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471. Chimeric Lentiviral Vectors with Safety Insurance
RNA VIRUS VECTORS I 469. Optimization of the Lentivirus Vector α Double Containing the MSCV-U3 and EF1α Internal Promoters
470. ABCB4 P-Glycoprotein Reduces Infectivity of Lentiviral Particles by Increasing Their Phosphatidylcholine Content
Naoya Uchida,1,2 Hideki Hanawa,1 Motoko Yamamoto,1 Takashi Shimada.1 1 Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2The Third Department of Internal Medicine, Nippon Medical School, Tokyo, Japan.
Niek P. van Til, Kirstin Heutinck, Ronald P. J. Oude Elferink, Jurgen Seppen. 1 AMC Liver Center, Academic Medical Center, Amsterdam, Netherlands.
The double gene vector carrying the expression units of the therapeutic gene and the reporter gene is widely used, since the reporter proteins such as the GFP and the DsRed are useful for measuring the titer of vector stock and the tracing gene modified cells. However, the activities of the double promoters in a single vector are often inhibited by the so called ‘promoter interference’. The internal ribosomal entry site (IRES) is being utilized to overcome this problem, but the expression of the second gene is not sufficient for the sensitive detection. In this study, we attempted to optimize the double gene lentivirus vector to achieve a high expression level of both genes. We generated a series of double gene lentivirus vector constructed of the MSCV-U3 promoter (Mp), the EF1α promoter (Ep), GFP cDNA (GFP), and DsRed cDNA (DsRed) in various configurations. In the first experiment, 293T cells were transduced with these lentivirus vectors and the expression levels of the two genes were monitored by the Northern blot analysis. A quantitative analysis of the Northern blots revealed that the Ep was relatively stable irrespective of its position, while the Mp activity was strongly inhibited when placed in the second position. In the second experiment, we further modified the Mp-GFP-Ep-DsRed vector to improve the expression level of the first gene. The flow cytometric analysis of transduced HeLa cells showed that the insertion of the 250 bp core insulator element (IR) upstream of the second Ep promoter (Mp-GFP-IR-Ep-DsRed) and the insertion of the 356 bp cytomegalovirus enhancer element (CMVe) upstream of the first Mp (CMVe-Mp-GFP-Ep-DsRed), in both vectors, significantly increased the GFP expression. The efficient expression of the double genes in this configuration was also observed when the GFP was replaced by a BMP2 or PTHR. These results indicated that optimization of the double promoter vector was important for the efficient gene expression. The combination of the CMVe-Mp and the IR-Ep promoters in this order may be useful for the tissue nonspecific expression of two genes.
Human Immunodeficiency Virus-1 (HIV-1) particles are assembled and released from membrane microdomains (lipid rafts). The membrane composition of the viral particle, which is important for viral integrity and infectivity, is therefore determined by the raft lipid composition. ATP binding cassette (ABC) transporters are transmembrane proteins, which require ATP to transport a broad array of compounds through membranes. The ABC transporter ABCB4 (MDR3 Pglycoprotein) is a phospholipid floppase that translocates phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of the cellular membrane. ABCB4 is located in the apical membrane of hepatocytes; translocation of PC by ABCB4 promotes PC release into bile, which neutralizes the toxic effect of bile salts. Lack of ABCB4 expression leads to the severe liver disease Progressive Familial Intrahepatic Cholestasis type 3 (PFIC3). In order to develop gene therapy for this disease, we created a third generation HIV-1 lentiviral vector with a cassette consisting of ABCB4 cDNA driven by a phosphoglycerokinase (PGK) promoter. Our attempts at producing high titer (>1 x 105 transducing units per mL) lentiviral ABCB4 expression vector were unsuccessful. Because during lentiviral vector production ABCB4 is also expressed, we proposed that the PC floppase activity of ABCB4 might have affected lentiviral vector production negatively. To test this, we co-expressed ABCB4 during GFP lentiviral vector production. We observed that co-expression of ABCB4 inhibited viral infectivity, but the production of viral particles was not affected. Co-expression of an inactive ABCB4 mutant did not reduce viral infectivity. In addition, the ratio of phosphatidylcholine to cholesterol of lentiviral particles increased significantly from 0.4 in producer cells expressing no ABCB4 or a mutant ABCB4 to 1.3 if the wildtype ABCB4 protein was co-expressed. We therefore conclude that the specific translocase activity of ABCB4 can affect viral infectivity negatively by changing the lipid membrane composition of the viral particles. This has implications for the strategy of development of a successful lentiviral gene therapy system for the treatment of PFIC3.
