Patent Application
20200172873
The present invention relates to a novel cell line for producing an adenovirus having a limited autoreplication capability; a method of preparing the same; and the use of the cell line.
Vectors are most commonly used as delivery systems in which a gene is delivered to cells and delivered in vivo to apply the gene to clinical trials for gene therapy.
The vectors generally include non-viral vectors in which nucleic acids such as DNA and RNA are used by themselves, and viral vectors including Adeno, Retro, pox, vaccinia, herpes, and the like. Among these, Adeno is most commonly used in clinical trials for gene therapy.
Adenoviruses carrying double-stranded DNA have a high expression level in both dividing and non-dividing cells. Also, the adenoviruses have an advantage in that they have few limitations in insertion of foreign genes.
However, the presence of replication-competent adenovirus (RCA) is one of the most serious problems caused when an adenovirus is used as a gene delivery vector. The RCA may be produced by recombination or contamination with host DNA sequences in various steps of a preparation process. When there is even a small number of an RCA, the RCA can replicate autonomously in human tissues and circulate to other sites, thereby causing serious side effects in immunodeficient patients.
Therefore, there is a need for production of a cell line producing a recombinant adenovirus having a limited autoreplication capability, in which no RCA is produced, thereby solving the above problems.
One aspect of the present invention is to provide a cell line for producing a recombinant adenovirus having a limited autoreplication capability, in which no RCA is produced.
Another aspect of the present invention is to provide a method of preparing a cell line for producing the adenovirus having a limited autoreplication capability, and the use of the cell line.
To achieve the above object, according to one aspect of the present invention, there are provided a cell line for producing an adenovirus having a limited autoreplication capability; a method of preparing the cell line for producing an adenovirus having a limited autoreplication capability; and various uses of the cell line for producing an adenovirus having a limited autoreplication capability.
One exemplary embodiment of the present invention provides a cell line for producing an adenovirus having a limited autoreplication capability, which includes:
at least one of an E1 protein and a nucleic acid sequence encoding the same; and
at least one of an E1A protein, a nucleic acid sequence encoding the E1A protein, an E1B protein, and a nucleic acid sequence encoding the E1B protein.
According to one exemplary embodiment of the present invention, the E1 protein may be an E1 protein of adenovirus type 5.
According to another exemplary embodiment of the present invention, the nucleic acid sequence encoding the E1A protein may be represented by SEQ ID NO: 1.
According to still another exemplary embodiment of the present invention, the nucleic acid sequence encoding the E1B protein may be represented by SEQ ID NO: 2.
According to one exemplary embodiment of the present invention, the cell line may be a human-derived cell line.
According to one exemplary embodiment of the present invention, the cell line may be an L132 cell line.
The cell line for producing an adenovirus according to the present invention is characterized in that the cell line may be safely used for therapeutic applications because the autoreplication capability of the adenovirus itself is lost, and an RCA is not produced.
Also, the present invention provides a packaging system for producing a recombinant adenovirus, which includes the cell line for producing an adenovirus having a limited autoreplication capability; and an adenoviral vector.
In addition, in order to prepare a cell line for producing an adenovirus having a limited autoreplication capability, the present invention provides a method of preparing a cell line for producing an adenovirus having a limited autoreplication capability, which includes:
(a) preparing each retrovirus vector comprising a nucleic acid sequences of adenoviral E1, E1A and E1B genes, respectively;
(b) transfecting the each retrovirus vector into a cell line for producing a retrovirus to produce retroviruses, respectively;
(c) infecting a cell line for producing an adenovirus with the retrovirus produced in (b), which includes the nucleic acid sequence of the E1 gene; and
(d) further infecting the cell line for producing an adenovirus of (c) with at least one of the retroviruses produced in (b), which includes the nucleic acid sequences of the E1A and E1B genes, to obtain the cell line for producing the adenovirus having a limited autoreplication capability.
According to an exemplary embodiment of the present invention, the E1A gene may be represented by SEQ ID NO: 1.
According to another exemplary embodiment of the present invention, the E1B gene may be represented by SEQ ID NO: 2.
According to still another exemplary embodiment of the present invention, the cell line for producing an adenovirus may be a human-derived cell line. As one preferred example, the cell line may be an L132 cell line.
Also, the present invention provides various uses of the adenovirus obtained from the cell line produced by the method.
According to one exemplary embodiment of the present invention, there is provided a packaging system for producing an adeno-associated virus (AAV), which includes:
an adenovirus obtained from the cell line produced by the method described above;
a cell line including a nucleic acid sequence of a rep gene of the AAV and a nucleic acid sequence of a cap gene of the AAV; and
an AAV vector.
According to another exemplary embodiment of the present invention, there is provided a gene therapeutic agent including the adenovirus obtained from the cell line produced by the method described above.
Further, the present invention provides various uses of the cell line described above.
According to one exemplary embodiment of the present invention, there is provided a cell line for producing an antibody, which further includes:
a nucleic acid sequence of an E1 gene; and
a nucleic acid sequence of at least one selected from nucleic acid sequences of E1A and E1B genes.
According to one exemplary embodiment of the present invention, there is provided a cell line for producing a vaccine, which further includes:
a nucleic acid sequence of an E1 gene; and
a nucleic acid sequence of at least one selected from nucleic acid sequences of E1A and E1B genes.
That is, the cell line of the present invention may be used to produce antibodies or vaccines.
