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In vitro and in vivo gene transfer in the rat: characterization of recombinant adenoviral vectors for rat interleukin-4 or interleukin-10 cDNA
ELSEVIER
In Vitro and In Vivo Gene Transfer in the Rat: Characterization Recombinant Adenoviral Vectors for Rat Interleukin-4 or Interleukin-10 cDNA A. David, A. Cassard, L. Tesson, J.-P. Soulillou, and I. Anegon
G. Blanche,
J. Sigalla,
G
ENE TRANSFER into grafts of molecules with potentially immunomodulating actions may prolong allograft survival and give a method of analyzing the mechanisms of allograft rejection and tolerance.’ Interleukin-4 (IL-4) and interleukin-10 (IL-lo) have the capacity to drive the immune response from a Thl to a Th2 profile and inhibit macrophage activation,* making them candidates to be overexpressed in the local graft microenvironment3 The purpose of this study was to analyse the feasibility and conditions for efficient recombinant adenovirus (rAd)-mediated gene transfer into rat liver, kidney, and pancreatic islets, and to validate rAds as efficient vectors for localized cytokine overexpression.
MATERIALS
AND
B. Charreau,
Y. Godfrin,
of
H. Smit, B. Le Mauff,
RESULTS Liver Transduction Parameters for efficient in vivo transduction of rat hepatocytes were tested with Ad 1acZ. Analysis of p-gal expression 3 days after injection showed a dose-dependent effect. The minimal dose required to detect transduced hepatocytes (12.5 ? 11.8 positive cells/mm* of liver section, n = 4) was 5 X lo9 pfu. No transduced hepatocytes were detected with lo9 (n = 1) or 5 X 10” pfu (n = 1). Optimal transduction (82 ? 68 positive cells/mm’ of liver section, n = 5) was obtained with 10’” pfu and higher doses induced toxicity. Most positive cells were localized around portal spaces. Delivery of Ad IL-4 or Ad IL-10 resulted in production of IL-4 and IL-10 in all animals tested.
METHODS
rAd Construction
Kidney
Ad IL-4 and IL-10 have been constructed by homologous recombination between the El and E3-deleted adenovirus 5 genome and a plasmid containing Ad sequences flanquing the rat cytokine cDNA under the transcriptional control of the elongation factor (EFla) promoter. Ad 1acZ has been used as control vector.
Transduction
Delivery with different doses (lo9 to 10” pfu) of Ad 1acZ into kidney through different routes (ureteral injection [n = 131, renal artery injection [n = 51 or renal artery perfusion before transplantation [n = 31) showed a very low number of P-gal positive cells.
In Vitro Transduction Animals
Male (250 g) Wistar injection.
rats were
sacrified
3 days after rAd
rAd Administration
rAds were injected through the portal vein. In Kidney: rAd were administered either by ureteral injection, renal artery injection, or ex vivo renal artery perfusion (RPMI, 30 min, 37°C) before transplantation. Pancreatic islets: pancreatic islets were isolated and infected in vitro during 48 hours.
/3-Galactosidase P-gal positive
(p-gal)
Detection
cells were revealed
by X-gal coloration.
Incubation of rat pancreatic islets with 2 X 10’ pfuiislet of Ad 1acZ provided very efficient transduction with 100% of positive islets. Nevertheless, cryostat sections showed that only peripheral cells were transduced. In addition, we transduced different rat cell lines in vitro (10 to 100 pfuicell) with high pourcentage of P-gal positive cells. Moreover, in vitro infection of these different rat cell lines with Ad IL-4 or Ad IL-10 allowed detection of high level of biologically active secreted cytokines in the culture media.
From INSERM U437, ITERT, Nantes, France. Address reprint requests to Anne David, INSERM U437, ITERT, 30, Boulevard Jean Monnet, 44 093 Nantes Cedex 01, France.
0041-1315/97/$17.00 PII so041 -1345(97)00042-0
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1750
Transplantation
Proceedings, 29, 1750-l 751 (1997)
IN VW0 AND IN VITRO GENE TRANSFER
IN THE RAT
1751
CONCLUSION
REFERENCES
We have described in vitro gene transfer of IL-4 and IL-10 using rAd vectors. In vivo, liver and pancreatic islets are more efficiently transduced by rAds than kidney. rAd-mediated gene transfer may be a feasible strategy to engineer grafts to produce locally potential immunosupressive molecules acting on cells directly involved in graft recognition.
