Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes

1990 ◽  
Vol 10 (6) ◽  
pp. 3268-3271
Author(s):  
B F Smith ◽  
R K Hoffman ◽  
U Giger ◽  
J H Wolfe
Keyword(s):  
Gene Transfer ◽  
Primary Cultures ◽  
Muscle Cells ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Enzyme Deficiency ◽  
Transfer Genes

Retroviral vectors were used to transfer genes efficiently into rat and dog myoblasts in primary cultures under conditions which permitted the transduced myoblasts to differentiate into myotubes expressing the transferred genes. The transduced myotubes expressed normal markers of differentiation and were morphologically indistinguishable from uninfected myotubes. Retroviral vector-mediated gene transfer was also used to correct a genetic enzyme deficiency in mutant canine muscle cells.

scholarly journals Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

1990 ◽  
Vol 10 (6) ◽  
pp. 3268-3271 ◽  
Author(s):  
B F Smith ◽  
R K Hoffman ◽  
U Giger ◽  
J H Wolfe
Keyword(s):  
Gene Transfer ◽  
Primary Cultures ◽  
Muscle Cells ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Enzyme Deficiency ◽  
Transfer Genes

Retroviral vectors were used to transfer genes efficiently into rat and dog myoblasts in primary cultures under conditions which permitted the transduced myoblasts to differentiate into myotubes expressing the transferred genes. The transduced myotubes expressed normal markers of differentiation and were morphologically indistinguishable from uninfected myotubes. Retroviral vector-mediated gene transfer was also used to correct a genetic enzyme deficiency in mutant canine muscle cells.

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

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 237-245 ◽  
Author(s):  
CD Jr Lothrop ◽  
ZS al-Lebban ◽  
GP Niemeyer ◽  
JB Jones ◽  
MG Peterson ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Autologous Bone Marrow ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Foreign Gene ◽  
Large Animal ◽  
Low Level ◽  
Autologous Bone

Abstract 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.

scholarly journals Mobilization of Full-Length Semliki Forest Virus Replicon by Retrovirus Particles

2006 ◽  
Vol 80 (19) ◽  
pp. 9889-9895 ◽  
Author(s):  
Eric Piver ◽  
Christine Collin ◽  
Noémie Renault ◽  
Thierry Bru ◽  
Jean-Christophe Pagès
Keyword(s):  
Gene Transfer ◽  
Semliki Forest Virus ◽  
Direct Consequence ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Full Length ◽  
Efficient Gene ◽  
Retroviral Transfer

ABSTRACT Conciliating biosafety with efficient gene transfer remains a constant concern in the development of retroviral vectors. Semliki Forest virus (SFV) replicons allow important retroviral vector production with interesting features. It is noteworthy that retroviruses have the ability to package Ψ+ and, to some extent, Ψ− cellular RNAs. Therefore, it was important to study the retroviral transfer of highly abundant SFV genomes expressing retroviral proteins. Here, we show that full-length SFV-vector replicons, with or without Ψ, are efficiently packaged into retrovirus particles. Mechanistically, our data suggest that SFV packaging is the sum of its retroviral nucleocapsid-dependent recruitment together with a passive hijacking of membrane-anchored SFV replicon. A direct consequence of this phenomenon is the formation of particles harboring autonomous replicative abilities and contaminating vector preparations. Importantly, we confirm that retroviral SFV mobilization is not an exclusive feature of murine gamma retroviruses, since it is also observed using lentivectors.

New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: No Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2108-2108
Author(s):  
Farastuk Bozorgmehr ◽  
Eike C. Buss ◽  
Stephanie Laufs ◽  
K. Zsuzsanna Nagy ◽  
Stephanie Sellers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multidrug Resistance ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Dna Sequences ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Hematopoietic Stem ◽  
Leukaemia Virus ◽  
Multidrug Resistance 1 Gene

Abstract It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking. One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites. In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector. We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

Synthetic Vascular Grafts Seeded with Genetically Modified Endothelium in the Dog: Evaluation of the Effect of Seeding Technique and Retroviral Vector on Cell Persistence in Vivo

1995 ◽  
Vol 4 (2) ◽  
pp. 219-235 ◽  
Author(s):  
Jill E. Sackman ◽  
Michael B. Freeman ◽  
Mark G. Petersen ◽  
Zuhair Allebban ◽  
Glenn P. Niemeyer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Gene Sequence ◽  
Genetically Modified ◽  
Vascular Grafts ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Marker Genes ◽  
Growth Hormone Gene

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) pre-clotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 103 to 1.5 × 105 cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

scholarly journals Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1289-1298 ◽  
Author(s):  
FM Rosenthal ◽  
K Cronin ◽  
R Bannerji ◽  
DW Golde ◽  
B Gansbacher
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Antitumor Immunity ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Ifn Gamma ◽  
Gm Csf

Abstract Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

scholarly journals Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 504-516 ◽  
Author(s):  
DA Dichek ◽  
SW Lee ◽  
NH Nguyen
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Specific Effect ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Wild Type ◽  
Intravascular Thrombosis ◽  
Primate System ◽  
Pai 1

Abstract Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Abstract Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

scholarly journals Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1289-1298
Author(s):  
FM Rosenthal ◽  
K Cronin ◽  
R Bannerji ◽  
DW Golde ◽  
B Gansbacher
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Antitumor Immunity ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Ifn Gamma ◽  
Gm Csf

Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

Sign in / Sign up

Export Citation Format

Share Document