AAV production in insect cells, methods and compositions therefor

Abstract

Compositions and methods are disclosed for producing adeno-associated virus (AAV) in insect cells in vitro. Recombinant baculovirus vectors include an AAV Capsid gene expression cassette (Cap), an AAV Rep gene expression cassette (Rep), and a baculovirus homologous region (hr) located up to about 4 kb from a start codon in an AAV expression cassette. Production levels of baculovirus and AAV in insect cells harboring recombinant baculovirus comprising a Cap, a Rep, and an hr are higher compared to controls comprising a Cap and a Rep but no hr. Furthermore, levels of baculovirus and AAV production in insect cells infected with recombinant baculovirus comprising a Cap, a Rep, and an hr are comparatively stable over serial passages of cells, whereas levels of baculovirus and AAV production decline over serial passages of insect cells comprising recombinant baculovirus comprising a Cap and a Rep, but no hr.

Claims

1. A baculovirus vector for adeno-associated virus (AAV) production, comprising: an AAV Cap expression cassette; an AAV Rep expression cassette; and a baculovirus homologous region 2 (hr2) located up to about 4 kb from a start codon of an AAV expression cassette, wherein the vector has no Rep binding element (RBE) sequence, no inverted terminal region (ITR) containing an RBE sequence, and no promoter containing an RBE sequence.

2. The vector in accordance with claim 1, wherein the hr2 region is between the Rep expression cassette and the Cap expression cassette, and wherein the Rep expression cassette and the Cap expression cassette are in a head to head (5′ to 5′) orientation.

3. An insect cell line comprising cells comprising a vector in accordance claim 1.

4. The insect cell line in accordance with claim 3, wherein the cells further comprise a second vector, said second vector comprising a transgene flanked by AAV ITRs.

5. A method of growing baculovirus in vitro, comprising: providing a culture of insect cells in accordance with claim 3; and incubating the cells.

6. The method in accordance with claim 5, wherein the incubating the cells comprises passaging the cells, and wherein AAV production yield at passage 7 is at least 2-fold greater compared to a control insect cell line comprising a baculovirus vector comprising an AAV Cap expression cassette and an AAV Rep expression cassette but no baculovirus hr.

7. The method in accordance with claim 5, wherein the titer at passage 7 of baculovirus comprising the AAV Cap expression cassette is greater than 21.5% of total baculovirus titer.

8. A method of growing AAV in vitro, comprising: providing a culture of insect cells; infecting or transfecting the insect cells with a baculovirus vector in accordance with claim 1; and incubating the cells.

9. The method in accordance with claim 8, wherein the yield at P7 of AAV from the insect cells is at least 50% greater than the yield at P7 of AAV from insect cells comprising a baculovirus vector without the hr.

10. The method in accordance with claim 8, wherein the yield at P7 of AAV from cells comprising the baculovirus hr is at least 20% greater than the yield of AAV from cells comprising a baculovirus vector without the hr.

11. The method in accordance with claim 8, wherein the baculovirus vector is exclusive of a Rep binding element (RBE).

12. A method of producing AAV in vitro, comprising growing an insect cell culture comprising a vector in accordance with claim 1, and a vector comprising a transgene flanked by AAV ITRs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a baculovirus shuttle plasmid of the present teachings, comprising an hr2 sequence between an AAV8 capsid gene and an AAV2 rep gene.

(2) FIG. 2 illustrates a baculovirus shuttle plasmid of the present teachings, comprising an AAV8 Cap gene, and an hr2 sequence between an AAV2 rep gene and a gentamicin resistance (GmR) gene.

(3) FIG. 3 illustrates a baculovirus shuttle plasmid of the present teachings, comprising an AAV9 Cap gene, and an hr2 sequence between an AAV2 rep gene and a gentamicin resistance (GmR) gene.

(4) FIG. 4 illustrates a baculovirus shuttle plasmid of the present teachings, comprising an AAV6 Cap gene, and an hr2 sequence between an AAV2 rep gene and a gentamicin resistance (GmR) gene.

(5) FIG. 5 illustrates a baculovirus shuttle plasmid of the present teachings, comprising an AAV1 Cap gene, and an hr2 sequence between an AAV2 rep gene and a gentamicin resistance (GmR) gene.

(6) FIG. 6 illustrates a baculovirus shuttle plasmid of the present teachings, comprising an AAV5 Cap gene, and an hr2 sequence between an AAV2 rep gene and a gentamicin resistance (GmR) gene.

(7) FIG. 7 illustrates the GFP expression cassette flanked by two ITRs in baculovirus shuttle plasmid V372-pFB-CMV-GFP-SV40 pA-full ITR.

