- Email: [email protected]
783. Systemic Comparison of Promoters Driving Transgene Expression in Airway Epithelia Using a Non-Primate Lentiviral Vector
781. The DNA-Binding Specificity of Designer Zinc Finger Nucleases Is a Major Determinant of Activity and Genotoxicity in Human Cells
Tatjana I. Cornu, I Stacey Thibodcau-Bcgannyj Eva Guhl, I Stephen Alwin; Magdalena Eichtinger,' J. Keith Joung,' Toni
Cathomen.'
'Charite Medical School. Institute 0/ Virology (CBFJ, Berlin, Germany; lMassaclmsells General Hospital, Molecular Pathology Unit, Charlestown. MA.
The engineering of proteins to manipulate cell genomes at will has developed intoa promisingtechnology for biomedicalresearch, including gene therapy, In particular, zinc fingernucleases (ZFNs), which consistof a non-specific DNAcleavagedomaintetheredto an engineeredzinc fingerDNA-bindingdomain, have provenvaluable for stimulatinghomologousrecombinationin a varietyof cell types. Previous studies have demonstrated that ZFNs can be associated with significantcytotoxicity, which has been hypothesizedto be due to unintended cleavage at off-target sites. Here, we compared the in vitro affinitiesand specificitiesof various engineered zinc finger DNA-binding domains with their performance when expressed as ZFNs in human cells. Using cell-based recombination and genotoxicity assays, we assessed nine different zinc finger domains that recognizetargetsiteseither in the BCR-ABLtranslocation sequence, the erbB2 locus, or the HIV-I promoter. Our results reveal a good correlation between DNA-bindingspecificity- rather than affinity - and ZFN-induced genotoxicity, Explicitly, we found that ZFNs containing highly specific DNA-binding domains exhibited both superior performance in stimulating homology-directed correction of an integratedmarkergene and significantlyreducedgenotoxicity. Furthermore, we show that a bacterial reporter assay ean reliably identity DNA-binding domains with excellent affinities and specificities. In summary, these data demonstrate that the specificity of DNA-bindingis a main determinantofZFN-associatedgenotoxicity and reveal the utility of a bacterial cell-based assay for identifying candidatezinc fingerdomainsto beexpressedas ZFNs intherapeutic applications. RNA VIRUS VECTORS: NOVEL VECTORS AND APPLICATIONS
782. Characterizing the Molecular Mechanism for Robust, Sustained Hepatic Expression from Episomal Lentiviral Vectors
Matthew A. Bayer,' Brian Zeithaml,I Boris Kantor,' Thomas MeCown.' Hong Ma, I Tal Kafri.' 'Gene Therapy Center; University ofNorth Carolina-Chapel Hill. Chapel Hill, NC; 'Department 0/Psychiatry. University ofNorth Carolina-Chapel Hill. Chapel Hill, NC.
Recentdevelopments have underscoredthe danger of insertional mutagenesis in elincal gene-therapy trials using retroviral vectors. Nonintegrating retroviral and Icntiviral vectors otTer a way to minimize this risk; however, unintegrated lentiviral and retroviral episomes may be subject to a variety of endogenous chromatinsilencing factors, which may contribute to the reduced expression seen in nonintegrating vectors. As previous results indicated that shorter U3 sequences in the vector genome's LTR correlate with increasedepisomal expression,we made a nonintegrating lentiviral vector with a U3 deletion much larger than the standard deletion seen in self-inactivating (SIN) vectors (I), Cultured mammalian cells transduced with this vector exhibited elevated episomal expression from the U3-deletion vector over its SIN parental vector. Furthermore, the U3-deletionvector generated significantlygreater episomal expression than did its SIN parental vector when transducing the rat brain, and also redirected the vector's tropism from Molecular Therapy Volume15. Supplement I. ~ "'r 2007
Copyright © T he American Soci ety o r G ene Therapy
glial cells to neurons. Most importantly, we showed for the first time that the U3-deletion vector yielded a strong upregulation of episomal expression in the mouse liver,and the increase in expression remained robust over 45 days, allaying concerns of reduced expression in the liver from dilution or inhibitory immuneresponse (2). To address the possibility that the increase in episomal expression seen from U3-deletion vectors corresponds to a change in episomal structure, we examined the relative abundances of the variousepisomal forms(linear episomes, I-LTRcircular episomes, 2-LTRcircular episornes, and mutant circular episomes) present in transduced cultured cells. In vitro analysis by Southern blotting and shuttle-vectoranalysisof