- Email: [email protected]
O 7 Isolated liver perfusion for liver-specific gene-delivery with replication-defective adenovirus vectors
Abstracts
06 SPRCIFIC RECOGNITION SITES FOR BINDING DEOXYNUCLEOTIDES ON LIVER CELLS
AND UPTAKE
OF 32P-OLIGO-
E.AL.Biesseo. I+. Vktsch nd Ih1.C. “an Berkel Division of Biophprmreutics. LeidcrJAmskrdamCenkr of Drug Reseat&. L&den Univemity. ll,e Nmbedands
In,roduc,ii Applicaion of antisense deoxymmleotides(ODN) may conaituk- an alumalive clinical appm.wJlfor spccificduymgulmiingur cxpwsiw Of geaK.9.“ourever, the ,herapcutic&xivl,y of ODN mokades is ofkn hnpaind by their susceptibilityu) untimely membolk hucdwica and by d-&r insbili to cmss the cell* membrmc. A nomisinn sua,a?y P circumvent bo,h problems in&s specific tagcting of ODN u) 6-e aimed c& ,yp. & this study, we have studiedmC in viw fee of “P-kbekd ODN in D1s in order to BFS~SS w-r ODN i&elf is pm m tissue-specific accumulation Methods and Resultr np-~DN ~1s rapidly cleated from the blood.sUFM upon inUavery)usinjemion into ram. Appmximakly 45% of Ur injected doseof QP-ODN was reccwemdin the liver within 5 min aR.?rinjecrion Non-parrnchymel liver cells. and Kupffer cells in panicular. v!em responsible for rhe helutic ue,ake of -P-oDN. Hemtic emeke of ‘=P-DDN a,o bc dew the miU; of ur om. as.li~er&kc of +oDN’WU greatly im&red efkr hert-induceddensuation. To elucidak the m&cular basis of Ur liver a&e of ‘+ODN. we have studied ,he intenetiw of “P.ODN with isolated nt endMhelial’(EC) and Kupfler cells KC) using in vitro bioding and u@e ah&?.. “P-0DN biw%,,g u) EC Md KC was sammbk (30.3 acd 26.7 r@& respnivcly) ad of moderae eflinity (Kd=31 snd 117 &I. respeaively). Binding wea ahnol abolishedby preincubating the cells with tqpsine (PC. I5 min) and afkr m acid wash @H 2.8). indicating that binding is mediated by a pmteidic reawdtion she. Binding u) EC and KC could be inhibited for 90% by ODN. polyirmsirdc acid. fucaidin ad nucleodde di- and viphosphaes whereas headenamrakd ODN. polycyddink acid, polyadenosinic acid. s.sDNA. and nucleotide or nuclmtide mompborphak amh%ues were complekk ineffecdve. Sumrisinnly. acuvlated lowdensity-limmm,ein. ee es&shed sutaram &or& scavengerreceptoron;&,helial and Kupffer c&.‘v/~s ,,o, able u) inhibit “P-0DN binding. Uptake Mldks at 37*C demonsaed &a, ligand binding was efficknlly coupled to inkrnalisvion ud lysosomll pm&wing. Moreover. infaft ODN could s6ll be recoveredin Ur lysosomalcampamnen,afw incubationof EC ami KC with “P-0DN.
mci~~
Condusions I, can be concluded,ha endmbelial nnd Kupffer cells pos.sess ODN-specific recognhicmsiks. The inhibition pmfile of 90~~ binding siks on bmb ~eu typs is ewwiauy eqtml: and comspondp closely m uul of the saveeger receptor.The faa mat in viva 9.0~~ is mainly ,ransconed u) mtwxe,xhvmaJ liver cells and tien UD via raumor-mediakd enkr$osis may have impli&ons f& potential in viva applicstionbf antisen& ODN in the regularionof gene expressionof endahelial amdKupffer ccl&spcifk gene pmducta.Leidee University, Leiden lnstiue of Cbemisby, GorlaeusIatommries.
