Means and methods to increase adenovirus production

Abstract

The invention relates to a method for increasing the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region, wherein the adenoviruses are generated in a production cell by: (a) expressing an adenoviral pIX polypeptide from a nucleic acid sequence encoding adenoviral pIX polypeptide under the control of at least a minimal endogenous pIX promoter and a heterologous promoter; and (b) expressing the elements necessary for the production and assembly of the adenoviruses, thereby increasing the yield of adenoviruses generated in the production cell in comparison to the yield in the absence of nucleic acid sequence encoding the adenoviral pIX polypeptide. Further, the invention relates to a method for constructing an adenovirus library, a production cell, and the use of an adenoviral pIX polypeptide for increasing the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region.

Claims

1. A method for increasing the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region, wherein said adenoviruses are generated in a production cell, the method comprising the steps of: (a) expressing in said production cell an adenoviral pIX polypeptide from a nucleic acid sequence encoding said adenoviral pIX polypeptide under the control of at least a minimal endogenous pIX promoter and at least one heterologous promoter, wherein said minimal endogenous pIX promoter is located downstream of said heterologous promoter; and (b) expressing in said production cell the elements necessary for the production and assembly of said adenoviruses from corresponding coding sequences, thereby increasing the yield of said adenoviruses generated in said production cell in comparison to the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region generated in said production cell in the absence of said nucleic acid sequence encoding said adenoviral pIX polypeptide, wherein the adenovirus is a human adenovirus of subgroup D.

2. A method for the construction of a replication-incompetent adenovirus library, wherein the adenoviruses have at least a partial deletion in the E1-region, comprising the steps of: (a) providing one or more nucleic acid sequences in expressible form comprising at least two partial adenoviral genomes, wherein each nucleic acid sequence of said one or more nucleic acid sequences comprises at least one partial adenoviral genome, each partial adenoviral genome further comprising at least one transgene, wherein the at least two partial adenovirus genomes and/or the two transgenes differ from each other; (b) introducing the one or more nucleic acid sequences of step (a) into production cells comprising one or more nucleic acid sequences in expressible form comprising a partial adenoviral genome which complements each partial adenoviral genome comprised by the one or more nucleic acid sequences of step (a) by transfection, wherein each complemented adenoviral genome encodes the elements necessary for the production and assembly of said different adenoviruses and comprises the coding sequence for an adenoviral pIX polypeptide under the control of at least a minimal endogenous pIX promoter and a heterologous promoter, wherein said minimal endogenous pIX promoter is located downstream of said heterologous promoter; and (c) culturing the production cells under conditions suitable for the assembly and production of said differing adenoviruses, thereby constructing said replication-incompetent adenovirus library.

3. The method of claim 1, wherein one or more of said coding sequences of step (b) are introduced into the production cell for expression (a) by transduction using the replication-incompetent adenoviruses having at least a partial deletion in the E1-region that are to be produced in the cell; or (b) by transfection.

4. The method of claim 1, wherein the subgroup D adenovirus is of serotype 19a.

5. The method of claim 1, further comprising the step of assessing the expression level of said pIX protein in the production cell and/or the increase in yield of said adenoviruses.

6. The method of claim 1, wherein the subgroup D adenovirus is selected from the group consisting of serotypes 8, 9, 13, 15, 17, 19, 20, 22 to 25, 27 to 30, 32, 33, 36 to 39, 42 to 49 and 51.

7. The method of claim 1, wherein the production cell is selected from the group consisting of a HEK293 production cell, a Per.C6 production cell, a CAP cell, a GH329 production cell and a pTG6559 production cell.

8. The method of claim 1, wherein the heterologous promoter is (a) a heterologous minimal pIX promoter; or (b) selected from the group consisting of a viral promoter, a cellular promoter, synthetic promoter and a hybrid promoter.

9. The method of claim 8, wherein the heterologous minimal pIX promoter of (a) originates from a human adenovirus serotype 5.

10. The method of claim 8, wherein the heterologous promoter of (b) is selected from the group consisting of CAG, CMV, PKG, SV40, EF1 alpha and RSV.

