- Email: [email protected]
92. Efficient Trangene Expression in Central Nervous System through a Non-Integrative Lentiviral Vector
RNA VIRUS VECTORS: GENE EXPRESSION up in an inactive, episomal, non-integrated, hypoacetylated form. These data raise concerns regarding in vivo use of vectors containing the cHS4-insulator. We are currently further investigating acetylation levels of histone H3 and H4 using chromatin immunoprecipitation in order to investigate if the cHS4-insulator actually is hyperacetylated in the context of a lentiviral vector and we intend to present these data at the meeting. References: 1. Emery et al. PNAS 2000. 2. Ramezani et al. Blood 2003. 3. Jakobsson et al. Exp Cell Res 2004.
92. Efficient Trangene Expression in Central Nervous System through a Non-Integrative Lentiviral Vector Stephanie Philippe,1 Che Serguera,1 Sebastien Bonnel,2 Christelle Vetu,2 Martine Barkats,1 Caroline Petit,3 Marc Abitbol,2 Chamsy Sarkis,1 Jacques Mallet.1 1 Laboratoire de Génétique Moleculaire de la Neurotransmission et des Processus Neurodégénératifs - Hopital de la Pitié Saplétrière, CNSR UMR7091, Paris, France; 2CERTO - Faculté Necker Enfants Malades, Paris, France; 3Unité d’oncologie virale, institut Cochin, Paris, France. Lentiviral vectors are widely used in experimental gene transfer and are a promising tool for clinical applications, particularly for the treatment of neurodegenerative diseases. However, one of the most important limits to the application of this technology to humans is the risk of insertional mutagenesis through the integration of the viral genome into the host cell chromatin. This risk remains poorly evaluated and uncontrolled. In order to eliminate this risk, we have developed a lentiviral vector derived from the human immunodeficiency virus type 1 lacking integration property. After cell transduction, the vector genome persists in an extrachromosomic circular form in the nucleus and allows an effective and stable transgene expression in vitro and in vivo. The episomal phenotype of the particles was induced by introducing mutations in the C terminal coding sequence of the integrase in the transcomplementation plasmid. This has been used to produce vector stocks carrying the coding sequence of the green fluorescent protein (GFP) under control of the immediate early promoter of the human cytomegalovirus (hCMVie). We have established in vitro that these integrase-mutant vectors are capable of transducing various cell lines. This expression gradually disappears through cell division, suggesting the episomal phenotype of the particles. This phenotype was confirmed with quantitative PCR analysis of the different forms of viral genomes (circular or integrated). The residual integration frequency was evaluated with vectors expressing the neo-resistance gene. Cellular clones obtained after transduction with these vectors and G418 selection were further analyzed by mean of LAM-PCR. We also showed the efficiency of these vectors to transduce primary quiescent neurons derived from rat cortex cultures where GFP expression was observed up to 25 days after transduction. Lastly, we showed the efficiency of these episomal vectors in vivo in the central nervous system. Particles were injected into either the brain or the retina and GFP expression was observed for at least two months and this expression was comparable with that obtained using the integrative vectors.
