Recombinant virus production using mammalian cells in suspension

Abstract

The invention generally provides methods for producing recombinant AAV viral particles using cells grown in suspension. The invention provides recombinant AAV particles for use in methods for delivering genes encoding therapeutic proteins, and methods for using the recombinant AAV particles in gene therapy.

1 . A method for producing recombinant AAV viral particles in a mammalian cell comprising:
 co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest; and   allowing the virus to infect the mammalian cell;   
       thereby producing recombinant AAV viral particles in a mammalian cell. 
     
     
         2 . The method of  claim 1 , wherein the gene of interest is a therapeutic gene. 
     
     
         3 . The method of  claim 2 , wherein the therapeutic gene is selected from the group consisting of: an inhibitor of anti-angiogenic genes, alpha-1 antitrypsin, retinoschisin, acid alpha glucosidase, RPE65, beta-subunit of the cone photoreceptor cGMP-gated channel (CNGB-3), alpha-subunit of the cone photoreceptor cGMP-gated channel (CNGA-3), cone photoreceptor G-protein alpha-subunit (GNAT2), Retinal pigment epithelium-specific 65 kDa (RPE65), X-linked juvenile retinoschisis (RS1), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Myotonic dystrophy protein kinase (DMPK), CCHC-type zinc finger, nucleic acid binding protein (known as CNBP or ZNF9), Retinitis pigmentosa GTPase regulator (RPGR), Acid α-glucosidase (GAA), Choroideremia (CHM), Rab escort protein-1 (REP1), Alpha-synuclein (SNCA), Coagulation factor VIII, procoagulant component (hemophilia A or F8), Coagulation factor IX (plasma thromboplastic component, Christmas disease, hemophilia B or F9), Aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), X-linked Inhibitor of Apoptosis Protein (XIAP), clarin-1 (CLRN1), Leber's hereditary neuropathy genes (MT-ND1, MT-ND4, MT-ND4L, and MT-ND6), alpha-galactosidase A (α-Gal A) or Alpha-L-iduronidase. 
     
     
         4 . The method of  claim 1 , wherein the AAV cap gene has a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, and AAV-8, AAV-9, and rh-AAV-10. 
     
     
         5 . A method for producing recombinant AAV viral particles in a mammalian cell comprising:
 co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep2 and an AAV cap1 or cap 2 gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising an AI gene or an alpha 1 antitrypsin gene, and a promoter operably linked to said gene; and   allowing the virus to infect the mammalian cell;   thereby producing recombinant AAV viral particles in a mammalian cell.   
     
     
         6 . The method of any of  claims 1 - 5 , wherein the mammalian cell is selected from the group consisting of: BHK, HEK-293 (293), Vero, RD, HT-1080, A549, Cos-7, ARPE-19, and MRC-5. 
     
     
         7 . A method for producing recombinant AAV viral particles in a BHK cell comprising:
 co-infecting a BHK cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest; and   allowing the virus to infect the BHK cell;   
       thereby producing recombinant AAV viral particles in a BHK cell. 
     
     
         8 . A method for producing recombinant viral particles in a BHK cell comprising:
 co-infecting a BHK cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep2 and an AAV cap1 or cap 2 gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising an AI gene or an alpha 1 antitrypsin gene, and a promoter operably linked to said gene of interest; and   allowing the virus to infect the BHK cell;   
       thereby producing recombinant viral particles in a BHK cell. 
     
     
         9 . The method of any one of  claim 1 ,  5 ,  7  or  8 , wherein the herpesvirus is a virus selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein barr virus (EBV). 
     
     
         10 . The method of  claim 9 , wherein the herpesvirus is replication defective. 
     
     
         11 . The method of  claim 7 , wherein the gene of interest is a therapeutic gene. 
     
