A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector

University of Florence, Florens, Tuscany, Italy
Hepatology (Impact Factor: 11.06). 08/2003; 38(1):114-22. DOI: 10.1053/jhep.2003.50265
Source: PubMed


Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy.

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Available from: Pierre Charneau, Jan 14, 2015
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    • "titers and better percent transduction . This is generally the case whether or not the targets are cell lines or primary cells , dividing or non - dividing , or transduced in vitro or in vivo ( Baekelandt et al . , 2002 ; Barry et al . , 2001 ; Breckpot et al . , 2003 ; Dardalhon et al . , 2001 ; De Rijck et al . , 2005 ; Follenzi et al . , 2000 ; Giannini et al . , 2003 ; Hungnes et al . , 1992 ; Manganini et al . , 2002 ; Nguyen et al . , 2002 ; Park and Kay , 2001 ; Seppen et al . , 2002 ; Sirven et al . , 2000 ; Van Maele et al . , 2003 ; Zennou et al . , 2000 , 2001 ) . The difference between cPPT - CTS ( þ ) and ( − ) HIV - 1 vectors has varied , but is generally on the order of 0 . 5 – 1 . 0 logs"
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    ABSTRACT: Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient "central DNA flap" in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade singe-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins, and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors.
    Preview · Article · Apr 2013 · Virology
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    • "The central DNA Flap and its importance for HIV-1 replication have been controversial for many years. Its role as a cis-acting determinant of nuclear import has proven all-important for gene transfer based on lentiviral vectors and in the context of replicative viruses [5,9-13,17-21,23,24,26-31,33,34,39]. This defect is independent of envelope tropism (R5, X4, VSV-G) or cell type used (cell lines, primary lymphocytes, APOBEC3G/F status). "
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    ABSTRACT: : The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS). The central DNA Flap is a determinant of HIV-1 nuclear import, however, neither cPPT nor CTS mutations entirely abolish nuclear import and infection. Therefore, to determine whether or not the DNA Flap is essential for HIV-1 nuclear import, we generated double mutant (DM) viruses, combining cPPT and CTS mutations to abolish DNA Flap formation. The combination of cPPT and CTS mutations reduced the proportion of viruses forming the central DNA Flap at the end of reverse transcription and further decreased virus infectivity in one-cycle titration assays. The most affected DM viruses were unable to establish a spreading infection in the highly permissive MT4 cell line, nor in human primary peripheral blood mononuclear cells (PBMCs), indicating that the DNA Flap is required for virus replication. Surprisingly, we found that DM viruses still maintained residual nuclear import levels, amounting to 5-15% of wild-type virus, as assessed by viral DNA circle quantification. Alu-PCR quantification of integrated viral genome also indicated 5-10% residual integration levels compared to wild-type virus. This work establishes that the central DNA Flap is required for HIV-1 spreading infection but points to a residual DNA Flap independent nuclear import, whose functional significance remains unclear since it is not sufficient to support viral replication.
    Full-text · Article · Nov 2011 · Retrovirology
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    • "Development of the humanized hCYP1A1_1A2_Cyp1a1/1a2(−/−)-Ahr b1 transgenic line has been detailed (Dragin et al., 2007). Chimeric mice bearing human hepatocytes were generated using uPA(+/+)/SCID mice as the host (Giannini et al., 2003) and characterized (Tateno et al., 2004; Katoh et al., 2008); their human hepatocyte-replacement rates were between 73% and 83%. All experiments involving mice adhered to the Guidelines for Animal Experiments and Use Committee of the Nihon University School of Medicine. "
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    ABSTRACT: Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how "human-like" can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.
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