(8) FIG. 8 illustrates comparative AAV production yields by recombinant baculoviruses (rBVs) with or without the hr2 sequence.

(9) FIG. 9A-H illustrates comparative recombinant baculovirus (rBV) titers with or without the hr2 sequence from passage 3 to passage 10.

(10) FIG. 10A-E illustrates comparative AAV production yields between recombinant baculoviruses (rBVs) with or without the hr2 sequence from passage 3 to passage 10 or at passage 10 between AAV strains.

(11) FIG. 11A-B illustrates Western blot expression of AAV capsid proteins in cells infected with recombinant baculoviruses with and without hr2 sequences.

(12) FIG. 12 A-B illustrates Western blot expression of AAV rep proteins in cells infected with recombinant baculoviruses with and without hr2 sequences.

DETAILED DESCRIPTION

(13) Methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), Cold Spring Harbor, N.Y., 2003 and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. As used in the present description and any appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise.

(14) The following materials and methods are also used in various aspects of the present teachings.

(15) Insect Cell Culture

(16) Spodoptera frugiperda Sf9 cells, Trichoplusia ni cells, and Estima acrea cells were cultured in coming storage bottles at 28° C. in ESF921 medium (Expression Systems, Davis, Calif.) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin. The cells were split 1:4 once the cell density reaches 8×10.sup.6 cells/mi for maintenance.

(17) Plasmid Construction and Recombinant Baculovirus Generation

(18) The hr2 sequence was PCR amplified from the baculovirus genome and cloned into plasmid V053-pFBD-inRepOpt-inCap8 to create V059-pFBD-inRepOpt-hr2-inCap8. In order to include the kozak sequence, plasmid V059 was cut with BstZ17I and AgeI to remove the BstZ17I-AgeI fragment and replaced with the BstZ17I-AgeI fragment with kozak sequence upstream of VP1 start codon from V150-pFB-inCap8-inRep-kozak to create V277-pFB-inCap8-hr2-inRep-kozak where the hr2 is located between the Rep and the Cap expression cassettes. To insert the hr2 sequence into another location after the polyA sequence of the Rep expression cassette, the hr2 sequence was PCR amplified with primers 3205F (5′-GCTTTACGAGTAGAATTCTACGTGT-3′ SEQ ID NO: 7) and 3206R (5′-GGCCTACGTAGTTTACACGTAGAATTCTACTCGT-3′ SEQ ID NO: 8) from V277. The pc promoter sequence was amplified with primers 3065F (5′-ATTTGACTTGGTCAGGGCCG-3′ SEQ ID NO: 9) and 3204R (5′-GAATTCTACTCGTAAAGCCCAGTTGACATAAGCCTGTTCG-3′ SEQ ID NO: 10) from V150. These hr2 and pc promoter PCR fragments were joined together through a second PCR reaction with primers 3065F and 3206R. The joined PCR fragment was digested with BsrG1 and SnaBI and ligated into the BsrG1 and SnaBI sites of V150, V212, V195, V188, and V146 to create V288-pFB-inCap8-inRep-hr2, V289-pFB-inCap9-inRep-hr2, V290-pFB-inCap6-inRep-hr2, V291-pFB-inCap1-inRep-hr2, and V295-pFB-inCap5-inRep-hr2 respectively. Examples of plasmids with an hr sequence insertion are illustrated in FIGS. 1-6.

(19) Plasmid pFB-CMV-GFP was constructed by PCR amplifying the GFP fragment which was then cloned into the multiple cloning sites of V032-pFB-CMV-SV40 pA (FIG. 7)

(20) The plasmids were used to generate bacmids according to manufacturer's protocol (Invitrogen). Briefly, plasmids were diluted to a concentration of 2 ng/μl and 2 μl of plasmid DNA was used to transform DH10Bac competent cells. After 2 days of incubation, white colonies were picked and miniprep bacmid DNAs were prepared. The miniprep bacmid DNAs were used to transfect S9 cells to generate recombinant baculoviruses.

(21) Plaque Purification and Passaging of Recombinant Baculovirus

(22) The generated recombinant baculoviruses were plaque purified in order to get homogenous clones. Briefly, Sf9 cells were plated on 6-well plates with cell density of 1.5e+6 cells % well in 2 ml ESF921 media and incubated at 28° C. for 30 min. The baculoviruses were each diluted to 10.sup.−2, 10.sup.−3, 10.sup.−4, 10.sup.−5, 10.sup.−6, and 10.sup.−7 in 1 ml volume. At the end of incubation, media were removed from the wells and 250 μl of each dilution was added to infect the Sf9 cells at 28° C. for 1 hour. At the end of the incubation, 3 ml of 1% agarose overlay (cool down to 42° C.) was added to the wells. When the agarose was solidified, 2 ml of ESF921 media was added to each well and the plates were incubated at 28° C. for 5 to 7 days. Well-formed plaques were picked and used to infect insect cells for passaging.