SS
At present,UP is hing anpoyed for !he hnmbepadcadministradonof high dosescbemotherapeuticagentsfor the wearmen,of imesectablecolorecLalliver melastases.We decided m explore u) potential of ILP for the in viw delivery of adetmvirus-based vecu)m. For Uris uwose. we have used adamviral vecu~scawing Ihe firefly-luciferase gene (Ad5:ChIVluc) or & E.coli 5galacmsidasegem (AdS:CMV&&z a Lbe&orexs in ad& W&r rats as the model. We used Ur Ad3:CMVhx vims u) comparethe cfficiencics of gene wansfer u) the liver afkr lLP and afkr inmaportalinfbsion (IPI). Borh ILP and W re.ud,edin cfficien, gene hansfer. h both gmups. equivalent expressionkvds of Uw luciferase repotter gene were obkbxd (2-7 x IO’ light tmits/mg pmtein). However. after IPI significant lucifemse activity lo’-IO’ ligh, unhs/mgpmkin) could alsa be dekckd in mea aher tisams (including kidney. spleen. inlestine. hem,. lung. testis). Although low levels of lucifemse activity could also bc deBned in some of or8ansin numberof aim& after adminis@adonof the vector aAer ILP. the exposureof the odw organsw&i very signhicandy reduced,u) in some casesundetectabk levels. These lesuhs show chatperfusionof a vaswlariy is&ted liver can be used UI achieveliverspecific gm transfer.and considerablyreducesIhe erposumof other tissuesIO ,he vec,ors.
08 LIPOSOMES AND CANCER IMMUNOTHERAPY DJA. Crommelin”. IJ. Bergers’,F. Koppenhagen’.0. Swam’ and W. den O,tc? for Pb&-maEeutical Sckxes. U,recb,*. li,e Ne,berlarv,s. ’ Dutfh N&anal lmtituk for Public Health and Envimnmentll Pmkctior,, ’ Vehxiwrian Faculty. UtlccM University
’ UmcM lMtit”te
In this mnuibution tvm immum%bempeutical awe&es will be discussedthat we presnlly hwaigak in cur gmup and where lipxomes me being used. The first one concam Ihe es,atdishmentof cmxmlkd mkase profiles of cytokiw wi,b liposoms; the seconddells wiul hlmor asociaed amigen pawenmdon(wilh immunestimulams). 1) Ccmmdkd mkase: commlkd releaseof e cytokine is desirable.if e concentrationsare U, be avoided and long exposi,ion is required. hurammoral or peri,umor.xl injection of cyt&h% loaded lipxomes is row under investigationas a way of optimizing local cymkiee therapy. Sina the cytoldw rekese kinetics dependon Ur liposcme fype. anentionshould be paid u) pmpdy designlhcsecytokine ha&d lipxomes. 2) Tumor associati anrigen pmsemmion (wi,b immune’ wimulsnu): Tumor assccived ,ramphmLyjon am&em (TATA) cm be tffeaively prrsenkd on Liposomes.e.g. in 1 lymphosnmma model in mia (SL2 in DBAR mice). The advaruages of Bposome1~ TATA PRsmtafion vehkks over Ur use of irradiakd cells for immunmhawv are: minimizahon of administration of nuckar maedal. kss diludon with inckvam ma&~. pmper chamcmrization nd skrilization pasibi1bk.s. opcimiution of am&n de&w aed, if required. comhination whh immwomcdulatom. As a example Our msuhswhh & SL2 mm& model will be discussed.ikwadng dte possibi1itie.s UI optimize Ihe immune respxse by using a dewgem solubiliufionlpanial purification/vesiculilationapproachin combination whh Ml%m-2 stimuk,ion. l
Par, of Ur Gmningen/U,rech, InstiNP for Drug Exploration,GUIDE
07 ISOLATED LIVER PERFUSION FOR LIVER-SPECIFIC REPLICATION-DEFECTIVE ADENOVIRUS VECTORS
GENE-DELIVERY
WITH
F. Fallnux’. W. de Ro&, A. MarineUi’. A. Lararis-Karauas’. B. Bouwman2.1. Nook.beom’. L. van der Bb’. 0. Te,~s,ra’ m-d R. Hoehen’ ‘De@ Medical Biochcmislry. Sect. Gene Nmpy. Sylvius Laboruories. Leiden Universily. Leiden ‘Ikp General Surgery, Univenity Hospiral. Ieiden University. L&den ‘Immgen BV. Rijswijlc. The Netherlands
