Retrovirus-producing Cell Line Research Articles

Detection of RD-114 virus by a reverse transcriptase assay based on product enhancement

RD-114 virus is a feline endogenous retrovirus that exists in the genome of all cats. It can be assumed that feline and canine live vaccines manufactured by culturing cells of feline origin are contaminated with the virus. The current study is attempted to develop a product enhanced reverse transcriptase (PERT) assay to detect replication-competent RD-114 virus. Since culture supernatants of Crandell-Rees feline kidney (CRFK) cells do not have detectable reverse transcriptase activity in case of do not passage on other cell lines, these results raise the possibility that RD-114 virus grow efficiently in other retrovirus producing cell lines. For the PERT assay, RD-114 virus isolated from CRFK cells may need to be passaged more than four times on 293T cells to be expressed at appreciable levels. The PERT assay described here provides an accurate method for determining the presence of RD-114 in culture supernatants and will help monitor live vaccines produced in endogenous virus producing cell lines.

Retrovirus producer cell line metabolism: implications on viral productivity

The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and alpha-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production.

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.

Transformation of the Adult Human Mesenchymal Stem Cells into Cardiomyocyte-Like Cells in Vivo

Myocardial cell transplantation is being explored as a potential therapy for treating heart failure. We examined the possibility whether genetically modified adult human mesenchymal stem cells (hMSCs) can be grafted safely into myocardium and whether they undergo “milieu-influenced differentiation” to cardiac cells. The β-gal gene was transduced into adult hMSCs by culturing with recombinant retrovirus producer cell line (FLY A4). The positively stained cells were transplanted into left ventricular free wall of the Severe Combined Immune Deficient (SCID) mice by infradiaphragmatic approach. Twenty-four mice were injected with hMSCs and nine mice with serum free media. The animals were sacrificed at different time intervals and tissue was processed for X-gal, H&E, and immunostaining for myosin heavy chain and troponin-I. Immunostaining was also done against vWF for angiogenesis and against CD3 for lymphocytes for rejection. There was 65 ± 7.90% transduction of the hMSCs. Seventeen of the 24 mice injected with hMSC expressed the lacZ gene in the myocardium. Cells stained positive for X-gal in the myocardium for up to 2 months confirming gene expression. The transplanted cells aligned themselves parallel to the host myocardium. No chronic rejection was observed. Positive staining against endothelial cells was increased compared to control heart. Immunostaining to β-myosin heavy chain was positive. This study showed that adult hMSCs could be transformed into cardiomyocyte-like cells. We also demonstrated that active gene expression was feasible and persisted in the myocardium for up to 2 months using the retroviral vector. These adult hMSCs can be transformed into cardiomyocyte-like cells, which may be a potential source of cells for autotransplantation in treating heart failure.

Preparation of a Specific Retrovirus Producer Cell Line

Establishing a cell line that produces high levels of a specific retrovirus construct involves several steps. First, the retroviral construct must be stably introduced into an appropriate packaging cell line. This can be accomplished either directly by transfection or by transfection to produce a transient virus stock followed by cross-infection of this stock into a separate packaging line; procedures for both approaches are described in this unit. After stable lines are produced, they must be characterized to identify lines that produce high titers of virus with an appropriate structure. A drug selection protocol (the most common method for determining virus titer) is provided along with a sample calculation of BAG virus titer. Sometimes, a short and direct method of estimating virus titer is available when the virus encodes a histochemically detectable gene such as lacZ. For this case, an Xgal staining protocol is described. Similarly, a protocol is provided to estimate the levels of virus being produced by a given cell line by quantitating any specific product of the virus (e.g., RNA) present within the producer line.

Repeated cycles of retrovirus-mediated HSVtk gene transfer plus ganciclovir increase survival of rats with peritoneal carcinomatosis.

Peritoneal carcinomatosis is a common clinical situation that requires novel therapeutic approaches. We investigated the efficiency of an HSVtk gene therapy for the treatment of peritoneal carcinomatosis induced in syngeneic rats by DHD/K12 colon carcinoma cells. In this setting, the efficiency of two different retrovirus producing cell lines (GP+AmEnv12 and FLYA13) was compared. Rats treated with a single injection of retrovirus producing cells followed by a 5-day course of ganciclovir treatment showed an increased survival as compared with control animals. Animals treated with three injections of producing cells, each followed by a 4-5-day course of ganciclovir treatment, showed an increased survival as compared with control rats and with those treated with a single cycle of retrovirus producing cells plus ganciclovir. However, only a few animals remained tumor-free after day 180. There was no difference between the two producing cell lines in any of the experiments. RT-PCR demonstrated a faint expression of the tk transgene in the liver, spleen, epiploon, bowels and the lung of the animals injected with the HSVtk producing cells, reflecting most likely the transduction of only a limited number of cells.