471. Chimeric Lentiviral Vectors with Safety Insurance Kritika Kachapati,1 Geetanjali Sachdeva,1 Suresh Arya.1,2 Center for Cancer Research, National Cancer Institute, Bethesda, MD; 2Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD. 1
Lentiviral vectors as presently designed are considered to be safe. No evidence for the generation of replication competent viruses has emerged for these vectors when transduced in vitro into cultured cells. Their behavior in vivo remains to be ascertained. Their propensity to cause insertional mutagenesis, if any, is also unknown. One major safety concern is the recombination between the components of the vector system during vector production in the laboratory and with the resident virus in the clinic during therapy of individuals with apparent or non-apparent HIV infection. As an additional safety insurance, we have created chimeric or hybrid lentiviral vectors derived from HIV-1 and HIV-2. This was done for both transfer vectors and packaging constructs. Our premise was that HIV-1 and HIV-2 are dissimilar enough in sequence to curtail recombination, but similar enough to functionally complement. Our S182
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
RNA VIRUS VECTORS I initial studies were done with HIV-2 vectors lacking the central polypurine track (PPT) and the woodchuck post-transcriptional enhancer element (WPRE), two regulatory elements reported to enhance transduction efficiency of HIV-1 vectors. This study includes HIV-2 vectors with these elements. A non-reciprocal interaction between conventional HIV-1 and HIV-2 vectors was observed in cross-packaging experiments. The titer of HIV-2 vectors was roughly the same whether packaged with the HIV-1 or HIV-2 packaging construct when titrated in cells in culture. In contrast, the titer of the HIV-1 vectors was twenty to thirty folds lower when packaged with HIV-2 than with the HIV-1 packaging construct. This could be due to the difference in the abundance of the transfer vector RNAs in the packaging cells, or due to the particular specificities of recognition and encapsidation. It was possible that the titer of HIV-1 vector was low when packaged with HIV-2 packaging construct because the Tat provided by the HIV-2 construct was poor in transactivating HIV-1 vector RNA. However, provision of additional HIV-1 or HIV-2 Tat did not change the outcome. This might imply that the reason for differential titer or non-reciprocacity lies in the interaction of the packaging signal and the gag determinants. With the chimeric packaging construct where the ‘gag-pol’ of the HIV-2 packaging constructs was replaced with the ‘gag-pol’ of HIV1, the titer of the HIV-1 vector increased and that of the HIV-2 vector decreased relative to homologous packaging. This was not the case for the ‘tat-rev’ chimeric packaging constructs. However, the exchange of ‘leader-gag’ of HIV-1 vector with that of the HIV-2 vector in transfer vector chimeras by itself was detrimental for vector titer in both cases. These results support the possibility of creating chimeric vectors with added safety features and without sacrificing efficiency. Funded by the US Government.
