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      Gene therapy: trials and tribulations

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      Nature Reviews Genetics

      Springer Science and Business Media LLC

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          Abstract

          The art and science of gene therapy has received much attention of late. The tragic death of 18-year-old Jesse Gelsinger, a volunteer in a Phase I clinical trial, has overshadowed the successful treatment of three children suffering from a rare but fatal immunological disease. In the light of the success and tragedy, it is timely to consider the challenges faced by gene therapy--a novel form of molecular medicine that may be poised to have an important impact on human health in the new millennium.

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          Most cited references 54

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          Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

           M Gossen,  H Bujard (1992)
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            Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity.

            Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P(tet) promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2(S)-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.
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              Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences.

              Gene-transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells. The HIV-1 proteins Matrix, Vpr and Integrase have been implicated in the nuclear import of the viral genome in non-dividing cells. Here we show that a sequence within pol is also required in cis. It contains structural elements previously associated with the progress of reverse transcription in target cells. We restored these elements in cis within late-generation lentiviral vectors. The new vector transduced to a much higher efficiency several types of human primary cells, when both growing and growth-arrested, including haematopoietic stem cells assayed by long-term repopulation of NOD/SCID mice. On in vivo administration into SCID mice, the vector induced higher plasma levels of human clotting factor IX (F.IX) than non-modified vector. Our results indicate that nuclear translocation of the genome is a rate-limiting step in lentiviral infection of both dividing and non-dividing cells, and that it depends on protein and nucleic acid sequence determinants. Full rescue of this step in lentivirus-based vectors improves performance for gene-therapy applications.
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                Author and article information

                Journal
                Nature Reviews Genetics
                Nat Rev Genet
                Springer Science and Business Media LLC
                1471-0056
                1471-0064
                November 2000
                November 2000
                : 1
                : 2
                : 91-99
                Article
                10.1038/35038533
                11253666
                © 2000

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