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Efficiency of in vitro adenoviral-mediated gene transfer in isolated pancreatic islets and effect on in vivo and in vitro islet function
72
Cell Transplantation • Volume 5, Number 5S-2, 1996
10.03 E F F I C I E N T
AND REPRODUCIBLE GENE TRANSFER INTO PORCINE ISLETS USING CATIONIC LIPOSOMES: STANDARDIZATION OF PROCEDURE. Benhamou P Y , M o r i s c o t C, P r e v o s t P, R o l l a n d E, Halimi S, C h r o b o c z e k J ; G r e n o b l e F R A N C E
IL-10 AND TGF-B GENE TRANSFER FOR XENOGENIqlC 10.04 ISILTI " TRANSPLANTATION: COMPARISON OF EFFECT IN
CONCORDANT VS. DISCORDANT SPECIES COMBINATIONS DenR S, Yang ZD, Kucher T, W~er M, Shaked A, Naji A, Ketchum RJ andBrayman KL; Philadelphia, U S A . Freshly isola~d canine islets transplanted to immunocompetem rats have a high rate of primary nonfunction (PNF), however PNF is not observed in the transplantation of freshly isolated dog or rat islets to immunocompetent mice. To dctenitine the role of local immunity in this processt we invesfiga_ted the transfer of genes for immunosuppressive cytokines, such as IL-10 and TGF-B, to isolated canine ann rat islets and examined its effect on islet xenograft survival in a concordant and discordant species combination. Isolated islets (canine and ra0 were exposed to adenoviras bearing lac-z marker gene, or cytokine genes (muTGF-B, vlL-10) at a muluplicity of infection (MOI) of 50 to 1, during culture (F-12, 10%FCS, 5%CO2/37"C). Islets were transplanted in concordant (rat to mouse) or discordant (dog to rat) combinations. Grafts to athymic nude mice se.~ea as controls. Geneproduct expression was demonstrated by xgaL staining in me-z wansfected islets_by RT-PCR demonstration of IL-10 and TGF-B mRNA in cytokine-tmnsfected islets, and by cytokine-specific ELISA of culture supernatants. Transplantation of islets, be~"ng lac-z, 1L-10, or TGF-B genes, to STZ-diabetic nude mice resmtecl ra recipient normogtycemta for z30 days (n--4/group). Decreased islet survival was observed in the rat-to-mouse com.bina.tion, while dramatically, improved early islet function resulted in the do,-to-rat combination. TxplGroups Rx n Survival MST P Value Rat-to-Mouse Control 12 9-35days 15.7±2.2 La'cZ 6 12-30 days 20.8~3.1 NS IL-10/TGF7 4-9 days 5.9x'O.7 p<0.01 Dog-to-Mouse Control 6 5-16 days 9.7±1.7 -DoR-to-Rat Control 6 0 days 0.0~.0 ** . IL-IOfI'GF5 2-7 days 5.2±1.0 p
N e w s t r a t e g i e s to i m p r o v e t h e o u t c o m e o f encapsulated porcine islets m a y involve the transfer of g e n e s e q u e n c e s to e n h a n c e islet viability. W h i l e a d e n o v i r a l v e c t o r s a p p e a r as the m o s t efficient gene t r a n s f e r s y s t e m so far e s t a b l i s h e d f o r islets, non-viral v e c t o r s are m o s t likely to fulfill microbiological safety criteria and be retained in the clinical setting. O u r aim w a s to standardize the procedures of gene transfer into adult porcine islets u s i n g cationic i i p o s o m e D O T A P . Porcine islets obtained by collagenase digestion were lipofected w i t h p l a s m i d c o d i n g f o r luciferase or B-galaetosidase. T h e following parameters were explored: e x p o s u r e time to vector (1 to 48 hours), D N A a m o u n t (1 to 15 p g / 500 islets), D O T A P / D N A ratio (2 to 16). R e p o r t e r g e n e e x p r e s s i o n w a s d e t e r m i n e d 48 to 72 h o u r s f o l l o w i n g lipofection. U n d e r optimal conditions, freshly isolated islets (n=5000) in s u s p e n s i o n were efficiently transduced and distribution of gene expressing islets was h o m o g e n o u s w h e n islets were subsequently plated in 500 islets aliquots. Luciferase gene e x p r e s s i o n w a s detected for at least 7 days f o l l o w i n g iipofection. X-gal staining revealed that 80% of islets were transfeeted. Islet viability lwas not adversely affected. T h e relevance of lipofeetion n experimental islet transplantation using genes targeting onspecific inflammation is n o w under investigation.