1. Anegon I, David A, Charreau B, et al: In Tilney NL, Strom TB, Paul LC (eds): Transplantation Biology: Cellular and Molecular Aspects. New York: Lippincott-Raven, 1992, Chapter 57 2. Mosmann 3. Dallman
TR, Sad S: Immunol
Today
MJ: Curr Opin Immunol
17:138, 1996
7:632, 1995
In Vitro and In Vivo Gene Transfer in the Rat: Characterization Recombinant Adenoviral Vectors for Rat Interleukin-4 or Interleukin-10 cDNA A. David, A. Cassard, L. Tesson, J.-P. Soulillou, and I. Anegon
G. Blanche,
J. Sigalla,
G
ENE TRANSFER into grafts of molecules with potentially immunomodulating actions may prolong allograft survival and give a method of analyzing the mechanisms of allograft rejection and tolerance.’ Interleukin-4 (IL-4) and interleukin-10 (IL-lo) have the capacity to drive the immune response from a Thl to a Th2 profile and inhibit macrophage activation,* making them candidates to be overexpressed in the local graft microenvironment3 The purpose of this study was to analyse the feasibility and conditions for efficient recombinant adenovirus (rAd)-mediated gene transfer into rat liver, kidney, and pancreatic islets, and to validate rAds as efficient vectors for localized cytokine overexpression.
MATERIALS
AND
B. Charreau,
Y. Godfrin,
of
H. Smit, B. Le Mauff,
RESULTS Liver Transduction Parameters for efficient in vivo transduction of rat hepatocytes were tested with Ad 1acZ. Analysis of p-gal expression 3 days after injection showed a dose-dependent effect. The minimal dose required to detect transduced hepatocytes (12.5 ? 11.8 positive cells/mm* of liver section, n = 4) was 5 X lo9 pfu. No transduced hepatocytes were detected with lo9 (n = 1) or 5 X 10” pfu (n = 1). Optimal transduction (82 ? 68 positive cells/mm’ of liver section, n = 5) was obtained with 10’” pfu and higher doses induced toxicity. Most positive cells were localized around portal spaces. Delivery of Ad IL-4 or Ad IL-10 resulted in production of IL-4 and IL-10 in all animals tested.
METHODS
rAd Construction
Kidney
Ad IL-4 and IL-10 have been constructed by homologous recombination between the El and E3-deleted adenovirus 5 genome and a plasmid containing Ad sequences flanquing the rat cytokine cDNA under the transcriptional control of the elongation factor (EFla) promoter. Ad 1acZ has been used as control vector.
Transduction
Delivery with different doses (lo9 to 10” pfu) of Ad 1acZ into kidney through different routes (ureteral injection [n = 131, renal artery injection [n = 51 or renal artery perfusion before transplantation [n = 31) showed a very low number of P-gal positive cells.
In Vitro Transduction Animals
Male (250 g) Wistar injection.
rats were
sacrified
3 days after rAd
rAd Administration
rAds were injected through the portal vein. In Kidney: rAd were administered either by ureteral injection, renal artery injection, or ex vivo renal artery perfusion (RPMI, 30 min, 37°C) before transplantation. Pancreatic islets: pancreatic islets were isolated and infected in vitro during 48 hours.
/3-Galactosidase P-gal positive
(p-gal)
Detection
cells were revealed
by X-gal coloration.
Incubation of rat pancreatic islets with 2 X 10’ pfuiislet of Ad 1acZ provided very efficient transduction with 100% of positive islets. Nevertheless, cryostat sections showed that only peripheral cells were transduced. In addition, we transduced different rat cell lines in vitro (10 to 100 pfuicell) with high pourcentage of P-gal positive cells. Moreover, in vitro infection of these different rat cell lines with Ad IL-4 or Ad IL-10 allowed detection of high level of biologically active secreted cytokines in the culture media.
From INSERM U437, ITERT, Nantes, France. Address reprint requests to Anne David, INSERM U437, ITERT, 30, Boulevard Jean Monnet, 44 093 Nantes Cedex 01, France.
0041-1315/97/$17.00 PII so041 -1345(97)00042-0
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1750
Transplantation
Proceedings, 29, 1750-l 751 (1997)
IN VW0 AND IN VITRO GENE TRANSFER
IN THE RAT
1751
CONCLUSION
REFERENCES
We have described in vitro gene transfer of IL-4 and IL-10 using rAd vectors. In vivo, liver and pancreatic islets are more efficiently transduced by rAds than kidney. rAd-mediated gene transfer may be a feasible strategy to engineer grafts to produce locally potential immunosupressive molecules acting on cells directly involved in graft recognition.
1. Anegon I, David A, Charreau B, et al: In Tilney NL, Strom TB, Paul LC (eds): Transplantation Biology: Cellular and Molecular Aspects. New York: Lippincott-Raven, 1992, Chapter 57 2. Mosmann 3. Dallman
TR, Sad S: Immunol
Today
MJ: Curr Opin Immunol
17:138, 1996
7:632, 1995