Viral vectors and line for gene therapy

Abstract

Novel viral vectors in which the expression of viral genes is regulated in such a way that it is functional in a complementation cell and non-functional in a host cell, as well as viral particles and host cells containing said novel vectors, are disclosed. A complementation cell including a viral gene expression regulator, and a method for preparing infectious viral particles, are also disclosed. Finally, a pharmaceutical composition containing said vectors, and the therapeutical use thereof, are disclosed.

1 . Viral vector, characterized in that it compris s an expression unit containing one or mor viral genes said expression unit being functional in a complementation cell and nonfunctional in a host call, and comprising one or more heterologous regulator sequence(s).  
     
     
         2 . Viral vector according to  claim 1 , characterized in that said expression unit comprises one or more regulatory sequence(s) enabling to activate the expression of said viral gene in the presence of an inducer and/or to inhibit the expression of said viral gene in the presence of a repressor.  
     
     
         3 . Viral vector according to  claim 1  or  2 , characterized in that said regulatory sequence can act at the level of transcription, elongation, transport or stability of the messenger or translation.  
     
     
         4 . Viral vector according to  claim 3 , characterized in that said regulatory sequence in placed at the level of the promoter of said unit, and more *specially up-stream of the TATA box.  
     
     
         5 . Viral vector according to one of  claims 1  to  4 , characterized in that said expression unit comprises one or more regulatory sequence(s) selected from the TAR, RRE, GRE, PRE, ERE and Gal4 UAS sequences and the regulatory sequences of the metallothionein gone and of the bacterial tryptophans lactose and tetracycline operons.  
     
     
         6 . Viral vector according to  claim 5 , characterized in that said expression unit comprises one or more regulatory sequence(s) derived from the tetracycline operon, placed upstream of the TATA box of said promoter, to give a promoter which in activable by an inducer of the tetracycline transactivator (tTA) type and repressible by tetracycline.  
     
     
         7 . Viral vector according to  claim 5 , characterized in that said expression unit comprises one or more regulatory sequence(s) derived from the tetracycline operon, placed downstream of the TATA box of said promoter, to give a promoter which is r pressible by the tetracycline repressor (T tR).  
     
     
         8 . Viral vector according to one of  claims 1  to  7 , derived from a virus selected from the herpesvirus, cytomegalovirus, AAV (adeno-associated virus), poxvirus and adenovirus.  
     
     
         9 . Adenoviral vector according to  claim 8 , derived from an adenovirus of human, canine, avian, bovine, murine, ovine, porcine or simian origin, or alternatively from a hybrid comprising adenoviral genome fragments of different origins.  
     
     
         10 . Viral vector according to  claim 8  or  9 , characterized in that it is defective for replication.  
     
     
         11 . Adenoviral vector according to  claim 10 , characterized in that it lacks at least all or part of the E1 region and, optionally, all or part of the E3 region.  
     
     
         12 . Adenoviral vector according to one of  claims 9  to  11 , characterized in that it comprises one or more expression unit(s) containing one or more viral genes of the E2, E4 or L1-L5 regions.  
     
     
         13 . Adenoviral vector according to  claim 12 , characterized in that it comprises an expression unit containing one or more regulatory sequence(s) derived from the tetracycline operon, placed upstream of the TATA box of the promoter and open reading frames (ORFs) 6 and 7 of the E4 region, so that the expression of said reading frames is activable by an inducer of the tetracycline transactivator (tTA) type and repressible by tetracycline.  
     
     
         14 . Viral vector according to one of  claims 1  to  13 , characterized in that it comprises an exogenous nucleotide sequence placed under the control of the elements needed for its expression in the host cell.  
     
     
         15 . Viral vector according to  claim 14 , characterized in that the exogenous nucleotide sequence is selected from the genes coding for a cytokine, a cell or nuclear receptor, a ligand, a coagulation factor, the CFTR protein, insulin, dystrophin, a growth hormone, an enzyme, an enzyme inhibitor, a polypeptide having an antitumor effect, a polypeptide capable of inhibiting a bacterial, parasitic or viral infection, and in particular HIV, an antibody, a toxin, an immunotoxin and a marker.  
     
     
         16 . Infectious viral particle comprising a viral vector according to one of  claims 1  to  15 .  
     
     
         17 . Eukaryotic host cell comprising a viral vector according to one of  claims 1  to  15  or an infectious viral particle according to  claim 16 .  
     
     
         18 . Complementation cell, characterized in that it comprises an inducer and/or a repressor.  
     
     
         19 . Complementation cell according to  claim 18 , characterized in that it comprises a DNA fragment coding for an inducer and/or a repressor.  
     
     
         20 . Complementation cell according to  claim 18  or  19 , characterized in that it is derived from line 293.  
     
     
         21 . Complementation call according to one of  claims 18  to  20 , for the complementation of an adenoviral vector which is defective for the E1 function and at least one second, late or early adenoviral function, characterized in that it comprises: 
 (i) a first cassette for the expression of all or part of the E1 region of an adenovirus, placed under the control of the elements necessary for its expression in said complementation cell, and  
 (ii) a second cassette for the expression of all or part of a late or early region of an adenovirus other than the E1 region, placed under the control of the elements necessary for its expression in said complementation cell, said elements comprising one or more regulatory sequences according to  claims 5  to  7 .  
 