11. The method of claim 1, wherein the coding sequence for said adenoviral pIX polypeptide is under the control of an additional heterologous promoter.

12. The method of claim 4, wherein the heterologous promoter is (a) a heterologous minimal pIX promoter that originates from a human adenovirus serotype 5; or (b) an SV40 viral promoter.

Description

(1) The FIGURE shows:

(2) FIG. 1:

(3) A schematic representation including vectors maps for the construction of recombinant adenovirus type 19a genome harbouring a GFP expression cassette with or without a heterologous promoter upstream of their respective Ad19a pIX coding sequence is shown in FIGS. 1A-D.

(4) The examples illustrate the invention:

(5) Example 1: Construction of Recombinant Adenovirus Type 19a BACs Using Site-Specific Recombination in E. coli Expressing Flp Recombinase

(6) For construction of a human type 19a recombinant adenovirus genome, a first Ad19a nucleic acid pDonorSir19aGFP containing a GFP expression cassette (pDonorSir19aGFP (SEQ ID NO.: 1), and the acceptor Ad19a nucleic acid molecule pBACSir19a_SV40 (SEQ ID NO.: 2), were combined and reacted in DH10B E. coli cells harbouring pBACSir19a-_SV40 and the plasmid pCP20 for conditional expression of FLP recombinase. The plasmid pDonorSir19aGFP was introduced into the DH10B E. coli cells by means of electroporation using a standard protocol. The nucleic acid Ad19a molecule pBACSir19a_SV40 was maintained in E. coli DH10B (or equivalent E. coli K12-derived strains lacking the F-factor) harbouring a conditional expression system for Flp. Here, in example 1, the DH10B cells harboured the adenovirus type 19a BAC pBACSir19a_SV40, and the Flp recombinase was provided by the plasmid pCP20, which replication is controlled by a temperature-sensitive origin of replication. DH10B cells harbouring pBACSir19a_SV40 and the pCP20 were maintained at 30 C. in the presence of ampicillin (50 g/ml) and chloramphenicol (25 g/ml). Next, these DH10B cells were electro-transformed with pDonorSir19aGFP and cultured for 60 minutes at 42 C. in the absence of any antibiotics. The expressed Flp induced site-specific recombination between FRT sites present on pDonorSir19aGFP and pBACSir19a_SV40, respectively. At the same time the elimination of Flp expression also started, since pCP20 cannot replicate in E. coli at elevated temperature. The transformed culture was plated onto agar plates which contained kanamycin (25 g/ml) and chloramphenicol (25 g/ml) as selecting agents. Under these conditions E. coli containing recombined recombinant adenovirus type 19a BACs (pRAB19aGFP_SV40 SEQ ID NO.: 3) were selected in which at least one pDonorSir19aGFP plasmid had recombined with pBACSir19a_SV40. DNA from growing cultures of DH10B cells was isolated and the integrity of the reaction products analysed by restriction digestion with Kpnl. All the recombination products analysed contained one copy of a contiguous Ad19a vector sequence flanked by the Ad19a ITRs (data not shown). A schematic representation and vectors maps for the construction of recombinant adenovirus type 19a genome harbouring a GFP expression cassette and a heterologous SV40 promoter is shown in FIG. 1A.

(7) Example 2: Reconstitution of and Production of Recombinant Human Adenovirus Type 19a Vectors in 293 Cells

