RECOMBINANT VIRUS PRODUCTION USING MAMMALIAN CELLS IN SUSPENSION
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 (RSI), 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.
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6 . The method of claim 1 , 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.
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9 . The method of claim 1 , 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.
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14 . The method of claim 1 , 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 claim 1 wherein the co-infection is simultaneous.
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24 . A method for producing recombinant viral particles in a mammalian cell according to claim 1 , 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 (RSI), 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.
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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 recombinant 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 method of claim 33 , wherein the recombinant herpesvirus is a virus selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein barr virus (EBV).
35 . The method of claim 34 , wherein the recombinant herpesvirus is replication defective.
36 . The method of claim 33 , wherein the gene of interest is a therapeutic gene.
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