Ex Vivo Adenovirus-Mediated Gene Transfer to Corneal Graft Endothelial Cells in Mice

Qian, Ying; Leong, Fee‐Lai; Kazlauskas, Andrius; Dana, M. Reza

doi:10.1167/iovs.03-0901

Cited by 19 publications

(14 citation statements)

References 30 publications

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“…In previous studies using adenovirus to transfect the cornea, we found that adenovirus selectively infected the endothelium but increased graft failure (24). Recent reports identified lentivirus as a less immunogenic corneal gene therapy vector (25,26).…”

Section: Lentivirus Effectively Transduced the Corneal Endotheliummentioning

confidence: 82%

“…Numerous studies using adenoviral delivery of genes to the corneal endothelium indicate that, though transduction efficiency is high, this vehicle carries harmful immunogenic effects and is not suitable for transducing corneas prior to transplantation (24,(33)(34)(35). More recently, lentiviral gene therapy was used to introduce endostatin (26) and IDO (32) in the cornea.…”

Section: Discussionmentioning

confidence: 99%

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Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Bárcia

Dana

Kazlauskas

2007

American Journal of Transplantation

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Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immuneprivileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium.

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Section: Lentivirus Effectively Transduced the Corneal Endotheliummentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Bárcia

Dana

Kazlauskas

2007

American Journal of Transplantation

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“…When the topographical distribution of conjunctiva lymphoid tissue is projected onto the ocular surface, it overlies the surface of the cornea during eye closure and is hence in a suitable position to assist corneal immunity and immunoregulation (43). On one hand, corneal inflammation often induces the development of organized conjunctival leukocytic aggregates (12,63). On the other hand, conjunctival immune cells have been shown to influence the corneal immune response and the course of HSK after corneal HSV-1 infection (1).…”

Section: Cd25mentioning

confidence: 99%

Functional Foxp3⁺CD4⁺CD25^(Bright+)“Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4⁺and CD8⁺Effector T Cells during Ocular Herpes Infection

Nesburn

Bettahi

Dasgupta

et al. 2007

J Virol

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We studied the phenotype and distribution of "naturally" occurring CD4 The immune system has evolved numerous mechanisms of peripheral T-cell immunoregulation, including a network of ␣␤ and ␥␦ CD4 ϩ and CD8 ϩ regulatory T (T reg ) cells, that modulate primary and memory effector T (T eff ) cell responses at various times and locations and in various inflammatory circumstances (32,33,37,39,46,59,77). Among the ␣␤ T reg cells, several subpopulations of CD4 ϩ T reg cells have been recently identified and shown to control T eff cell function in various animal species and in humans (reviewed in references 2, 61, and 68). Some are considered to be natural regulators, the so-called thymus-derived "natural" CD4 ϩ CD25 ϩ T reg cells (CD4 ϩ CD25 ϩ nT reg cells) (21), that constitutively express a specific set of "molecular markers" including CD25 (the interleukin-2 [IL-2] receptor ␣-chain), forkhead/winged-helix transcription factor (Foxp3), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA4 or CD152) (reviewed in references 50 and 62). Other extrathymically generated CD4 ϩ CD25 ϩ T reg (iT reg ) cells, designated "adaptive"؉antigen-specific T reg cells, produce and mediate their suppression via IL-10 and/or transforming growth factor ␤ (TGF-␤), such as Tr1 and T helper type 3 (Th3) cells (11,69,81,86). Converging evidence shows that CD4 ϩ CD25 ϩ T reg cells are important not only in the pathogenesis of herpes simplex virus type 1 (HSV-1) and HSV-2 infections but also in regulating herpes infection and immunity (4,18,75,79). However, work in this area has been focused primarily on systemic CD4 ϩ

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“…This explains the speed of translation of the neoproteins, from the third hour after electrotransfer, unlike low-temperature preservation at +4 ° C [42] . These transgene expression kinetics are comparable to those obtained in animals after electroporation of corneal epithelial cells [43] or of stromal keratocytes [43,44] .…”

Section: Discussionmentioning

confidence: 99%

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

Hé¹,

Pipparelli²,

Manissolle³

et al. 2009

Ophthalmic Res

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Aims: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. Methods: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (β-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas β-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions’ integrity. Results: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with β-gal ranged from 0.1 to 54% of the transfected ECs (mean ± SD: 7 ± 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. Conclusions: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.

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Ex Vivo Adenovirus-Mediated Gene Transfer to Corneal Graft Endothelial Cells in Mice

Abstract: Adenoviral vector can selectively and efficiently deliver exogenous gene(s) to the endothelium of corneal grafts during hypothermic organ preservation. Gene expression is retained in vivo in corneal syngeneic grafts for longer periods than are allogeneic grafts.

Cited by 19 publications

References 30 publications

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Functional Foxp3⁺CD4⁺CD25^(Bright+)“Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4⁺and CD8⁺Effector T Cells during Ocular Herpes Infection

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

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Ex Vivo Adenovirus-Mediated Gene Transfer to Corneal Graft Endothelial Cells in Mice

Abstract: Adenoviral vector can selectively and efficiently deliver exogenous gene(s) to the endothelium of corneal grafts during hypothermic organ preservation. Gene expression is retained in vivo in corneal syngeneic grafts for longer periods than are allogeneic grafts.

Cited by 19 publications

References 30 publications

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Functional Foxp3+CD4+CD25(Bright+)“Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4+and CD8+Effector T Cells during Ocular Herpes Infection

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

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Product

Resources

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Functional Foxp3⁺CD4⁺CD25^(Bright+)“Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4⁺and CD8⁺Effector T Cells during Ocular Herpes Infection