(8) DNA from cultures of DH10B cells containing the pRAB19aGFP_SV40 was isolated and purified from saturated E. coli overnight cultures (100 ml) in LB medium using a kit for plasmid preparation. Here, the NUCLEOBOND PC-100 kit from Macherey and Nagel, Germany was used according to the manufacturer's recommendations. For virus reconstitution purified pRAB19aGFP_SV40 DNA was treated with 10 U PacI per g DNA for 2 h according to the manufacture's recommendations. Subsequently the PacI-digested DNA was purified using phenol-chloroform according to standard protocols prior to transfection into 293 cells. In brief, 10 g pRAB19a DNA was digested in a volume of 100 l for 1.5 h at 37 C. in a water bath. Subsequently 50 l phenol/chloroform (1:1 mixture) was added to the reaction tube (Eppendorff cup size 1.5 ml, Eppendorf AG, Hamburg, Germany) and vortexed for 20 sec. Here, the Vortexer MS-3 basic was used (IKA Werke GmbH & Co. KG, Staufen, Germany). The tube was centrifuged in a table top centrifuge at maximum speed (20000.times.g) for 5 min at room temperature and 80 l of the aqueous upper phase was transferred into a fresh tube and 10 l 3 M NaAc (pH 4.5) and 200 l EtOH was added. All reagents and chemicals were purchased from CARL ROTH GMBH+CO. KG, Karlsruhe. The tube was mixed with the finger tips until the precipitated DNA became visible. Moreover, the tube was incubated for 5 min at room temperature and the DNA was pelleted in a table top centrifuge at maximum speed for 15 min at room temperature. The supernatant was quantitatively removed and the pellet immediately dissolved in 20 l sterile deionized water.

(9) HEK-293 cells plated in DMEM (PAA)+10% FCS+2 mM L-Glutamine+1% PS (standard culture media) in Ewell plates the day before were transfected with the adenoviral DNA by using JETPEI (Polyplus, Illkirch Cedex, France) according to manufacturer's guidelines and incubated for 3 days at 37 C.

(10) Cells were then flushed off using standard culture media, centrifuged for 5 min at 100 g and resuspended in 400 l standard culture media. The viral particles were released by 3 rounds of freeze-thaw. Briefly the cell suspension was frozen in liquid nitrogen for 2 min until the suspension solidified and then thawed in a water bath at 37 C. for 2 min. This procedure was repeated 3 times and cell debris was removed by centrifugation at 3500 g for 10 minutes at 4 C.

(11) Fresh HEK-293 cells plated the day before on 6 well plates were then infected with the resulting cell lysate and incubated at 37 C. When cells became confluent they were expanded from Ewell to 10 cm dishes. Infected cells were cultivated until the cytopathic effect was completed (CPE). Harvest was performed as previously described. The cell pellet was resuspended in 1000 l culture media. Subsequently, 3 rounds of freeze-thaw was performed as described above.

(12) 5E+06 HEK293 cells were seeded in a 15 cm dish and infected the next day with 150 l of the virus inoculum received from the second 6 well plate. Harvest was performed when cpe was almost completed. Finally cells were harvested as described above and cell pellet was resuspended in 400 l culture media. The titer of the obtained recombinant human Ad19a adenovirus vector expressing GFP (hAd19aGFP_SV40) was determined as fluorescence forming units (IU) 48 h after 293 cells had been infected with limited dilutions with purified adenovirus. The genomic titer in vector genomes was determined by means of QPCR according. The titer was 1.36E+08 IU/ml and the yield 6.79E+07 IU in total.
Example 3: Effect of Promoter Choice for pIX Expression on Virus Reconstitution

(13) A series of recombinant human Ad19a adenovirus vector genomes containing an expression cassette for GFP were constructed applying the same method as described in example 1. The first recombinant adenovirus vector genome contained a heterologous human adenovirus type 5 pIX promoter upstream of the pIX coding sequence and a GFP expression cassette (pRAB.sub. pRAB19aGFP_5pIX, SEQ ID NO.: 4). This vector was constructed by combining the donor plasmid pDonorSir19aGFP_5pIX (SEQ ID NO.: 5) and the acceptor vector pBACSir19aGFP (SEQ ID NO.: 7). The second recombinant adenovirus vector genome contained a heterologous SV40 promoter upstream of the pIX coding sequence and a GFP expression cassette (pRAB.sub. pRAB19aGFP_SV40 (SEQ ID NO.: 3)). This vector was constructed by combining the donor plasmid pDonorSir19aGFP (SEQ ID NO.: 1) and the acceptor vector pBACSir19a_SV40 (SEQ ID NO.: 2) containing an SV40 promoter sequence upstream of pIX coding sequence. The third recombinant adenovirus vector genome contained no heterologous promoter upstream of the pIX coding sequence (pRAB19aGFP, SEQ ID NO.: 7). This vector was constructed by combining the donor plasmid pDonorSir19aGFP and the acceptor vector pBACSir19a (SEQ ID NO.: 6). The fourth recombinant adenovirus vector genome contained both the adenovirus type 5 pIX promoter and the SV40 promoter upstream of the pIX coding sequence pRAB19aGFP_p5IX+SV40 (SEQ ID NO.: 8). This vector was constructed by combining the donor plasmid pDonorSir19aGFP_5pIX (SEQ ID NO.: 5) and the acceptor vector pBACSir19a_SV40 (SEQ ID NO.: 2). A schematic representation including vectors maps for the construction of recombinant adenovirus type 19a genome harboring a GFP expression cassette with or without a heterologous promoter upstream of their respective Ad19a pIX coding sequence is shown in FIGS. 1A-D.