episome formation in cells transduced with the U3-deletionvector revealed little variance fromthat of the parental vector. Next, we explored the possibility that the notable increase in episomal expression in vivo reflects differences in episome formation secondary to the fact that the targeted cell types, ineluding neurons and hepatocytes, are in G I or GO phase of the cell cycle. We found that episome formation was equivalent in cycling and Gl-arrested cells, and that a variety of cell lines deficient in homologous recombination, including Xrcc3, ATM, and Ercc1, exhibited thc normal ratio of episome forms. These data suggest that nonintegrating lentiviral vectorsarea safe and efficacious means of delivering and robustly expressing therapeutic transgencs and provides new insight on the formation of I-LTR circular lentiviral vectorepisomes.Acknowledgments: Wethank R.S. Nairn and L.I-I. Thompson for providing cell lines. The study was supported by the UNC Center for AIDS Research developmental core. References I. Miyoshi I-I et al. 1998. 1. Virol. 72(1998), pp. 8150-8157. 2. Brown BD et al. 2006. Blood. Dec. 19.
783. SystemiC Comparison of Promoters Driving Transgene Expression in Airway Epithelia Using a Non-Primate Lentiviral Vector Erin R. Burnight,' Patrick L. Sinn,' Paul B. Mcf'ray,' I Pediatrics, University 0/iowa, iowa City.
Gene transfer using viral vectors has potential as a therapy for many genetic disorders ineluding cystic fibrosis (CF). The major cause of morbidity and mortality in CF patients is chronic inflammation and bacterial infectionin the airways. Thus, gene transferof the Cystic FibrosisTransmembraneConductanceRegulator(CFTR) to the airway epithelia is a goal of many gene therapy strategies to treat the disease. Lentiviral gene transfer offers advantages over other viral vectors for CFTR gene transfer. Lentiviruses can transduce a variety of cell types in both quiescent and dividing cells. Additionally, they integrate into the host genome to achieve stable and persistent expression. However, there are some barriers to successful gene transfer to respiratory epithelia using lentiviral vectors, ineluding expression in non-epithelial cell types and low transgeneexpressionlevels.The development oflentiviral constructs with promoters of varying strengths and cell specificity would aid in our selection of optimal reagents for CFTR gene transfer. In the present study we contrasted several constructs in the context of a FIV lentiviral vector. Viral promoters such as Cytomegalovirus (CMV) confer strong expression. Additionally, the Jaagsiekte Sheep Respiratory Virus promoter is hypothesized to drive strong expression in the lungas its native target is respiratoryepithelia. We have previouslydemonstratedthat the RSV promoterhas the ability to persist up to one year post transduction in vivo following nasal deliveryin mice. The RSVpromoterservesas a basisof comparison. In addition to viral promoters that drive expression ubiquitiously, it would be beneficial to generate vectors with promoters that are active specifically in the airway epithelia. Using transcript profiling in human airway epithelia we found that two members of the Palate, Lung, Nasal Epithelium Clone (PLUNC) family are highly expressed in human airway epithelia. We hypothesize that these S301
promoters-PLUNC and Long Palate, Lung, Nasal Epithelium Clone (LPLUNCI}-will confer sufficientand persistent activity in the airway. Furthermore, to restrict expression to epithelia we aim to use the cell-type specific promoters FOX]) (specific to ciliated cells) and E-Cadherin (specific to epithelia) in the FIV vector. We constructed FIV vectors containing the aforementioned internal promoters driving the firefly lueiferase reporter gene. Using these vectors we transduced murine nasal epithelia in vivo and monitored reporter activity using bioluminescence imaging. In addition, we evaluated promoter activity in vitro using plasmidscontaining various promotersdriving luciferaseexpression. In experimentsto date, luciferase reporter activity from the JSRVand CMV promoters was higher than reporter activity from the cell-type specific E-Cadherin and FOXJI promoters in vivo. Additionally, in vitro data suggest that the PLUNC promoter drives high reporter expression. Determining the persistence and level ofexpression from ubiquitous and tissue specific promoters will aid in developing optimal vectors for sufficient, sustained transgene expression in the airway epithelia. This work is supported by NIH HL075363 and HL5I670.
