Antigen presentation in retroviral vector-mediated gene transfer in vivo

Abstract Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.

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Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽

10.1182/blood-2002-10-3050 ◽

2003 ◽

Vol 101 (10) ◽

pp. 3924-3932 ◽

Cited By ~ 73

Author(s):

Lingfei Xu ◽

Cuihua Gao ◽

Mark S. Sands ◽

Shi-Rong Cai ◽

Timothy C. Nichols ◽

...

Keyword(s):

Gene Therapy ◽

Growth Factor ◽

Gene Transfer ◽

Hepatocyte Growth Factor ◽

Retroviral Vector ◽

Factor Ix ◽

Hemophilia B ◽

Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

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Generation of high-titer retroviral vector-producing macrophages as vehicles for in vivo gene transfer

Gene Therapy ◽

10.1038/sj.gt.3301405 ◽

2001 ◽

Vol 8 (6) ◽

pp. 431-441 ◽

Cited By ~ 14

Author(s):

S Pastorino ◽

S Massazza ◽

M Cilli ◽

L Varesio ◽

M C Bosco

Keyword(s):

Gene Transfer ◽

High Titer ◽

Retroviral Vector ◽

In Vivo Gene Transfer

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Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo

Science China Life Sciences ◽

10.1007/s11427-015-4931-3 ◽

2015 ◽

Vol 59 (1) ◽

pp. 49-58 ◽

Cited By ~ 1

Author(s):

Jing Lu ◽

Feng Chen ◽

Zhen Xu ◽

Lingling Zhang ◽

Peng Xu ◽

...

Keyword(s):

Gene Transfer ◽

B Cell ◽

Cell Receptor ◽

Retroviral Vector ◽

B Cell Receptor ◽

Partial Function ◽

Membrane Immunoglobulin ◽

Vector Gene Transfer

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In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors

Science ◽

10.1126/science.1317968 ◽

1992 ◽

Vol 256 (5063) ◽

pp. 1550-1552 ◽

Cited By ~ 1131

Author(s):

K. Culver ◽

Z Ram ◽

S Wallbridge ◽

H Ishii ◽

E. Oldfield ◽

...

Keyword(s):

Gene Transfer ◽

Brain Tumors ◽

Retroviral Vector ◽

Experimental Brain Tumors ◽

In Vivo Gene Transfer

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Retrovirus-mediated Gene Transfer into Rat Salivary Gland Cells In Vitro and In Vivo

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549704501110 ◽

1997 ◽

Vol 45 (11) ◽

pp. 1533-1545 ◽

Cited By ~ 9

Author(s):

Tibor Barka ◽

Hendrika M. van der Noen

Keyword(s):

Enzyme Activity ◽

Salivary Gland ◽

Gene Transfer ◽

Cell Line ◽

Submandibular Gland ◽

Acinar Cells ◽

Retroviral Vector ◽

Placental Alkaline Phosphatase

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the β-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector. (J Histochem Cytochem 45:1533–1545, 1997)

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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

Cell Transplantation ◽

10.1177/096368979500400206 ◽

1995 ◽

Vol 4 (2) ◽

pp. 219-235 ◽

Cited By ~ 4

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.

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

Blood ◽

10.1182/blood.v83.5.1289.1289 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1289-1298 ◽

Cited By ~ 58

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.

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

Blood ◽

10.1182/blood.v84.2.504.504 ◽

1994 ◽

Vol 84 (2) ◽

pp. 504-516 ◽

Cited By ~ 18

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.

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

Blood ◽

10.1182/blood.v83.5.1289.bloodjournal8351289 ◽

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.

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