472. Biodistribution of Semliki Forest Virus Vectors and Effect of Preimmunizations in Mice Juan R. Rodriguez,1 Erkuden Casales,1 Jesus Prieto,1 Cristian Smerdou.1 1 Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Navarra, Spain. Semliki Forest virus (SFV) based vectors have a very broad tropism, being able to infect cells from many different origins, including tumor cells. These vectors have been used to express cytokines and other antitumoral molecules in different tumor models, leading to the induction of efficient antitumoral immune responses. It was of great interest to study if SFV vectors were able to target tumor nodules in vivo when given by different routes of inoculation, as well as to analyze the biodistribution of these vectors in the organism. In this study we used a CCD camera to analyze in vivo distribution of an SFV vector expressing firefly luciferase (SFV-luc) after intravenous, intraperitoneal, or intratumoral administration in either mice bearing different types of tumors or in mice without tumors. Specific infection of the tumoral tissue was only found when the virus was inoculated intratumorally, without spreading of the vector to other tissues. Analysis of luciferase activity in tissue homogenates from mice systemically inoculated with SFV-luc showed a high infectivity in heart and lung and a medium infectivity in kidney, spleen, and gonads without significant gender influence. In this study we also investigated the effect of preimmunizations with SFV vectors over subsequent vector administrations. Systemic immunization with a single dose of 107 pv or two doses of 106 pv of SFV-LacZ given with a 2-week interval were able to significantly
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
inhibit expression of luciferase in animals inoculated systemically with SFV-luc. This inhibition correlated with a high presence of neutralizing antibodies in animal sera. However, these immunizations only had a moderate effect on luciferase activity in animals inoculated intratumorally, indicating that although SFV vectors are immunogenic, the generated antivector immune response is not able to prevent tumor infectivity. Finally, intratumoral preimmunization with SFVluc also generated an antivector immune response, but only when doses up to 107 pv were used.
473. Semliki Based Systems for the Production of Retroviral Vectors: A Safety Concern Eric Piver,1 Christine Collin,1 Noémie Renault,1 Thierry Bru,2 Jean-Christophe Pages.1 1 ESPRI 3856 IFR 136, The Vector Group, Université François Rabelais, Faculté de Medecine 10 Bd Tonnelle, Tours, France; 2 Cancer Research UK Chromosome Replication Research Group, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, Scotland, United Kingdom. The development of efficient and safe production procedures are an essential goal conditioning viral vectors usefulness. For retroviral vectors production, two groups have suggested that Semliki forest virus (SFV) derived systems might generate interestingly elevated yields. The system relies on the transfection of three complementary SFV replicons. It is noteworthy that Muriaux and collaborator have shown that RNA is an important component for retroviral particles and that some SFV RNA could be packaged into retroviral particles. Furthermore, Lebedeva et al. and Rolls et al. have shown the arising of autonomous replication replicon from SFV vectors expressing various viral glyco-proteins envelopes. Thus, these data suggested that retroviral vectors might be vehicles for all sorts of SFV derived replicons. We designed a study aimed at thoroughly examining the packaging of full length SFV replicon into retroviral vectors. For this purpose we constructed two GFP expressing SFV replicons. One contained a prototypic Ψ sequence from MLV, while the other was missing this sequence. As SFV replicons normally give rise to genomic as well as subgenomic RNAs, we also modified their structure to prevent the formation of the later. This allowed us to set simple mobilization assays with straightforward read out based on the counting of GFP positive cells. In a first series of experiments we separately followed the mobilization of the modified SFV-GFP vectors in three conditions: using standard retroviral producing cells (PhoenixA cells); using the Li and Garoff Semliki based retroviral producing system and, lastly, in a tri-transfection lentiviral vector production. We observed mobilization events with all three conditions, albeit at different efficiencies. Using a secondary mobilization assay, we next showed that, in the Li and Garoff system, the MLV particles not only contained the GFP Semliki vector but also the Gagpol and the Env full length SFV replicons. Furthermore, a western blot analysis suggested that the retroviral particles might also contained uncleaved replicase. Finally we have shown that, in producing cells, both the retroviral proteins and the Semliki replicase were colocalised. All together, our data clearly demonstrate that retroviral particles have the ability to package genomic and subgenomic SFV RNAs, containing or not a retroviral Ψ. Therefore, these observations strongly advocate against the use of the Semliki system for clinical grade retroviral or lentiviral vector production.