(14) Subsequently the four recombinant adenovirus type 19a vector genomes were reconstituted and produced in 293 cells according to the method provided in example 2. To define the effect of the presence of heterologous promoters upstream of the pIX open reading frame on reconstitution all vectors were harvested at the same timepoint independently on the cpe state. 300 l of the 6 well lysate was used to transduce 5E+06 cells seeded in 15 cm dishes. When cells became confluent, a 1:2 split was performed. The total yield of recombinant adenovirus vectors was dependent on the presence of heterologous promoters, and increased by 39-fold for the adenovirus with the combined 5pIX+SV40 promoter (hAd19aGFP_5pIX+SV40) compared to the adenovirus vector without any promoter hAd19aGFP_delta. The yield of adenoviruses with the SV40 promoter only hAd19aGFP_SV40 was increased 22-fold over hAd19aGFP_delta, and the yield of the adenovirus vector with the 5pIX promoter hAd19aGFP_5pIX was increased 13-fold over hAd19aGFP_delta (Table 1). The adenovirus serotype 19a vectors all contained a minimal endogenous pIX promoter sequence.

(15) TABLE-US-00001 TABLE 1 Effect of promoter choice for pIX expression on virus reconstitution Factor Titer relative to Virus Promoter (ifu/ml) Total ifu no promoter hAd19aGFP_ SV40 + 5pIX 1.97E+09 1.57E+09 38.88 5pIX + SV40 hAd19aGFP_delta no promoter 5.06E+07 4.04E+07 1.00 hAd19aGFP_5pIX 5pIX 6.76E+08 5.41E+08 13.37 hAd19aGFP_SV40 SV40 1.10E+09 8.81E+08 21.79 Table 1: The duration until the 2.sup.nd rescue was harvested was 6 days. Cells were seeded in 15 cm dishes at 5.00E+06 and an inoculum of 300 l was used per 15 cm dish. Duration for virus amplification on 15 cm dish until cpe completed was 6 days with partial cpe on all dishes observed for all virus constructs. The 15 cm dishes were split 1:2 and the total duration of the experiment was 15 days.
Example 4: Effect of Promoter Choice For pIX Expression on Virus Amplification

(16) To define the effect of promoter choice on the virus amplification, the adenovirus vectors described in Example 3 were produced in HEK293 the same way as described there but harvest was performed when all cells showed complete cpe which resulted in various harvest times. The improving effect of SV40 promotor in hAd19aGFP_SV40 and hAd19aGFP_5pIX+SV40 compared to hAd19aGFP_delta was 2.69 for the presence of the SV40 promoter alone, and 2.44-fold for the SV40 promoter in combination with the 5pIX promoter. hAd19aGFP_5pIX+SV40 and hAd19aGFP_SV40 were harvested after 2 days, hAd19aGFP_delta was harvested after 5 days (Table 2).

