieved in vivo expression of recombinant low-density-lipoprotein (LDL) receptors in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for the human disease familial hypercholesterolemia. A retroviral vector was constructed containing the human LDL receptor cDNA and was used to stably transduce primary skin fibroblasts from WHHL rabbits. The integrity and function of the introduced LDL receptor was established by immunoprecipitation, by a fluorescent LDL binding assay, and by the ability of the transduced cells to suppress 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase activity in response to exogenous cholesterol. Autologous transduced fibroblasts were reimplanted into donor rabbits; in vivo LDL receptor expression and the survival of the transduced cells were analyzed by immunohistochemistry and by LDL binding assays performed on cells recovered from the implants. LDL receptor-bearing cells could be identified on tissue sections and recovered from implants for up to four weeks. Total and LDL cholesterol levels decreased significantly after implantation of the transduced cells; however, control experiments indicated that the decreases were not mediated through the recombinant LDL receptor. While in vivo stable expression of recombinant LDL receptors in Watanabe rabbits is possible, consequent changes in lipid levels must be interpreted with caution. This system of site-specific in vivo expression of recombinant LDL receptors permits further evaluation of the role of LDL receptor-gene replacement in the therapy of hypercholesterolemia.

Animals↗

PubMed1991

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells.

Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterial neoR gene. The presence of the neoR gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of the neoR gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4+ and CD8+ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4+ and CD8+ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4+ or CD8+ cells. In other experiments highly purified CD4+ and CD8+ T cell subpopulations from either TIL or PBMC could be successfully transduced with the neoR gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity of vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4+ and CD8+ gene-modified TIL.

Blotting, Southern↗