gent
Hepk dwapy may otxe offmat lppaling akmadve for &e weahnen, of various inhcriti disotden of bepadc fmxtion such ss funilial hypenholestEmkmia by LDL deficimcy. phenyl Lelonuria. Cringle‘ N&r’s disease. and haemophilia This appmach wxdd involve Ur genetic modiflcatian of (a number of) the patient’s primary liver cells. Two wekgles can be envisaged.On the nr hand. liver cells cm be isolmed. urd gae,icaUy modified u-viva. fokwed by reimplantationof the modified cells. In this approach ule genetic modhicadcmcan be prformed under urehrlly ConuOUedconditions.In addition. Uu gurtic modificadm is suicdy limited m tk isolved c&S: budvermm gene ,mnsfer inI the cells of dx gmmdsthat muld fheoreticelly resti in germ-lint rransmissionof the inh-oduced gem?(s).cm be fully excluded.However. a majar hurdle in the developmentOf u “iw gent therapy stnkgies is the current inabiB,y m achieve efficient grafting of Ur genetiCpUy modified c&s. Furlhennore. wben it is essentialU, main& ,be integrity of the rarget tissue (e.g. ,he liver of a haenmphlliac)ulc geneticmcdiflca,ion has u1 be performedin viva
Currendy. recombinant human adewvimres (rAdV) representm of r&e most powerful v&or-systems for in viva gene delivery. Effkien, adewirus-mediekd in-viva gene uvlsfer has been achieved in several large-mimal medeb. e.g. info the liver of h’aemophilicdogs. However. a major cnrem in ,he in-viva s,r;uegiesis tha ,he vedor gents could ether tbeomtieallv Ihe zermline. and es a cmueaumce. would be inherited by Ilu patient’s Therefore, impmved in viva gene &livety mzrhod.5ore highly &&bIe.‘Iselakd oerfusioe of the liver (ILP) with venorcontairdng buffers may provide such a new method. .With this technique.completevascularisolationoi thF liver is establishedsurgically
offspring.
Poster Preseatations Pl CACO.2 CELLS AS AN IN VfFRO MODEL FOR ADENOVIRAL TO THE GUT EPITHELIUM
GENE TRANSFER
E. Waker’. MA. Cmyk’. B.L. David&m’. BJ. Roesslcr’.I.M. Hilfmge+. G.L. Amidon’ ’ Colkge of Phamwy. 7he UniVemily of Micbigm, Am, Artor. Michigan 48109-1065 1 College of Medicine. Univenily of Iowa, Deparhnem of Internal Medicine. low Chy. Iows~2242
’ Ixpamamt of Inkma! Medicine. Division of Rheumu0logy. Univershy of Michigan Medical Cramer.Arm Arbor. Mkhigm 48109 ’ Tsi?L. lncorporaed. AM Amor. Michigan 48108
Imerkukk 1 (U-1) is dnught to mediate the inflammaloy respase presau in intlammamry bowel diseasz(IBD). The mcem dllry of 1 receptora”Qords, specific for IL-1 i&-Ire) has generaed a gma, deaJof hwres, in this pmcess.espxially becaw U-In can suppmw Uu pm-inflammawy setivity of U-l found in experimental colitis in rabblLp [I]. Ihc discoveryof IL-lra also suggestsP polentinl sh’amgyutilizing gew ,berapy for Vcatmen, of IBD: the urgeled dellvery of remmbinvl, ldermvims encodingIhe IL-It-am gut epithelium. In order to titime gent VMSfer in ,he ieksdnal wet, m adcnoviml vector should have sufickn, smbility u) infect hnestinalcells in vitro in a simulakd hmdnal envimnmen,.Using an ademvims mntiti,,g mC -g&awidase gem from E. coli lhat is specifically tarBet& for the nucleus (Ad.RSVntlocZ), WC show lhnr exposureof ,be adaovims u) gasMim&lul pmhxszs and bile acids only slightly affected Ihe inteftivity of ti virus over Ur ks,i”g period. Fwhermore. variation of ulc luminrl pH in ule range of 5.5 u) 7.4 did IW, ,eed m ctwgca in inIectivity.
