Inhibition of Cell Cytolysis Which Combines Treatment with Soluble DR5 Soluble Fas and Soluble TNFR1

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A major factor limiting prolonged transgene expression is due to the elimination of the cells infected by the adenovirus gene therapy that expresses the gene therapy product. Elimination of cells by either nonspecific "bystander" mediators or specific induction of cell death by T cells and other inflammatory cells is carried out by the process of either necrosis or apoptosis. Necrosis results in lysis of the cell and is mediated by TNFct as well as other cytokines. Apoptosis results in elimination of the cell by triggering programmed cell death followed by phagocytosis of the cell into nearby reticulo-endothelial cells. The primary molecules that contain an intracellular death domain include Fas, TNF receptor (TNFR), and death domain receptor (DR3, -4, and -5) mediated by TRAIL (Fig. 4).

The relative contribution of TNFa-mediated necrosis compared to Fas ligand (FasL) or TRAIL-mediated apoptosis was investigated using systemic treatment or adenovirus that expressed soluble forms of receptors capable of neutralizing these factors. We have previously shown that Fas-Fc is capable of neutralizing Fas ligand and preventing Fas ligand-mediated apoptosis [47], Similarly, a soluble form of the death domain receptor 5 (sDR5-Fc) can neutralize TRAIL and inhibit TRAIL-mediated apoptosis [48]. Adenoviruses were constructed that expressed soluble forms of DR5-Fc (sDR5), AdsDR5, and soluble forms of Fas-Fc (sFas), AdsFas. The pretreatment with AdsFas can prevent liver cell apoptosis after iv administration of anti-Fas Jo-2 [49], AdsDR5 was shown to protect TRAIL-mediated apoptosis of Jurkat T cells.

DcRl/TRID

DcRl/TRID

Figure 4 Death domain receptor family. Death domain family members are exemplified by Fas and TNF receptor 1 (TNFR1). These have three and four extracellular cystine-rich repeat domains, respectively. There is an 9- to 31 -amino-acid linker between the extracellular domain and the transmembrane domain. Both molecules have a homologous intracellular death domain represented by a rectangle. Other members include cytotoxic apoptosis receptor 1 (CAR-1) and also death domain receptors that bind TNF-related apoptosis-inducing ligand (TRAIL). These receptors include death domain receptor 3 (DR3), DR4, and DR5. Also, there is a decoy receptor (DcRl) that can bind TRAIL but lacks the intracellular death domain and therefore binds to TRAIL but does not introduce apoptosis.

Figure 4 Death domain receptor family. Death domain family members are exemplified by Fas and TNF receptor 1 (TNFR1). These have three and four extracellular cystine-rich repeat domains, respectively. There is an 9- to 31 -amino-acid linker between the extracellular domain and the transmembrane domain. Both molecules have a homologous intracellular death domain represented by a rectangle. Other members include cytotoxic apoptosis receptor 1 (CAR-1) and also death domain receptors that bind TNF-related apoptosis-inducing ligand (TRAIL). These receptors include death domain receptor 3 (DR3), DR4, and DR5. Also, there is a decoy receptor (DcRl) that can bind TRAIL but lacks the intracellular death domain and therefore binds to TRAIL but does not introduce apoptosis.

BAdsDR5 SAdsFas

0 AdsDRS+AdsFas AdsDR5+sTNFR1 ■ AdsFas+sTNR1

7 14

Figure 5 Inhibition of liver apoptosis by soluble TNFR1, soluble Fas, and soluble DR5. Mice were treated with adenovirus-expressing soluble Fas (AdsFas) and soluble DR5 (AdsDR5) as well as soluble TNFR1 (100 ng/mouse, iv). Mice were also given an Ad-luciferase. The luciferase activity was measured at 3, 7, 14, 30, and 50 days in mice treated with AdsDR5, AdsFas, sTNFRI protein, AdsDR5 plus AdsFas, AdsDR5 pi us sTNFRI, or AdsFas plus sTNFRI. As a control, mice were given Ad-luciferase plus PBS.

BAdsDR5 SAdsFas

0 AdsDRS+AdsFas AdsDR5+sTNFR1 ■ AdsFas+sTNR1

7 14

Time (days)

Figure 5 Inhibition of liver apoptosis by soluble TNFR1, soluble Fas, and soluble DR5. Mice were treated with adenovirus-expressing soluble Fas (AdsFas) and soluble DR5 (AdsDR5) as well as soluble TNFR1 (100 ng/mouse, iv). Mice were also given an Ad-luciferase. The luciferase activity was measured at 3, 7, 14, 30, and 50 days in mice treated with AdsDR5, AdsFas, sTNFRI protein, AdsDR5 plus AdsFas, AdsDR5 pi us sTNFRI, or AdsFas plus sTNFRI. As a control, mice were given Ad-luciferase plus PBS.

To determine if adenovirus gene expression can be prolonged by protecting liver cells from apoptosis, mice were pretreated with either AdsDR5, AdsFas, or sTNFRI, or different combinations of these cytoprotective therapies. Pre-treatment with AdsDR5 alone resulted in a greatly prolonged expression by the liver after subsequent administration of an Ad vector expressing luciferase (AdLuc) (Fig. 5). Consistent with our previous results, the second most effective molecule to prolong luciferase expression was treatment with sTNFRI. Pretreatment with AdsFas provided only modest prolongation of the luciferase gene expression. Combined treatment with AdsDR5 and sTNFRI provided the greatest protective effect after administration of AdLuc and the greatest prolongation of gene expression. These results indicate that liver gene therapy is limited primarily by expression of TRAIL by either infected liver cells or the immune response to this adenovirus gene therapy and TNF and FasL play a lesser role.

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Confident Kids

Confident Kids

Although nobody gets a parenting manual or bible in the delivery room, it is our duty as parents to try to make our kids as well rounded, happy and confident as possible. It is a lot easier to bring up great kids than it is to try and fix problems caused by bad parenting, when our kids have become adults. Our children are all individuals - they are not our property but people in their own right.

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