784. Differential Regulation of Interferon AlphaResponsive Antiviral Pathways Determines Tumor Selectivity of Newcastle Disease Virus
Subbiah Elankumaran.P Daniel D. Rockemann,' Siba K. Samal.' I Virginia-Maryland Regional College ofVeterinary Medicine, University ofMaryland, College Park, MD; 1/1rginia-Maryland Regional College ofVeterinary Medicine, Virginia Polytechnic Institute and State University. Blacksburg, VA.
Newcastle disease virus (NOV), an avian paramyxovirus, is a promising broad-spectrum oncolytic agent with a high therapeutic index. However, the mechanisms governing the permissiveness of tumor cells to NOV infection remain to be fully characterized. Tumor selectivity of oncolytic viruses is ascribed to the expression ofcomponents ofthe constitutively activated ras and/or inhibited or response pathways or dysregulated alpha/beta interferon (IFN-a/~) a deficiency of p53. We hypothesized that tumor selective replication and spread ofNOV is dependent on the differential regulation of type IIFN antiviral pathways. lf'so, NOV with a defect in type I IFN antagonism should fail to replicate and spread in normal human cells as against tumor cells. Further, viruses that have the capability to replicate and spread by syncytium formation without the need forspecific proteases should be able to replicate and spread better in human cells. To analyze this, we used recombinant NOV (rNDV) strains with the following properties (a) a low-pathogenic strain to the natural host (chickens) with a mutation in the fusion (F) protein to allow replication and spread in cell culture without exogenous trypsin (rLaSota V.F.), (b) a moderately pathogenic strain to chickens with intact IFN antagonistic function (rBC), (c) a moderately pathogenic strain to chickens with a defect in IFN antagonism (rBC-edit), and (d) a moderately pathogenic strain to chickens expressing enhanced green fluorescent protein (rBCEGFP). Weused a combination of assays to study the cytotoxicity, virus replication and spread of these viruses in human tumor cells of ccto-, cndo-, and mesodermal origin. In most of the tumor cells, virus spread and replicationofrNDV occurred with release of'infectious progeny while in normal human cells, virus replication was restricted.The rBC-edit virus with a defect in interferonantagonism induced a robust IFN response and failed to grow in normal human cells, but grew to very high titers in tumor cells that lack an IFN- a was produced in virus infected response. Upon infection, IFN-~ normal human and tumor cells at comparable levels with no effect on virus replication by all rNDV. But, the tumor selective replication ofrNDV was found to be mainly dependent on the differential regulation ofIFN-a mediated antiviral pathways: In normal human cells, the robust IFN-a mediated antiviral responses limited virus S302
replication, while in tumor cells, the inability to mount an effective IFN-a mediated responses permitted virus replication. Differential regulation of IFN-a and downstream antiviral genes induced by IFN-a after infection, including differential activation of STAT genes, STAT independent 2'5 'A, and p38 mitogen-activated protein kinase-mediated antiviral pathways determined the outcome of rNDV infection in normal and tumor cells. Further, the failure to activate IRF-7, a master regulator of type IIFN, in tumor cells, may be a key mechanistic event permitting tumor selectivity and oncolysis by NOV.