Ruthenium Red Preserves Glycoprotein Peplomers of C-Type Retroviruses for Transmission Electron Microscopy

Peplomers, the glycoprotein projections of the outer viral envelope, are distinctive for many viruses. Peplomers of retroviral C-type particles are fragile and are not preserved in standard preparations for transmission electron microscopy of thin sections, whereas the peplomers of B-and D-type retroviruses are usually preserved. Ruthenium red, extensively used in transmission electron microscopy to enhance the preservation of glycosylated proteins, was used in the preparation of three retrovirus-producing lymphoblastoid cell lines: murine SC-1 cells producing the C-type murine leukemia retrovirus LP-BM5 that causes immunodeficiency, human DG-75 cells producing a murine leukemia retrovirus, and human C5/MJ cells producing human T-cell lymphotropic virus type I (HTLV-I). Fixation of cells was carried out with ruthenium red present in the glutaraldehyde, osmium tetroxide, and the ethanol dehydration through the 70% ethanol step. The detailed structure of peplomers of these three different viruses was well preserved.

Antimurine retroviral effect of doxycycline.

The tetracycline family is composed of several molecules whose antibacterial properties are due to the fixation on the bacterial ribosomes. Among those, doxycycline is one of the most potent antibiotics for which additional features have been recently discovered. Doxycycline has been found to inhibit metalloproteinases, to decrease gelatinolytic and metastatic activities of cancer cells, to have a "chondroprotective" effect in inflammatory arthritides, and to have strong antimalarial properties. In this study, a murine retrovirus producing cell line (psi CRIP-pXT1) was incubated in variable concentrations of doxycycline. The retroviral titer of this cell line was measured by the ability to transfer resistance to G418 to NIH/3T3 cells. The retroviral titer was significantly decreased by 70% when the packaging cells had been incubated with 25 microM of doxycycline at 37 degrees C. The ID50 was around 8 micrograms/ml. Astonishingly, this effect was not observed at 32 degrees C. The mechanism of this effect is still to be determined. It may be useful to be aware of this effect for uncovering all of the possible antiviral qualities of doxycycline and its related molecules, such as glycylcyclines or anthracyclines.

Adoptive transfer of in vitro-targeted, activated T lymphocytes results in total tumor regression.

Adoptive immunotherapy of cancer uses the transfer of tumor-reactive immune cells. The success of this procedure is dependent upon the specificity of the transferred immune cells, their number, and their ability to reach their target cells. We genetically modified T lymphocytes and equipped them with the ability to specifically recognize tumor cells. Tumor cells overexpressing the ErbB-2 receptor served as a model. The target cell recognition specificity is conferred to T lymphocytes by transduction of a chimeric gene encoding the zeta-chain of the TCR and a single chain antibody (scFv(FRP5)) directed against the human ErbB-2 receptor. The chimeric scFv(FRP5)-zeta gene was introduced into primary mouse T lymphocytes via retroviral gene transfer. Naive T lymphocytes were activated and infected by cocultivation with a retrovirus-producing packaging cell line. The scFv(FRP5)-zeta fusion gene was expressed in >75% of the T cells. These T cells lysed ErbB-2-expressing target cells in vitro with high specificity. We tested the antitumor efficacy of scFv(FRP5)-zeta-expressing T cells in a syngeneic BALB/c model. The mice were treated with autologous, transduced T cells. The adoptively transferred scFv(FRP5)-zeta-expressing T cells caused total regression of ErbB-2-expressing tumors. The presence of the transduced T lymphocytes in the tumor tissue was monitored. No humoral response directed against the transduced T cells was observed. Abs directed against the ErbB-2 receptor were detected upon tumor lysis.