(17) TABLE-US-00002 TABLE 2 Effect of promoter choice for pIX expression on virus amplification Duration for virus amplification on 15 cm dish until cpe Total Titer Total Factor relative to Virus Promoter completed duration (ifu/ml) ifu no promoter hAd19aGFP_5pIX + SV40 + 2 days 13 days 1.23E+08 6.16E+07 2.44 SV40 5pIX hAd19aGFP_delta no 5 days 16 days 5.06E+07 2.53E+07 1.00 promoter hAd19aGFP_SV40 SV40 2 days 13 days 1.36E+08 6.79E+07 2.69 Table 2: The duration until the 2.sup.nd rescue was harvested was 8 days. Cells were seeded in 15 cm dishes at 5.00E+06 and an inoculum of 250 l was used per 15 cm dish. Complete cpe on all dishes was observed for all virus constructs. The 15 cm dishes were not split.
Example 5: Effect of Promoter Choice for pIX Expression on Virus Yield after Inoculation with a Defined Virus Amount

(18) To simulate virus batch production, 2.5E+06 HEK293 cells were seeded in 15 cm dishes and inoculated with the vectors hAd19aGFP_delta, hAd19aGFP_5pIX, hAd19aGFP_SV40, hAd19aGFP_5pIX+SV40 with a defined amount of infectious viral particles. A multiplicity of infection of 5 (MOI) was used. Harvest was performed at the time point when cells showed a CPE associated with rounding up of cells and beginning cell detachment. Accordingly, hAd19aGFP_5pIX+SV40 and hAd19aGFP_SV40 were harvested 4 days after inoculation, and the viruses hAd19aGFP_delta and hAd19aGFP_5pIX harvested after 5 days with only partially completed cpe. The total yield of recombinant virus was increased in the presence of heterologous promotor 5pIX+SV40 by 2.26 fold compared to the promotor of hAd19aGFP_delta. The hAd19aGFP_5pIX resulted in a less significant increase of 1.89 followed by an 1.59 increase of the hAd19aGFP_SV40 promotor (Table 3a). The total yield of recombinant virus particles was increased in the presence of heterologous promotor 5pIX+SV40 by 6.89-fold compared to the promotor of hAd19aGFP_delta. The hAd19aGFP_5pIX resulted in a less significant increase of 6.17-fold followed by an 6-fold increase of the hAd19aGFP_SV40 promotor (Table 3b).

(19) TABLE-US-00003 TABLE 3a Effect of promoter choice for pIX expression on virus yield after defined virus amplification Duration for virus Cells amplification Factor seeded on 15 cm relative in 15 cm dish until cpe Titer Total to no Virus Promoter dish completed (ifu/ml) ifu promoter hAd19aGFP_5pIX + SV40 + 2.50E+06 4 days 1.93E+08 7.71E+07 2.26 SV40 5pIX hAd19aGFP_delta no 2.50E+06 5 days 8.53E+07 3.41E+07 1.00 promoter hAd19aGFP_5pIX 5pIX 2.50E+06 5 days 1.61E+08 6.45E+07 1.89 hAd19aGFP_SV40 SV40 2.50E+06 4 days 1.36E+08 5.44E+07 1.59 Cells were seeded in 15 cm dishes at 2.50E+06 and a volume inoculum of MOI5 was used per 15 cm dish. Partial cpe on dishes was observed for all virus constructs, except for construct hAd19aGFP_5pIX + SV40 which showed complete cpe. The 15 cm dishes were not split.

(20) TABLE-US-00004 TABLE 3b Duration for virus Factor ampli- in- fication crease on 15 vg Cells cm dish relative seeded until to no in 15 cm cpe com- pro- Virus Promoter dish pleted vg/ml moter hAd19aGFP_ SV40 + 2.50E+06 4 days 8.13E+10 6.89 5pIX + SV40 5pIX hAd19aGFP_ no 2.50E+06 5 days 1.18E+10 1 delta promoter hAd19aGFP_ 5pIX 2.50E+06 5 days 7.28E+10 6.17 5pIX hAd19aGFP_ SV40 2.50E+06 4 days 7.09E+10 6.00 SV40 Cells were seeded in 15 cm dishes at 2.50E+06 and a volume inoculum of MOI5 was used per 15 cm dish. Partial cpe on dishes was observed for all virus constructs, except for construct hAd19aGFP_5pIX + SV40 which showed complete cpe. The 15 cm dishes were not split.