(23) To passage the plaque purified recombinant baculoviruses, insect cells were infected with about 1 moi of the viruses for 3 days at 28° C. and supernatant was harvested. The harvested viruses were used to infect fresh insect cells again and so on until passage 10.

(24) Real-Time Quantitative PCR (qPCR) Quantification of Recombinant Baculoviruses and AAV Vectors

(25) To determine the titers of recombinant baculoviruses and AAV, a qPCR method was employed. It was empirically determined that one plaque-forming unit (pfu) comprises about 20 genome copies of the virus. Briefly, harvested viruses were diluted in qPCR dilution buffer and heated to 95° C. for 30 min to break the virus particles. The treated virus samples were then assayed in the CHROMO4™ system (Bio-Rad Laboratories, Inc., Hercules, Calif.) together with a known standard. The Ct values were converted to pfu and used to guide the baculovirus passaging and AAV production.

(26) AAV Vector Production and Quantification

(27) Recombinant baculoviruses were used to infect insect cells to produce AAV vectors. Briefly, 10 moi of recombinant baculovirus containing the Rep and Cap genes with or without the hr2 sequence were co-infected with 5 moi of recombinant baculovirus containing the GFP marker gene flanked by AAV ITRs for 3 days at 28° C. Cell pellets were collected by centrifugation at 3000 rpm for 10 min. The cell pellets were lysed in SF9 lysis buffer (50 mM Tris-HCl, pH7.8, 50 mM NaCl, 2 mM MgCl.sub.2, 1% Sarkosyl, 1% Triton X-100, and 140 units/mi Benzonase) by sonication. Cell debris was removed by centrifugation at 8000 rpm for 20 min. The cleared lysates were used for quantification of AAV productivity as follows: the lysates were diluted with qPCR dilution buffer and contaminating DNA was destroyed by incubating with DNase I enzyme at 37° C. for 1 hour. The DNase I enzyme was inactivated by heating at 95° C. for 30 min. in the presence of 100 mM EDTA. The treated AAV samples were further diluted and assayed in the Chromo4 qPCR machine. The Ct values were converted to AAV vector genome copies.

(28) Western Blot Analysis

(29) Recombinant baculoviruses containing the rep and cap genes were used to infect Sf9 cells for three days and cell pellets were harvested by centrifugation at 3,000 rpm for 10 min. The cell pellets with about 2×10.sup.6 cells were first resuspended in 300 μl of PBS buffer and then vortexed with 100 μl of 4×LDS sample buffer to lyse the cells. The lysates were heated at 95° C. for 5 min and then sonicated for 20 seconds to shear the genomic DNA. After brief centrifugation, the lysates were then loaded onto a 10% SDS-gel to separate the proteins. The proteins were then transferred from the gel to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were probed with specific antibodies against the rep or cap proteins. A second antibody coupled with horseradish peroxidase (HRP) against the first antibody was used to detect the rep or cap proteins through color matrix reaction.

EXAMPLES

(30) The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or aspect. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

(31) This example illustrates a shuttle vector of the present teachings.

(32) In this plasmid (FIG. 1), an hr2 sequence is located between an AAV8 capsid gene cassette and an AAV2 rep gene cassette. The vector includes the Cap and Rep genes in head-to-head orientation. This shuttle plasmid was used to generate recombinant baculovirus (rBV), which in turn was used to produce AAV8 in insect cells.

Example 2

(33) This example illustrates a shuttle vector of the present teachings.

(34) In this plasmid (FIG. 2), an hr2 sequence is located between an AAV2 rep gene cassette and a gentamicin resistance (GmR). The vector also includes an AAV8 capsid gene cassette. The Cap and Rep genes are in a head-to-tail orientation. This plasmid was used to generate recombinant baculovirus (rBV), which in turn was used to produce AAV8 in insect cells.

Example 3

(35) This example illustrates a shuttle vector of the present teachings.

(36) In this plasmid (FIG. 3), an hr2 sequence is located between an AAV2 rep gene cassette and a gentamicin resistance (GmR). The vector also includes an AAV9 capsid gene cassette. The Cap and Rep genes are in a head-to-tail orientation. This plasmid was used to generate recombinant baculovirus (rBV), which in turn was used to produce AAV9 in insect cells.