10.05 TRANSFER OF GENES FOR IL-10 AND TGF-fi TO ISOLATED
HUMAN PANCREATIC ISLETS. Denn S, Yan~ ZD Ketchum RJ, Shaked A, Barker CF, Naji A and l~rayman KL; Plliladelphia, U S A. Production of immunosuppressive cytokines at the site of a cellular graft may reduce _g~aft-specific host immunity, pr?mote engraftment andprolong gr',ift survival. In this study, wansfer of vlL-10 and muTGF-B genes to human islets, using adenoviral transfection vectors was investigated in vitro and in vivo. Human islets, isolated by collagenase digestion and purified by Euro-Ficoll gradient centrifugation were cultured for =lShrs (F-12, w/10% FCS, 37"C/5%CO2). Pure islets were exposed to adenovirus bearing marker gene (ad-lac-z), or cytokine genes (ad-muTGF-B, ad-vlL-10) at varying multipliciues of rafection (MOi). Following adenoviral co-culture, islets were washed, cultured24hr, and assayed for gene transfer by X-gal staining or RT-PCR. Islet function, posttransfection, was assessed ra vitro, by perifusion, and in vivo by xenotransplantation. Gene product expression in Wans-fected human islets was detemained to be optimal at MOI 100:1 with 48-72hr incubation. At this density, gene transfer to --23% of dispersed islet cells (ad-lac-z vector and x-gal staining) was obtained. Messenger RNA from IL-10 or TGF-IJ W a n s f ~ islets (MOI 50:1) was to generate cDNA (reverse transcriptasc), which was amplified by PCR. Demonstration of gene products matching IL-10 ahd TGF-B controls (1% agarose_gel electrophoresis, ethidium bromide staining) indicamd successful transfection andgene product expression. Production of IL-10 and TGF-B was coniirmedby cy(okine-specitic ELISA of culture supematants. Human islets, j~erifused 7 da~ post-ad-lac-z exposure, demonstrated glucose-stunnlated insniln release which di~l not differ significantly from untreated controls. Transplantation of islets following lac-z gene transfer, to STZdiabcuc nude mice, resulted in normalizauon of recipient blood glucose in all cases (n=6). Islets transfected with IL-10, or TGF-B reversed STZ-iudoced hyperglycemia in 50% of nude mouse recipients (n---g). These results demonstrate that IL-10 and TGF-fl are produced by transfected cells within isolated human islets. Further, human islet viability and function were preserved following viral exposure and gene (marker or cytokine) transfer. Thus, a model has been developed, using isolated islets bearing novel genes to investigate the local delivery of agents, such as immunosuppressive cytokines, to modulate host immunoreactivity and affect gralt survival. Further studies will examine the ability of gene transfer to modify host allo- and xenoimmunity to cellular grdts.
] 0.06
EFFICIENCY OF IN VITRO ADENOVIRAL-MEDIATED GENE TRANSFER IN ISOLATED PANCREATIC ISLETS AND EFFECT ON IN VIVO AND IN VITRO ISLET FUNCTION. Sigalla J, David A, Fiche M, Cassard A, Boeffard F, Soulillou JP, Le M a u f f B, Anegon I ; Nantes, F R A N C E
We investigated the efficiency and functional consequences of adenoviral-mediated gene transfer into murine pancreatic islets with a recombinant adenovirus encoding for the E. Coil B-Gal reporter gene. Over 90% of islets were transduced with 4.10 s pfu/islet. Analysis of frozen islet sections showed that transduced cells were always located at the periphery of islets. Transduced and control islets were functionnally assessed in vitro by static incubation, and showed a similar insulin secretion. Viability was assessed in vivo by transplantation ofsyngeneie islets in STZ- diabetic recipients. All mice receiving either transduced (n=5) or control (n=7) islets recovered normoglycemia at 4 + 4.1 vs 3.8 + 2.9 days and remained euglycemic over 120 days. Staining of grafted transduced islets with X-Gal showed that B-Gal expression was lost in the tissues between day 30 and 50. Control (n=10) or transduced islets (n=5) transplanted in an allogeneic combination (DBA2 into C57BL/6) showed equivalent kinetics of glycemia normalisation and rejection (16.2 + 4 vs 16.3 + 4 days). Cellular expression of adenoviral proteins following Ad transduction is known to induce host immunisation and local inflammation. However, and in contrast to 293 cells, transduced islets did not express detectable levels of adenoviral hexon proteins. Nevertheless, mice grafted with syngeneie transduced islets showed a moderate leukocyte infiltrate and weak levels of antiadenovirus antibodies. In summary, Ad-mediated gene transfer into islets was highly efficient and did not impair their function. In spite of a transient expression, Ad-mediated gene transfer in islets could be an effective tool for modifying the local environment early alter transplantation with antiinflarnmatory or immunosuppressive molecules.