93. Diminished Mobilization of SelfInactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat Hideki Hanawa,1 Derek A. Persons,2 Takashi Shimada,1 Arthur W. Nienhuis.2 1 Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TN. Hematopoietic stem cell gene therapy using an oncoretroviral vector has been shown to have clear clinical benefit (N Engl J Med 346:1185, 2002) (Science 296:2410, 2002) but long term follow-up revealed a side effect of the vector from insertional mutagenesis in patients having severe combined immunodeficiency. Retroviral vectors developed from lentivirus such as HIV-1 or SIV may have a similar risk, although the risk may be less because lentiviral vectors do not show the predilection for integration near promoter exhibited by oncoretroviral vectors and leukemogenesis has not yet been shown in any animal model or clinical trial. Another possible risk of HIV-1 vector is interaction between wild type HIV-1 and integrated vector genome, so we have been shown that (1) a SIN HIV-1 vector could be mobilized by HIV-1 packaging constructs after transfection, (2) a tat coding SIN vector without an internal promoter for tat could express detectable level of tat from approximately 1 in 3,000 integrants, (3) the frequency of genome transcription was diminished by removal of the MSCV LTR enhancer or by addition of an insulator element from the chicken globin locus to the SIN LTR, and (4) proviral transcripts are derived from cryptic promoters on the vector and genomes (Blood 102:249a, 2003). The mobilized vector genomes re-integrated without rearrangement and with intact LTRs as shown by analysis of the proviral genome in eight single copy clones. We then evaluated the effect of globin regulatory elements on mobilization of SIN lentiviral vector genomes. Initially, we substituted the LTR GFP cassette in vector MSCV-U3 for one in which 1.7Kb β-globin LCR were linked to the β-globin promoter driving GFP in the reverse transcriptional orientation (d432βGFPim). Two additional vectors were also studied; the globin LCR β-promoter GFP cassette was reversed to derive Fd432βGFP and then it was modified by substituting larger LCR elements to derive FmLARβV5GFP. Three separate polyclonal 293T cell populations transduced 3 times at high vector concentration were established. Vector mobilization was evaluated by transfection of these populations with packaging plasmids after which conditioned medium was titered on HeLa cells. The mobilized titers were normalized based on vector copy number (100 copies/cells). The mobilized titer of the MSCV-U3 was 38,000±3500 and that of d432βGFPim was only 810±100 (p=0.0004). The mobilized titers of the vectors in which the globin regulatory elements were in the forward orientation were also significantly lower than MSCV-U3; Fd432βGFP was 2100±250 (p=0.005) and FmLARβV5GFP was 1600±120 (p=0.0005). Those data suggest that the globin LCR elements are less likely than the retroviral LTR to induce transcription of the integrated vector genome in nonerythroid cells.
94. Transgene Expression with IntegrationDeficient HIV-1 Based Lentiviral Vectors Expressing Class I Integrase Mutants G. Joseph,1 J. Marsh,2 T. Johnson,2 K. Cornetta.1,2 1 Microbiology & Immunology, Indiana University School of Medicine; 2Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN. Despite the benefit of achieving stable transgene expression with retroviral-based vectors, adverse effects associated with the administration of these vectors include the potential for insertional
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Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
92. Efficient Trangene Expression in Central Nervous System through a Non-Integrative Lentiviral Vector Stephanie Philippe,1 Che Serguera,1 Sebastien Bonnel,2 Christelle Vetu,2 Martine Barkats,1 Caroline Petit,3 Marc Abitbol,2 Chamsy Sarkis,1 Jacques Mallet.1 1 Laboratoire de Génétique Moleculaire de la Neurotransmission et des Processus Neurodégénératifs - Hopital de la Pitié Saplétrière, CNSR UMR7091, Paris, France; 2CERTO - Faculté Necker Enfants Malades, Paris, France; 3Unité d’oncologie virale, institut Cochin, Paris, France. Lentiviral vectors are widely used in experimental gene transfer and are a promising tool for clinical applications, particularly for the treatment of neurodegenerative diseases. However, one of the most important limits to the application of this technology to humans is the risk of insertional mutagenesis through the integration of the viral genome into the host cell chromatin. This risk remains poorly evaluated and uncontrolled. In order to eliminate this risk, we have developed a lentiviral vector derived from the human immunodeficiency virus type 1 lacking integration property. After cell transduction, the vector genome persists in an extrachromosomic circular form in the nucleus and allows an effective and stable transgene expression in vitro and in vivo. The episomal phenotype of the particles was induced by introducing mutations in the C terminal coding sequence of the integrase in the transcomplementation plasmid. This has been used to produce vector stocks carrying the coding sequence of the green fluorescent protein (GFP) under control of the immediate early promoter of the human cytomegalovirus (hCMVie). We have established in vitro that these integrase-mutant vectors are capable of transducing various cell lines. This expression gradually disappears through cell division, suggesting the episomal phenotype of the particles. This phenotype was confirmed with quantitative PCR analysis of the different forms of viral genomes (circular or integrated). The residual integration frequency was evaluated with vectors expressing the neo-resistance gene. Cellular clones obtained after transduction with these vectors and G418 selection were further analyzed by mean of LAM-PCR. We also showed the efficiency of these vectors to transduce primary quiescent neurons derived from rat cortex cultures where GFP expression was observed up to 25 days after transduction. Lastly, we showed the efficiency of these episomal vectors in vivo in the central nervous system. Particles were injected into either the brain or the retina and GFP expression was observed for at least two months and this expression was comparable with that obtained using the integrative vectors.