785. Lentiviral Protein Transduction and Intracellular PIC Visualization by Vector Incorporated HIV-1 Integ rase Fusion Proteins
Diana A. M. Schenkwein.F Vesa P.Turkki,'> Hanna P. Lesch," KarkkainenA. Hanna-Riikka.F Mahonen J. Anssi.P Airenne J. Karl,' Yla-Herttuala Seppo.P:' I Department ofBiotechnology and Molecular Medicine, Al.Vlnstttute, University ofKuopio, Kuopio, Finland; lArk Therapeutics Oy. Ark Therapeutics Oy. Kuopio, Finland; 'Department ofMedicine and Gene Therapy Unit, UniversityofKuopio, Kuopio, Finland; "University Hospital Kuopio, University Hospital Kuopio, Kuopio, Finland. Adetailedunderstanding ofthe lifecycle of HIV-I based lentivirus vectors is an importantstarting point in designing futuregenerations of new lentivirus vectors. Differently labeled Hlv-l virions have provided a method to visually examine the interactions between the virus and target cell during various steps of infection. With the aim of visualizing intracellular traffickingof third generation lentivirus vector prcintegration eomplcxcs (PICs), wc have developed a fluorescently labeled I-IIV-I integrase(IN) fusion protein that is directly incorporated into vector particles devoid of all unnecessary viral proteins such as Vpr. IN was fused to mCherry, which was eloned into the vector packaging plasmid C-terminal to the IN open reading frame.The IN-mCherryfusionsare thus incorporatedinto thc newly forming vector particles as parts of the large gag-pol-polyprotein and become correctly proecsscd at the natural reverse transcriptasc (KOliN interphase by the viral protease. Vectorswere produced by a standard four plasmid transfection of 2931' cells and verified for the correct packaging ofthe IN fusionprotein by western blots. p24titers ofthe vectors were comparable to control vectors bearing a wt IN, but the functionaltiters of'the IN-mCherryvectorssuITered from a slight decrease. IntracellularIN-mCherrycontainingparticleswere detected in infectedcells by confocal microscopy.The functionality of the fusion proteins to catalyze vector integration was verified by cell culture assays using HeLa and 2931' cells. Our method is thus an effective means to incorporate foreign proteins into lentiviral vectors independent ofthe Vpr dependent trans-packaging method formerly used for the same objective.
786. Optimizing Systemic Delivery of Oncolytic Measles Virus by Using Virus Infected Cell Carriers Kah-Whye Peng,' Hooi T Ong,' Allan B. Dietz,' Stephen J. Russell.1 lMolecular Medicine Program, Mayo Clinic College ofMedicine, Rochester, MN; "Iransfusion Medicine, Mayo Clinic College of Medicine, Rochester, MN.
Live attenuated measles virus (MV) has potent and tumor-selective oncolytic activity against a variety cancer cells. It causes extensive intercellular fusion between the infected cell and the neighboring cells, greatly increasing the bystander killing of the virus. Two recombinant MVs expressing monitoring proteins are being evaluated for intraperitoneal, intratumoral or intravenous adMolecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C AmericanSocietyo f Gene Tllcr.lpr
Tatjana I. Cornu, I Stacey Thibodcau-Bcgannyj Eva Guhl, I Stephen Alwin; Magdalena Eichtinger,' J. Keith Joung,' Toni
Cathomen.'
'Charite Medical School. Institute 0/ Virology (CBFJ, Berlin, Germany; lMassaclmsells General Hospital, Molecular Pathology Unit, Charlestown. MA.
The engineering of proteins to manipulate cell genomes at will has developed intoa promisingtechnology for biomedicalresearch, including gene therapy, In particular, zinc fingernucleases (ZFNs), which consistof a non-specific DNAcleavagedomaintetheredto an engineeredzinc fingerDNA-bindingdomain, have provenvaluable for stimulatinghomologousrecombinationin a varietyof cell types. Previous studies have demonstrated that ZFNs can be associated with significantcytotoxicity, which has been hypothesizedto be due to unintended cleavage at off-target sites. Here, we compared the in vitro affinitiesand specificitiesof various engineered zinc finger DNA-binding domains with their performance when expressed as ZFNs in human cells. Using cell-based recombination and genotoxicity assays, we assessed nine different zinc finger domains that recognizetargetsiteseither in the BCR-ABLtranslocation sequence, the erbB2 locus, or the HIV-I promoter. Our results reveal a good correlation between DNA-bindingspecificity- rather than affinity - and ZFN-induced genotoxicity, Explicitly, we found that ZFNs containing highly specific DNA-binding domains exhibited both superior performance in stimulating homology-directed correction of an integratedmarkergene and significantlyreducedgenotoxicity. Furthermore, we show that a bacterial reporter assay ean reliably identity DNA-binding domains with excellent affinities and specificities. In summary, these data demonstrate that the specificity of DNA-bindingis a main determinantofZFN-associatedgenotoxicity and reveal the utility of a bacterial cell-based assay for identifying candidatezinc fingerdomainsto beexpressedas ZFNs intherapeutic applications. RNA VIRUS VECTORS: NOVEL VECTORS AND APPLICATIONS
782. Characterizing the Molecular Mechanism for Robust, Sustained Hepatic Expression from Episomal Lentiviral Vectors
Matthew A. Bayer,' Brian Zeithaml,I Boris Kantor,' Thomas MeCown.' Hong Ma, I Tal Kafri.' 'Gene Therapy Center; University ofNorth Carolina-Chapel Hill. Chapel Hill, NC; 'Department 0/Psychiatry. University ofNorth Carolina-Chapel Hill. Chapel Hill, NC.
