Lentiviral vectors derived from SIVagm, methods for their preparation and their use for gene transfer into mammalian cells

Abstract

The preparation and use of novel lentiviral SIVagm-derived vectors for gene transfer and in particular a method of preparation of the lentiviral vectors containing capsid particles of the simian immunodeficiency virus SIVagm and envelope proteins of SIVagm and other retroviruses such as the human immunodeficiency viruses (HIV), other simian immunodeficiency viruses (SIV), other retroviruses such as the murine leukemia virus (MLV) or the “gibbon ape leukemia virus” (GaLV) or the porcine endogenous or exogenous retrovirus (PERV), the “vesicular stomatitis virus” (VSV-G) are described. The vectors and corresponding packaging cells may be employed for packaging and transfer of genes which are not packaged by other lentiviral, retroviral and other vectors or which show an inefficient gene transfer with other vector particles. These vectors may be used for gene transfer into selected cell types, specifically into proliferatively active and resting human cells.

1 . SIVagm vectors comprising virus cores and virus envelopes characterized in that the virus cores are derived from simian immunodeficiency virus (SIVagm) of the African vervet monkey (Chlorocebus, formerly  Cercopithecus aethiops ) and the virus envelopes are derived from SIVagm or other viruses.  
     
     
         2 . SIVagm vectors according to    claim 1    characterized in that the virus envelopes are derived from the human immunodeficiency virus 1 or 2 (HIV-1 or HIV-2, respectively) or simian immunodeficiency virus  Cercopithecus aethiops  (SIVagm) or  Macaca mulatta  (SIVmac) or  Pan troglodytes  (SIVcpz) or  Cercopithecus mitis  (SIVsyk) or  Papio sphinx  (SIVmnd) or  Cercocebus atys  (SIVsm) or  Macaca nemestrina  (SIVmne).  
     
     
         3 . SIVagm vectors according to    claim 1    or    2    characterized in that the viral envelopes of the murine ecotropic or amphotropic leukemia virus (MLV), the avian spleen necrosis virus (SNV), the GaLV (“gibbon ape leukemia virus”), the porcine endogenous or porcine exogenous retrovirus (PERV) are used.  
     
     
         4 . SIVagm vectors according to any of    claims 1    to    3    characterized in that the viral envelopes have been modified by single chain antibodies (scFv) or other ligands (e.g. cytokins) of cell surface molecules.  
     
     
         5 . SIVagm vectors according to any of    claims 1    to    4    characterized in that the viral envelopes are derived from different viruses.  
     
     
         6 . SIVagm vectors according to any of    claims 1    to    5    suitable for packaging and transfer of genes into mammalian cells which may not be packaged or transduced by other lentiviral or retroviral vectors, e.g. in the context of gene therapy strategies for the treatment or prevention of a HIV infection in man.  
     
     
         7 . Method for the preparation of packaging cells forming SIVagm-derived vectors according to any of    claims 1    to    6    comprising transfecting a recipient cell or a packaging cell (env- and expression construct-positive) which does or does not produce vial envelopes (env-negative) with a gag/pol gene containing the genetic information for SIVagm capsid particles.  
     
     
         8 . Method for the preparation of packaging cells forming lentiviral vectors according to any of    claims 1    to    6    comprising transfecting a cell with expression genes for gag and pol and/or an expression construct comprising a packaging signal and the genetic information to be transduced, and an expression gene containing the genetic information for envelope proteins.  
     
     
         9 . Method according to any of    claims 7    to    8    wherein pSgfp or pSlacZ (see FIG. 1) are used as expression vector genes.  
     
     
         10 . Packaging cells obtainable by the method according to any of    claims 7    to    8   .  
     
     
         11 . Use of the lentiviral vectors according to any of    claims 1    to    6    or the packaging cells according to    claim 10    as a medicament.  
     
     
         12 . Use of the lentiviral vectors according to any of    claims 1    to    6    or the packaging cells according to    claim 10    for the preparation of a medicament for transduction of genes into cells.  
     
     
         13 . Use according to    claim 12    wherein said cells are human cells.  
     
     
         14 . Use of the lentiviral vectors according to any of    claims 1    to    6    and of corresponding packaging cells according to    claim 10    for the preparation of a medicament for modification of cells by genetic engineering or to provide an active substance in the context of a gene therapy.