     
         12 . The method of  claim 11 , wherein the therapeutic gene is selected from the group consisting of: anti-angiogenic genes, alpha-1 antitrypsin, retinoschisin, acid alpha glucosidase, RPE65, beta-subunit of the cone photoreceptor cGMP-gated channel (CNGB-3), alpha-subunit of the cone photoreceptor cGMP-gated channel (CNGA-3), cone photoreceptor G-protein alpha-subunit (GNAT2), Retinal pigment epithelium-specific 65 kDa (RPE65), X-linked juvenile retinoschisis (RS1), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Myotonic dystrophy protein kinase (DMPK), CCHC-type zinc finger, nucleic acid binding protein (known as CNBP or ZNF9), Retinitis pigmentosa GTPase regulator (RPGR), Acid α-glucosidase (GAA), Choroideremia (CHM), Rab escort protein-1 (REP1), Alpha-synuclein (SNCA), Coagulation factor VIII, procoagulant component (hemophilia A or F8), Coagulation factor IX (plasma thromboplastic component, Christmas disease, hemophilia B or F9), Aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), X-linked Inhibitor of Apoptosis Protein (XIAP), clarin-1 (CLRN1), Leber's hereditary neuropathy genes (MT-ND1, MT-ND4, MT-ND4L, and MT-ND6), alpha-galactosidase A (α-Gal A) or Alpha-L-iduronidase. 
     
     
         13 . The method of  claim 7 , wherein the AAV cap gene has a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and rhAAV-10. 
     
     
         14 . The method of any one of  claims 1 - 13 , further comprising the step of determining multiplicity of infection (MOI). 
     
     
         15 . The method of  claim 14 , wherein the MOI is between: 3 and 14. 
     
     
         16 . The method of any one of  claims 1 - 15  wherein the co-infection is simultaneous. 
     
     
         17 . A method for producing recombinant viral particles in a BHK cell comprising:
 simultaneously co-infecting a BHK cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest;   allowing the virus to infect the BHK cell; and   purifying the viral particles;   
       thereby producing recombinant viral particles in a BHK cell. 
     
     
         18 . A method for producing recombinant viral particles in a BHK cell comprising:
 simultaneously co-infecting a BHK cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising an AI gene or an alpha 1 antitrypsin gene, and a promoter operably linked to said gene of interest;   allowing the virus to infect the BHK cell; and   purifying the viral particles;   
       thereby producing recombinant viral particles in a BHK cell. 
     
     
         19 . The method of  claim 17  or  18 , wherein the herpesvirus is a virus selected from the group consisting of: HSV-1, HSV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8. 
     
     
         20 . The method of  claim 19  wherein the recombinant herpesvirus is replication defective. 
     
     
         21 . The method of  claim 17 , wherein the gene of interest is a therapeutic gene. 
     
     
         22 . The method of  claim 21 , wherein the therapeutic gene is selected from the group consisting of: AI, alpha-1 antitrypsin, retinoschisin, acid alpha glucosidase, and RPE65. 
     
     
         23 . The method of  claim 17  or  18 , wherein the AAV cap gene has a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and rhAAV-10. 
     
     
         24 . A method for producing recombinant viral particles in a mammalian cell according to any one of  claims 1 - 23 , whereby the number of viral particles produced is equal to or greater than the number of viral particles grown in an equal number of cells under adherent conditions. 
     
     
         25 . A recombinant AAV viral particle produced in a mammalian cell by the method comprising
 co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest; and   allowing the virus to infect the mammalian cell;   
       thereby producing recombinant AAV viral particles in a mammalian cell. 
     
     
         26 . The recombinant viral particle of  claim 25 , wherein the herpesvirus is a virus selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein barr virus (EBV). 
     
     
         27 . The recombinant viral particle of  claim 26 , wherein the recombinant herpesvirus is replication defective. 
     
     
         28 . The recombinant viral particle of  claim 26 , wherein the gene of interest is a therapeutic gene. 
     
