KR101606910B1 - A549 Based Cell Line for Preparing Adenovirus Vector - Google Patents

Abstract

The present invention relates to a production cell line for producing adenovirus vector, and more particularly to an adenovirus-producing cell line based on A549 cell line containing adenovirus-derived E1a gene and E1b gene.
According to the present invention, it is possible to provide a safe adenovirus-producing cell line that is excellent in adenovirus production efficiency and low in the ability to infect wild-type virus.

Description

A549-based cell line for producing adenovirus vector {A549 Based Cell Line for Preparative Adenovirus Vector}

The present invention relates to a production cell line for producing adenovirus vector, and more particularly to an adenovirus-producing cell line based on A549 cell line containing adenovirus-derived E1a gene and E1b gene.

Adenoviruses exhibit certain properties that are particularly advantageous for use as vectors for gene delivery in gene therapy. Specifically, they have a fairly wide range of hosts, are able to infect dormant cells, are not integrated into the genome of infected cells, and are not associated with major pathology in the human body to date. Thus, adenovirus has been used to deliver the gene of interest to muscle, liver, nervous system, etc. (Ragot et al. , Nature 361: 647, 1993; Jaffe et al. , Nature genetics 1: 372, 1992; Akli et al . , Nature genetics 3: 224, 1993).

The adenovirus is a virus with linear double helix DNA having a size of about 36 kb. Its genome includes an inverted repeat sequence (ITR) at each end, an encapsidation sequence (Psi), an early gene and a late gene. Important early genes are contained in the E1, E2, E3 and E4 genes. Of these, genes contained in the E1 region are essential for virus multiplication. Important late genes are included within the L1 to L5 region. The genome of Ad5 adenovirus is fully sequenced and available on database (Genebank M73260). Likewise, some or even all of the other adenoviral genomes (Ad2, Ad7, Ad12, etc.) are also sequenced.

For use in their gene therapy, various vectors derived from adenovirus have been produced by integrating various genes (β-gal, OTC, α-1AT, cytokine, etc.). In each of these constructs, the adenovirus was modified to be non-replicable in infected cells. Thus, the constructs described in the prior art are adenoviruses in which the E1 region essential for viral replication is deleted and to which extent heterologous DNA sequences have been inserted (Levrero et al., Gene 101: 195, 1991; Gosh-Choudhury et al. , Gene 50: 161, 1986). These adenoviruses are produced in a complementary cell line 293, into which a portion of the adenoviral genome is integrated.

However, a vector lacking the E1 region (E1-vector, designated as the first generation vector) exhibits certain disadvantages for therapeutic use. Specifically, they may not be a complete defect for in vivo replication, particularly because of the presence of certain complementarity-shifting cellular functions. Thus, E1 homologous transcriptional activity was detected in F9-derived carcinoma cells (Imperiale et al ., Mol. Cell. Biol. 4: 867-874, 1984). The same type of activity regulated by interleukin-6 was also detected (Spergel et al. , J. Virol . 66: 1021-1030, 1992). Other disadvantages associated with these vectors are that there are a number of viral genes that can be expressed in vivo after gene delivery and that can cause immune or tumor responses.

To overcome these disadvantages, it has been proposed to generate other deletions or modifications within the adenoviral genome. Thus, a sitonal point mutation was introduced into the ts125 mutant to inactivate the 72 kDa DNA binding protein (DBP) (Van der Vliet et Sussenbach, Virology 67: 415-426, 1975). These vectors can also be produced at high temperatures in cells of 293 cells at an authorized temperature (32 ° C). However, this type of vector also has many disadvantages such as overexpression of the E4 region in vivo, the presence of point mutations that can be mutated back, the risk of partial activation at 37 < 0 > C,

In addition, a variety of cell lines such as HEK293 and PERC6, which are conventional adenovirus producing cell lines, have been developed with a strategy to minimize the occurrence of RCA (Replication Competent Adenovirus) in safety, but the most commonly used HEK293 cell line There is no problem about royalty payment for use, but RCA is somewhat higher, and the use of PERC6 (Crucell), which resolves the RCA problem to some extent, is used.

Therefore, the inventors of the present invention have made efforts to develop original technology for producing adenovirus vector by developing adenovirus producing cell line with improved safety, and as a result, found that the adenovirus vector producing adenovirus vector, which is a site of occurrence of RCA which is a problem of stability based on A529 cell line, It was confirmed that adenovirus-producing cell lines expressing the virus-derived genes E1a and E1b were able to produce adenoviruses with a low RCA and a low efficiency of the A529-based adenovirus-producing cell lines, thereby completing the present invention.

It is an object of the present invention to provide an adenovirus producing cell line having high stability and a method for producing the same.

In order to achieve the above object, the present invention provides a method for producing a lentivirus vector, comprising: (a) preparing a lentivirus vector comprising a lentivirus vector comprising an adenovirus-derived E1a gene and an adenovirus-derived E1b gene; (b) transfecting a lentivirus vector comprising a lentivirus vector comprising the adenovirus-derived E1a gene and an adenovirus-derived E1b gene into an A549 cell line (ATCC CLL 185); (c) culturing the A549 cell line transfected with the lentiviral vector; And (d) obtaining an A549-based adenovirus producing cell line expressing the E1a gene and the E1b gene.

The present invention also provides an A549-based adenovirus-producing cell line, which is produced by the above method and is characterized in that the adenovirus-derived E1a gene and E1b gene are inserted into the genome of the A549 (ATCC CLL 185) cell line.

According to the present invention, it is possible to provide a safe adenovirus-producing cell line that is excellent in adenovirus production efficiency and low in the ability to infect wild-type virus.

1 shows a process for producing a pLenti-PGK vector of the present invention.
Fig. 2 shows a process for producing the pLenti-PGKP-E1a vector of the present invention.
Fig. 3 shows a process for producing the pLenti-PGKP-E1a-Neo vector of the present invention.
Fig. 4 shows a process for producing the pLenti-E1b-Neo vector of the present invention.
FIG. 5 shows sequence maps of restriction enzymes of E1a and E1b expression vectors for constructing adenovirus-producing cell lines and results of confirmation after restriction enzyme treatment.
Figure 6 shows the results of quantification of a lenti-copGFP viral vector using the FACS analysis method.
Fig. 7 shows the results of lentiviral vector quantification using a real-time PCR method.
Fig. 8 shows the stable clones of adenovirus-producing cell lines obtained in the screening of production lines.
FIG. 9 shows the results of analysis of adenovirus production efficiency (72 hours) of A549 cells and control QBI-293A cells.
Fig. 10 shows the results of adenovirus production efficiency analysis (72 hours) of adenovirus producing cell line clones.
Figure 11 shows the results of an adenovirus production efficiency analysis (72 hours) of single cell clones derived from # 54 clones.
12 shows the results of analysis of the stability of the E1a gene and the E1b gene of the production cell line # 54-1 clone.
13 shows the results of Western blot analysis of the E1a protein expression stability of the # 54-1 clone.
Fig. 14 shows the results of RT-PCR analysis of E1b expression stability of the # 54-1 clone.
15 shows the results of analysis of the presence or absence of a wild-type lentiv vector of the # 54-1 clone.
Figure 16 shows the results of the mass production efficiency analysis of adenovirus of the # 54-1 clone.
FIG. 17 shows a photograph of CPE observation. CPE observation was performed during the fifth amplification, and the plate in which the CPE was observed was indicated as CPE (+).

(A) preparing a lentivirus vector comprising a lentivirus vector comprising an adenovirus-derived E1a gene and an adenovirus-derived E1b gene; (b) transfecting a lentivirus vector comprising a lentivirus vector comprising the adenovirus-derived E1a gene and an adenovirus-derived E1b gene into an A549 cell line (ATCC CLL 185); (c) culturing the A549 cell line transfected with the lentiviral vector; And (d) obtaining an A549-based adenovirus-producing cell line expressing the E1a gene and the E1b gene, and a method for producing an A549-based adenovirus producing cell line.

In the present invention, A549 cell line (ATCC CCL-185) -based production cell line was used to develop adenovirus-producing cell line with improved safety and to develop original technology for adenovirus production.

In order to establish stable cell line of A549-based production cell line, it is necessary to maintain the expression of Ela and E1b introduced, and to continuously insert and maintain the host cell chromosome. Most of the stable cell lines prepared by the transfection method have a disadvantage in that the introduced gene is maintained on the chromosome but not expressed well, whereas the lentiviral vector is inserted into the chromosome regardless of the cell cycle the E1 and E1b genes are inserted into the A549 cell line using the lentiviral vector in the present invention.

Since RCA is mainly caused by homologous recombination between adenovirus vector and the endogenous E1a promoter (5 'ψ) and the adenovirus vector (E1a promoter), the vector used for cell line construction contains phosphoglycerate kinase promoter (P _PGK ). The adenovirus E1b promoter was used for E1b expression, and the E1a and E1b genes were cloned into each expression cassette and used for the production of two types of lentive vectors.

In the present invention, the E1a gene may have the nucleotide sequence of SEQ ID NO: 1, and the E1b gene may have the nucleotide sequence of SEQ ID NO: 2.

In the present invention, the lentiviral vector comprising the adenovirus-derived E1a gene may be characterized by containing a human PGK promoter for E1a gene expression.

In the present invention, the lentivirus vector comprising the adenovirus-derived E1a gene and the lentivirus vector comprising the adenovirus-derived E1b gene may each be characterized by containing different antibiotic resistance genes.

The promoter for E1a expression in the E1a expression lentiviral vector was synthesized using a human PGK promoter. The E1a gene was amplified by PCR from the wild-type 5 adenovirus genome and used for cloning. The puromycin resistance gene was selected Was used as a marker.

In the lentiviral vector for E1b expression, E1b was PCR amplified from the wild type 5 adenovirus genome and cloned. The promoter used was a self-promoter possessed by the E1b gene, and the antibiotic resistance was selected as a neomycin resistance gene as a selection marker Respectively.

Each of the prepared lentiviral vectors was infected with the A259 vector, and then a clone expressing E1a gene and E1b gene was screened. Among the selected clones, expression of E1a gene and E1b gene in 10 passage and 20 passage system Was selected as the final adenovirus producing cell line.

In another aspect, the present invention provides a method for producing a 549-based adenovirus-producing cell line, which comprises the step of culturing the A549-based adenovirus (ATCC CLL 185) cell line, which is characterized by the insertion of the E1a gene and the E1b gene derived from adenovirus into the genome of the A549 Production cell lines.

The expression of the E1a gene and the E1b gene in the adenovirus producing cell line clone (# 54-1) finally selected in the present invention was maintained at 0 passage and 20 passage by Western blot and RT-PCR method Respectively.

In another embodiment of the present invention, it was confirmed that no wild-type replication-competent lentivirus (RCL) vector exists in the # 54-1 cell line.

In addition, it was confirmed that the adenovirus-producing cell line of the present invention has high adenovirus production efficiency and low occurrence of RCA (Replication competent adenovirus) and thus is a highly stable adenovirus producing cell line.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the present invention is not construed as being limited by these embodiments.

Example 1: Construction of construct for lentiviral vector production for constructing adenovirus producing cell line

A feline-based lentiviral vector system (pCDF cDNA Cloning and Expression Lentives, Cat # CD100A-1, CD111B-1, System Biosciences, www.systembio.com) was used to construct adenovirus producing cell lines. Since RCA is mainly caused by homologous recombination between adenovirus vector and the endogenous E1a promoter (5 'ψ) and the adenovirus vector (E1a promoter), the vector used for cell line construction contains phosphoglycerate kinase The promoter (P _PGK ) was used, the adenovirus E1b promoter was used for E1b expression, and E1a and E1b were cloned into each expression cassette to construct two kinds of lentiviral vectors.

The E1a promoter was synthesized from 1 to 518 nt of the human PGK promoter (GenBank accession no. AH002937), and E1a was used to amplify 548 to 1575 nts of the wild type 5 adenovirus (GenBank accession No. X02996.1) Amplification, and cloning. E1a was expressed under the control of the PGK promoter and the puromycin resistance gene was used as selection marker.

E1b was PCR amplified and cloned from 1672 to 4070 nt of wild-type 5 adenovirus (GenBank accession no. X02996.1), using its own promoter possessed by the E1b gene, and antibiotic resistance was confirmed by the neomycin resistance gene Were used as selection markers.

(1) Production of pLenti-PGKP-E1a

To construct a lenti-shuttle vector (pLenti-PGKP-E1a) expressing E1a, 1 to 518 nt of human PGK promoter (GenBank accession no. AH002937) was first synthesized and Cla I and BamH I restriction enzyme -shuttle vector pLenti-PGK promoter was constructed as shown in FIG.

CD100A-1 pCDF1-MCS1 cDNA Cloning and Expression Vector Cla I restriction endonuclease cleavage site that is used to remove the CMV7 promoter in (SBI, Cat # CD100A-1 , USA) Since there are more one to FIV dLTR area triple ligation . The pLenti-PGK promoter was constructed using the restriction enzymes Afl II, BamH I, and Pac I. The promoter of the CMV7 was deleted and the PGK promoter was inserted. The vector was named pLenti-PGKP vector.

The E1a gene was amplified by PCR using the following primers from 548 to 1575 nt of the wild type 5 adenovirus (GenBank accession no. X02996.1) and submitted to Bioneer Inc. for sequencing to confirm that there was no mutation Respectively.

Forward primer, 5'-ATG CGG ATC CCC GGG ACT GAA AAT GAG ACA TAT TAT CTG-3 '(SEQ ID NO: 3)

Reverse primer, 5'-ATG CGG ATC CTA ACC ACA CAC GCA ATC ACA GGT TTA C-3 '(SEQ ID NO: 4)

The prepared pLenti-PGKP vector and the obtained E1a PCR product were made into the same shape at both ends using BamH I restriction enzyme, and the E1a gene fragment was inserted into the pLenti-PGKP vector using T4 DNA ligase , pLenti-PGKP-E1a were prepared and confirmed to be inserted in the correct direction using BamH I, Hind III and Xba I (FIG. 2).

(2) Production of pLenti-E1b-Neo

In order to construct a lentiviral vector containing the E1b gene and the neomycin resistance gene (Neo), the puromycin hypothermia gene (puro) was amplified with the PCR-amplified Neomycin resistance in the pLenti-PGKP-E1a vector prepared in (1) gene (Neo) and inserted the E1b gene.

First, the puro gene was removed by treatment with Sal I and Not I in the pLenti-PGKP-E1a plasmid, and the Neo region was amplified by PCR using the following primers: pCI-neo (Promega, Cat. # E1841, USA) And then cloned into pLenti-PGKP-E1a-Neo.

Forward primer, 5'-ATG CGC GGC CGC TTT CTC CTT ACG CAT CTG TGC GGT-3 '(SEQ ID NO: 5)

Reverse primer, 5'-ATG TGT CGA CCG CTA TCG ATT CAC ACA AAA AAC C-3 '(SEQ ID NO: 6)

Wherein the pLenti-PGKP-E1a-Neo Sal it, Kpn I to confirm it was confirmed that the DNA of the desired size, there was treated with Not I double digestion (Fig. 3).

The E1b gene was amplified by PCR using the following primers from 1672 to 4070 nt of wild type 5 adenovirus (GenBank accession No. X02996.1).

Forward primer, 5'-ATG CGA ATT CTA TAT AAT GCG CCG TGG GCT AAT CTT G GT-3 '(SEQ ID NO: 7)

Reverse primer, 5'-ATG CGA ATT CGA TCC AAA TCC AAA CAG AGT CTG GTT TT-3 '(SEQ ID NO: 8)

The above-prepared pLenti-PGKP-E1a-Neo was digested with EcoR I restriction enzyme to remove the PGK promoter and E1a gene region and ligated with the E1b PCR product to construct pLenti-E1b-Neo. EcoR I, Hind III and Afl II, respectively, to confirm the insertion of the E1b gene (Fig. 4).

Sequence analysis confirmed the same as E1b, and normal E1b expression analysis was confirmed by RT-PCR using mRNA because E1b-specific antibody was not easily available.

FIG. 5 shows vector maps of the lentiviral vectors pLenti-PGKP-E1a and pLenti-E1b-Neo expressing E1a and E1b prepared in the examples, and the results of confirming the vectors by restriction enzyme treatment.

Example 2: Establishment of stable cell lines after lentivirus vector production, quantification and lentivector introduction

(1) Lentiviral vector production for stable cell line production of adenovirus

To construct stable adenovirus producing cell lines, lentivirus vectors were produced using pLenti-PGKP-E1a and pLenti-E1b-Neo constructs containing E1a and E1b genes, respectively. A pCDF1-MCS2-EF1-Puro Vector containing a lentiviral vector expressing a GFP protein and a puromycin antibiotic gene using a plasmid DNA of pSIF1-H1-siLuc-copGFP ( SBI, Cat. # LV201B-1, USA) Were used to produce lentiviral vectors.

Production of lentiviral vectors was performed using pPACKF1 ^™ Lentivector Packaging Kit (SBI Cat, # LV100A-1). The 293TN cell line (SBI, Cat. # LV900A- 1, USA) on the day before transfection were passaged by infecting a cell density of 3 x 10 ⁶ cells / dish in 100-mm dish. The pLenti-PGKP-E1a and pLenti-E1b-Neo constructs and control constructs (MCS2 / copGFP) containing E1a and E1b, To produce lentiviral vectors.

48 hours after the transfection, the cell culture was collected and stored at -80 ° C. The transfection efficiency was confirmed by analyzing the expression of GFP protein in the control group pSIF1-H1-siLuc-copGFP.

The lentivirus vector was quantitatively analyzed by real-time PCR using the Global UltraRapid Lentiviral Titer Kit (SBI, Cat. # LV961A-1) and the lenti-copGFP viral vector using FACS analysis.

A549 cells and NCI-H1299 (ATCC, Cat. # CRL-5803, USA) cells for stable cell line construction were transferred to a 12-well plate at the cell density of 1 × 10 ⁵ cells / well the day before infection. Polybrene was replaced with complete medium supplemented with 5 μg / mL, and each lentiviral vector was infected with 1, 10, or 100 μL. Cells were harvested at 48 hours after the infection, and quantified by FACS analysis and real-time PCR.

A549 and NCI-H1299 cells were infected with 1, 10, and 100 μL of lenti-copGFP vector. Cells were collected and fixed with 4% paraformaldehyde. FACS analysis was performed to measure the number of GFP- Quantitative results of copGFP vector were predicted. As shown in FIG. 6, FACS analysis showed 6.44%, 49.75%, and 99.74% transduction efficiency in NCI-H1299 cells and 0.58%, 10.38%, and 79.44% in A549 cells Respectively. NCI-H1299 was able to predict 7 x 10 ⁶ IFU / mL and A549 could predict 1.32 x 10 ⁶ IFU / mL using transduction efficiency and MOI correlation, and compared with real-time PCR method And final quantification results were obtained.

A549 and NCI-H1299 cells were infected with 1, 10, and 100 μL of each lentiviral vector, and genomic DNA of transduced cells was isolated using Global UltraRapid Lentiviral Titer Kit (SBI, Cat. # LV961A-1) The WPRE gene contained in the lentiviral vector and the internal UCR1 gene existing in animal cells were amplified and quantified as shown in FIG. 7 by real-time PCR.

ΔCt = Average Ct of WPRE - Average Ct of UCR1 of the same standard of sample

Number of virus particles (IFU / mL) = (MOI of the sample) x (number of cells in the well infected) x 1000 / (μL of viral suspension added to the well for infection)

The lentivirus vector was quantitatively determined by the real-time PCR method as follows: Lenti-E1a was 8.23 × 10 ⁵ IFU / mL, Lenti-E1b was 7.12 × 10 ⁵ IFU / mL, lenti-MCS 2 was 8.54 × 10 ⁵ IFU / mL , And Lenti-copGFP showed the result of 6.42 x 10 ⁵ IFU / mL.

In comparison with the quantitative results of lenti-copGFP using the above FACS analysis, the quantitative results obtained by the real-time PCR method were about 4.86 times higher than the quantitative results of the FACS analysis, and the results of the FACS analysis In order to obtain the same result as the efficiency, it is expected that the same result can be obtained by infecting with approximately 7 MOI on the basis of lenti-copGFP.

Example 3: Infection and screening of lentivirus vector for construction of adenovirus producing cell line (A549-based)

(1) Construction of A549-based production cell line for stable cell line Screening method: Antibiotic resistance test

Since Lenti-PGKP-E1a contains the Puromycin resistance gene and Lenti-E1b-Neo contains the neomycin resistance gene, two Lentivirus vectors were introduced to construct the A549-based production cell line. lines screening method was established as follows.

In this study, A549 cells were cultured in a 6-well plate at 3 × 10 ⁵ cells / well in the presence of puromycin and 300 μg / mL of puromycin and G418 (neomycin), respectively. . After 24 hours, puromycin was treated at a concentration of 1.25 to 20 μg / mL, G418 was treated at a concentration of 125 to 2000 μg / mL, and antibiotic resistance analysis was conducted at every 3 to 4 days by changing the antibiotic medium .

In the case of Puromycin treated with antibiotics, A549 cells were killed under all conditions of 1.25 to 20 μg / mL. Antibiotic resistance experiments were performed at lower treatment concentrations (0.07813 to 1.25 μg / mL) In the range of 0.625 μg / mL, 100% of A549 cells died or 100% survived.

In the case of G418, 100% of A549 cells died or 100% survived in the range of 500 to 1000 μg / mL.

As a result, more than 90% of A549 cells were killed at 0.5 μg / mL of puromycin and 1000 μg / mL of G418. Therefore, the concentration of antibiotics for screening the stable cell lines among the production lines into which lentiviral vectors were introduced, 0.5 μg / mL, and G418 was determined as the concentration of 1000 μg / mL.

(2) Construction of adenovirus-producing cell line by infecting the lentivirus vector

A549 cells were plated on a 6-well plate at 1 × 10 ⁵ cells / well, and Lenti-E1a and Lntiviral-E1b vectors were co-infected at various ratios after 24 hours, Respectively. After 24 hours, 6-well cells were trypsinized and transferred to a 100-mm dish. Cells transferred to a 100-mm dish were cultured for 24 hours to express E1a / E1b protein, and puromycin (0.5 μg / mL) and G418 (1000 μg / mL) antibiotics were treated together and replaced with a new antibiotic medium every 3 days. Stable cell lines Clones were selected and cultured in a 24-well plate containing the same concentration of puromycin and G418. The cells were transferred to a 100 mm dish at 80-90% confluency and cultured for 80-90% confluency (Fig. 8). ≪ tb >< TABLE >

Example 4 Analysis of Cell Line Clone, Stable Cell Lines Producing A549-Based Adenovirus

 (1) Production efficiency analysis of adenovirus producing cell line clone, stable cell lines

Example 3 To analyze the possibility of adenovirus production of production cell lines in order to establish clones were subcultured with 0.5 x 10 ⁵ cells / well cell density for each cell clones in 12-well plate. Each clone was infected with recombinant adenovirus (rAd-GFP) stock expressing GFP protein, and the expression of cytopathic effect (CPE) and green fluorescence protein (GFP) at 24, 48, To predict and analyze adenovirus production efficiency. As a control group, QBI-293A cells (Qbiogene, Cat. # AES0503, USA) were used as a conventional adenovirus producing cell line.

The CPE phenomenon and the degree of GFP expression at 72 hours after infection with the recombinant adenovirus (rAd-GFP) stock are shown in FIGS. 9 and 10. Many production cell clones showed 100% CPE and strong GFP protein expression similar to the control QBI-293A cells.

(2) Single cell clone progress and production efficiency analysis of # 54 clone with the highest production efficiency

As a result of the above experiment, # 54 clone having the highest efficiency of adenovirus production was selected and single cell cloning was performed from # 54 as follows.

The frozen stocks of # 54 clones were diluted to 1 cell / well (96-well plate) and cultured in antibiotic medium (10% FBS + 1% penicillin / streptomycin + 1000 μg / mL G418 + 0.5 μg / mL puromycin) . Cells were cultured in a 6-well plate. Cells were partially subcultured at 1 × 10 ⁵ cells / well (12-well plate) to obtain recombinant adenovirus (rAd- GFP) stocks were infected to analyze the CPE phenomenon and GFP expression to confirm the possibility of adenovirus production of a single cell clone. The remaining cells were transferred to a 100 mm dish and cultured. Frozen stocks were prepared for each clone. Single cell clone # 54-1 with the highest efficiency of adenovirus production was selected by comparing CPE and GFP expression at 24, 48, and 72 hours after infection with QBI-293A cells as a control (Figure 11) .

(3) E1a and E1b gene stability analysis of single cell clone # 54-1

In order to maintain productivity in single cell clone # 54-1 having the best adenovirus productivity selected in (2), the genetic stability of E1a and E1b inserted into the cell line is important. Therefore, Expression of E1a and E1b in passage 20 was analyzed. Genomic DNA was extracted from passage 10 and passage 20 cells of single cell clone # 54-1. The stability of E1a gene and E1b gene was analyzed by using 0.5 μg of extracted genomic DNA as template DNA.

PCR primers and reaction conditions PCR condition Primer sequence 95 ° C, 5 min 1 cycle E1a Forward: 5'-ATG CGG ATC CCC GGG ACT GAA AAT GAG ACA TAT TAT CTG-3 '(SEQ ID NO: 9)
Reverse: 5'-ATG CGG ATC CTA ACC ACA CAC GCA ATC ACA GGT TTA C-3 '(SEQ ID NO: 10) 94 ° C, 30 sec
60 ° C, 30 sec
70 [deg.] C, 2 min 30 cycles E1b Forward: 5'-GCT CTA GAA TGG AGG CTT GGG AGT GTT TGG AAG ATT TTT CTG CTG-3 '(SEQ ID NO: 11)
Reverse: 5'-CGG GAT CCT CAA TCT GTA TCT TCA TCG CTA GAG CCA AAC TCA GC-3 '(SEQ ID NO: 12) 72 ° C, 5 min 1 cycle

As shown in Fig. 12, it was confirmed that the E1a gene and the E1b gene were stably maintained in both the control group HEK293 and the production cell line # 54-1 clone. On the other hand, as expected, it was confirmed that E1a gene and E1b gene were not amplified in the A549 cell used as a control cell line. In addition, the E1a and E1b gene bands in the # 54-1 clone were found to remain constant in the passages 10 and 20 without any difference in cell density, and the E1a gene and the E1b gene were stably maintained even under continuous passage culture .

(4) Stability analysis of E1a, E1b protein expression of single cell clone # 54-1

Production cell line Clone # 54-1 cells were transfected with the E1a gene inserted into the host chromosomal DNA to obtain stable protein expression in passage 10 and passage 20 and analyzed by Western blot method. HEK293 cells expressing all E1a / E1b proteins were used as a positive control, and 20 μg of each cell lysate protein was electrophoresed and then labeled with Anti-E1a (Cat. # Ab33183, abcam, UK) Were used to analyze protein expression.

As a result, as shown in Fig. 13, expression of the E1a gene protein was confirmed in both the production cell line clone # 54-1 and the positive control HEK293 cell line. In addition, the results of E1a protein expression in passage 10 and 20 showed that E1a gene was stably expressed in the state of continuous cultivation.

Since the antibody that can confirm the protein expression of the E1b gene is not presently produced, the expression of E1b protein was analyzed by extracting RNA and performing RT-PCR. As in the E1a assay, production cell clone cells were recovered from passage 10 and passage 20, purified and then cDNA was synthesized and PCR was performed using E1b specific primers.

PCR conditions for E1b gene identification PCR condition Primer sequence 95 ° C, 5 min 1 cycle E1b Forward: 5'-GCT CTA GAA TGG AGG CTT GGG AGT GTT TGG AAG ATT TTT CTG CTG-3 '(SEQ ID NO: 13)
Reverse: 5'-CGG GAT CCT CAA TCT GTA TCT TCA TCG CTA GAG CCA AAC TCA GC-3 '(SEQ ID NO: 14) 94 ° C, 30 sec
60 ° C, 30 sec
70 [deg.] C, 2 min 30 cycles 72 ° C, 5 min 1 cycle

As a result, as shown in Fig. 14, expression of the E1b gene was confirmed in both the production cell line clone # 54-1 and the positive control HEK293 cell line. In addition, the results of E1b expression in passage 10 and passage 20 indicate that the E1b gene inserted into the host cell chromosomal DNA is stably expressed even when the culture is continuously performed.

(5) Safety verification by analysis of presence of wild-type lentiviral vector in # 54-1 clone

In order to verify safety by analyzing the presence of replication-competent lentivirus (RCL) vector in # 54-1 clone, lentiviral vectors expressing E1a and E1b used to construct adenovirus producing cell lines lack replication ability The presence of the wild-type lentiviral vector was confirmed using a FIV p24 ELISA kit (Cell Biolabs, INC. Cat. # VPK-108-F) for measuring the concentration of p24 protein Respectively.

Assuming that one RCL (replication-competent lentivirus) vector is present in the first cloned # 54-1, it is assumed that a number of RCLs were amplified in the passage of passage 10 and passage 20. Therefore, the # 54-1 clone was cultured to determine the presence of RCL vector (wild type lentiv vector) by measuring the p24 concentration in the cell culture medium of passage 10 and passage 20.

FIV p24 standard was prepared at concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL, respectively. Cells were prepared from passage 10 and passage 20 Respectively. In addition, the cell culture medium and FIV p24 g standard protein were mixed at a concentration of 12.5 ng / mL to confirm the FIV p24 detection interference effect by the cell culture medium.

First, the cell culture medium (control) and the cell culture solution obtained in passage 20 were analyzed first through the first experiment. The absorbance of 0.811 and 1.098, which showed similar tendency to that of standard 0 ng / mL, in the control and # 54-1 clone, respectively, when the undiluted solution was used as is, but the FIV p24 standard protein concentration was 12.5 ng / mL The values of 1.041 and 1.660 were lower than those of the standard 25 ng / mL absorbance of 2.644 in the mixed stock solution, and it was confirmed that there was FIV p24 detection interference effect (Table 1 and FIG. 15).

On the other hand, in the wells using a 10-fold diluted sample, the absorbance was similar to that of the standard 0 ng / mL in which FIV p24 protein was not detected. Thus, in both of the control culture media and the # 54-1 clone It was confirmed that there is no RCL vector. In addition, in the experimental group to analyze the FIV p24 detection interference effect, the absorbance of 2.236 and 2.533 similar to that of standard 25 ng / mL absorbance was similar to 2.644, showing no interference effect by the medium.

(6) Analysis of mass production efficiency of single cell clone # 54-1 adenovirus

In order to analyze the mass production efficiency of adenovirus, 100 plates (passage 20) of # 54-1 cells and HEK293 (existing adenovirus producing cell line, control group) were prepared with 5 × 10 ⁶ cells / The CMV-GFP viral vector was infected at a concentration of 10 μL / 100 mm dish. After 48 hours, the cells and cell culture were recovered and centrifuged at 1,000 g, 20 min, and 4 ° C to recover the cell pellet. The cell pellet was recovered three times by freezing / thawing and centrifuged at 1,000 g, 20 min, After separation, the supernatant containing the adenovirus was separated and placed in the supernatant to a final concentration of 50 units / mL of Benzonase nuclease (Novagen, Cat. No. 70746-3, 25 Units / mL) Lt; / RTI >

8 mL of a 1.4 CsCl solution and 6 mL of a 1.2 CsCl solution were added to a tube for ultracentrifuge, and the supernatant containing the obtained adenovirus was overlaid and subjected to primary centrifugation at 25,000 rpm, 196 min and 4 ° C. After centrifugation, only the portion of the adenovirus band was transferred to a new tube, and the total volume was adjusted to 10 mL by adding 50 mM Tris-HCl (pH 7.8) and 10 mM MgCl ₂ buffer. In the same manner as the primary centrifugation method, 12 mL of a 1.4 CsCl solution and 14 mL of a 1.2 CsCl solution were added, and the adenovirus obtained by primary centrifugation was overlaid thereon. Then, the cells were overlaid on the adenovirus at 22,500 rpm, 20 hrs, Separation was carried out.

After the second centrifugation, the portion of the adenovirus band was removed and the buffer was exchanged three times with dialysis buffer (2.5% glycerol, 25 mM NaCl, 20 mM Tris, pH 8.0, 4 ° C) . The dialyzed adenoviruses were filtered using a 0.22 μM filter and stored at -80 ° C.

The purified virus was diluted with a certain amount of virus, and the absorbance was measured three times at OD ₂₆₀ , and the effective range was set to OD ₂₆₀ = 0.1 to 1.0. The number of virus particles (vp, vp) was quantified using the following virus quantification equation.

Viral titer (vp / mL) = OD ₂₆₀ x viral dilution x 1.1 x 10 ¹²

The results of the mass production efficiency analysis of the adenovirus of the clone # 54-1 of the production cell line are shown in FIG. HEK293 cells showed a production efficiency of 1.356 × 10 ¹³ vp and a # 54-1 clone showed a production efficiency of 6.339 × 10 ¹² vp. These results confirm that the production efficiency of HEK293 cells is about 50%.

(7) Analysis of the occurrence of replication competent adenovirus (RCA) in single cell clone # 54-1

The RCA test was performed according to the SOP established by the SCT (Note), which became the specificity and detection limit according to the International Conference on Harmonization (ICH) Q2 (R1) guidelines (Table 4).

RCA analysis tests correspond to the limits of Testing for impurities, and validation parameters are specificity and detection limit.

The RCA assay was performed as follows to detect 1 RCA at 3 x 10 ¹⁰ vp in accordance with US FDA and KFDA regulations. That is, an RCA analysis test was carried out using adenovirus-producing cell line (# 54-1) and rAd-CMV-GFP adenovirus produced in HEK293 cell line as a sample. The amount of the used sample was 3 x 10 ¹⁰ vp, and the CPE was observed through the fifth amplification. The number of CPE observed in all five repeated conditions is shown in Table 5.

 RCA analysis of adenovirus produced using A549 # 54-1 and HEK293 Groups
A B C D E F rAd (HEK293) rAd
(# 54-1) ARM
(1 NIU) rAd (HEK293) + ARM (1 NIU) rAd
(# 54-1) + ARM (1 NIU) Sham Primary amplification 0/5 0/5 0/5 0/5 0/5 0/5 Secondary amplification 0/5 0/5 2/5 0/5 0/5 0/5 Third amplification 0/5 0/5 4/5 2/5 1/5 0/5 4th amplification 0/5 0/5 5/5 2/5 1/5 0/5 5th amplification 1/5 1/5 5/5 3/5 3/5 0/5

The RCA assay was found to meet all of the criteria of detection (specificity, detection limit, specificity) and that rAd-CMV-GFP produced by both cell types met the criteria of 3 x 10 ¹⁰ vp <1 NIU RCA. The newly constructed adenovirus producing cell line # 54-1 was evaluated as safe in terms of RCA generation.

The CPE (cytopathic effect) was determined on the 7th day of the 5th amplification as shown in the figure below. CPE was not observed in cultured Group F (Sham, negative control), CPE was observed in all plates in Group C (ARM 1 NIU), and 3 CPEs were observed in Spiked Control Groups D and E And were validated for validity.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> SCT          Industry Academy Coopteration Groop of Chungbuk Health & Science University <120> A549 Based Cell Line for Preparing Adenovirus Vector <130> P13-B079 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 1028 <212> DNA <213> type 5 Adenovirus <400> 1 ccgggactga aaatgagaca tattatctgc cacggaggtg ttattaccga agaaatggcc 60 gccagtcttt tggaccagct gatcgaagag gtactggctg ataatcttcc acctcctagc 120 cattttgaac cacctaccct tcacgaactg tatgatttag acgtgacggc ccccgaagat 180 cccaacgagg aggcggtttc gcagattttt cccgactctg taatgttggc ggtgcaggaa 240 gggattgact tactcacttt tccgccggcg cccggttctc cggagccgcc tcacctttcc 300 cggcagcccg agcagccgga gcagagagcc ttgggtccgg tttctatgcc aaaccttgta 360 ccggaggtga tcgatcttac ctgccacgag gctggctttc cacccagtga cgacgaggat 420 gaagagggtg aggagtttgt gttagattat gtggagcacc ccgggcacgg ttgcaggtct 480 tgtcattatc accggaggaa tacgggggac ccagatatta tgtgttcgct ttgctatatg 540 aggacctgtg gcatgtttgt ctacagtaag tgaaaattat gggcagtggg tgatagagtg 600 gtgggtttgg tgtggtaatt ttttttttaa tttttacagt tttgtggttt aaagaatttt 660 gtattgtgat ttttttaaaa ggtcctgtgt ctgaacctga gcctgagccc gagccagaac 720 cggagcctgc aagacctacc cgccgtccta aaatggcgcc tgctatcctg agacgcccga 780 catcacctgt gtctagagaa tgcaatagta gtacggatag ctgtgactcc ggtccttcta 840 acacacctcc tgagatacac ccggtggtcc cgctgtgccc cattaaacca gttgccgtga 900 gagttggtgg gcgtcgccag gctgtggaat gtatcgagga cttgcttaac gagcctgggc 960 aacctttgga cttgagctgt aaacgcccca ggccataagg tgtaaacctg tgattgcgtg 1020 tgtggtta 1028 <210> 2 <211> 2399 <212> DNA <213> type 5 adenovirus <400> 2 tatataatgc gccgtgggct aatcttggtt acatctgacc tcatggaggc ttgggagtgt 60 ttggaagatt tttctgctgt gcgtaacttg ctggaacaga gctctaacag tacctcttgg 120 ttttggaggt ttctgtgggg ctcatcccag gcaaagttag tctgcagaat taaggaggat 180 tacaagtggg aatttgaaga gcttttgaaa tcctgtggtg agctgtttga ttctttgaat 240 ctgggtcacc aggcgctttt ccaagagaag gtcatcaaga ctttggattt ttccacaccg 300 gggcgcgctg cggctgctgt tgcttttttg agttttataa aggataaatg gagcgaagaa 360 acccatctga gcggggggta cctgctggat tttctggcca tgcatctgtg gagagcggtt 420 gtgagacaca agaatcgcct gctactgttg tcttccgtcc gcccggcgat aataccgacg 480 gaggagcagc agcagcagca ggaggaagcc aggcggcggc ggcaggagca gagcccatgg 540 aacccgagag ccggcctgga ccctcgggaa tgaatgttgt acaggtggct gaactgtatc 600 cagaactgag acgcattttg acaattacag aggatgggca ggggctaaag ggggtaaaga 660 gggagcgggg ggcttgtgag gctacagagg aggctaggaa tctagctttt agcttaatga 720 ccagacaccg tcctgagtgt attacttttc aacagatcaa ggataattgc gctaatgagc 780 ttgatctgct ggcgcagaag tattccatag agcagctgac cacttactgg ctgcagccag 840 gggatgattt tgaggaggct attagggtat atgcaaaggt ggcacttagg ccagattgca 900 agtacaagat cagcaaactt gtaaatatca ggaattgttg ctacatttct gggaacgggg 960 ccgaggtgga gatagatacg gaggataggg tggcctttag atgtagcatg ataaatatgt 1020 ggccgggggt gcttggcatg gacggggtgg ttattatgaa tgtaaggttt actggcccca 1080 attttagcgg tacggttttc ctggccaata ccaaccttat cctacacggt gtaagcttct 1140 atgggtttaa caatacctgt gtggaagcct ggaccgatgt aagggttcgg ggctgtgcct 1200 tttactgctg ctggaagggg gtggtgtgtc gccccaaaag cagggcttca attaagaaat 1260 gcctctttga aaggtgtacc ttgggtatcc tgtctgaggg taactccagg gtgcgccaca 1320 atgtggcctc cgactgtggt tgcttcatgc tagtgaaaag cgtggctgtg attaagcata 1380 acatggtatg tggcaactgc gaggacaggg cctctcagat gctgacctgc tcggacggca 1440 actgtcacct gctgaagacc attcacgtag ccagccactc tcgcaaggcc tggccagtgt 1500 ttgagcataa catactgacc cgctgttcct tgcatttggg taacaggagg ggggtgttcc 1560 taccttacca atgcaatttg agtcacacta agatattgct tgagcccgag agcatgtcca 1620 aggtgaacct gaacggggtg tttgacatga ccatgaagat ctggaaggtg ctgaggtacg 1680 atgagacccg caccaggtgc agaccctgcg agtgtggcgg taaacatatt aggaaccagc 1740 ctgtgatgct ggatgtgacc gaggagctga ggcccgatca cttggtgctg gcctgcaccc 1800 gcgctgagtt tggctctagc gatgaagata cagattgagg tactgaaatg tgtgggcgtg 1860 gcttaagggt gggaaagaat atataaggtg ggggtcttat gtagttttgt atctgttttg 1920 cagcagccgc cgccgccatg agcaccaact cgtttgatgg aagcattgtg agctcatatt 1980 tgacaacgcg catgccccca tgggccgggg tgcgtcagaa tgtgatgggc tccagcattg 2040 atggtcgccc cgtcctgccc gcaaactcta ctaccttgac ctacgagacc gtgtctggaa 2100 cgccgttgga gactgcagcc tccgccgccg cttcagccgc tgcagccacc gcccgcggga 2160 ttgtgactga ctttgctttc ctgagcccgc ttgcaagcag tgcagcttcc cgttcatccg 2220 cccgcgatga caagttgacg gctcttttgg cacaattgga ttctttgacc cgggaactta 2280 atgtcgtttc tcagcagctg ttggatctgc gccagcaggt ttctgccctg aaggcttcct 2340 cccctcccaa tgcggtttaa aacataaata aaaaaccaga ctctgtttgg atttggatc 2399 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgcggatcc ccgggactga aaatgagaca tattatctg 39 <210> 4 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atgcggatcc taaccacaca cgcaatcaca ggtttac 37 <210> 5 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgcgcggcc gctttctcct tacgcatctg tgcggtatgc gcggccgctt tctccttacg 60 catctgtgcg gt 72 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atgtgtcgac cgctatcgat tcacacaaaa aacc 34 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atgcgaattc tatataatgc gccgtgggct aatcttggt 39 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atgcgaattc gatccaaatc caaacagagt ctggtttt 38 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atgcggatcc ccgggactga aaatgagaca tattatctg 39 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 atgcggatcc taaccacaca cgcaatcaca ggtttac 37 <210> 11 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gctctagaat ggaggcttgg gagtgtttgg aagatttttc tgctg 45 <210> 12 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cgggatcctc aatctgtatc ttcatcgcta gagccaaact cagc 44 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gctctagaat ggaggcttgg gagtgtttgg aagatttttc tgctg 45 <210> 14 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cgggatcctc aatctgtatc ttcatcgcta gagccaaact cagc 44

Claims (7)

A method for preparing a host cell line for producing adenovirus comprising the steps of:
(a) preparing a lentivirus vector comprising a lentivirus vector comprising an adenovirus-derived E1a gene and an adenovirus-derived E1b gene;
(b) transfecting an A549 cell line (ATCC CLL 185) with a lentivirus vector comprising a lentivirus vector comprising the adenovirus-derived E1a gene and an adenovirus-derived E1b gene; And
(c) culturing the transfected A549 cell line to obtain a host cell line for adenovirus production.
[Claim 2] The method according to claim 1, wherein the E1a gene has the nucleotide sequence of SEQ ID NO: 1.
[Claim 2] The method according to claim 1, wherein the E1b gene has the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 1, wherein the lentiviral vector comprising the adenovirus-derived E1a gene contains a human PGK promoter for E1a gene expression.
2. The adenovirus producing host according to claim 1, wherein the lentivirus vector comprising the adenovirus-derived E1a gene and the lentivirus vector comprising the adenovirus-derived E1b gene each contain a different antibiotic resistance gene &Lt; / RTI &gt;
A host cell line for adenovirus production, which is produced by the method according to any one of claims 1 to 5 and is characterized in that the adenovirus-derived E1a gene and the E1b gene are inserted into the genome of the A549 (ATCC CLL 185) cell line.
A method of producing an adenovirus comprising the steps of:
(a) infecting the adenovirus-producing host cell of claim 6 with an adenovirus;
(b) culturing the adenovirus-infected adenovirus-producing cell line to produce adenovirus; And
(c) obtaining the resulting adenovirus.

Images