Cell Transplantation • Volume 5, Number 5S-2, 1996
10.03 E F F I C I E N T
AND REPRODUCIBLE GENE TRANSFER INTO PORCINE ISLETS USING CATIONIC LIPOSOMES: STANDARDIZATION OF PROCEDURE. Benhamou P Y , M o r i s c o t C, P r e v o s t P, R o l l a n d E, Halimi S, C h r o b o c z e k J ; G r e n o b l e F R A N C E
IL-10 AND TGF-B GENE TRANSFER FOR XENOGENIqlC 10.04 ISILTI " TRANSPLANTATION: COMPARISON OF EFFECT IN
CONCORDANT VS. DISCORDANT SPECIES COMBINATIONS DenR S, Yang ZD, Kucher T, W~er M, Shaked A, Naji A, Ketchum RJ andBrayman KL; Philadelphia, U S A . Freshly isola~d canine islets transplanted to immunocompetem rats have a high rate of primary nonfunction (PNF), however PNF is not observed in the transplantation of freshly isolated dog or rat islets to immunocompetent mice. To dctenitine the role of local immunity in this processt we invesfiga_ted the transfer of genes for immunosuppressive cytokines, such as IL-10 and TGF-B, to isolated canine ann rat islets and examined its effect on islet xenograft survival in a concordant and discordant species combination. Isolated islets (canine and ra0 were exposed to adenoviras bearing lac-z marker gene, or cytokine genes (muTGF-B, vlL-10) at a muluplicity of infection (MOI) of 50 to 1, during culture (F-12, 10%FCS, 5%CO2/37"C). Islets were transplanted in concordant (rat to mouse) or discordant (dog to rat) combinations. Grafts to athymic nude mice se.~ea as controls. Geneproduct expression was demonstrated by xgaL staining in me-z wansfected islets_by RT-PCR demonstration of IL-10 and TGF-B mRNA in cytokine-tmnsfected islets, and by cytokine-specific ELISA of culture supernatants. Transplantation of islets, be~"ng lac-z, 1L-10, or TGF-B genes, to STZ-diabetic nude mice resmtecl ra recipient normogtycemta for z30 days (n--4/group). Decreased islet survival was observed in the rat-to-mouse com.bina.tion, while dramatically, improved early islet function resulted in the do,-to-rat combination. TxplGroups Rx n Survival MST P Value Rat-to-Mouse Control 12 9-35days 15.7±2.2 La'cZ 6 12-30 days 20.8~3.1 NS IL-10/TGF7 4-9 days 5.9x'O.7 p<0.01 Dog-to-Mouse Control 6 5-16 days 9.7±1.7 -DoR-to-Rat Control 6 0 days 0.0~.0 ** . IL-IOfI'GF5 2-7 days 5.2±1.0 p
N e w s t r a t e g i e s to i m p r o v e t h e o u t c o m e o f encapsulated porcine islets m a y involve the transfer of g e n e s e q u e n c e s to e n h a n c e islet viability. W h i l e a d e n o v i r a l v e c t o r s a p p e a r as the m o s t efficient gene t r a n s f e r s y s t e m so far e s t a b l i s h e d f o r islets, non-viral v e c t o r s are m o s t likely to fulfill microbiological safety criteria and be retained in the clinical setting. O u r aim w a s to standardize the procedures of gene transfer into adult porcine islets u s i n g cationic i i p o s o m e D O T A P . Porcine islets obtained by collagenase digestion were lipofected w i t h p l a s m i d c o d i n g f o r luciferase or B-galaetosidase. T h e following parameters were explored: e x p o s u r e time to vector (1 to 48 hours), D N A a m o u n t (1 to 15 p g / 500 islets), D O T A P / D N A ratio (2 to 16). R e p o r t e r g e n e e x p r e s s i o n w a s d e t e r m i n e d 48 to 72 h o u r s f o l l o w i n g lipofection. U n d e r optimal conditions, freshly isolated islets (n=5000) in s u s p e n s i o n were efficiently transduced and distribution of gene expressing islets was h o m o g e n o u s w h e n islets were subsequently plated in 500 islets aliquots. Luciferase gene e x p r e s s i o n w a s detected for at least 7 days f o l l o w i n g iipofection. X-gal staining revealed that 80% of islets were transfeeted. Islet viability lwas not adversely affected. T h e relevance of lipofeetion n experimental islet transplantation using genes targeting onspecific inflammation is n o w under investigation.
