Showing papers by "Luigi Naldini published in 2000"

Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences.

Abstract: 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.

Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy.

Abstract: Lentiviral vectors are tools for gene transfer derived from lentiviruses. From their first application to now they have been strongly developed in design, in biosafety and in their ability of transgene expression into target cells. Primate and non-primate derived lentiviral vectors are now available and with both types of systems a lot of studies tuned to improve their performances in a large number of tissues are ongoing. Here we review the state of the art of lentiviral vector systems discussing their potential for gene therapy.

Efficient lentiviral transduction of liver requires cell cycling in vivo

Abstract: Human-immunodeficiency-virus (HIV)-based lentiviral vectors are a promising tool for in vivo gene therapy. Unlike Moloney-murine-leukaemia-based retroviruses (MLV), lentiviruses are believed to stably transduce quiescent (non-cycling) cells in various organs. No previous studies, however, have directly established the cell-cycle status of any transduced cell type at the time of vector administration in vivo. In vitro studies using wild-type HIV or HIV-based vectors have shown that, in some cases, cell-cycle activation is required for infection, even though cellular mitosis is not an absolute requirement for integration. Even if the block in reverse transcription is overcome in quiescent T cells, productive infection by HIV cannot be rescued in the absence of cell-cycle activation. The potential use of these vectors for gene therapy prompted our study, which establishes a cell-cycle requirement for efficient transduction of hepatocytes in vivo.

Method and means for producing high titer, safe, recombinant lentivirus vectors

Abstract: Lentiviral vectors modified at the 5′ LTR or both the 5′ and 3′ LTR's are useful in the production of recombinant lentivirus vectors. Such vectors can be produced in the absence of a functional tat gene. Multiple transformation of the host cell with the vector carrying the transgene enhances virus production.

Intracerebral Gene Transfer Using Viral Vectors

Abstract: The recent development of recombinant viral vectors that are capable of transducing postmitotic cells may provide a powerful new tool for studying brain function, as well as ameliorative strategies in models of neurological disease. Some of these vectors have recently demonstrated direct expression of biologically relevant levels of protein expression (Mandel et al., 1997, 1998; Left et al., 1999; B16mer et al., 1998) for varying periods of time of up to 1 yr after direct intracerebral injection (Mandel et al., 1997, 1998; B16mer et al., 1997; Naldini et al., 1996a). Moreover, vectors injected into the nervous system have been shown, not only to be capable of expressing de novo transgenes, but also to be able to regionally suppress gene expression by delivery of antisense oligonucleotides (Xiao et al., 1997), by expression of a dominant-negative protein, by local knock-out of genes in transgenic animals carrying a transgene surrounded by lox-P sites (Donello et al., 1998), or by the production of molecules capable of inhibiting specific mRNAs, called ribozymes (Lewin et al., 1998). Thus, the use of viral vectors has great potential, not only for the study of gene therapy (GT), but also for the study of the molecular basis of certain brain functions. This chapter provides information about a range of practical issues involved in the use of recombinant vectors, and may be used as a guide for laboratories that intend to use viral vectors in their research. This chapter focuses on the three recombinant vectors that are among the most commonly currently used in the literature, i.e., recombinant adeno-associated virus (rAAV) vectors, recombinant adenovirus (rAd), and recombinant lentivirus (rLV) vectors. Other promising vector systems are under development for direct gene transfer, e.g., vectors based on herpes simplex virus (HSV) (During et al., 1994; Bowers et al., 1997). HSV vectors will not be discussed here, but this does not reflect any judgement on the quality or the potential of HSV vectors

Procede et moyens de production de vecteurs lentiviraux recombinants surs et a titre eleve

Abstract: L'invention concerne des vecteurs lentiviraux modifies au niveau de 5' LTR ou au niveau de 5' et 3' LTR utiles dans la production de vecteurs lentiviraux recombinants (voir fig. 1). Ces vecteurs peuvent etre obtenus en l'absence d'un gene fonctionnel tat. Les multiples transformations de la cellule hote avec le vecteur porteur du transgene contribuent a l'amelioration de la production de virus. Les vecteurs peuvent contenir des promoteurs inductibles ou conditionnels.