A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector.
ABSTRACT 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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ABSTRACT: Ex vivo liver gene therapy may be a future alternative to orthotopic liver transplantation for the treatment of some liver diseases. We previously described the transduction in suspension with lentiviral vectors and immediate hepatocyte transplantation (SLIT) protocol and its high transduction rate with normal human hepatocytes. We also reported SLIT efficiency in the animal model of Crigler-Najjar type 1 syndrome (CN-1), the Gunn rat. Here, we evaluated SLIT efficiency with diseased human hepatocytes. Hepatocytes of the liver from a 4-year-old patient presenting CN-1 were isolated. They were transduced with liver-specific lentiviral vectors expressing uridine-diphosphate-glucuronosyltransferase (hUGT1A1) or green fluorescent protein, and then analyzed in vitro for transduction efficiency and hUGT1A1 expression, or transplanted in nonobese diabetic/severe combined immunodeficiency (SCID) mice to evaluate long-term survival of transplanted cells. More than 90% of CN-1 hepatocytes were transduced. Hepatocytes produced hUGT1A1 protein after lentiviral transduction. After having been subjected to the SLIT, lentivirally transduced CN-1 hepatocytes engrafted long term (up to 26 weeks posttransplantation) in recipient livers and expressed green fluorescent protein or hUGT1A1 vector. The SLIT protocol allowed for a high transduction of CN-1 hepatocytes and restoration of the expression of the deficient protein. Furthermore, long-term survival of lentivirally transduced CN-1 hepatocytes in the liver of immunodeficient mice was demonstrated. This study is therefore an important step toward human application of lentiviral gene therapy.Transplantation 05/2009; 87(7):1006-12. · 3.78 Impact Factor
Article: Lentiviral vectors.[show abstract] [hide abstract]
ABSTRACT: Vectors based on lentiviruses have reached a state of development such that clinical studies using these agents as gene delivery vehicles have now begun. They have particular advantages for certain in vitro and in vivo applications especially the unique capability of integrating genetic material into the genome of non-dividing cells. Their rapid progress into clinical use reflects in part the huge body of knowledge which has accumulated about HIV in the last 20 years. Despite this, many aspects of viral assembly on which the success of these vectors depends are rather poorly understood. Sufficient is known however to be able to produce a safe and reproducible high titre vector preparation for effective transduction of growth-arrested tissues such as neural tissue, muscle and liver.Journal of Biomedical Science 08/2013; 11(4):439-49. · 2.46 Impact Factor
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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.Retrovirology 11/2011; 8:92. · 5.66 Impact Factor