S183
470. ABCB4 P-Glycoprotein Reduces Infectivity of Lentiviral Particles by Increasing Their Phosphatidylcholine Content
Naoya Uchida,1,2 Hideki Hanawa,1 Motoko Yamamoto,1 Takashi Shimada.1 1 Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2The Third Department of Internal Medicine, Nippon Medical School, Tokyo, Japan.
Niek P. van Til, Kirstin Heutinck, Ronald P. J. Oude Elferink, Jurgen Seppen. 1 AMC Liver Center, Academic Medical Center, Amsterdam, Netherlands.
The double gene vector carrying the expression units of the therapeutic gene and the reporter gene is widely used, since the reporter proteins such as the GFP and the DsRed are useful for measuring the titer of vector stock and the tracing gene modified cells. However, the activities of the double promoters in a single vector are often inhibited by the so called ‘promoter interference’. The internal ribosomal entry site (IRES) is being utilized to overcome this problem, but the expression of the second gene is not sufficient for the sensitive detection. In this study, we attempted to optimize the double gene lentivirus vector to achieve a high expression level of both genes. We generated a series of double gene lentivirus vector constructed of the MSCV-U3 promoter (Mp), the EF1α promoter (Ep), GFP cDNA (GFP), and DsRed cDNA (DsRed) in various configurations. In the first experiment, 293T cells were transduced with these lentivirus vectors and the expression levels of the two genes were monitored by the Northern blot analysis. A quantitative analysis of the Northern blots revealed that the Ep was relatively stable irrespective of its position, while the Mp activity was strongly inhibited when placed in the second position. In the second experiment, we further modified the Mp-GFP-Ep-DsRed vector to improve the expression level of the first gene. The flow cytometric analysis of transduced HeLa cells showed that the insertion of the 250 bp core insulator element (IR) upstream of the second Ep promoter (Mp-GFP-IR-Ep-DsRed) and the insertion of the 356 bp cytomegalovirus enhancer element (CMVe) upstream of the first Mp (CMVe-Mp-GFP-Ep-DsRed), in both vectors, significantly increased the GFP expression. The efficient expression of the double genes in this configuration was also observed when the GFP was replaced by a BMP2 or PTHR. These results indicated that optimization of the double promoter vector was important for the efficient gene expression. The combination of the CMVe-Mp and the IR-Ep promoters in this order may be useful for the tissue nonspecific expression of two genes.
Human Immunodeficiency Virus-1 (HIV-1) particles are assembled and released from membrane microdomains (lipid rafts). The membrane composition of the viral particle, which is important for viral integrity and infectivity, is therefore determined by the raft lipid composition. ATP binding cassette (ABC) transporters are transmembrane proteins, which require ATP to transport a broad array of compounds through membranes. The ABC transporter ABCB4 (MDR3 Pglycoprotein) is a phospholipid floppase that translocates phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of the cellular membrane. ABCB4 is located in the apical membrane of hepatocytes; translocation of PC by ABCB4 promotes PC release into bile, which neutralizes the toxic effect of bile salts. Lack of ABCB4 expression leads to the severe liver disease Progressive Familial Intrahepatic Cholestasis type 3 (PFIC3). In order to develop gene therapy for this disease, we created a third generation HIV-1 lentiviral vector with a cassette consisting of ABCB4 cDNA driven by a phosphoglycerokinase (PGK) promoter. Our attempts at producing high titer (>1 x 105 transducing units per mL) lentiviral ABCB4 expression vector were unsuccessful. Because during lentiviral vector production ABCB4 is also expressed, we proposed that the PC floppase activity of ABCB4 might have affected lentiviral vector production negatively. To test this, we co-expressed ABCB4 during GFP lentiviral vector production. We observed that co-expression of ABCB4 inhibited viral infectivity, but the production of viral particles was not affected. Co-expression of an inactive ABCB4 mutant did not reduce viral infectivity. In addition, the ratio of phosphatidylcholine to cholesterol of lentiviral particles increased significantly from 0.4 in producer cells expressing no ABCB4 or a mutant ABCB4 to 1.3 if the wildtype ABCB4 protein was co-expressed. We therefore conclude that the specific translocase activity of ABCB4 can affect viral infectivity negatively by changing the lipid membrane composition of the viral particles. This has implications for the strategy of development of a successful lentiviral gene therapy system for the treatment of PFIC3.
