Recombinant virus production using mammalian cells in suspension

1. A method for producing recombinant AAV viral particles, the method comprising:

co-infecting a suspension BHK cell 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 a second recombinant herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest;

wherein said cell is infected at a combined multiplicity of infection (MOI) of between 3 and 14; and

allowing the cell to produce the recombinant AAV viral particles, thereby producing the recombinant AAV viral particles.

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, an alpha-1 antitrypsin gene, a retinoschisin gene, an acid alpha glucosidase gene, a RPE65 gene, a beta-subunit of the cone photoreceptor cGMP-gated channel (CNGB-3) gene, an alpha-subunit of the cone photoreceptor cGMP-gated channel (CNGA-3) gene, a cone photoreceptor G-protein alpha-subunit (GNAT2) gene, a Retinal pigment epithelium-specific 65 kDa (RPE65) gene, a X-linked juvenile retinoschisis (RS1) gene, a Brain-derived neurotrophic factor (BDNF) gene, a Glial cell-derived neurotrophic factor (GDNF) gene, a Myotonic dystrophy protein kinase (DMPK) gene, a CCHC-type zinc finger gene, a nucleic acid binding protein (known as CNBP or ZNF9) gene, a Retinitis pigmentosa GTPase regulator (RPGR) gene, an Acid α-glucosidase (GAA) gene, a Choroideremia (CHM) gene, a Rab escort protein-1 (REP1) gene, an Alpha-synuclein (SNCA) gene, a Coagulation factor VIII gene, a procoagulant component (hemophilia A) gene, a procoagulant component (F8) gene, a Coagulation factor IX gene, a plasma thromboplastic component gene, a Coagulation factor IX of Christmas disease gene, a Coagulation factor IX of hemophilia B gene, a Coagulation factor IX of F9 gene, an Aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) gene, a X-linked Inhibitor of Apoptosis Protein (XIAP) gene, a clarin-1 (CLRN1) gene, a Leber's hereditary neuropathy gene, a MT-ND1 gene, a MT-ND4 gene, a MT-ND4L gene, a MT-ND6 gene, an alpha-galactosidase A (α-Gal A) gene and an Alpha-L-iduronidase gene.

4. The method of claim 1 , wherein the cap gene is selected from an AAV with 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 rh-AAV-10.

5. The method of claim 1 , wherein the first herpesvirus and the second herpesvirus are viruses selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein barr virus (EBV).

6. The method of claim 5 , wherein the herpesvirus is replication defective.

7. The method of claim 1 wherein the co-infection is simultaneous.

8. A method for producing recombinant AAV viral particles in a mammalian cell, the method comprising:

co-infecting a suspension BHK cell 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 a second recombinant herpesvirus comprising a gene of interest, and a promoter operably linked to said gene of interest;

wherein said cell is infected at a combined multiplicity of infection (MOI) of between 3 and 14; and

allowing the cell to propagate,

thereby producing the recombinant AAV viral particles, 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.

9. A method for delivering a nucleic acid sequence encoding a therapeutic protein to a suspension BHK cell, the method comprising:

co-infecting the BHK cell 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 a second herpesvirus comprising a gene of interest, wherein the gene of interest comprises a therapeutic protein coding sequence, and a promoter operably linked to said gene of interest; and

wherein said BHK cell is infected at a combined multiplicity of infection (MOI) of between 3 and 14; and

allowing the virus to infect the BHK cell and express the therapeutic protein,

thereby delivering the nucleic acid sequence encoding the therapeutic protein to the BHK cell.