Novel compositions and methods for production of recombinant virus

Abstract

This invention relates to novel nonmammalian carrier vectors and viruses useful in the production of high titers of recombinant viruses which may contain foreign DNA inserts or which may be point-mutated or deleted viruses, and methods of producing those viruses. The nonmammalian carrier vector (“carrier vector”) is a chimeric vector which includes those portions of a nonmammalian virus backbone which allow replication in a nonmammalian host cell. The carrier vector includes various nucleic acid cassettes, which may include an embedded recombinant viral genome containing a desired transgene, components necessary for production of a replication-defective recombinant virus containing the transgene, and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant virus as a substantially homogeneous. preparation, compositions to produce the recombinant virus, and novel recombinant viruses.

1 . A carrier vector for the manufacture of a recombinant virus, wherein the carrier vector comprises a nonmammalian virus backbone, or a portion or a modification thereof which is capable of replication in a nonmammalian cell.  
     
     
         2 . The carrier vector of  claim 1  further comprising one or more of the following elements: 1) an embedded recombinant viral genome; 2) nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant viral genome, 3) nucleic acid sequences encoding helper functions, 4) nucleic acid sequences that encode a ligand that can interact with a mammalian cell, and 5) regulatory control sequences that regulate sequences in the nonmammalian virus backbone or in a replication-proficient portion or modification thereof.  
     
     
         3 . The carrier vector according to  claim 2  which comprises nucleic acid sequences that encode a ligand that can bind to a mammalian cell receptor.  
     
     
         4 . The carrier vector according to  claim 1  which further comprises an embedded recombinant viral genome.  
     
     
         5 . The carrier vector according to  claim 1  which further comprises nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant viral genome.  
     
     
         6 . The carrier vector according to  claim 1  which further comprises nucleic acid sequences encoding helper functions.  
     
     
         7 . The carrier vector according to  claim 1  which further comprises regulatory control sequences that regulate expression of nucleic acid sequences in the nonmammalian virus backbone or in a replication-proficient portion or modification thereof.  
     
     
         8 . The carrier vector according to  claim 1  which further comprises nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant viral genome and nucleic acid sequences encoding helper functions.  
     
     
         9 . The carrier vector according to  claim 1  which further comprises an embedded recombinant viral genome and nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant viral genome and/or nucleic acid sequences encoding helper functions.  
     
     
         10 . The carrier vector according to  claim 1  which comprises all those elements which are required by a mammalian host cell to produce an infectious recombinant virus.  
     
     
         11 . The carrier vector according to  claim 2  wherein the nucleic acid sequences that encode a ligand that can interact with a mammalian cell are under the regulatory control of a non-mammalian promoter.  
     
     
         12 . The carrier vector according to  claim 11 , wherein the ligand can bind to a mammalian cell receptor.  
     
     
         13 . The carrier vector according to  claim 2  wherein the embedded recombinant viral genome comprises flanking sequences derived from an adeno-associated virus (AAV), an adenovirus, a retrovirus or a herpesvirus.  
     
     
         14 . The carrier vector according to  claim 13 , wherein the embedded recombinant viral genome comprises flanking sequences derived from AAV and the carrier vector further comprises helper sequences encoding a protein providing a helper function required for replication of AAV.  
     
     
         15 . The carrier vector according to  claim 14 , wherein said helper sequences are derived from adenovirus (Ad) DNA, herpes simplex virus (HSV) type I or type II DNA, pseudorabies virus (PRV), cytomegalovirus (CMV) or vaccinia virus.  
     
     
         16 . The carrier vector according to  claim 15 , wherein said helper sequences encode at least one gene product selected from the group consisting of adenoviral genes E1A, E1B, E2A, E4orf6 and VAI, or at least one gene product selected from the group consisting of HSV type 1 genes UL5, UL8, UL52, and UL29.  
     
     
         17 . The carrier vector according to  claim 14 , further comprising a nucleic acid sequence encoding the AAV rep and cap proteins.  
     
     
         18 . The carrier vector according to  claim 13 , wherein the embedded recombinant viral genome comprises flanking sequences and packaging signals derived from adenovirus.  
     
     
         19 . The carrier vector according to  claim 13 , wherein the embedded recombinant viral genome comprises a herpesvirus “a” packaging sequence and a herpesvirus origin of replication.  
     