     
         29 . The recombinant viral particle of  claim 26 , wherein the therapeutic gene is selected from the group consisting of: anti-angiogenic genes, alpha-1 antitrypsin, retinoschisin, acid alpha glucosidase, RPE65, beta-subunit of the cone photoreceptor cGMP-gated channel (CNGB-3), alpha-subunit of the cone photoreceptor cGMP-gated channel (CNGA-3), cone photoreceptor G-protein alpha-subunit (GNAT2), Retinal pigment epithelium-specific 65 kDa (RPE65), X-linked juvenile retinoschisis (RS1), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Myotonic dystrophy protein kinase (DMPK), CCHC-type zinc finger, nucleic acid binding protein (known as CNBP or ZNF9), Retinitis pigmentosa GTPase regulator (RPGR), Acid α-glucosidase (GAA), Choroideremia (CHM), Rab escort protein-1 (REP1), Alpha-synuclein (SNCA), Coagulation factor VIII, procoagulant component (hemophilia A or F8), Coagulation factor IX (plasma thromboplastic component, Christmas disease, hemophilia B or F9), Aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), X-linked Inhibitor of Apoptosis Protein (XIAP), clarin-1 (CLRN1), Leber's hereditary neuropathy genes (MT-ND1, MT-ND4, MT-ND4L, and MT-ND6), alpha-galactosidase A (α-Gal A) or Alpha-L-iduronidase. 
     
     
         30 . The recombinant viral particle of  claim 26 , wherein the gene of interest is a reporter gene. 
     
     
         31 . The recombinant viral particle of  claim 26 , wherein the AAV cap gene has a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and rhAAV-10. 
     
     
         32 . A recombinant AAV viral particle produced in a BHK cell comprising:
 co-infecting a BHK cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest; and   allowing the virus to infect the BHK cell;   
       thereby producing recombinant AAV viral particles in a BHK cell. 
     
     
         33 . A method for delivering a nucleic acid sequence encoding a therapeutic protein to a target cell, the method comprising:
 co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second herpesvirus comprising a gene of interest, wherein the gene of interest comprises a therapeutic gene, and a promoter operably linked to said gene of interest; and   allowing the virus to infect the mammalian cell and express the nucleic acid sequence encoding a therapeutic protein;   
       thereby delivering a nucleic acid sequence encoding a therapeutic protein to the target cell. 
     
     
         34 . The recombinant viral particle of  claim 32  or  33 , wherein the herpesvirus is a virus selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein ban virus (EBV). 
     
     
         35 . The recombinant viral particle of  claim 34 , wherein the recombinant herpesvirus is replication defective. 
     
     
         36 . The recombinant viral particle of  claim 33 , wherein the gene of interest is a therapeutic gene. 
     
     
         37 . The recombinant viral particle of  claim 34  or  36 , wherein the therapeutic gene is selected from the group consisting of: anti-angiogenic genes, alpha-1 antitrypsin, retinoschisin, acid alpha glucosidase, RPE65, beta-subunit of the cone photoreceptor cGMP-gated channel (CNGB-3), alpha-subunit of the cone photoreceptor cGMP-gated channel (CNGA-3), cone photoreceptor G-protein alpha-subunit (GNAT2), Retinal pigment epithelium-specific 65 kDa (RPE65), X-linked juvenile retinoschisis (RS1), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Myotonic dystrophy protein kinase (DMPK), CCHC-type zinc finger, nucleic acid binding protein (known as CNBP or ZNF9), Retinitis pigmentosa GTPase regulator (RPGR), Acid α-glucosidase (GAA), Choroideremia (CHM), Rab escort protein-1 (REP1), Alpha-synuclein (SNCA), Coagulation factor VIII, procoagulant component (hemophilia A or F8), Coagulation factor IX (plasma thromboplastic component, Christmas disease, hemophilia B or F9), Aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), X-linked Inhibitor of Apoptosis Protein (XIAP), clarin-1 (CLRN1), Leber's hereditary neuropathy genes (MT-ND1, MT-ND4, MT-ND4L, and MT-ND6), alpha-galactosidase A (α-Gal A) or Alpha-L-iduronidase. 
     
     
         38 . The recombinant viral particle of  claim 33  or  34 , wherein the AAV cap gene has a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and rhAAV-10. 
     
     
         39 . A kit for making a recombinant viral particle in a mammalian cell that is capable of growing in suspension, and instructions for use. 
     
     
         40 . A kit for delivering a nucleic acid sequence encoding a therapeutic protein to a target cell according to  claim 33 , and instructions for use.