Recentdevelopments have underscoredthe danger of insertional mutagenesis in elincal gene-therapy trials using retroviral vectors. Nonintegrating retroviral and Icntiviral vectors otTer a way to minimize this risk; however, unintegrated lentiviral and retroviral episomes may be subject to a variety of endogenous chromatinsilencing factors, which may contribute to the reduced expression seen in nonintegrating vectors. As previous results indicated that shorter U3 sequences in the vector genome's LTR correlate with increasedepisomal expression,we made a nonintegrating lentiviral vector with a U3 deletion much larger than the standard deletion seen in self-inactivating (SIN) vectors (I), Cultured mammalian cells transduced with this vector exhibited elevated episomal expression from the U3-deletion vector over its SIN parental vector. Furthermore, the U3-deletionvector generated significantlygreater episomal expression than did its SIN parental vector when transducing the rat brain, and also redirected the vector's tropism from Molecular Therapy Volume15. Supplement I. ~ "'r 2007
Copyright © T he American Soci ety o r G ene Therapy
glial cells to neurons. Most importantly, we showed for the first time that the U3-deletion vector yielded a strong upregulation of episomal expression in the mouse liver,and the increase in expression remained robust over 45 days, allaying concerns of reduced expression in the liver from dilution or inhibitory immuneresponse (2). To address the possibility that the increase in episomal expression seen from U3-deletion vectors corresponds to a change in episomal structure, we examined the relative abundances of the variousepisomal forms(linear episomes, I-LTRcircular episomes, 2-LTRcircular episornes, and mutant circular episomes) present in transduced cultured cells. In vitro analysis by Southern blotting and shuttle-vectoranalysisof episome formation in cells transduced with the U3-deletionvector revealed little variance fromthat of the parental vector. Next, we explored the possibility that the notable increase in episomal expression in vivo reflects differences in episome formation secondary to the fact that the targeted cell types, ineluding neurons and hepatocytes, are in G I or GO phase of the cell cycle. We found that episome formation was equivalent in cycling and Gl-arrested cells, and that a variety of cell lines deficient in homologous recombination, including Xrcc3, ATM, and Ercc1, exhibited thc normal ratio of episome forms. These data suggest that nonintegrating lentiviral vectorsarea safe and efficacious means of delivering and robustly expressing therapeutic transgencs and provides new insight on the formation of I-LTR circular lentiviral vectorepisomes.Acknowledgments: Wethank R.S. Nairn and L.I-I. Thompson for providing cell lines. The study was supported by the UNC Center for AIDS Research developmental core. References I. Miyoshi I-I et al. 1998. 1. Virol. 72(1998), pp. 8150-8157. 2. Brown BD et al. 2006. Blood. Dec. 19.
783. SystemiC Comparison of Promoters Driving Transgene Expression in Airway Epithelia Using a Non-Primate Lentiviral Vector Erin R. Burnight,' Patrick L. Sinn,' Paul B. Mcf'ray,' I Pediatrics, University 0/iowa, iowa City.