Example 4

(37) This example illustrates a shuttle vector of the present teachings.

(38) In this plasmid (FIG. 4), an hr2 sequence is located between an AAV2 rep gene cassette and a gentamicin resistance (GmR). The vector also includes an AAV6 capsid gene cassette. The Cap and Rep genes are in a head-to-tail orientation. This plasmid was used to generate recombinant baculovirus (rBV), which in turn was used to produce AAV6 in insect cells.

Example 5

(39) This example illustrates a shuttle vector of the present teachings.

(40) In this plasmid (FIG. 5), an hr2 sequence is located between an AAV2 rep gene cassette and a gentamicin resistance (GmR). The vector also includes an AAV1 capsid gene cassette. The Cap and Rep genes are in a head-to-tail orientation. This plasmid was used to generate recombinant baculovirus (rBV), which in turn was used to produce AAV1 in insect cells.

Example 6

(41) This example illustrates a shuttle vector of the present teachings.

(42) In this plasmid (FIG. 6), an hr2 sequence is located between an AAV2 rep gene cassette and a gentamicin resistance (GmR). The vector also includes an AAV5 capsid gene cassette. The Cap and Rep genes are in a head-to-tail orientation. This plasmid was used to generate recombinant baculovirus (rBV), which in turn was used to produce AAV5 in insect cells.

Example 7

(43) This example illustrates that an hr sequence enhances yields of AAV production.

(44) In these experiments, the hr2 sequence was cloned either between the Rep and the Cap expression cassettes in plasmid V277 (FIG. 1) or after the poly A sequence of the Rep expression cassette in plasmids V289 (FIG. 3) and V295 (FIG. 6) and recombinant baculoviruses were prepared. These recombinant baculoviruses were used to coinfect Sf9 cells with recombinant baculovirus carrying GFP gene for AAV production. The results are shown in FIG. 8. The data indicate that hr2 sequence enhances the AAV productivity irrespective of the location of the hr2 sequence and AAV serotypes. The increase of AAV productivity ranged from 2- to 4-fold compared to controls lacking an hr.

Example 8

(45) This example illustrates that an hr sequence enhances stability of recombinant baculoviruses containing the AAV rep and cap genes.

(46) In these experiments, to further analyze the stability of recombinant baculoviruses with or without the hr2 sequence, plaque purification and passaging of the recombinant baculoviruses multiple times were employed. A pair of qPCR primers—gp64F (5′-CCCTCTGTGTACTTGGCTCTAACG-3′ SEQ ID NO: 11) and gp64R (5′-CGGTGAAACGCAAAGTCGAGCACCG-3′ SEQ ID NO: 12)—corresponding to the gp64 gene (present in all recombinant baculoviruses of the present teachings) was used to determine total baculovirus titer. For baculoviruses comprising the Rep and Cap expression cassettes, a pair of qPCR primers—Rep2F (5′-ATTCATOCTCCACCTCAACC-3′ SEQ ID NO: 13) and Rep2R (5′-GCCGTCTGGATCATGACTIT-3′ SEQ ID NO: 14)-corresponding to the Rep sequence was used to determine titer of these baculoviruses.