10.05 TRANSFER OF GENES FOR IL-10 AND TGF-fi TO ISOLATED
HUMAN PANCREATIC ISLETS. Denn S, Yan~ ZD Ketchum RJ, Shaked A, Barker CF, Naji A and l~rayman KL; Plliladelphia, U S A. Production of immunosuppressive cytokines at the site of a cellular graft may reduce _g~aft-specific host immunity, pr?mote engraftment andprolong gr',ift survival. In this study, wansfer of vlL-10 and muTGF-B genes to human islets, using adenoviral transfection vectors was investigated in vitro and in vivo. Human islets, isolated by collagenase digestion and purified by Euro-Ficoll gradient centrifugation were cultured for =lShrs (F-12, w/10% FCS, 37"C/5%CO2). Pure islets were exposed to adenovirus bearing marker gene (ad-lac-z), or cytokine genes (ad-muTGF-B, ad-vlL-10) at varying multipliciues of rafection (MOi). Following adenoviral co-culture, islets were washed, cultured24hr, and assayed for gene transfer by X-gal staining or RT-PCR. Islet function, posttransfection, was assessed ra vitro, by perifusion, and in vivo by xenotransplantation. Gene product expression in Wans-fected human islets was detemained to be optimal at MOI 100:1 with 48-72hr incubation. At this density, gene transfer to --23% of dispersed islet cells (ad-lac-z vector and x-gal staining) was obtained. Messenger RNA from IL-10 or TGF-IJ W a n s f ~ islets (MOI 50:1) was to generate cDNA (reverse transcriptasc), which was amplified by PCR. Demonstration of gene products matching IL-10 ahd TGF-B controls (1% agarose_gel electrophoresis, ethidium bromide staining) indicamd successful transfection andgene product expression. Production of IL-10 and TGF-B was coniirmedby cy(okine-specitic ELISA of culture supematants. Human islets, j~erifused 7 da~ post-ad-lac-z exposure, demonstrated glucose-stunnlated insniln release which di~l not differ significantly from untreated controls. Transplantation of islets following lac-z gene transfer, to STZdiabcuc nude mice, resulted in normalizauon of recipient blood glucose in all cases (n=6). Islets transfected with IL-10, or TGF-B reversed STZ-iudoced hyperglycemia in 50% of nude mouse recipients (n---g). These results demonstrate that IL-10 and TGF-fl are produced by transfected cells within isolated human islets. Further, human islet viability and function were preserved following viral exposure and gene (marker or cytokine) transfer. Thus, a model has been developed, using isolated islets bearing novel genes to investigate the local delivery of agents, such as immunosuppressive cytokines, to modulate host immunoreactivity and affect gralt survival. Further studies will examine the ability of gene transfer to modify host allo- and xenoimmunity to cellular grdts.
] 0.06
EFFICIENCY OF IN VITRO ADENOVIRAL-MEDIATED GENE TRANSFER IN ISOLATED PANCREATIC ISLETS AND EFFECT ON IN VIVO AND IN VITRO ISLET FUNCTION. Sigalla J, David A, Fiche M, Cassard A, Boeffard F, Soulillou JP, Le M a u f f B, Anegon I ; Nantes, F R A N C E
We investigated the efficiency and functional consequences of adenoviral-mediated gene transfer into murine pancreatic islets with a recombinant adenovirus encoding for the E. Coil B-Gal reporter gene. Over 90% of islets were transduced with 4.10 s pfu/islet. Analysis of frozen islet sections showed that transduced cells were always located at the periphery of islets. Transduced and control islets were functionnally assessed in vitro by static incubation, and showed a similar insulin secretion. Viability was assessed in vivo by transplantation ofsyngeneie islets in STZ- diabetic recipients. All mice receiving either transduced (n=5) or control (n=7) islets recovered normoglycemia at 4 + 4.1 vs 3.8 + 2.9 days and remained euglycemic over 120 days. Staining of grafted transduced islets with X-Gal showed that B-Gal expression was lost in the tissues between day 30 and 50. Control (n=10) or transduced islets (n=5) transplanted in an allogeneic combination (DBA2 into C57BL/6) showed equivalent kinetics of glycemia normalisation and rejection (16.2 + 4 vs 16.3 + 4 days). Cellular expression of adenoviral proteins following Ad transduction is known to induce host immunisation and local inflammation. However, and in contrast to 293 cells, transduced islets did not express detectable levels of adenoviral hexon proteins. Nevertheless, mice grafted with syngeneie transduced islets showed a moderate leukocyte infiltrate and weak levels of antiadenovirus antibodies. In summary, Ad-mediated gene transfer into islets was highly efficient and did not impair their function. In spite of a transient expression, Ad-mediated gene transfer in islets could be an effective tool for modifying the local environment early alter transplantation with antiinflarnmatory or immunosuppressive molecules.