Helper viruses for the preparation of recombinant viral vectors

Abstract

Novel helper vectors are provided for complementing defective recombinant viral vectors, characterized in that they are provided with recombination sequences recognized by a recombinase. A complementation cell expressing the recombinase, and a method for preparing recombinant viral vectors as infectious viral particles for transferring and expressing genes of interest in a host organism or cell, are also provided. The invention is particularly suitable for use in gene therapy, especially in humans.

1 . A helper virus for the production of a recombinant viral vector defective for replication, characterized in that it comprises a first recombination sequence at 5′ and a second recombination sequence at 3′ of a region essential to the propagation of said helper virus; said recombination sequences being recognized by a recombinase.  
     
     
         2 . A helper virus according to  claim 1 , characterized in that it is deficient for replication.  
     
     
         3 . A helper virus according to  claim 1  or  2 , characterized in that said recombination sequences, are positioned at 5′ and 3′ of the encapsidation region of said virus.  
     
     
         4 . A helper virus according to one of  claims 1  to  3 , characterized in that said first and second recombination sequences are positioned in the same orientation with respect to one another.  
     
     
         5 . A helper virus according to one of  claims 1  to  4 , characterized in that said first and second recombination sequences are selected from the group formed by the sequences loxP, FRT and R.  
     
     
         6 . A helper virus according to one of  claims 1  to  5 , for the production of a recombinant adenoviral vector defective for replication, which derives from the genome of an adenovirus and comprises the ITRs 5′ and 3′, an encapsidation region and at least one viral gene selected from the genes of the E1, E2, E4 and L1-L5 regions, said gene being defective in said recombinant adenoviral vector, characterized in that it comprises a first recombination sequence at 5′ and a second recombination sequence at 3′ of said encapsidation region.  
     
     
         7 . A helper virus according to  claim 6 , characterized in that it is devoid of all or part of the E1 and/or E4 region.  
     
     
         8 . A helper virus according to one of  claims 1  to  7 , characterized in that it additionally comprises a DNA fragment coding for a recombinase.  
     
     
         9 . A complementation cell line comprising a DNA fragment coding for a recombinase.  
     
     
         10 . A complementation cell line according to  claim 9  and a helper virus according to  claim 8 , in which said DNA fragment codes for a recombinase selected from the group formed by CRE, CRE-ER, FLP and R.  
     
     
         11 . A complementation cell line according to  claim 9  or  10 , deriving from the 293 line.  
     
     
         12 . A procedure for preparing a viral particle comprising a recombinant viral vector, which comprises the following steps: 
 (a) Preparing a recombinant viral vector-deficient for replication;    (b) Preparing a helper virus according to one of  claims 1  to  8  and  10 ;    (c) Introducing the recombinant viral vector and the helper virus into an appropriate cell line;    (d) Culturing said cell line under appropriate conditions to allow the production of the viral particle in the presence of a functional recombinase able to recognize said first and second recombination sequences; and    (e) Recovering the viral particle in the cell culture.    
     
     
         13 . A procedure according to  claim 12 , which additionally comprises an amplification step, probably in step (d).  
     
     
         14 . A procedure according to  claim 12  or  13 , according to which, in the course of step (d), the recombinase is added to the cell culture.  
     
     
         15 . A procedure according to  claim 12  or  13 , according to which said helper virus is according to  claim 8  and said cell line is the 293 complementation cell line.  
     
     
         16 . A procedure according to  claim 15 , which comprises the following steps: 
 (a) Preparing a recombinant adenoviral vector defective for all of the adenoviral functions with the exception of E4;    (b) Preparing a helper virus defective for the E1 and E4 functions and comprising (i) the LoxP sequences at 5′ and at 3′ of the encapsidation region and (ii) a DNA fragment coding for the CRE-ER hybrid recombinase;    (c) Introducing the recombinant viral vector and the helper virus into a 293 cell line;    (d) Culturing said cell line under appropriate conditions to allow the production of the viral particle and, after a sufficiently long time, continuing the culture in a medium comprising estradiol; and    (e) Recovering the viral particle in the cell culture.    
     
     
         17 . A procedure according to  claim 12  or  13 , according to which said helper virus is according to one of  claims 1  to  7  and said cell line according to one of  claims 9  to  11 .  
     
     
         18 . A procedure according to  claim 17 , which comprises the following steps: 
 (a) Preparing a recombinant adenoviral vector defective for all of the adenoviral functions with the exception of E4;    (b) Preparing a helper virus defective for the E1 and E4 functions and comprising the LoxP sequences at 5′ and 3′ of the encapsidation region;    (c) Introducing the recombinant viral vector and the helper virus into a 293 cell line comprising a DNA fragment coding for the CRE-ER hybrid recombinase.    (d) Culturing said cell line under appropriate conditions to allow the production of the viral particle and, after a sufficiently long time, continuing the culture in a medium comprising estradiol; and    (e) Recovering the viral particle in the cell culture.    
     
     
         19 . A procedure according to one of  claims 12  to  18 , which additionally comprises a step of purification of the recombinant viral vector.  
     
     
         20 . A procedure for preparing a viral particle comprising a recombinant viral vector by means of a helper virus, according to which the ratio viral particles of recombinant viral vector to helper virus is greater than 50%, advantageously greater than 60%, preferably greater than 70% and, in a very preferred manner greater than 80%.  
     
     
         21 . A viral particle obtained by a procedure according to one of  claims 12  to  20 .  
     
     
         22 . A eukaryotic host cell comprising a viral particle according to  claim 21 .  
     
     
         23 . A pharmaceutical composition comprising as therapeutic or prophylactic agent a viral particle according to  claim 21  or a eukaryotic host cell according to  claim 22 , in combination with a carrier acceptable from a pharmaceutical point of view.  
     
     
         24 . Viral particle according to  claim 21  or eukaryotic host cell according to  claim 22  for use as medicament.