06 SPRCIFIC RECOGNITION SITES FOR BINDING DEOXYNUCLEOTIDES ON LIVER CELLS
AND UPTAKE
OF 32P-OLIGO-
E.AL.Biesseo. I+. Vktsch nd Ih1.C. “an Berkel Division of Biophprmreutics. LeidcrJAmskrdamCenkr of Drug Reseat&. L&den Univemity. ll,e Nmbedands
In,roduc,ii Applicaion of antisense deoxymmleotides(ODN) may conaituk- an alumalive clinical appm.wJlfor spccificduymgulmiingur cxpwsiw Of geaK.9.“ourever, the ,herapcutic&xivl,y of ODN mokades is ofkn hnpaind by their susceptibilityu) untimely membolk hucdwica and by d-&r insbili to cmss the cell* membrmc. A nomisinn sua,a?y P circumvent bo,h problems in&s specific tagcting of ODN u) 6-e aimed c& ,yp. & this study, we have studiedmC in viw fee of “P-kbekd ODN in D1s in order to BFS~SS w-r ODN i&elf is pm m tissue-specific accumulation Methods and Resultr np-~DN ~1s rapidly cleated from the blood.sUFM upon inUavery)usinjemion into ram. Appmximakly 45% of Ur injected doseof QP-ODN was reccwemdin the liver within 5 min aR.?rinjecrion Non-parrnchymel liver cells. and Kupffer cells in panicular. v!em responsible for rhe helutic ue,ake of -P-oDN. Hemtic emeke of ‘=P-DDN a,o bc dew the miU; of ur om. as.li~er&kc of +oDN’WU greatly im&red efkr hert-induceddensuation. To elucidak the m&cular basis of Ur liver a&e of ‘+ODN. we have studied ,he intenetiw of “P.ODN with isolated nt endMhelial’(EC) and Kupfler cells KC) using in vitro bioding and u@e ah&?.. “P-0DN biw%,,g u) EC Md KC was sammbk (30.3 acd 26.7 r@& respnivcly) ad of moderae eflinity (Kd=31 snd 117 &I. respeaively). Binding wea ahnol abolishedby preincubating the cells with tqpsine (PC. I5 min) and afkr m acid wash @H 2.8). indicating that binding is mediated by a pmteidic reawdtion she. Binding u) EC and KC could be inhibited for 90% by ODN. polyirmsirdc acid. fucaidin ad nucleodde di- and viphosphaes whereas headenamrakd ODN. polycyddink acid, polyadenosinic acid. s.sDNA. and nucleotide or nuclmtide mompborphak amh%ues were complekk ineffecdve. Sumrisinnly. acuvlated lowdensity-limmm,ein. ee es&shed sutaram &or& scavengerreceptoron;&,helial and Kupffer c&.‘v/~s ,,o, able u) inhibit “P-0DN binding. Uptake Mldks at 37*C demonsaed &a, ligand binding was efficknlly coupled to inkrnalisvion ud lysosomll pm&wing. Moreover. infaft ODN could s6ll be recoveredin Ur lysosomalcampamnen,afw incubationof EC ami KC with “P-0DN.
mci~~
Condusions I, can be concluded,ha endmbelial nnd Kupffer cells pos.sess ODN-specific recognhicmsiks. The inhibition pmfile of 90~~ binding siks on bmb ~eu typs is ewwiauy eqtml: and comspondp closely m uul of the saveeger receptor.The faa mat in viva 9.0~~ is mainly ,ransconed u) mtwxe,xhvmaJ liver cells and tien UD via raumor-mediakd enkr$osis may have impli&ons f& potential in viva applicstionbf antisen& ODN in the regularionof gene expressionof endahelial amdKupffer ccl&spcifk gene pmducta.Leidee University, Leiden lnstiue of Cbemisby, GorlaeusIatommries.