471. Chimeric Lentiviral Vectors with Safety Insurance Kritika Kachapati,1 Geetanjali Sachdeva,1 Suresh Arya.1,2 Center for Cancer Research, National Cancer Institute, Bethesda, MD; 2Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD. 1
Lentiviral vectors as presently designed are considered to be safe. No evidence for the generation of replication competent viruses has emerged for these vectors when transduced in vitro into cultured cells. Their behavior in vivo remains to be ascertained. Their propensity to cause insertional mutagenesis, if any, is also unknown. One major safety concern is the recombination between the components of the vector system during vector production in the laboratory and with the resident virus in the clinic during therapy of individuals with apparent or non-apparent HIV infection. As an additional safety insurance, we have created chimeric or hybrid lentiviral vectors derived from HIV-1 and HIV-2. This was done for both transfer vectors and packaging constructs. Our premise was that HIV-1 and HIV-2 are dissimilar enough in sequence to curtail recombination, but similar enough to functionally complement. Our S182
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
RNA VIRUS VECTORS I initial studies were done with HIV-2 vectors lacking the central polypurine track (PPT) and the woodchuck post-transcriptional enhancer element (WPRE), two regulatory elements reported to enhance transduction efficiency of HIV-1 vectors. This study includes HIV-2 vectors with these elements. A non-reciprocal interaction between conventional HIV-1 and HIV-2 vectors was observed in cross-packaging experiments. The titer of HIV-2 vectors was roughly the same whether packaged with the HIV-1 or HIV-2 packaging construct when titrated in cells in culture. In contrast, the titer of the HIV-1 vectors was twenty to thirty folds lower when packaged with HIV-2 than with the HIV-1 packaging construct. This could be due to the difference in the abundance of the transfer vector RNAs in the packaging cells, or due to the particular specificities of recognition and encapsidation. It was possible that the titer of HIV-1 vector was low when packaged with HIV-2 packaging construct because the Tat provided by the HIV-2 construct was poor in transactivating HIV-1 vector RNA. However, provision of additional HIV-1 or HIV-2 Tat did not change the outcome. This might imply that the reason for differential titer or non-reciprocacity lies in the interaction of the packaging signal and the gag determinants. With the chimeric packaging construct where the ‘gag-pol’ of the HIV-2 packaging constructs was replaced with the ‘gag-pol’ of HIV1, the titer of the HIV-1 vector increased and that of the HIV-2 vector decreased relative to homologous packaging. This was not the case for the ‘tat-rev’ chimeric packaging constructs. However, the exchange of ‘leader-gag’ of HIV-1 vector with that of the HIV-2 vector in transfer vector chimeras by itself was detrimental for vector titer in both cases. These results support the possibility of creating chimeric vectors with added safety features and without sacrificing efficiency. Funded by the US Government.
