Stable pseudotyped lentiviral particles and uses thereof

Abstract

The present invention relates to a method for obtaining stable pseudotyped lentiviral particles including a heterologous gene of interest, comprising the following steps:a) transfecting at least one plasmid in appropriate cell lines, wherein said at least one plasmid comprises the gene of interest, the rev, gag and pol genes, and a sequence coding for an ERV syncytin, wherein the rev, gag and pol genes are retroviral genes;b) incubating the transfected cells obtained in a), so that they produce the stable pseudotyped lentiviral particles in the supernatant; andc) harvesting and concentrating the stable lentiviral particles obtained in b).The present invention also relates to a method to transduce immune cells using lentiviral vectors pseudotyped with an ERV syncytin glycoprotein. The method can be performed on non-stimulated blood cells or on cells stimulated briefly with IL7, and the cells can be expanded.The stable pseudotyped lentiviral particles obtained are particularly useful in gene therapy.

1 . Stable lentiviral particles pseudotyped with an endogenous retroviral syncytin (ERV syncytin) and packaging a heterologous gene of interest, presenting a high physical and/or infectious titer(s). 
     
     
         2 . A method for obtaining the stable pseudotyped lentiviral particles of  claim 1 , comprising the following steps:
 a) transfecting at least one plasmid in appropriate cell lines, wherein said at least one plasmid comprises the gene of interest, the retroviral rev, gag and pol genes, and a sequence coding for an ERV syncytin;   b) incubating the transfected cells obtained in a), so that they produce the stable lentiviral particles pseudotyped with an ERV syncytin, respectively, and packaging the heterologous gene of interest; and   c) harvesting and concentrating the stable lentiviral particles obtained in b), preferably wherein the concentration of step c) comprises centrifugating and/or purifying the harvested stable lentiviral particles obtained in b).   
     
     
         3 . The method according to  claim 2 , wherein the retroviral rev, gag and pol genes are lentiviral rev, gag and pol genes; preferably HIV-1 rev, gag and pol genes. 
     
     
         4 . Particles obtained by the process of any one of  claim 2  or  3 . 
     
     
         5 . Particles according to  claim 1  or  4 , or the method according to any one of  claims 2  to  4 , wherein the ERV syncytin is selected from the group consisting of HERV-W, HERV-FRD, murine syncytin-A, murine syncytin-B, syncytin-Ory1, syncytin-Car1 and syncytin-Rum1, preferably the ERV syncytin is selected from the group consisting of HERV-W, HERV-FRD and murine syncytin-A and even more preferably the ERV syncytin is HERV-W or HERV-FRD. 
     
     
         6 . Particles according to  claim 1  or  5 , or the method according to any one of  claims 2  to  4 , wherein the high infectious titer is a titer of infectious particles produced at the end of step c), higher than 2 E+04 TU/ml, preferably higher than 1 E+05 TU/ml, preferably higher than 1 E+06 TU/ml, preferably higher 5 than 2 E+06 TU/ml, and/or wherein the high physical titer is a titer of particles produced at the end of step c) higher than 0.7×10 5  ng p24/mL, notably higher than 1×10 5  ng p24/mL, preferably higher than 1.1×10 5  ng p24/mL, more preferably higher than 1.5×10 5  ng p24/mL. 
     
     
         7 . Particles according to any one of  claim 1 ,  5  or  6 , for use as a medicament, preferably for use in gene therapy or immunotherapy or as a vaccine or in immunoprophylaxis. 
     
     
         8 . In vitro use of the particles according to any one of  claim 1 ,  5  or  6 , for the transduction of immune cells, preferably B cells or myeloid cells. 
     
     
         9 . In vitro use of the particles according to any one of  claim 1 ,  5 ,  6  or  8 , for biotechnology engineering, preferably for producing immunoglobulins. 
     
     
         10 . Particles according to any one of  claims 1  or  5  to  7 , for use for therapy by transducing immune cells, preferably for use for treating immune deficiencies, auto-immunities, infectious diseases or B cell-related cancers. 
     
     
         11 . A sample of immune cells infected with the particles according to any one of  claim 1 ,  5  or  6 , for use as a medicament, or for a diagnosis purpose. 
     
     
         12 . An ex vivo process for obtaining immune cells modified to express a heterologous gene of interest, comprising a step of infecting immune cells, preferably naïve immune cells optionally previously stimulated, with the particles according to any one of  claim 1 ,  5  or  6 . 
     
     
         13 . The ex vivo process of  claim 12 , wherein it is performed in the presence of a LAH4 peptide or a functional derivative thereof, preferably the LAH4-A4 vectofusin-1, and/or wherein the immune cells are chosen from B cells, T cells, dendritic cells, monocytes and macrophages, preferably are B cells. 
     
     
         14 . The ex vivo process of any one of  claim 12  or  13 , wherein it comprises the following steps:
 optionally stimulating naïve immune cells by incubating them in a medium comprising IL-7, and 
 infecting the naïve immune cells, stimulated or not, with said particles according to any one of  claim 1 ,  5  or  6 , in the presence of a LAH4 peptide or a functional derivative thereof. 
 
     
     
         15 . The immune cells, preferably B cells, more preferably naïve B cells, comprising said heterologous gene of interest obtainable by the ex vivo process according to any one of  claims 12  to  14 .