(47) Recombinant baculoviruses carrying Cap1-Rep (FIG. 5), Cap6-Rep (FIG. 4), Cap9-Rep (FIG. 3), and Cap8-Rep (FIG. 2) were selected for these experiments. For each passage, the total baculovirus pfu was determined with the gp64 primers, and set to 100%. The specific baculovirus for each passage was determined using the Rep primers set as percentage of total baculovirus. The results are shown in FIG. 9A-H for recombinant baculovirus (rBV) titers with or without the hr2 sequence. FIG. 9A illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-inCap1-inRep (Cap1 VI88) expressing AAV1 capsid and AAV2 rep genes without the hr2 sequence. FIG. 9B illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-inCap1-inRep-hr2 (Cap1 V291 expressing AAV1 capsid and AAV2 rep genes with the hr2 sequence). FIG. 9C illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV expressing AAV6 capsid and AAV2 rep genes without the hr2 sequence. FIG. 9D illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-inCap6-inRep (Cap6 V195) expressing AAV6 capsid and AAV2 rep genes with the hr2 sequence. FIG. 9E illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-inCap9-inRep (Cap9 V212) expressing AAV9 capsid and AAV2 rep genes without hr2 sequence. FIG. 9F illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-inCap9-inRep-hr2 (Cap9 V289) expressing AAV9 capsid and AAV2 rep genes with hr2 sequence. FIG. 9G illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-Cap8-inRep (Cap8 V150) expressing AAV8 capsid and AAV2 rep genes without hr2 sequence. FIG. 9H illustrates rBV titers determined with gp64 and rep2 qPCR primers in cells harboring rBV-inCap8-inRep-hr2 (Cap8 V288) expressing AAV8 capsid and AAV2 rep genes with hr2 sequence. rBVs were produced and passaged in Sf9 cells (FIG. 9A-D), Tni pro cells (FIG. 9E-F), and E4a cells (FIG. 9G-H). The data indicate that specific baculovirus titer decreased with increasing number of passages when the baculovirus did not contain an hr2 sequence (FIG. 9A, FIG. 9C, FIG. 9E, and FIG. 9G), whereas there was a smaller decrease in specific baculovirus titer when the baculovirus contained the hr2 sequence near the rep expression cassette (FIG. 9B, FIG. 9D, FIG. 9F, and FIG. 9H). These changes held true regardless of whether SF9 (FIG. 9A-9D). Tni Pro (FIG. 9E-F), or E4a cells (FIG. 9G-H) were the host cells. By passage P10, baculovirus with AAV Cap1 or AAV Cap 6 were below 10% of total rBV titer when the vectors contained no hr (FIG. 9A, FIG. 9C, FIG. 9E, and FIG. 9G), but baculovirus with AAV Cap1 or AAV Cap 6 were about 20% of total rBV titer when the vectors contained hr2 (FIG. 9B, FIG. 9D, FIG. 9F, FIG. 9H).

Example 9

(48) This example illustrates that an hr sequence enhances AAV productivity of recombinant baculoviruses (rBV) containing the AAV rep and cap genes.

(49) In these experiments, the rBVs with or without the hr sequence from passage 3 to passage 10 were used to co-infect Sf9, Tni Pro, and E4a cell lines with the rBV containing GFP to produce AAV vectors. After three days of co-infection, the cell pellets were harvested and AAV production yields were determined. As shown in FIG. 10A-D, the production yield of AAV1 in Sf9 cells (FIG. 10A), AAV6 in Sf9 cells (FIG. 10B), AAV9 in Tni pro cells (FIG. 10C) and AAV8 in E4a cells (FIG. 10D) was maintained over passages 3-10 when the baculovirus vector included an hr2 sequence. In contrast, production yield of AAV1, AAV6, AAV9, and AAV8 declined dramatically over passages 3-10 in the absence of hr2 in the rBV. To further confirm this observation, S9 cells were co-infected with passage 10 rBVs with hr2 (V290, V288, and V289) or without hr2 (V195, V150, and V212) sequence to produce AAV6 (V195 and V290), AAV8 (V150 and V288), and AAV9 (V212 and V289) vectors. The results show substantially higher AAV production yields from S9 cells infected with rBV containing hr2 than those infected with rBV without hr2 sequence (FIG. 10E) (compare hr.sup.+ rBVs V290, V288 and V289, to hr rBVs V195, V150 and V212).

Example 10

(50) This example illustrates that AAV rep and cap expression directly correlates with rBV stability through multiple passages.

(51) In these experiments, Western blots were performed to determine the expression level of rep and cap proteins. FIG. 11A-B illustrate the expression of AAV6 capsid proteins VP1, VP2, and VP3 after infection of Sf9 cells with recombinant baculoviruses rBV-inCap6-inRep (V195) without hr2 sequence (FIG. 11A) and rBV-inCap6-inRep-hr2 (V290) with hr2 sequence (FIG. 11B) from passage 3 to passage 10 respectively. M, protein size markers; lanes 1 to 8, cell lysates prepared from Sf9 cells infected with rBVs from passages 3 to 10.

(52) FIG. 12A-B illustrate the expression of AAV2 rep proteins REP78 and REP52 after infection of Sf9 cells with recombinant baculoviruses rBV-inCap8-inRep (V150) without hr2 sequence (FIG. 12A) and rBV-inCap8-inRep-hr2 (V288) with hr2 sequence (FIG. 12B). M, protein size markers; lanes 1-5, cell lysates prepared from Sf9 cells infected with rBVs from passages 6 to 10. The results in FIG. 11 and FIG. 12 indicate that rBVs with an hr sequence express higher levels of rep and cap proteins throughout multiple passages, including later passages, compared to rBVs lacking an hr sequence.

(53) All cited references are incorporated by reference, each in its entirety. Applicant reserves the right to challenge any conclusions presented by the authors of any reference.