     
     
         22 . Complementation cell according to  claim 21 , characterized in that said elements of the second expression cassette comprise a minimal promoter equipped at its 5′ end with 1 to 20 tet O sequences.  
     
     
         23 . Complementation cell according to  claim 22 , characterized in that said elements of the second expression cassette comprise a minimal promoter derived from the CMV virus (cytomegalovirus) equipped at its 5′ and with 7 tet O sequences.  
     
     
         24 . Complementation cell according to one of  claims 18  to  23  for the complementation of an adenoviral vector which is defective for the E1 and E4 functions, characterized in that the second expression cassette in a cassette for the expression of all or part of the E4 region of an adenovirus.  
     
     
         25 . Complementation cell according to  claim 24 , characterized in that said second expression cassette is a cassette for the expression of the sequences coding for open reading frames 6 and 7 (ORFs 6/7) of the E4 region of an adenovirus.  
     
     
         26 . Complementation cell according to one of  claims 18  to  23  for the complementation of an adenoviral vector which is defective for the E1 and E2 functions, characterized in that said second expression cassette is a cassette for the expression of all or part of the E2 region of an adenovirus.  
     
     
         27 . complementation cell according to  claim 26 , characterized in that said second expression cassette is a cassette for the expression of the sequences coding for the DBP protein (DNA binding protein) of the E2 region of an adenovirus.  
     
     
         28 . Complementation cell according to  claim 26 , characterized in that said second expression cassette is a cassette for the expression of the sequences coding for a temperature-sensitive mutant of the DBP protein of the E2 region of an adenovirus.  
     
     
         29 . Complementation cell according to one of  claims 18  to  28  for the complementation of an adenoviral vector which is defective for the E1 function and at least two other, late or early adenoviral functions, characterized. in that it comprises, in addition, a third cassette for the expression of all or part of a late or early region of an adenovirus other than the E1 region and the adenoviral region of the second expression cassette, placed under the control of the elements necessary for its expression in said complementation cell, and preferably of a promoter as defined in  claim 5 ,  6  or  7 .  
     
     
         30 . complementation cell according to  claim 29  for the complementation of an adenoviral vector which is defective for the E1, E2 and E4 functions, comprising: 
 (i) a first cassette for the expression of all or part of the E1 region of an adenovirus, placed under the control of the elements necessary for its expression in said complementation cell,  
 (ii) a second cassette for the expression of all or part of the E4 region of an adenovirus, placed under the control of the elements necessary for its expression in said complementation cell, and  
 (iii) a third cassette for the expression of all or part of the E2 region of an adenovirus, placed under the control of the elements necessary for its expression in said complementation call,  
 said elements of the second and/or third expression cassette comprising a promoter equipped with at least one tet O sequence, and more especially a minimal promoter derived from the C virus (cytomegalovirus), equipped. at its 5′ end with 7 tet O sequences.  
 
     
     
         31 . Complementation cell according to one of  claims 18  to  30 , characterized in that the titer of viral particles produced by said complementation cell is greater than 5×10 8  pfu (plaque forming units)/ml.  
     
     
         32 . Complementation cell according to one of  claims 18  to  31 , characterized in that the titer of viral particles produced by said complementation cell is greater than 1×10 10  ifu (infectious units)/ml.  
     
     
         33 . Method of preparation of an infectious viral particle according to  claim 16 , according to which: 
 (i) a viral vector according to one of  claims 1  to  15  in introduced into a complementation call capable of complementing in trans said viral vector, to obtain a transfected complementation cell;    (ii) said transfected complementation cell is cultured under suitable conditions to permit the expression of the viral genes and the production of said infectious viral particle; and    (iii) said infectious viral particle is recovered in the cell culture.    
     
     
         34 . Method according to  claim 33 , characterized in that said viral vector in an adenoviral vector and said complementation cell is according to  claim 20 .  
     
     
         35 . Method of preparation of an infectious adenoviral particle, according to which: 
 (i) an adenoviral vector is introduced into a complementation cell according to one of  claims 21  to  32 , to obtain an infected complementation cell,    (ii) said transfected [sic] complementation cell is cultured under suitable conditions to permit the expression of the viral genes and the production of said infectious viral particle; and    (iii) said infectious viral particle is recovered in the cell culture.    
     
     
         36 . Pharmaceutical composition comprising a viral vector according to one of  claims 1  to  15 , an infectious viral particle according to  claim 16  or obtained employing a preparation method according to one of  claims 33  to  35 , a eukaryotic host call according to  claim 17  or a complementation cell according to one of  claims 18  to  32 , in combination with a vehicle which is acceptable from a pharmaceutical standpoint.  
     
     
         37 . Therapeutic or prophylactic use of a viral vector according to one of  claims 1  to  15 , of an infectious viral particle according to  claim 16  or obtained employing a preparation method according to one of  claims 33  to  35 , of a eukaryotic host call according to  claim 17  or of a complementation cell according to one of  claims 18  to  32 , for the preparation of a medicinal product intended for the treatment of the human or animal body by gone therapy.  
     
     
         38 . Use according to  claim 37 , in combination with a repressor.