SS
At present,UP is hing anpoyed for !he hnmbepadcadministradonof high dosescbemotherapeuticagentsfor the wearmen,of imesectablecolorecLalliver melastases.We decided m explore u) potential of ILP for the in viw delivery of adetmvirus-based vecu)m. For Uris uwose. we have used adamviral vecu~scawing Ihe firefly-luciferase gene (Ad5:ChIVluc) or & E.coli 5galacmsidasegem (AdS:CMV&&z a Lbe&orexs in ad& W&r rats as the model. We used Ur Ad3:CMVhx vims u) comparethe cfficiencics of gene wansfer u) the liver afkr lLP and afkr inmaportalinfbsion (IPI). Borh ILP and W re.ud,edin cfficien, gene hansfer. h both gmups. equivalent expressionkvds of Uw luciferase repotter gene were obkbxd (2-7 x IO’ light tmits/mg pmtein). However. after IPI significant lucifemse activity lo’-IO’ ligh, unhs/mgpmkin) could alsa be dekckd in mea aher tisams (including kidney. spleen. inlestine. hem,. lung. testis). Although low levels of lucifemse activity could also bc deBned in some of or8ansin numberof aim& after adminis@adonof the vector aAer ILP. the exposureof the odw organsw&i very signhicandy reduced,u) in some casesundetectabk levels. These lesuhs show chatperfusionof a vaswlariy is&ted liver can be used UI achieveliverspecific gm transfer.and considerablyreducesIhe erposumof other tissuesIO ,he vec,ors.
08 LIPOSOMES AND CANCER IMMUNOTHERAPY DJA. Crommelin”. IJ. Bergers’,F. Koppenhagen’.0. Swam’ and W. den O,tc? for Pb&-maEeutical Sckxes. U,recb,*. li,e Ne,berlarv,s. ’ Dutfh N&anal lmtituk for Public Health and Envimnmentll Pmkctior,, ’ Vehxiwrian Faculty. UtlccM University
’ UmcM lMtit”te
In this mnuibution tvm immum%bempeutical awe&es will be discussedthat we presnlly hwaigak in cur gmup and where lipxomes me being used. The first one concam Ihe es,atdishmentof cmxmlkd mkase profiles of cytokiw wi,b liposoms; the seconddells wiul hlmor asociaed amigen pawenmdon(wilh immunestimulams). 1) Ccmmdkd mkase: commlkd releaseof e cytokine is desirable.if e concentrationsare U, be avoided and long exposi,ion is required. hurammoral or peri,umor.xl injection of cyt&h% loaded lipxomes is row under investigationas a way of optimizing local cymkiee therapy. Sina the cytoldw rekese kinetics dependon Ur liposcme fype. anentionshould be paid u) pmpdy designlhcsecytokine ha&d lipxomes. 2) Tumor associati anrigen pmsemmion (wi,b immune’ wimulsnu): Tumor assccived ,ramphmLyjon am&em (TATA) cm be tffeaively prrsenkd on Liposomes.e.g. in 1 lymphosnmma model in mia (SL2 in DBAR mice). The advaruages of Bposome1~ TATA PRsmtafion vehkks over Ur use of irradiakd cells for immunmhawv are: minimizahon of administration of nuckar maedal. kss diludon with inckvam ma&~. pmper chamcmrization nd skrilization pasibi1bk.s. opcimiution of am&n de&w aed, if required. comhination whh immwomcdulatom. As a example Our msuhswhh & SL2 mm& model will be discussed.ikwadng dte possibi1itie.s UI optimize Ihe immune respxse by using a dewgem solubiliufionlpanial purification/vesiculilationapproachin combination whh Ml%m-2 stimuk,ion. l
Par, of Ur Gmningen/U,rech, InstiNP for Drug Exploration,GUIDE
07 ISOLATED LIVER PERFUSION FOR LIVER-SPECIFIC REPLICATION-DEFECTIVE ADENOVIRUS VECTORS
GENE-DELIVERY
WITH
F. Fallnux’. W. de Ro&, A. MarineUi’. A. Lararis-Karauas’. B. Bouwman2.1. Nook.beom’. L. van der Bb’. 0. Te,~s,ra’ m-d R. Hoehen’ ‘De@ Medical Biochcmislry. Sect. Gene Nmpy. Sylvius Laboruories. Leiden Universily. Leiden ‘Ikp General Surgery, Univenity Hospiral. Ieiden University. L&den ‘Immgen BV. Rijswijlc. The Netherlands
