Recombinant herpes viruses for preparing recombinant adeno-associated viruses

Abstract

This document describes a recombinant herpesvirus which comprises the AAV rep and cap genes and also a process for producing high-titer, highly infectious adeno-associated virus vector preparations.

1 . A recombinant herpesvirus, which contains a rep and a cap gene derived from adeno-associated viruses (AAVs) and operatively linked to an expression control sequence.  
   
   
       2 . A recombinant herpesvirus as claimed in  claim 1  which does not exhibit any reversion to the wild type.  
   
   
       3 . A recombinant herpesvirus as claimed in  claim 1 , which additionally comprises a reporter gene.  
   
   
       4 . A recombinant herpesvirus as claimed in  claim 1 , which is selected from the group of Herpesviridae comrprising herpes simplex virus (HSV), cytomegalovirus (CMV), pseudorabies virus (PRV) and Epstein-Barr virus (EBV) and other members of the herpesvirus family.  
   
   
       5 . A recombinant herpesvirus as claimed in  claim 4 , which is a herpes simplex virus (HSV).  
   
   
       6 . A recombinant herpesvirus as claimed in  claim 5 , which is the HSV-1 mutant 1802.  
   
   
       7 . A recombinant herpesvirus as claimed in  claim 1 , which is a mutant which is completely or partially replication-deficient.  
   
   
       8 . A recombinant herpesvirus as claimed in  claim 1 , wherein the insertion does not encompass the complete AAV ITR sequence.  
   
   
       9 . A recombinant herpesvirus as claimed in  claim 1 , wherein the AAV rep gene and the AAV cap gene are inserted in the U L  or the U S  region of the herpesvirus.  
   
   
       10 . A process for preparing a recombinant herpesvirus as claimed in  claim 1 , wherein the AAV rep gene and the AAV cap gene are stably integrated into the genome of a herpesvirus.  
   
   
       11 . The process as claimed in  claim 10 , wherein the rep gene and the cap gene are integrated into the herpes genome by restriction cleavage/ligation or by homologous recombination.  
   
   
       12 . The process as claimed in  claim 10 , wherein use is made of an HSV mutant which possesses a unique restriction site.  
   
   
       13 . The process as claimed in  claim 11 , wherein use is made of an HSV mutant which is completely or partially replication-deficient.  
   
   
       14 . A nucleic acid which comprises the helper functions of a herpesvirus genome which are required for replicating AAV viruses and, inserted therein, a rep gene and a cap gene derived from adeno-associated viruses (AAVs), in each case operatively linked to an expression control sequence.  
   
   
       15 . A vector, which comprises a nucleic acid as claimed in  claim 14 .  
   
   
       16 . A viral composition which comprises a recombinant herpervirus as claimed in  claim 1 .  
   
   
       17 . A composition as claimed in  claim 16 , which is free of wild-type herpesvirus.  
   
   
       18 . A process for preparing infectious AAV vector preparations, comprising the steps of: 
 a) preparing a viral vector which is based on adeno-associated viruses (AAVs),    b) preparing a recombinant herpesvirus as claimed in  claim 1 ,    c) introducing the AAV vector from (a) and the recombinant herpesvirus from (b) into a cell,    d) replicating the AAV vector, and    e) obtaining an infectious AAV vector preparation.    
   
   
       19 . The process as claimed in  claim 18 , wherein the AAV vector and the recombinant herpesvirus are introduced into the cell by infection.  
   
   
       20 . The process as claimed in  claim 18 , wherein an encapsulated rAAV preparation is obtained.  
   
   
       21 . The process as claimed in claims  18 , wherein use is made of a replicatable recombinant herpesvirus.  
   
   
       22 . The process as claimed in claims  18 , wherein use is made of a non-replicatable recombinant herpesvirus.  
   
   
       23 . A cell, which contains a recombinant herpesvirus as claimed in  claim 1  or a vector as claimed in  claim 15 .  
   
   
       24 . A cell as claimed in  claim 23 , wherein the recombinant herpesvirus or the vector has been introduced by infection.  
   
   
       25 . A cell as claimed in  claim 23 , which additionally contains a recombinant AAV vector.  
   
   
       26 . A cell as claimed in  claim 25 , wherein the AAV vector contains a heterologous DNA insert which encodes a therapeutically active polypeptide.  
   
   
       27 . A cell as claimed in  claim 23 , which is a BHK cell, a Vero cell or a HeLa cell.  
   
   
       28 . A process for producing infectious AAV vector preparations, with an AAV vector and a helper virus being introduced into a cell, the AAV vector being replicated and an infectious AAV vector preparation being obtained from the cell and/or the culture supernatant, wherein the AAV vector and the helper virus are introduced into the cell by infection.  
   
   
       29 . A recombinant herpesvirus, which contains a rep and a cap gene derived from adeno-associated viruses (AAVs) and operatively linked to an expression control sequence, with the rep gene and cap gene being located on an insert which is integrated in the genome of the herpesvirus.  
   
   
       30 . A nucleic acid which comprises the helper functions of a herpesvirus genome which are required for replicating AAV viruses and, inserted therein, a rep gene and a cap gene derived from adeno-associated viruses (AAVs), in each case operatively linked to an expression control sequence, with the rep gene and the cap gene being located on an insert which is integrated in the genome of the herpesvirus.