472. Biodistribution of Semliki Forest Virus Vectors and Effect of Preimmunizations in Mice Juan R. Rodriguez,1 Erkuden Casales,1 Jesus Prieto,1 Cristian Smerdou.1 1 Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Navarra, Spain. Semliki Forest virus (SFV) based vectors have a very broad tropism, being able to infect cells from many different origins, including tumor cells. These vectors have been used to express cytokines and other antitumoral molecules in different tumor models, leading to the induction of efficient antitumoral immune responses. It was of great interest to study if SFV vectors were able to target tumor nodules in vivo when given by different routes of inoculation, as well as to analyze the biodistribution of these vectors in the organism. In this study we used a CCD camera to analyze in vivo distribution of an SFV vector expressing firefly luciferase (SFV-luc) after intravenous, intraperitoneal, or intratumoral administration in either mice bearing different types of tumors or in mice without tumors. Specific infection of the tumoral tissue was only found when the virus was inoculated intratumorally, without spreading of the vector to other tissues. Analysis of luciferase activity in tissue homogenates from mice systemically inoculated with SFV-luc showed a high infectivity in heart and lung and a medium infectivity in kidney, spleen, and gonads without significant gender influence. In this study we also investigated the effect of preimmunizations with SFV vectors over subsequent vector administrations. Systemic immunization with a single dose of 107 pv or two doses of 106 pv of SFV-LacZ given with a 2-week interval were able to significantly
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
inhibit expression of luciferase in animals inoculated systemically with SFV-luc. This inhibition correlated with a high presence of neutralizing antibodies in animal sera. However, these immunizations only had a moderate effect on luciferase activity in animals inoculated intratumorally, indicating that although SFV vectors are immunogenic, the generated antivector immune response is not able to prevent tumor infectivity. Finally, intratumoral preimmunization with SFVluc also generated an antivector immune response, but only when doses up to 107 pv were used.
473. Semliki Based Systems for the Production of Retroviral Vectors: A Safety Concern Eric Piver,1 Christine Collin,1 Noémie Renault,1 Thierry Bru,2 Jean-Christophe Pages.1 1 ESPRI 3856 IFR 136, The Vector Group, Université François Rabelais, Faculté de Medecine 10 Bd Tonnelle, Tours, France; 2 Cancer Research UK Chromosome Replication Research Group, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, Scotland, United Kingdom. The development of efficient and safe production procedures are an essential goal conditioning viral vectors usefulness. For retroviral vectors production, two groups have suggested that Semliki forest virus (SFV) derived systems might generate interestingly elevated yields. The system relies on the transfection of three complementary SFV replicons. It is noteworthy that Muriaux and collaborator have shown that RNA is an important component for retroviral particles and that some SFV RNA could be packaged into retroviral particles. Furthermore, Lebedeva et al. and Rolls et al. have shown the arising of autonomous replication replicon from SFV vectors expressing various viral glyco-proteins envelopes. Thus, these data suggested that retroviral vectors might be vehicles for all sorts of SFV derived replicons. We designed a study aimed at thoroughly examining the packaging of full length SFV replicon into retroviral vectors. For this purpose we constructed two GFP expressing SFV replicons. One contained a prototypic Ψ sequence from MLV, while the other was missing this sequence. As SFV replicons normally give rise to genomic as well as subgenomic RNAs, we also modified their structure to prevent the formation of the later. This allowed us to set simple mobilization assays with straightforward read out based on the counting of GFP positive cells. In a first series of experiments we separately followed the mobilization of the modified SFV-GFP vectors in three conditions: using standard retroviral producing cells (PhoenixA cells); using the Li and Garoff Semliki based retroviral producing system and, lastly, in a tri-transfection lentiviral vector production. We observed mobilization events with all three conditions, albeit at different efficiencies. Using a secondary mobilization assay, we next showed that, in the Li and Garoff system, the MLV particles not only contained the GFP Semliki vector but also the Gagpol and the Env full length SFV replicons. Furthermore, a western blot analysis suggested that the retroviral particles might also contained uncleaved replicase. Finally we have shown that, in producing cells, both the retroviral proteins and the Semliki replicase were colocalised. All together, our data clearly demonstrate that retroviral particles have the ability to package genomic and subgenomic SFV RNAs, containing or not a retroviral Ψ. Therefore, these observations strongly advocate against the use of the Semliki system for clinical grade retroviral or lentiviral vector production.
S183