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

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

Blood ◽

10.1182/blood.v84.2.504.bloodjournal842504 ◽

1994 ◽

Vol 84 (2) ◽

pp. 504-516 ◽

Cited By ~ 2

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

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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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 ◽

10.1016/0963-6897(95)90034-9 ◽

1995 ◽

Vol 4 (2) ◽

pp. 219-235 ◽

Cited By ~ 18

Author(s):

J SACKMAN

Keyword(s):

Genetically Modified ◽

Vascular Grafts ◽

Retroviral Vector

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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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Abstract 3426: The Generation Of Biosynthetic Small Caliber Grafts With Improved Patency Using Autologous Genetically Modified Endothelial Cells.

Circulation ◽

10.1161/circ.116.suppl_16.ii_774-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Meir Preis ◽

Jacob Schneiderman ◽

Belly Koren ◽

Tzafra Cohen ◽

Dana Levin Ashkenazi ◽

...

Keyword(s):

Endothelial Cells ◽

Transgene Expression ◽

Carotid Arteries ◽

Genetically Modified ◽

Vascular Grafts ◽

Retroviral Vectors ◽

Synthetic Polymer ◽

Histological Studies ◽

Systemic Side Effects ◽

Blood Vessel Engineering

Introduction: Seeding polymer-based synthetic vascular grafts with endothelial cells (EC) improves grafts biocompatibility and consequently grafts patency. EC adhesion and survival on the synthetic polymer remains a considerable challenge. We used pseudo-typed retroviral vectors to modify EC to co-express fibulin-5 and VEGF 165 and created a unique EC phenotype with increased adhesion and enhanced proliferation and used these cells to seed vascular grafts. Methods: Polymer-based grafts seeded with autologous venous EC transduced to co-express fibulin-5 and VEGF 165 were implanted in the carotid arteries of the donor sheep. 18 grafts (8 seeded grafts with EC co-expressing fibulin-5 and VEGF, 6 seeded with control EC and 4 bare grafts) were implanted for two week in nine sheep. Six months studies were performed on 18 grafts (6 study, 6 seeded with control EC and 6 bare) implanted in 18 sheep. Histological studies for cell adhesion, intra-luminal thrombosis and transgene expression were tested in implanted grafts after angiography. Results: At two weeks all 8 grafts seeded with EC co-expressing fibulin-5 and VEGF were patent with no significant thrombosis and 73% coverage by seeded EC, while 3/10 control grafts were thrombosed and only 30% of seeded unmodified EC remained on grafts luminal surface. At 6 months, 5/6 grafts seeded with EC co-expressing fibulin-5 and VEGF were patent while 9/12 control grafts (6 bare, 6 seeded with unmodified EC, p<0.04) were occluded. At 6 months, the genetically modified cells were identified on the graft surface and these grafts had improved EC coverage and reduced intra-luminal neo-intima and thrombosis. No local or systemic side effects were attributed to the implanted grafts. Conclusion : Polymer-based vascular grafts seeded with EC transduced to co-express fibulin-5 and VEGF have lower propensity to thrombosis and improved patency. Use of grafts seeded with EC co-expressing fibulin-5 and VEGF provides a potentially clinically relevant method for small caliber blood vessel engineering.

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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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Appropriate In Vivo Expression of a Muscle-Specific Promoter by Using Avian Retroviral Vectors for Gene Transfer

Journal of Virology ◽

10.1128/jvi.66.8.5175-5175.1992 ◽

1992 ◽

Vol 66 (8) ◽

pp. 5175-5175

Keyword(s):

Gene Transfer ◽

Retroviral Vectors ◽

In Vivo Expression

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Enhanced In Vivo Antithrombotic Effects of Endothelial Cells Expressing Recombinant Plasminogen Activators Transduced With Retroviral Vectors

Circulation ◽

10.1161/01.cir.93.2.301 ◽

1996 ◽

Vol 93 (2) ◽

pp. 301-309 ◽

Cited By ~ 67

Author(s):

David A. Dichek ◽

Johanna Anderson ◽

Andrew B. Kelly ◽

Stephen R. Hanson ◽

Laurence A. Harker

Keyword(s):

Endothelial Cells ◽

Plasminogen Activators ◽

Retroviral Vectors ◽

Antithrombotic Effects

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3268-3271.1990 ◽

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.

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