Ex vivo gene transfer into myocardium using replication-defective retrovirus

Heart transplantation is the most effective therapy for chronic severe heart failure, but there is an extreme shortage of hearts available. We examined the possibility that cardiomyocytes can be modified genetically prior to being grafted to the heart. We used a replicationdefective retrovirus carrying the β-galactosidase (β-gal) reporter gene. The β-gal gene was transduced into murine fetal cardiac myocytes by culturing a recombinant retrovirus-producing cell line in a Transwell plate hung into the primary cardiomyocyte culture. The cultured cells were stained with the di-β- d-galactopyranoside (FDG) and were sorted by fluorescence-activated cell sorting (FACS). FACS analysis showed that 25.5 ± 4.3% of the cardiomyocytes in a primary culture were positive for β-gal activity. These cells were transplanted into the hearts of syngeneic adult mice. Expression of the β-gal gene in the grafted cells was demonstrated by staining with 5-bromo-4-chloro-3-indoyl-β- d-galactoside (β-gal). Gene expression was recognized as long as 6 mo after cell transplantation. Histologic analysis showed neither inflammation nor fibrous scar tissue on the host myocardium. This study demonstrated that genetically modified cardiac cells were transplantable to the heart.

Hepatocyte Growth Factor Expressed by a Retrovirus-Producing Cell Line Enhances Retroviral Transduction of Primary Hepatocytes: Implications forin VivoGene Transfer

Hepatocyte Growth Factor (HGF) is the more potent mitogen of mature hepatocytes. We have examined the effect of human HGF expression by a recombinant retroviral cell line (MFG-LacZ) on retroviral transduction of primary mouse and human hepatocytes. The HGF in the supernatant of MFG-LacZ cell line was correctly processed and biologically active. Transduction of mouse and human hepatocytes with the supernatant of transfected cells was increased 5-fold, as determined by β-galactosidase activity. The production of HGF was stable and did not interfere with the viral titers of the producer cells. This study provides evidence that expression of HGF within a retrovirus-producer cell line increases the transduction rate of primary hepatocytes. Since the number of corrected cells is a limiting step for phenotypic correction of liver deficiencies, our approach should improve hepatic gene therapy efficiency. Furthermore this cell line should be useful forin vivoliver gene therapy.

Efficient Retrovirus-Mediated Gene Transduction Into Murine Hematopoietic Stem Cells and Long-Lasting Expression Using a Transwell Coculture System

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus-producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.

Retrovirus-mediated gene transfer into rhesus monkey hematopoietic stem cells: the effect of viral titers on transduction efficiency.

We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until > 1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for > 2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.

Purification and characterization of retrovirally transduced hematopoietic stem cells.

We have developed a method for the purification of retrovirally transduced hematopoietic stem cells, based on a modification of the stem cell purification protocols developed by Spangrude et al. [Spangrude, G., Heimfeld, S. & Weissman, I. (1988) Science 241, 58-64] and Spangrude and Scollay [Spangrude, G. & Scollay, R. (1990) Exp. Hematol. 18, 920-926], that depends upon the use of bone marrow cells isolated from 5-fluorouracil-treated mice that have been subsequently cocultivated with recombinant retrovirus-producing cell lines. We found that purified cell populations bearing a Sca 1+ Lin- Thy 1- surface phenotype represent a 50-100% pure population of spleen colony-forming cells (CFU-S day 12). Animals injected with 300 or more purified cells were consistently radioprotected and reconstituted in multiple lineages with donor cells. Sca 1+ Lin- Thy 1- CFU-S day 12 stem cells were shown to be efficiently (100%) transduced by the recombinant retroviruses used in the study. Gene transfer into long-term reconstituting stem cells, as evidenced by Southern blot analysis of mature hematopoietic cell types 3 months after transplantation, was observed only in recipients injected with large numbers (approximately 4000-5000) of the purified cells. The development of methods for purifying retrovirally transduced stem cells should prove extremely useful for various studies in which it is of interest to characterize the activity of a specific gene product (e.g., growth factor, receptor, oncogene) specifically in primitive hematopoietic cell types.

Development of a High‐Titer Retrovirus Producer Cell Line and Strategies for Retrovirus‐mediated Gene Transfer into Rhesus Monkey Hematopoietic Stem Cells

Retroviral-mediated gene transfer into pluripotent hematopoietic stem cells has been difficult to achieve in large animal models. We have compared several infection protocols in a murine model system and concluded that bone marrow can be maintained and infected in vitro for 2-6 days. We have also developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (CFU) per milliliter of culture medium. Autologous rhesus monkey bone marrow cells were co-cultured with either high- (2 x 10(10) CFU/ml) or low- (5 x 10(6) CFU/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone marrow cells from all three animals reconstituted with cells co-cultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow co-cultured with the low-titer producer clone exhibited no evidence of gene transfer.

Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets

Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV-associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.

Internalization of Human Immunodeficiency Virus Type I and Other Retroviruses by Megakaryocytes and Platelets

Abstract Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV- associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.

Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells.

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.