Gene transfer using viral vectors has potential as a therapy for many genetic disorders ineluding cystic fibrosis (CF). The major cause of morbidity and mortality in CF patients is chronic inflammation and bacterial infectionin the airways. Thus, gene transferof the Cystic FibrosisTransmembraneConductanceRegulator(CFTR) to the airway epithelia is a goal of many gene therapy strategies to treat the disease. Lentiviral gene transfer offers advantages over other viral vectors for CFTR gene transfer. Lentiviruses can transduce a variety of cell types in both quiescent and dividing cells. Additionally, they integrate into the host genome to achieve stable and persistent expression. However, there are some barriers to successful gene transfer to respiratory epithelia using lentiviral vectors, ineluding expression in non-epithelial cell types and low transgeneexpressionlevels.The development oflentiviral constructs with promoters of varying strengths and cell specificity would aid in our selection of optimal reagents for CFTR gene transfer. In the present study we contrasted several constructs in the context of a FIV lentiviral vector. Viral promoters such as Cytomegalovirus (CMV) confer strong expression. Additionally, the Jaagsiekte Sheep Respiratory Virus promoter is hypothesized to drive strong expression in the lungas its native target is respiratoryepithelia. We have previouslydemonstratedthat the RSV promoterhas the ability to persist up to one year post transduction in vivo following nasal deliveryin mice. The RSVpromoterservesas a basisof comparison. In addition to viral promoters that drive expression ubiquitiously, it would be beneficial to generate vectors with promoters that are active specifically in the airway epithelia. Using transcript profiling in human airway epithelia we found that two members of the Palate, Lung, Nasal Epithelium Clone (PLUNC) family are highly expressed in human airway epithelia. We hypothesize that these S301
promoters-PLUNC and Long Palate, Lung, Nasal Epithelium Clone (LPLUNCI}-will confer sufficientand persistent activity in the airway. Furthermore, to restrict expression to epithelia we aim to use the cell-type specific promoters FOX]) (specific to ciliated cells) and E-Cadherin (specific to epithelia) in the FIV vector. We constructed FIV vectors containing the aforementioned internal promoters driving the firefly lueiferase reporter gene. Using these vectors we transduced murine nasal epithelia in vivo and monitored reporter activity using bioluminescence imaging. In addition, we evaluated promoter activity in vitro using plasmidscontaining various promotersdriving luciferaseexpression. In experimentsto date, luciferase reporter activity from the JSRVand CMV promoters was higher than reporter activity from the cell-type specific E-Cadherin and FOXJI promoters in vivo. Additionally, in vitro data suggest that the PLUNC promoter drives high reporter expression. Determining the persistence and level ofexpression from ubiquitous and tissue specific promoters will aid in developing optimal vectors for sufficient, sustained transgene expression in the airway epithelia. This work is supported by NIH HL075363 and HL5I670.
784. Differential Regulation of Interferon AlphaResponsive Antiviral Pathways Determines Tumor Selectivity of Newcastle Disease Virus
Subbiah Elankumaran.P Daniel D. Rockemann,' Siba K. Samal.' I Virginia-Maryland Regional College ofVeterinary Medicine, University ofMaryland, College Park, MD; 1/1rginia-Maryland Regional College ofVeterinary Medicine, Virginia Polytechnic Institute and State University. Blacksburg, VA.
Newcastle disease virus (NOV), an avian paramyxovirus, is a promising broad-spectrum oncolytic agent with a high therapeutic index. However, the mechanisms governing the permissiveness of tumor cells to NOV infection remain to be fully characterized. Tumor selectivity of oncolytic viruses is ascribed to the expression ofcomponents ofthe constitutively activated ras and/or inhibited or response pathways or dysregulated alpha/beta interferon (IFN-a/~) a deficiency of p53. We hypothesized that tumor selective replication and spread ofNOV is dependent on the differential regulation of type IIFN antiviral pathways. lf'so, NOV with a defect in type I IFN antagonism should fail to replicate and spread in normal human cells as against tumor cells. Further, viruses that have the capability to replicate and spread by syncytium formation without the need forspecific proteases should be able to replicate and spread better in human cells. To analyze this, we used recombinant NOV (rNDV) strains with the following properties (a) a low-pathogenic strain to the natural host (chickens) with a mutation in the fusion (F) protein to allow replication and spread in cell culture without exogenous trypsin (rLaSota V.F.), (b) a moderately pathogenic strain to chickens with intact IFN antagonistic function (rBC), (c) a moderately pathogenic strain to chickens with a defect in IFN antagonism (rBC-edit), and (d) a moderately pathogenic strain to chickens expressing enhanced green fluorescent protein (rBCEGFP). Weused a combination of assays to study the cytotoxicity, virus replication and spread of these viruses in human tumor cells of ccto-, cndo-, and mesodermal origin. In most of the tumor cells, virus spread and replicationofrNDV occurred with release of'infectious progeny while in normal human cells, virus replication was restricted.The rBC-edit virus with a defect in interferonantagonism induced a robust IFN response and failed to grow in normal human cells, but grew to very high titers in tumor cells that lack an IFN- a was produced in virus infected response. Upon infection, IFN-~ normal human and tumor cells at comparable levels with no effect on virus replication by all rNDV. But, the tumor selective replication ofrNDV was found to be mainly dependent on the differential regulation ofIFN-a mediated antiviral pathways: In normal human cells, the robust IFN-a mediated antiviral responses limited virus S302
replication, while in tumor cells, the inability to mount an effective IFN-a mediated responses permitted virus replication. Differential regulation of IFN-a and downstream antiviral genes induced by IFN-a after infection, including differential activation of STAT genes, STAT independent 2'5 'A, and p38 mitogen-activated protein kinase-mediated antiviral pathways determined the outcome of rNDV infection in normal and tumor cells. Further, the failure to activate IRF-7, a master regulator of type IIFN, in tumor cells, may be a key mechanistic event permitting tumor selectivity and oncolysis by NOV.