     
         20 . The carrier vector according to  claim 2  wherein the embedded recombinant viral genome comprises a transgene whose expression is regulated by expression regulatory sequences operably linked to said transgene.  
     
     
         21 . A method for producing a carrier virus, comprising the steps of: 
 (a) modifying a nonmammalian virus backbone DNA, or a replication-proficient portion or modification thereof, by inserting one or more nucleic acid inserts comprising 
 (i) a recombinant viral genome comprising a transgene operably linked to expression regulatory sequences and flanked by flanking elements,  
 (ii) nucleic acid sequences encoding helper functions operably linked to expression regulatory sequences,  
 (iii) nucleic acid sequences encoding replication and/or encapsidation functions for the recombinant virus,  
 (iv) a ligand DNA operably linked to expression regulatory sequences that are active in nonmammalian cells, and  
 (v) regulatory control sequences that regulate sequences in the nonmammalian virus backbone, a modified nonmammalian virus backbone or a replication-proficient portion of the backbone or modified backbone;  
   (b) transducing the resulting carrier vector into nonmammalian host cells;    (c) growing the nonmammalian host cells under conditions in which carrier virus is produced; and    (d) collecting the carrier virus from the nonmammalian host cells.    
     
     
         22 . The method according to  claim 21 , wherein the nonmammalian virus backbone is derived from baculovirus and the nonmammalian host cells are insect cells.  
     
     
         23 . A lysate or supernatant comprising the carrier virus produced by the method according to  claim 21 .  
     
     
         24 . The method according to  claim 21  further comprising the step of purifying the carrier virus.  
     
     
         25 . A purified preparation of carrier virus produced by the method according to  claim 21 .  
     
     
         26 . A method for producing a recombinant virus, comprising the steps of: 
 a) infecting mammalian host cells with a carrier virus, wherein the carrier virus optionally expresses a ligand on its surface;    b) growing the infected mammalian host cells under conditions in which the embedded recombinant viral genome is replicated, excised and encapsidated; and    c) collecting the recombinant virus from the mammalian host cells.    
     
     
         27 . The method according to  claim 26 , wherein the mammalian host cells are selected from CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, W138, MRC5, A549, HT1080, 293, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7 or HeLa cells.  
     
     
         28 . A lysate or supernatant comprising the recombinant virus produced by the method according to  claim 26 .  
     
     
         29 . The method according to  claim 26  further comprising the step of purifying the recombinant virus from the mammalian host cells.  
     
     
         30 . A purified recombinant virus produced by the method according to  claim 26 .  
     
     
         31 . A pharmaceutical composition comprising the carrier virus according to  claim 25  or the recombinant virus according to  claim 30  and further comprising a pharmaceutically acceptable carrier.  
     
     
         32 . A method for transient or stable gene transfer of a desired transgene to a mammalian cell, comprising the step of infecting the mammalian cell with the recombinant virus according to  claim 30 .  
     
     
         33 . The method according to  claim 32 , wherein said transient or stable gene transfer is for genetic immunization, correction of genetic defects or production of proteins in vitro, in vivo, or ex vivo.  
     
     
         34 . A method of using a recombinant virus comprising a point mutation or deletion as a vaccine, comprising the steps of producing an attenuated replication-proficient recombinant virus or a replication-deficient recombinant virus by the method of  claim 26  and administering the recombinant virus to a patient in a dose effective to induce an immunogenic response.  
     
     
         35 . The pharmaceutical composition according to  claim 31 , wherein the carrier virus further comprises adenoviral sequences required for replication and encapsidation of the recombinant adenovirus.  
     
     
         36 . A pharmaceutical composition comprising the recombinant virus according to  claim 30  and further comprising a pharmaceutically acceptable carrier.  
     
     
         37 . A pharmaceutical composition comprising the recombinant virus produced by the method according to  claim 26  and further comprising a pharmaceutically acceptable carrier.  
     
     
         38 . A pharmaceutical composition comprising the carrier virus produced by the method according to  claim 21  and further comprising a pharmaceutically acceptable carrier.  
     
     
         39 . A method for transient or stable gene transfer of a desired transgene to a mammalian cell, comprising the step of infecting the mammalian cell with the carrier virus produced by the method according to  claim 25.