gent
Hepk dwapy may otxe offmat lppaling akmadve for &e weahnen, of various inhcriti disotden of bepadc fmxtion such ss funilial hypenholestEmkmia by LDL deficimcy. phenyl Lelonuria. Cringle‘ N&r’s disease. and haemophilia This appmach wxdd involve Ur genetic modiflcatian of (a number of) the patient’s primary liver cells. Two wekgles can be envisaged.On the nr hand. liver cells cm be isolmed. urd gae,icaUy modified u-viva. fokwed by reimplantationof the modified cells. In this approach ule genetic modhicadcmcan be prformed under urehrlly ConuOUedconditions.In addition. Uu gurtic modificadm is suicdy limited m tk isolved c&S: budvermm gene ,mnsfer inI the cells of dx gmmdsthat muld fheoreticelly resti in germ-lint rransmissionof the inh-oduced gem?(s).cm be fully excluded.However. a majar hurdle in the developmentOf u “iw gent therapy stnkgies is the current inabiB,y m achieve efficient grafting of Ur genetiCpUy modified c&s. Furlhennore. wben it is essentialU, main& ,be integrity of the rarget tissue (e.g. ,he liver of a haenmphlliac)ulc geneticmcdiflca,ion has u1 be performedin viva
Currendy. recombinant human adewvimres (rAdV) representm of r&e most powerful v&or-systems for in viva gene delivery. Effkien, adewirus-mediekd in-viva gene uvlsfer has been achieved in several large-mimal medeb. e.g. info the liver of h’aemophilicdogs. However. a major cnrem in ,he in-viva s,r;uegiesis tha ,he vedor gents could ether tbeomtieallv Ihe zermline. and es a cmueaumce. would be inherited by Ilu patient’s Therefore, impmved in viva gene &livety mzrhod.5ore highly &&bIe.‘Iselakd oerfusioe of the liver (ILP) with venorcontairdng buffers may provide such a new method. .With this technique.completevascularisolationoi thF liver is establishedsurgically
offspring.
Poster Preseatations Pl CACO.2 CELLS AS AN IN VfFRO MODEL FOR ADENOVIRAL TO THE GUT EPITHELIUM
GENE TRANSFER
E. Waker’. MA. Cmyk’. B.L. David&m’. BJ. Roesslcr’.I.M. Hilfmge+. G.L. Amidon’ ’ Colkge of Phamwy. 7he UniVemily of Micbigm, Am, Artor. Michigan 48109-1065 1 College of Medicine. Univenily of Iowa, Deparhnem of Internal Medicine. low Chy. Iows~2242
’ Ixpamamt of Inkma! Medicine. Division of Rheumu0logy. Univershy of Michigan Medical Cramer.Arm Arbor. Mkhigm 48109 ’ Tsi?L. lncorporaed. AM Amor. Michigan 48108
Imerkukk 1 (U-1) is dnught to mediate the inflammaloy respase presau in intlammamry bowel diseasz(IBD). The mcem dllry of 1 receptora”Qords, specific for IL-1 i&-Ire) has generaed a gma, deaJof hwres, in this pmcess.espxially becaw U-In can suppmw Uu pm-inflammawy setivity of U-l found in experimental colitis in rabblLp [I]. Ihc discoveryof IL-lra also suggestsP polentinl sh’amgyutilizing gew ,berapy for Vcatmen, of IBD: the urgeled dellvery of remmbinvl, ldermvims encodingIhe IL-It-am gut epithelium. In order to titime gent VMSfer in ,he ieksdnal wet, m adcnoviml vector should have sufickn, smbility u) infect hnestinalcells in vitro in a simulakd hmdnal envimnmen,.Using an ademvims mntiti,,g mC -g&awidase gem from E. coli lhat is specifically tarBet& for the nucleus (Ad.RSVntlocZ), WC show lhnr exposureof ,be adaovims u) gasMim&lul pmhxszs and bile acids only slightly affected Ihe inteftivity of ti virus over Ur ks,i”g period. Fwhermore. variation of ulc luminrl pH in ule range of 5.5 u) 7.4 did IW, ,eed m ctwgca in inIectivity.