785. Lentiviral Protein Transduction and Intracellular PIC Visualization by Vector Incorporated HIV-1 Integ rase Fusion Proteins
Diana A. M. Schenkwein.F Vesa P.Turkki,'> Hanna P. Lesch," KarkkainenA. Hanna-Riikka.F Mahonen J. Anssi.P Airenne J. Karl,' Yla-Herttuala Seppo.P:' I Department ofBiotechnology and Molecular Medicine, Al.Vlnstttute, University ofKuopio, Kuopio, Finland; lArk Therapeutics Oy. Ark Therapeutics Oy. Kuopio, Finland; 'Department ofMedicine and Gene Therapy Unit, UniversityofKuopio, Kuopio, Finland; "University Hospital Kuopio, University Hospital Kuopio, Kuopio, Finland. Adetailedunderstanding ofthe lifecycle of HIV-I based lentivirus vectors is an importantstarting point in designing futuregenerations of new lentivirus vectors. Differently labeled Hlv-l virions have provided a method to visually examine the interactions between the virus and target cell during various steps of infection. With the aim of visualizing intracellular traffickingof third generation lentivirus vector prcintegration eomplcxcs (PICs), wc have developed a fluorescently labeled I-IIV-I integrase(IN) fusion protein that is directly incorporated into vector particles devoid of all unnecessary viral proteins such as Vpr. IN was fused to mCherry, which was eloned into the vector packaging plasmid C-terminal to the IN open reading frame.The IN-mCherryfusionsare thus incorporatedinto thc newly forming vector particles as parts of the large gag-pol-polyprotein and become correctly proecsscd at the natural reverse transcriptasc (KOliN interphase by the viral protease. Vectorswere produced by a standard four plasmid transfection of 2931' cells and verified for the correct packaging ofthe IN fusionprotein by western blots. p24titers ofthe vectors were comparable to control vectors bearing a wt IN, but the functionaltiters of'the IN-mCherryvectorssuITered from a slight decrease. IntracellularIN-mCherrycontainingparticleswere detected in infectedcells by confocal microscopy.The functionality of the fusion proteins to catalyze vector integration was verified by cell culture assays using HeLa and 2931' cells. Our method is thus an effective means to incorporate foreign proteins into lentiviral vectors independent ofthe Vpr dependent trans-packaging method formerly used for the same objective.
786. Optimizing Systemic Delivery of Oncolytic Measles Virus by Using Virus Infected Cell Carriers Kah-Whye Peng,' Hooi T Ong,' Allan B. Dietz,' Stephen J. Russell.1 lMolecular Medicine Program, Mayo Clinic College ofMedicine, Rochester, MN; "Iransfusion Medicine, Mayo Clinic College of Medicine, Rochester, MN.
Live attenuated measles virus (MV) has potent and tumor-selective oncolytic activity against a variety cancer cells. It causes extensive intercellular fusion between the infected cell and the neighboring cells, greatly increasing the bystander killing of the virus. Two recombinant MVs expressing monitoring proteins are being evaluated for intraperitoneal, intratumoral or intravenous adMolecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C AmericanSocietyo f Gene Tllcr.lpr