93. Diminished Mobilization of SelfInactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat Hideki Hanawa,1 Derek A. Persons,2 Takashi Shimada,1 Arthur W. Nienhuis.2 1 Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TN. Hematopoietic stem cell gene therapy using an oncoretroviral vector has been shown to have clear clinical benefit (N Engl J Med 346:1185, 2002) (Science 296:2410, 2002) but long term follow-up revealed a side effect of the vector from insertional mutagenesis in patients having severe combined immunodeficiency. Retroviral vectors developed from lentivirus such as HIV-1 or SIV may have a similar risk, although the risk may be less because lentiviral vectors do not show the predilection for integration near promoter exhibited by oncoretroviral vectors and leukemogenesis has not yet been shown in any animal model or clinical trial. Another possible risk of HIV-1 vector is interaction between wild type HIV-1 and integrated vector genome, so we have been shown that (1) a SIN HIV-1 vector could be mobilized by HIV-1 packaging constructs after transfection, (2) a tat coding SIN vector without an internal promoter for tat could express detectable level of tat from approximately 1 in 3,000 integrants, (3) the frequency of genome transcription was diminished by removal of the MSCV LTR enhancer or by addition of an insulator element from the chicken globin locus to the SIN LTR, and (4) proviral transcripts are derived from cryptic promoters on the vector and genomes (Blood 102:249a, 2003). The mobilized vector genomes re-integrated without rearrangement and with intact LTRs as shown by analysis of the proviral genome in eight single copy clones. We then evaluated the effect of globin regulatory elements on mobilization of SIN lentiviral vector genomes. Initially, we substituted the LTR GFP cassette in vector MSCV-U3 for one in which 1.7Kb β-globin LCR were linked to the β-globin promoter driving GFP in the reverse transcriptional orientation (d432βGFPim). Two additional vectors were also studied; the globin LCR β-promoter GFP cassette was reversed to derive Fd432βGFP and then it was modified by substituting larger LCR elements to derive FmLARβV5GFP. Three separate polyclonal 293T cell populations transduced 3 times at high vector concentration were established. Vector mobilization was evaluated by transfection of these populations with packaging plasmids after which conditioned medium was titered on HeLa cells. The mobilized titers were normalized based on vector copy number (100 copies/cells). The mobilized titer of the MSCV-U3 was 38,000±3500 and that of d432βGFPim was only 810±100 (p=0.0004). The mobilized titers of the vectors in which the globin regulatory elements were in the forward orientation were also significantly lower than MSCV-U3; Fd432βGFP was 2100±250 (p=0.005) and FmLARβV5GFP was 1600±120 (p=0.0005). Those data suggest that the globin LCR elements are less likely than the retroviral LTR to induce transcription of the integrated vector genome in nonerythroid cells.
94. Transgene Expression with IntegrationDeficient HIV-1 Based Lentiviral Vectors Expressing Class I Integrase Mutants G. Joseph,1 J. Marsh,2 T. Johnson,2 K. Cornetta.1,2 1 Microbiology & Immunology, Indiana University School of Medicine; 2Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN. Despite the benefit of achieving stable transgene expression with retroviral-based vectors, adverse effects associated with the administration of these vectors include the potential for insertional
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Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy