JP3713038B2 - Recombinant adenovirus - Google Patents

Description

  The present invention relates to a recombinant promoter comprising a cytomegalovirus enhancer, a chicken β-actin promoter, and a hybrid promoter (CAG promoter) comprising a splicing acceptor of rabbit β globin, a poly A sequence, and a DNA sequence encoding a predetermined polypeptide. It relates to adenoviruses, their production methods and their use, in particular gene therapy.

  Adenovirus vectors have been studied as vectors for exogenous gene introduction experiments because they exhibit introduction efficiency close to 100% in various animal cultured cells. In addition, its usefulness has been recognized because the function of a foreign gene can be examined without killing the introduced cells, and the available animal species cover a wide range including mice and rats.

  However, the value of the adenovirus vector has come to be recognized in 1992 as a method of gene therapy for cystic fibrosis. Since 1993, reports such as being useful as an expression method in the nervous system were successively (Non-Patent Documents 2 to 5), and the possibility of application to gene therapy was shown. Thus, nowadays, adenovirus expression vectors not only exhibit introduction efficiency close to 100% to many differentiated and undifferentiated cells including the nervous system, but also can express genes by direct injection / administration to individual animals. As shown, it is expected to be applied to gene therapy.

  Until now, retroviruses have often been used as viral vectors for gene transfer, but this virus can be introduced only into dividing cells and is integrated into the host cell chromosomes. In gene therapy, it is considered that there is a problem from the viewpoint of safety, and its application range is considered to be narrow.

Unlike retroviruses, adenoviruses do not have an active mechanism for chromosomal integration, and have the advantage that gene transfer can be performed even in resting cells. The range of applications is extremely wide, and it will be established as a major gene therapy technology in the near future. It seems to be.
Nature Genetics, VOL.3.1-2, 1993 Science, VOL.259, 988-990, 1993 Nature Genet.VOL.3, 219-223, 1993 ibid, VOL.3, 224-228, 1993 ibid, VOL.3, 229-234, 1993

  Adenoviruses are originally known to cause Kaze symptoms in humans. Therefore, there is a concern that a large amount of administration may cause inflammation due to the administration. In fact, the Crystal et al. Group reportedly reported that during the initial stages of patient treatment, lung administration occurred in patients who had been administered to the lung at high concentrations and the plan was suspended. Even in experiments using cultured cells, when a high concentration of virus is infected, direct side reactions caused by virus particles are observed, such as cells becoming rounded and raised. Therefore, in actual gene therapy, there are concerns about side effects caused by the administration of high-concentration virus solution. Therefore, a combination with a high-expression promoter that ensures a sufficient expression level with a low-concentration virus solution is absolutely necessary. It is thought to lead to problem solving when adenovirus is applied to treatment.

  With the advancement of gene recombination technology, production of useful substances using gene recombination has advanced rapidly in recent years. When a foreign gene is expressed using a gene recombination technique, an appropriate host cell and an expression vector having a foreign gene expression promoter corresponding thereto are used. As an expression system using an animal cell as a host known so far, many animal virus gene promoters and systems using animal cell gene promoters have been reported. The former includes SV40 early gene promoter, adenovirus major late gene promoter and the like, and the latter includes thymidine kinase gene promoter, metallothionein gene promoter, immunoglobulin gene promoter and the like. However, these promoter activities are measured in cells that are actively dividing and have a high gene transfer rate by the calcium phosphate method, DEAE-dextran method, electroporation method, etc., such as mouse-derived L cells and hamster-derived CHO cells. And African green monkey-derived COS cells. Therefore, such a study has not been conducted on differentiated cells. When considering application to gene therapy, expression in differentiated cells such as nerve cells, muscle cells, liver cells, and blood cells is important.

  The inventors of the present invention have focused on the fact that adenoviruses that have been rarely used for gene transfer in the past can infect quiescent cells widely, and in a wide range of animal cells by incorporating a strong promoter into the adenovirus genome. We thought that it would be possible to allow gene expression, and prepared recombinant adenoviruses incorporating various strong promoters and examined them using a wide range of host cells. Successfully obtained the adenovirus shown. The present invention has been completed by further research based on this fact.

  An object of the present invention is to provide a recombinant adenovirus incorporating a promoter exhibiting strong activity in a wide range of animal cells, particularly a recombinant adenovirus incorporating a nucleotide sequence encoding a foreign polypeptide of interest in such a recombinant adenovirus, Among others, it is to provide a recombinant adenovirus for gene therapy incorporating a human defective gene. Another object of the present invention is to provide a simple method for producing such a recombinant adenovirus.

That is, the present invention
[1] The adenovirus genome consists of a nucleotide sequence encoding a foreign target polypeptide sequence, a cytomegalovirus enhancer that controls the expression of this sequence, a chicken β-actin promoter, and a rabbit β globin splicing acceptor. A hybrid promoter (CAG promoter), as well as a recombinant adenovirus characterized in that it has a poly A sequence in the left direction ,
[2] The recombinant adenovirus according to the above [1], wherein the adenovirus genome has a 1.3% to 9.3% fragment containing the E1A gene region deleted from the E1 gene region ,
[3] A nucleotide sequence encoding a foreign target polypeptide sequence and a CAG promoter are inserted into a site where the 1.3% to 9.3% fragment containing the E1A gene region is deleted. The recombinant adenovirus according to the above [2],
[4] The recombinant adeno described in [2] or [3] above, wherein the adenovirus genome further lacks a 79.6% to 84.8% fragment of the genome from the E3 gene region. Virus,
[ 5 ] When a nucleotide sequence encoding a foreign polypeptide sequence of interest is expressed under the control of a CAG promoter in a natural host cell, it encodes a polypeptide that is secreted into the medium of the host cell. above, wherein the has [1] to [4] the recombinant adenovirus according to any one, and [6] is for gene therapy, the [1] to [4] the recombinant adenovirus according to any one ,
About.

  The present invention provides a recombinant adenovirus incorporating a promoter exhibiting strong activity in a wide range of animal cells, and in particular, a recombinant adenovirus useful for the treatment of genetic diseases incorporating a human gene.

The present invention is described in detail below.
The human adenovirus used in the present invention is a virus having human as a natural host. The adenovirus genome is a double-stranded linear DNA of about 36 kbp, which has an inverted repeat base sequence consisting of approximately 100 bp at both ends of the DNA strand, and the E2B gene product is cleaved at the 5 ′ ends of both ends of the DNA strand. The unique 55k protein is covalently bonded. The fact that this unique genomic structure has been a major obstacle when using adenovirus as a vector is as described below, and the core of the present invention is a novel and useful product completed by overcoming such an obstacle. The object is to provide a recombinant adenovirus and a method for producing the same.

  The recombinant adenovirus of the present invention is a kind of recombinant vector suitable for transformation of a eukaryotic cell line capable of infecting an adenovirus, particularly a human or animal cell line, and comprising an E1 gene region, particularly a cell. E1A gene region involved in canceration is deleted, and thus cells that continuously express E1A and further E1B genes, such as human fetal kidney-derived cell lines (293 cells), are excluded in host cells. It has the characteristic that it cannot proliferate.

The recombinant adenovirus particles of the present invention grow to a high titer of 10 ⁸ to 10 ⁹ pfu (plaque forming units) / ml, as in the wild type strain, when inoculated into the above-mentioned 293 cells. However, when inoculated into other cells or animal tissues, the virus particles enter the cells at a high rate, and the viral genome is injected into the nucleus, but the E1A gene region is deleted, so this gene product All other adenoviral promoters that are transcriptionally activated by can not work. On the other hand, the target gene in the adenovirus genome can be transcribed and expressed from a foreign promoter integrated in the genome. Therefore, if the recombinant adenovirus particle of the present invention is used, the gene of interest can be expressed in a wide range of animal cells while minimizing the influence of the adenovirus genome as a vector.

  Although the wild-type human adenovirus can proliferate after infection is limited to human cells, the cell types and tissues that can be expressed by the recombinant adenovirus of the present invention are far wider. This is because even a cell in which the original adenovirus cannot grow can function sufficiently as an expression vector in the case of the recombinant adenovirus of the present invention as long as the virus particles can infect and enter.

  In addition, although the genome of the recombinant adenovirus of the present invention does not replicate in an extrachromosomal state, it exists in the nucleus for 2 weeks to 2 months, and the expression of the target gene is considerably sustained. It is advantageous as a vector.

  The genome of the recombinant adenovirus of the present invention includes a hybrid promoter (CAG promoter) comprising a cytomegalovirus enhancer, a chicken β-actin promoter, and a rabbit β globin splicing acceptor, and a poly A derived from rabbit β globin. It has the feature that the sequence is incorporated.

  This hybrid promoter (CAG promoter) is disclosed in JP-A-3-16887 as a high expression vector, and its preparation is pCAGGS (JP-A-3-16887, page 13, lines 20 to 20) described in the publication. (Page 14 and page 22, line 1 to page 25, line 6) can be performed by cutting out with restriction enzymes SalI and HindIII, and can be used in the present invention. The inventors of the present invention have found that the above hybrid promoter is superior when several promoters called high expression promoters are used on recombinant adenovirus and the expression levels of the lacZ gene are compared (comparative example). This is incorporated into the adenovirus of the present invention.

  By incorporating this hybrid promoter, it is possible to reduce the side effects on infected human cells that are observed when a high concentration virus solution is used in the treatment using recombinant adenovirus. Because the expression level is large, it is sufficient to use a low concentration virus solution.

  The nucleotide sequence encoding the foreign target polypeptide sequence incorporated in the adenovirus genome of the present invention is particularly limited as long as it is a nucleotide sequence that can be expressed by the above hybrid promoter (CAG promoter). Although not intended, from the viewpoint of usefulness, normal gene sequences corresponding to human deficient genes, DNA sequences encoding cytokines such as interleukins and interferons, cancer suppressor gene sequences, cancer gene antisenses An arrangement or the like is particularly preferable.

  The LacZ gene also expresses β-galactosidase, which is useful for non-lactose diseases. Thus, the nucleotide sequence encoding the foreign polypeptide sequence of interest incorporated in the recombinant adenovirus of the present invention is expressed under the control of the CAG promoter in the natural host cell, and the host cell It is characterized in that the polypeptide encoded by it is secreted into the medium.

  The genome of the adenovirus of the present invention lacks the E1 gene region, particularly the E1A gene region. This is because the E1A gene region involved in adenovirus cell canceration activity is deleted, thereby detoxifying adenovirus and expressing only foreign nucleotide sequences integrated into the genome. It is not always necessary to delete the entire E1 gene region, and the object can be achieved by removing 1.3 to 9.3% of the fragment containing the E1A gene region.

  The adenovirus genome of the present invention may also have the E3 gene region deleted. In particular, those obtained by deleting a 79.6 to 84.8% fragment from the adenovirus genome are preferred. This is because it is unnecessary for the replication of the foreign nucleotide sequence.

  The chicken β-actin promoter integrated in the adenovirus genome of the present invention itself exhibits strong promoter activity, but removes the downstream splicing acceptor sequence from the middle of its intron region, and instead replaces the rabbit β globin. Incorporating a splicing acceptor sequence contained in the structural gene significantly increases its activity.

  As a polyA sequence incorporated into the genome of the adenovirus of the present invention, a gene derived from SV40 can be used, but a gene derived from a rabbit β globin structural gene is preferred.

  The adenovirus genome of the present invention is characterized in that a cytomegalovirus enhancer sequence is incorporated in order to enhance the promoter activity of the chicken β-actin promoter.

  When such a recombinant adenovirus of the present invention is infected with animal cells, the adenovirus genome is injected into the cells, and even in non-proliferating cells, it remains stable for a long period of time from half a month to 2 months outside the chromosome. However, the polypeptide encoded by the foreign nucleotide sequence integrated into the genome can continue to be expressed. At that time, the protein encoded by the genome itself is not produced due to the deletion of the E1 gene, and side effects due to the adenovirus itself are minimized. Therefore, the recombinant adenovirus of the present invention is considered extremely useful for gene therapy.

  Examples of such animal cells include human or mammalian lung epithelial cells, gastrointestinal epithelial cells, nervous system cells, liver, muscles (skeletal muscle, cardiac muscle, etc.) and the like.

Next, a method for producing the recombinant adenovirus of the present invention will be described.
The production of the recombinant adenovirus of the present invention is generally very difficult because the protein is covalently bound to both ends of the viral genome as described above.

Therefore, in the present invention, the following method is used.
(1) First, about 31 kb of genomic DNA from which the E3 gene region (1.9 kb) and E1A / E1B gene region (2.9 kb) unnecessary for replication are deleted from the full length of the adenovirus genome (36 kb). A target gene expression unit comprising an exogenous promoter at the E1A / E1B deletion site, a nucleotide sequence encoding an exogenous target polypeptide sequence to be expressed in a cell line, and a polyA sequence Incorporate The expression unit is preferably incorporated to the left. Here, the leftward direction refers to a direction opposite to the original transfer direction of E1A and E1B.

  The cosmid is prepared using a lambda in vitro packaging kit GigapackXL (Stratagene).

  Examples of nucleotide sequences encoding a foreign target polypeptide sequence to be expressed in a cell line include, for example, interleukin-1 to 12, interferon-α, β or γ, tumor necrosis factor-α, or β, Granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, erythropoietin, cytokines such as growth hormone, insulin and insulin-like growth factor, genetic disease-causing genes adenosine deaminase, dystrophin, brain-derived neurotrophic factor, thymidine kinase Nucleotide sequence encoding low density lipoprotein receptor, alpha-1 antitrypsin, blood coagulation factor 8, blood coagulation factor 9, galactosidase, non-self antigen gene allo HLA (HLA-B7), virus antigen, etc. , Cancer suppressor heredity In a p53, RB, WT-1, NM23, NF-1, etc., Ras antisense sequences, and the like an oncogene.

  (2) On the other hand, since the adenovirus genome replicates with the terminal protein encoded by the virus at both ends, genomic DNA with this terminal protein (DNA-terminal protein complex, DNA-TPC) is prepared. Then, it is cleaved with a restriction enzyme that frequently cleaves only the E1 gene region of the genome, such as EcoT22I (Takara Shuzo Co., Ltd.) or NsiI and AvaIII, which have the same recognition sequence, and used as the parent virus genome.

  (3) Next, the cosmid containing the target expression unit and the cleaved parent viral DNA-TPC are mixed, and 293 cells (ATCC No. CRL-1573) are transfected by the calcium phosphate method.

  In 293 cells, homologous recombination first occurred in a common sequence spanning 21 kb between both molecules, and then the molecule that started replication from the right end (opposite the E1 region) The plasmid-derived sequence is isolated and repaired. This is because, as described above, 102 base pairs at both ends of the adenovirus genome are completely the same sequence (terminal reverse sequence). By this mechanism, adenovirus is thought to repair itself with the opposite sequence even if one end is attacked by exonuclease.

  (4) The target recombinant adenovirus can be obtained from adenovirus grown in 293 cells by the above operation. In the method of the present invention, since the appearance frequency of the recombinant adenovirus is high, it is not necessary to use a selection marker, and it can be purified by a simple method as described below.

  293 cells co-transfected with the cosmid containing the target expression unit and the cleaved parent virus DNA-TPC as described above were cultured at 37 ° C. for about 1 day, and three 96-well dishes were used. Re-roll to 10-fold dilution and 100-fold dilution. By this method, the desired recombinant adenovirus is selected from among the proliferating viruses obtained from wells containing a single virus.

  In the selection method, 293 cells are infected with each virus solution, DNA is extracted from a culture solution containing cells killed by the growth of the virus, fragments obtained by cleavage with the restriction enzyme XhoI are subjected to electrophoresis, and the pattern is examined. Select the one in which the band from the break point in the expression unit to the left end of the adenovirus genome appears correctly. This solution can be used as a target recombinant adenovirus solution.

  In order to confirm that the deletion virus or the parent virus is not mixed in this solution, a part of this solution is infected with 293 cells, DNA is extracted from the propagated virus, and the cleavage pattern by the restriction enzyme XhoI is used. Check out. Discard this solution if you suspect that the deletion virus or parent virus is mixed.

  In order to clarify the characteristics of the present invention, it is as follows when compared with the conventional method (Melissa A. Rosenfeld et al., Cell, vol. 68, 143-155 (1992)).

  In the method of the present invention, a cosmid containing almost the entire length of adenovirus DNA is used. In the conventional method, a cassette of about 5 kb in the vicinity of the E1A / E1B gene region has been used. Therefore, the efficiency of homologous recombination was remarkably improved in the method of the present invention.

  In the method of the present invention, genomic DNA (DNA-TPC) to which a terminal protein is bound is used as the parent virus DNA, but in the conventional method, DNA from which the terminal protein has been removed has been used. For this reason, the efficiency of several tens of times has been achieved in the method of the present invention.

  Furthermore, most of the virus strains obtained by the conventional method were parent viruses, but in the method of the present invention, the E1 gene region (left side) of DNA-TPC is cleaved frequently (at 3 to 10 sites). By using the restriction enzyme, about half of the obtained virus strains became the target recombinant virus, and it became unnecessary to use a selection marker. As such a restriction enzyme, for example, EcoT22I or NsiI and AvaIII having the same recognition sequence can be preferably used.

  The high-titer virus solution obtained as described above by the method of the present invention is appropriately diluted for local injection (central nervous system, portal vein, etc.), oral (using intestinal solvent) administration, airway administration, transdermal Depending on the administration method such as administration, it can be used for treatment of various diseases including genetic diseases.

  The recombinant adenovirus obtained by the method of the present invention is also effectively used as a means for expressing foreign genes and vaccinating humans and animals using various cell culture systems.

  EXAMPLES Hereinafter, although an Example, a reference example, an experiment example, and a comparative example demonstrate this invention in more detail, this invention is not limited at all by these Examples.

  Unless otherwise specified, various procedures for handling phages, plasmids, DNA, various enzymes, Escherichia coli, cultured cells, etc. in the Examples are described in “Molecular Cloning, A Laboratory Manual, edited by T. Maniatis et al., Second Edition (1989). ), Cold Spring Harbor Laboratory ".

Example 1
<Production of recombinant adenovirus>
The production of recombinant adenovirus is roughly divided into three steps. That is, a step of inserting an expression unit into a cosmid, a step of preparing a parental virus DNA-TPC, and a step of co-transfection / separation and purification into 293 cells with cosmid and DNA-TPC. Each step will be described below.

(1) Insertion of expression unit into cosmid
(1) First, 0.2 μg of the expression unit fragment whose both ends were smoothed and 1 μg of pAdex1w DNA cut with SwaI were mixed.

  In this case, as the expression unit, a hybrid promoter (CAG promoter) comprising a cytomegalovirus enhancer, a chicken β-actin promoter, and a rabbit β globin splicing acceptor, a poly A sequence, and a LacZ gene set were used. For smoothing, Klenow enzyme was used, and after the treatment, phenol extraction was performed once and chloroform extraction was performed twice, followed by precipitation with ethanol for complete purification. Expression units were added in a molar ratio of 2-3 times that of cosmid (44 kb). In general, if there is a SwaI site in the expression unit, it cannot be cloned, but SwaI is rarely present in the expression unit because it recognizes 8 bases. There was no problem with this expression unit.

  PAdex1w was used as the adenovirus genome fragment. 10 μg of this was cut with SwaI, phenol extracted once, and 1 μg after centrifugation gel filtration described later was used. In addition to the SwaI (pAdex1w) listed here, the cosmid includes a ClaI (pAdex1c), and any of these can be used by using a corresponding restriction enzyme.

  (2) Next, ethanol is added to the mixed solution to precipitate cosmids. The precipitate was obtained by centrifugation and dissolved in a 5-fold dilution of a solution (TE) in which 1 mM EDTA was added to 10 mM Tris-hydrochloric acid (pH 7.5).

  (3) The obtained cosmid was added with ATP and T4 DNA ligase in a ligase reaction buffer and allowed to bind overnight in a final volume of 7 μl. Subsequently, sterilized water and a Swal reaction buffer were added to make 48 μl, and then the ligase was heat-inactivated at 70 ° C. for 10 minutes.

  At this time, unlike a plasmid, a cosmid efficiently packages macromolecules bound to a linear tandem rather than a circle.

(4) 2 μl of SwaI (manufactured by Boehringer) was added and cut at 25 ° C. for 1 hour.
The meaning of cleaving SwaI is that, when the cosmid recombines without adding the expression unit, the SwaI recognition sequence is regenerated, so in this step, the cosmid that does not incorporate the expression unit is recut and a colony is not formed. It is. This method is a powerful way to select only cosmids with inserts.

  (5) According to a conventional method (Molecular Cloning vol.3 E.34), phenol extraction of cosmid, centrifugation, and gel filtration were performed.

  (6) SwaI was cut again. That is, 5 μl of SwaI was added to the SwaI reaction buffer and cleaved at 25 ° C. for 2 hours. The reason is as described above.

(7) 1 μl of the obtained cosmid was subjected to in vitro packaging.
That is, Gigaback XL (manufactured by Stratagene), which is a lambda in vitro packaging kit, was used at ¼ scale, and the rest was frozen at −80 ° C. Since GIGABACK XL has a low packaging efficiency for cosmids of 42 kb or less, it is possible to select cosmids that have become larger with inserts to some extent. In this experiment, when 10 colonies were picked up, most of them contained inserts, and clones with the desired orientation (leftward) could be easily obtained.

  The handling of cosmids was carried out according to the conventional method (Izumi Saito et al., Experimental Medicine: 7: 183-187, 1989).

(8) The packaged cosmid was infected with DH1 (ATCC 33849).
That is, three Ap ⁺ (ampicillin-added) agar plates and 5 ml of Ap ⁺ LB (pool) were inoculated with 1/200, 1/20, 1/2, and the remaining total, respectively, and cultured overnight.
Pool miniprep DNA was extracted and prepared, and the ratio of the insert containing the whole enzyme was examined. Whole colonies were picked and cultured in 1.5 ml of Ap ⁺ LB overnight to prepare miniprep DNA.

(9) Next, the orientation and structure of the expression unit were confirmed by restriction enzyme digestion.
In addition, using NruI and ligase, a plasmid containing an expression unit but lacking most adenovirus DNA was prepared, DNA was prepared, and final confirmation of cDNA cloning was performed. The expression of the LacZ gene, which is the target gene, was confirmed by target expression. (Cosmid is used instead of this plasmid for the production of recombinant virus.)

(2) Preparation of adenovirus DNA-protein complex (Ad5 dlX DNA-TPC)
(1) Ad5 dlX (I. Saito et al., J. of Virology, vol. 54, 711-719 (1985)) was used as adenovirus DNA. Ad5 dlX was infected to HeLa cells (for 10 Roux) and cultured.

Specifically, the virus solution of Ad5-dlX (-10 ⁹ PFU / ml) was infected with 0.2 ml / Roux, and 3 days later, the detached cells were collected by centrifugation at 1500 rpm for 5 minutes. Most of the adenovirus particles are in the cell nucleus, not in the medium, which has the advantage that the virus can be purified from infected cells. (The following operations were performed non-aseptically.)

(2) The obtained cells are suspended in 20 ml of 10 mM Tris-HCl (pH 8.0), and the cells are crushed at 200 W for 2 minutes (30 seconds × 4) using a sealed sonicator, and the virus is intracellularized. Released from.
In order to release the virus from the cell, 5 ml or less may be used for freezing and thawing, but a sonicator is convenient for a volume larger than that. However, be sure to use a sealed type (with a special cup). The normal throw type is dangerous even in a safety cabinet.

  (3) After removing the precipitate from the obtained crushed material by centrifugation (10 krpm, 10 minutes), put 15 ml of cesium chloride solution (specific gravity 1.43) in the ultracentrifuge SW28 tube, and put the supernatant on it. The layers were layered and concentrated by cushion centrifugation (25 krpm, 1 hour, 4 ° C.).

(4) The virus layer just below the interface was transferred to a SW50.1 tube. The virus layer directly under the interface was usually visible, and 5 ml of the virus layer and cesium chloride under the virus layer were collected. At the same time, the other was filled with a cesium chloride solution (specific gravity 1.34).
These were ultracentrifuged overnight at 35 krpm and 4 ° C. Next, the white virus band was separated and transferred to a tube with a gradient. Further, ultracentrifugation was performed at 35 krpm and 4 ° C. for 4 hours or more.

  (5) A white virus band was collected, mixed with an equal amount of 8M guanidine hydrochloride at room temperature, and 4M guanidine hydrochloride saturated cesium chloride was added to fill a VTi65 tube. The particle protein was denatured and dissociated by 4M guanidine hydrochloride, and DNA-TPC was released. Ethidium promide was not available because a method for later removal has not been established.

  (6) The above tube was ultracentrifuged at 55 krpm and 15 ° C. overnight, fractionated by 0.2 ml, 1 μl of each was mixed with 20 μl of 1 μg / ml ethidium bromide aqueous solution, and the DNA was stained by fluorescence staining. The presence or absence was confirmed. A few fractions containing DNA were collected.

(7) Dialyzed overnight in 500 ml TE (twice) and stored at -80 ° C. The amount of Ad5dlX DNA-TPC thus obtained was calculated from OD _{260 in the same} manner as normal DNA.

(8) The obtained Ad5dlX DNA-TPC was cleaved with a sufficient amount of EcoT221 for 2 hours for preparation of the recombinant adenovirus in the third step, and stored at -80 ° C.
DNA-TPC could be digested with restriction enzymes, dialyzed, and gel filtered, but could not be electrophoresed, treated with phenol, or precipitated with ethanol. Since the concentration method was only cesium chloride equilibrium centrifugation, it was kept as thick as possible. About 300 μg of DNA-TPC could be obtained from 10 Roux infected cells.

(9) A part was collected, 10 μl of BPB buffer for electrophoresis was added, 1 μl of proteinase K (10 mg / ml) was added, and the mixture was reacted at 37 ° C. for 10 minutes to digest the terminal protein. Phenol was extracted and the supernatant was separated by agarose gel electrophoresis to confirm complete cleavage.
The restriction enzyme buffer in EcoT221-cleaved DNA-TPC was removed by centrifugal gel filtration, and then dispensed and stored at -80 ° C.

(3) Isolation of recombinant virus and preparation of high titer virus solution
(1) One 6 cm and 10 cm dishes of 293 cells cultured in 10% FCS-added DME were prepared.

(2) 8 μg (preferably 3 to 9 μg) of pAdex1w DNA incorporating the expression unit and 1 μg of Ad5dlX DNA-TPC cleaved with EcoT22I were mixed, and a 6 cm Petri dish was prepared using a Cellfect (Pharmacia) kit. The plates were transfected by the calcium phosphate method. The mixed solution was dropped from above the medium of 6 cm petri dish and the culture was continued.
Incubate overnight (about 16 hours), change the culture medium in the morning, and in the evening, add 3% collagen-coated 96-wells (stock solution, 10-fold dilution, 100-fold dilution) to each well using 5% FCS-added DME. Re-rolled with 0.1 ml per unit. In order to prevent the number of cells from greatly differing between each plate, 293 cells of a 10 cm dish were mixed and spread on two dilutions.

(3) After 3-4 days and 8-10 days, 50 μl of 10% FCS-added DME was added to each well. When 293 cells became thin, it was added early.
Wells with virus growth and cell death appeared between 7 and 15 days. Each time the cells in the well were completely killed, the culture solution (every dead cells) was aseptically transferred to a sterilized 1.5 ml tube with a sterile Pasteur pipette, snap frozen with dry ice and stored at −80 ° C.

(4) The determination was completed in 15-18 days. Select about 10 culture tubes collected from wells where cells died relatively late, freeze and thaw 6 times, and then centrifuge at 5 krpm for 10 minutes and store the supernatant at -80 ° C. as the first virus solution (first seed) did.
This is because a well in which virus growth has occurred earlier is likely to be a mixed infection of a plurality of virus strains.

  (5) Prepare 293 cells in a 24-well plate, and add 2% each of 5% FCS-DME (0.4 ml / well) and 10 μl of the primary virus solution.

(6) When the cells are completely killed in about 3 days, the supernatant is obtained by freezing and thawing and centrifuging 6 times in the same well as the preparation of the primary virus solution, and this is used as the secondary virus solution (second seed) − Stored at 80 ° C. The titer of the secondary virus solution was about 10 ⁷ to 10 ⁸ PFU / ml. Another 1-well dead cell was centrifuged at 5 krpm for 5 minutes, the supernatant was discarded, and only the cell mass was stored at -80 ° C. When cell clusters of 10 types of virus strains were collected, the total DNA of infected cells was extracted by the following method. To the cell mass, 400 μl of TNE for cell DNA (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA), 4 μl of proteinase K (10 mg / ml) and 4 μl of 10% SDS were added.

  (7) After treatment at 50 ° C. for 1 hour, the nucleic acid obtained by phenol / chloroform extraction twice, chloroform extraction twice, and ethanol precipitation was dissolved in 50 μl of TE (containing 20 μg RNase / ml).

  15 μl of the expression unit was cleaved with XhoI, an enzyme containing CG as a recognition sequence among the enzymes that cleave the expression unit, and the expression cosmid was digested with XhoI and electrophoresed overnight on an agarose gel with a length of about 15 cm. Compared. A band in which a band from the break point in the expression unit to the left end of the adenovirus genome appeared correctly was selected. In addition, clones in which unexplainable bands appear thin were discarded because they could be mixed with viruses with deletions.

  Since adenoviral DNA grows to 10,000 copies per cell, the total DNA can be extracted together with the cellular DNA, and the viral DNA band can be observed by restriction enzyme digestion. Enzymes containing CG in the recognition sequence, such as Xhol, do not cleave cellular DNA, so the pattern is easy to see. When using other enzymes, it was necessary to keep uninfected 293 cell DNA in control. (A band derived from a repetitive sequence of human cells appeared).

(8) 0.1 ml of the secondary virus solution of the target virus strain identified by Xhol cleavage was infected with 293 cells in a collagen-coated 150 cm ² bottle (medium was 25 ml).

  When the cells died after 3 days, 25 ml of the medium with dead cells was aseptically disrupted in a sealed sonicator 200w with a maximum output of 2 minutes (30 seconds × 4 times) to release the virus.

The precipitate was removed by centrifuging at 3 krpm and 4 ° C for 10 minutes, dispensed into 13 ml each of 2 ml in a 5 ml freezing tube, rapidly frozen with dry ice and stored at -80 ° C to prepare a tertiary virus solution. The tertiary virus solution was a solution containing the recombinant adenovirus of the present invention and had a high titer of about 10 ⁹ PFU / ml.

  In addition, 5 μl of the tertiary virus solution was infected to one well of 293 cells in a 24-well plate, and the enzyme cleavage pattern of the grown viral DNA was confirmed by the above method. If it is suspected to be a deletion virus or a mixture with the parent virus, the deletion virus that had already been slightly mixed in the secondary virus solution stage may have appeared due to its rapid growth. All the tertiary seeds were discarded, and the target virus was purified from another primary virus solution by redoing again or by limiting dilution from the primary virus solution.

Reference example 1
<Simple titration method for recombinant adenovirus of the present invention>
The recombinant adenovirus of the present invention can be easily measured by the following method.
(1) Prepare 293 cells for each 10 cm dish.
Recombinant recombinant adenovirus solution (tertiary virus solution) is serially diluted from 10 ^-1 to 10 ^-4 using DME supplemented with 5% FCS. For example, 0.9 ml DME + 0.1 ml virus solution. Change all the tips.

(2) Add 50 μl of 5% FCS-added DME to all wells of one 96-well collagen coat.
In the first row, 25 μl of recombinant virus diluted to 10 ⁻⁴ is added.
Transfer 25 μl to the second row of wells using an 8-well multichannel pipette. Thereafter, the same operation is repeated up to the 11th row, and the last 25 μl is discarded. As a result, 3 ⁿ serial dilution series can be prepared up to 3 ¹¹ × 10 ^-4 . The 12th row serves as a control for uninfected cells.
The chip used at this time is changed each time.

(3) Peel 293 cells of 10 cm petri dish with PBS-EDTA and resuspend in 6 ml of DME solution containing 5% FCS. Add 50 μl of this cell solution to 96 wells.
After 3-4 days and 6-7 days, 50 μl (10% FCS-added DME) is gently added to each well using a cell saver sterilization tip.
After 12 days, the end point of cell degeneration is determined with a microscope. If the cells can be maintained until 14th, the determination is obvious to anyone, but if the cells hurt, the determination becomes difficult.
The 50% cytopathic end point (TCID ₅₀ ) is calculated statistically using Kelver's equation (1) below.
Kelver's formula: If the desired TCID ₅₀ is 10 ^X ,
X = log a− (number of denatured wells at each dilution stage / total number of samples at each dilution stage−0.5) × log (dilution ratio)
However, a: dilution in the first row (10 ⁻⁴ × 3 ^{−1 in} this protocol)

Experimental Example After 12 days, the end point of cell degeneration was determined with a microscope, and the results shown in Table 1 were obtained. The titer in this case is as follows.
Since the diluted virus solution volume is 50 μl, assuming that this concentration is 50 μl and 1 PFU, the titer of the virus stock solution is 1 ml ÷ 50 μl ÷ 10 ^−7.817 = 20 × 10 ^7.817 = 10 ^9.118 = 1.3 × 10 ⁹ (PFU / ml). In other words, if the eighth row is half denatured, it is 1.3 × 10 ⁹ PFU / ml, and if it is half of the seventh row, it becomes one third of 4.4 × 10 ⁸ PFU / ml.

Reference example 2
<Construction of pAdex1c and pAdex1w>
(1) Preparation of a plasmid (pUAF0-17D) containing 17% of the left end of the adenovirus genome lacking the E1 gene region Type 5 adenovirus DNA was treated with S1 to make blunt ends and recovered by phenol extraction and ethanol precipitation . A BamH linker was attached to the blunt end, then digested with HindIII and separated by agarose gel electrophoresis. The fragment of interest (2.8 kb, corresponding to 8% of the left end of the adenovirus genome) was electrically extracted from the gel, recovered and inserted into the BamHI / HindIII site of pUC19 digested with BamHI / HindIII. The obtained plasmid of interest was named pUAF0-8.

  Adenovirus DNA was digested with HindIII and separated by agarose gel electrophoresis. The desired 3.4 kb fragment (corresponding to 8-17% of the left end of the adenovirus genome) was recovered from the gel and inserted into the HindIII site of pUC19 (named pUAF8-17).

  The base number of pUAF0-8 (the base number here is derived from adenovirus DNA) The 454th PvuII site was converted to a ClaI site using a ClaI linker. This plasmid was digested with BamHI / ClaI, and a 454 bp BamHI-ClaI fragment was recovered by agarose gel electrophoresis.

  The BglII site at base number 3328 of pUAF8-17 was converted to a ClaI site using a ClaI linker. This plasmid was digested with HindIII / ClaI, and a 2.9 kb HindIII-ClaI fragment was recovered by agarose gel electrophoresis.

  The 454 bp BamHI-ClaI fragment from pUAF0-8 and the 2.9 kb HindIII-ClaI fragment from pUAF8-17 were ligated and inserted into the BamHI / HindIII sites of pUC19. The resulting plasmid was named pUAF0-17D. This plasmid contains 17% of the left end of the adenovirus genome lacking the E1 gene region.

(2) Preparation of Bst1107-EcoRI fragment (21.6 kb) of adenovirus genome Type 5 adenovirus DNA was digested with Bst1107 and EcoRI and separated by agarose gel electrophoresis, and the desired 21.6 kb fragment was recovered. .

(3) Preparation of EcoRI-SalI fragment (6.5 kb) of adenovirus genome The SalI site of pX2S (I. Saito et. Al., J. of Virology, vol. 54, p711-719, 1985) was replaced with a SwaI linker. To convert to a SwaI site to obtain pX2W. After digesting pX2W with EcoRI and SwaI and separating by agarose gel electrophoresis, the desired 6.5 kb fragment was recovered.

(4) Preparation of Shalomide (chdRBR7-11)
To remove KpnI, SmaI and BamHI of charomid 9-11 (I. Saito & G. Stark, Proc. Natl. Acad. Sci. USA, vol. 83, p8664-8668, 1986) After digestion, blunting with Klenow fragment, self-ligation. Using this, it was transformed, and the desired shalomid was isolated and named charomid 6-11. A BamHI linker was inserted into the EcoRI site of charomid 6-11, and the resulting Shalomid was named chdRBR7-11.

(5) Preparation of pAdex1c BamHI-Bst1107 fragment (2.9 kb) of pUAF0-17D, Bst1107-EcoRI fragment (21.6 kb) of adenovirus genome, and EcoRI-SwaI fragment (6.5 kb) of pX2W with EcoRI and Eco36I Ligated with digested chdRBR7-11. Thereafter, it was packaged in vitro and infected with DH5α. A transformant having the desired fragment was isolated and named pAdex1c.

(6) Preparation of pAdex1w The ClaI site of pAdex1c was converted to a SwaI site using a SwaI linker to obtain pAdex1w.

Comparative Example 1
<Effect of various promoters on the expression of LacZ gene in various cell lines by recombinant adenovirus>
A recombinant adenovirus was prepared by replacing the promoter of the recombinant adenovirus of the present invention (CAG promoter) with the SRα promoter (SV40 early promoter + HTLV-ILTR) and the EF-1α promoter (derived from the human polypeptide elongation factor gene). The activity of the recombinant adenovirus of the present invention and the expression of the LacZ gene was examined.

The SRα promoter has been disclosed by Takebe et al. (Molecular and Cellular Biology, vol. 8, 466-472, 1991). The EF-1α promoter has also been disclosed by Kim et al. (Gene, vol. 91, 217-223, 1990).
The expression unit was introduced into the recombinant adenovirus genome as follows.

(1) In order to incorporate the CAG promoter and LacZ gene into the adenovirus genome, first, the LacZ gene was introduced into pCAGGS. That is, pCAGGS was digested with EcoRI, blunted with Klenow fragment, and a SwaI linker was inserted into this part. On the other hand, pMC1871 (Shapiro et al., Gene, vol. 25, 71-82, 1983) was used as a material for the LacZ gene. In LacZ of pMC1871, the base sequence of N-terminal 7 amino acids unnecessary for enzyme activity is a polylinker sequence. This N-terminal part was cleaved with SmaI, and a NotI linker was ligated. Then, it was ligated with a synthetic DNA containing an initiation codon, namely (PstI end) -CAGACCGTGCCATCATGA- (NotI end). It was cleaved with PstI after the termination codon, smoothed and introduced into the SwaI site. This resulted in a plasmid with the LacZ gene introduced downstream of the CAG promoter. Subsequently, this plasmid was excised with SalI and HindIII, and both ends were smoothed.
This was combined with pAdex1w cleaved with SwaI to prepare an expression cosmid.

  (2) For the incorporation of the SRα promoter and the LacZ gene into the adenovirus genome, pMC1871 was first used as a material for the LacZ gene, as in the case of the CAG promoter. This N-terminal part was cleaved with SmaI, and a NotI linker was ligated. Then, it was cleaved with PstI after the termination codon, smoothed, and a KpnI linker was ligated. This LacZ fragment was ligated with the above synthetic DNA containing the initiation codon and cloned between the PstI and KpnI sites of the expression vector pCD-SRα-296 having the SRα promoter (pSRLacZ).

  To create a recombinant adenovirus (Adex1SRLacZ) that expresses LacZ from the SRα promoter, the SRα-LacZ-polyA expression unit is cut out from pSRLacZ using HindIII and Tth111I to form a blunt end, and the SwaI site of the dex creation cassette pAdex1w And used for the production of recombinant virus.

  (3) A recombinant virus that expresses LacZ from the EF1α promoter is obtained by extracting the SV40T antigen portion of the expression vector pEF321-T (kim et al., Gene, vol. 91, 217-223, 1990) with HindIII and HpaI. First, pEF321w linked with a synthetic linker was prepared, and the coding region containing the initiation codon of pSRLacZ in the SwaI site was excised with PstI and KpnI, blunt-ended, and inserted into the SwaI site of pEF321w to prepare pEFlacZ. From here, the expression unit of EF1α-LacZ-polyA was excised with NheI and KpnI, inserted into the SwaI site of pAdex1w, and used for preparation of a recombinant virus.

The cell lines infected with recombinant adenovirus are HeLa cells, HepG2 cells, IMR-32 cells, EB-transformed Bcell-clone 1, EB-transformed Bcell-clone 2, Jurkat cells, CV-1 cells, CRFK cells. , MYA-1 cells, SHOK cells, NIH3T3 cells, LTK ⁻ cells, Ba / F3 cells.

The activity of the expressed β-galactosidase was measured as follows.
3 × 10 ⁵ cells were cultured in each well of a 24-well plate, and the prepared recombinant adenovirus was added to m. o. i. (Multiplicity of infection) Infected at 10 and incubated for 2 days. Thereafter, the cells were collected in an Eppendorf tube and washed twice with PBS. To this, 0.5 ml of extraction buffer was added, and ultrasonically disrupted at intervals of 30 seconds for a total of 90 seconds. Then, 0.5 ml of 80% glycerol was added and centrifuged at 15000 rpm for 10 minutes, and the resulting supernatant was subjected to cell extraction. Liquid.

To 850 μl of a reaction mixture consisting of 1.0 ml of 0.5 M sodium phosphate buffer (pH 7.8), 1.0 ml of 0.5 M β-mercaptanol, 1.0 ml of 10 mM MgCl ₂ and 5.5 ml of distilled water, 50 μl of cell extract, extraction buffer (for blank measurement) or β-galactosidase solution (for control) was added, preincubated for 5 minutes, and then the substrate solution was added. As a substrate, o-nitrophenyl β-galactoside was used.

  After incubating for 30 minutes, 400 μl of a reaction stop solution was added, the absorbance at 420 nm was measured, and the activity was calculated from the following formula. The result is shown in FIG. From this result, it can be seen that the CAG promoter of the present invention shows much stronger expression activity in all cells tested compared to the SRα promoter and the EF-1α promoter.

One unit is defined as the amount of catalyst that hydrolyzes 1 μmol of o-nitrophenyl β-galactoside to o-nitrophenol and D-galactose per minute under the above conditions.
When the absorbance exceeds 1, it is desirable to dilute the cell extract using an extraction buffer and measure it again.
Units / ml = (absorbance of sample-absorbance of blank) /4.51 ^a) x1.4ml ^b) x1 / 30min ^c) x1.0ml / 0.05ml ^d)
Here, a), b), c), and d) each represent the following meaning.
a) The concentration μmol / ml of the enzyme reaction product is determined by dividing by the extinction coefficient of o-Nitrophenol, that is, the absorbance of a 1 mM aqueous solution.
b) vol. Is applied to obtain the reaction product amount μmol.
c) Determine the amount of reaction per minute by dividing by the reaction time.
d) vol. Is to convert the reaction amount per 0.05 ml / ml.

Comparative Example 2
<Comparison of insertion direction of LacZ gene or hepatitis C virus (HCV) gene expression unit and virus production>
Using different promoters and genes, recombinant adenoviruses whose insertion directions were reversed were prepared, and the virus production was compared.

(1) Construction of HCV-expressing recombinant adenovirus A 2.2 kb fragment corresponding to base numbers 307 to 2554 of HCV was prepared by PCR and inserted into the PstI-KpnI site of pcDL-SRα in the same manner as in LacZ. Recombinant adenovirus was prepared in the same manner as in Comparative Example 1.

(2) Construction of LacZ-expressing recombinant adenovirus Prepared in the same manner as in Comparative Example 1.
The production amount of the recombinant virus in 293 cells is shown in Table 2.
From Table 2, 3 out of 4 cases showed a virus production amount about 10 times higher in the leftward direction than in the rightward direction, and in one case it was comparable. It was also found that this tendency was not due to promoters and genes. From this result, it was found that this recombinant adenovirus shows much higher virus production when the insertion direction is leftward.

  The present invention can provide a recombinant adenovirus capable of gene expression in a wide range of animal cells. The recombinant adenovirus of the present invention is useful for the treatment of genetic diseases.

FIG. 1 is a graph showing the effect of these promoters on the expression of LacZ gene in various cell lines by recombinant adenoviruses incorporating various promoters. FIG. 2 is a diagram illustrating the structure of pIndex1w.

Claims (6)

A hybrid promoter in which the adenovirus genome consists of a nucleotide sequence encoding a foreign polypeptide sequence of interest, a cytomegalovirus enhancer that controls the expression of this sequence, a chicken β-actin promoter, and a splice acceptor of rabbit β globin. CAG promoter), and a recombinant adenovirus characterized by having a poly A sequence in the left direction . The recombinant adenovirus according to claim 1, wherein the adenovirus genome has a 1.3% to 9.3% fragment containing the E1A gene region deleted from the E1 gene region .   A nucleotide sequence encoding a foreign target polypeptide sequence and a CAG promoter are inserted into a site where a 1.3% to 9.3% fragment containing the E1A gene region is deleted. The recombinant adenovirus according to claim 2.   The recombinant adenovirus according to claim 2 or 3, wherein the adenovirus genome further lacks a 79.6% to 84.8% fragment of the genome from the E3 gene region. A nucleotide sequence encoding a foreign polypeptide sequence of interest encodes a polypeptide that is secreted into the medium of the host cell when expressed under the control of the CAG promoter in the native host cell. The recombinant adenovirus according to any one of claims 1 to 4, wherein The recombinant adenovirus according to any one of claims 1 to 4 , which is used for gene therapy.

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