The present invention relates to a cell line for producing an adenovirus having a limited autoreplication capability. Therefore, the cell line of the present invention can be used to provide a cell line for producing a recombinant adenovirus having a limited autoreplication capability, in which an RCA is not produced, and can also be used to provide a cell line having a superior ability to produce an adenovirus (hereinafter referred to as a ‘adenovirus-producing ability’) having a limited autoreplication capability, compared to the existing cell lines for producing an adenovirus having a limited autoreplication capability, in which an RCA is not produced.
The terms typically used in the present invention are defined as follows.
The term “E1 protein” used in the present invention refers to a portion of a viral envelope protein. In the present invention, an adenoviral E1 protein has an adenoviral autoreplication capability.
The terms “limited autoreplication capability” and “limited autoreplication” used in the present invention mean that cells have no viral proliferation ability. In the case of adenovirus, in the present invention, the autoreplication capability of adenovirus is lacking due to partial or complete deletion of the E1 gene.
The term “cell line” used in the present invention refers to a clone of cultured cells capable of carrying on a family line by continuously dividing and proliferating cells by means of cell culture, that is, a series of cell lineages whose genetic traits are preserved even when the cells are subcultured.
The term “promoter” used in the present invention refers to a regulatory sequence that is a nucleic acid sequence region in which transcription initiation and rate are regulated. Such a promoter may contain a genetic element which may bind to regulatory proteins and molecules such as RNA polymerases and other transcription factors to initiate specific transcription of a nucleic acid sequence. The terms “effectively aligned,” “effectively bound,” “under the control,” and “under the transcriptional control” mean that a promoter is aligned at a proper position and/or orientation of action with regard to the nucleic acid sequence regulating transcription initiation and expression of the sequence.
The term “operably linked” used in the present invention means that one nucleic acid fragment is bound to another nucleic acid fragment so that the function or expression of the one nucleic acid fragment are affected by the another nucleic acid fragment. Also, an expression control sequence refers to a DNA sequence that regulates expression of an operably linked nucleic acid sequence in certain host cells. Such a control sequence includes any operator sequence for controlling the transcription, a sequence encoding a proper mRNA ribosome binding site, and a sequence for controlling termination of the transcription and reading.
The term “vector” used in the present invention refers to a DNA that transfers a genetic material from one cell to another. To construct the vector, a process of linking a desired DNA to another DNA should be performed. In this case, the DNA thus formed is referred to as a recombinant DNA.
The term “recombination” used in the present invention refers to a process of linking a DNA fragment of any organism to another DNA molecule. In the present invention, the resulting construct may be used as a recombinant vector or a recombinant virus.
The term “homologous recombination” used in the present invention refers to a strand exchange between two homologous DNA chains.
The term “introduction” used in the present invention means that a foreign DNA is allowed to flow in cells by means of transfection or transduction. The transfection may be performed using various methods known in the related art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Also, the transduction is delivery of a gene into cells by means of infection using a virus or a viral vector particle.
The term “nucleic acid” used in the present invention refers to a polymeric material consisting of purine bases and pyrimidine bases/sugars/phosphate groups. In this case, the nucleic acid is a polymeric organic matter assembled from unit materials called nucleotides. A pentose sugar is a type of carbohydrate having 5 carbon atoms (hereinafter abbreviated as ‘sugar’). Such sugars are divided into riboses and deoxyriboses. There are five bases: adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
The term “packaging system for producing a virus” used in the present invention refers to a system for producing a virus. In the present invention, the packaging system may be used to produce an adenovirus or a retrovirus. The packaging system for producing a virus includes nucleic acid molecules and proteins necessary for virus production.
The term “treatment” refers to an approach for obtaining beneficial or desirable clinical results. For the purpose of the present invention, the beneficial or desirable clinical results are non-limiting, and encompass relieving symptoms, reducing a range of diseases, stabilizing (i.e., not worsening) a disease status, delaying progression of a disease or reducing a progression rate of the disease, ameliorating or temporarily relieving and (partially or fully) mitigating a disease status, and determining whether the disease status is detectable or not.
The term “treatment” refers to both therapeutic and prophylactic treatments or measures. The treatments include treatments required for disorders to be prevented, as well as disorders which have been already developed. The term “palliating a disease” refers to reducing a range of the disease status and/or undesirable clinical signs and/or delaying or lengthening a time course of progression of the clinical signs, compared to when a subject undergoes no treatment.
The term “approximately” indicates an amount, level, value, number, frequency, percent, dimension, size, quantity, weight or length changed by 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference amount, level, value, number, frequency, percent, dimension, size, quantity, weight or length.
Throughout this specification, it will be further understood that the terms “comprises,” “comprising,” “includes” and/or “including” specify the presence of stated steps or elements, and/or groups thereof, but do not preclude the presence or addition of one or more other steps or elements, and/or groups thereof, unless the context clearly indicates otherwise.
Hereinafter, the present invention will be described in detail.
The present invention provides a cell line for producing an adenovirus having a limited autoreplication capability, which expresses an adenoviral E1 protein; and further expresses at least one of E1A and E1B proteins.
Also, the present invention provides a method of preparing a cell line for producing an adenovirus having a limited autoreplication capability; and use of the cell line.
I. Cell Line
One aspect of the present invention is directed to a cell line having a virus-producing ability.
The term “virus-producing ability” refers to a state in which the virus-producing ability is increased, enhanced, or improved by artificially engineering (e.g., deleting) a certain factor (e.g., a gene, a protein, or the like) for the purpose of treating or preventing a disease in order to prevent a certain function (e.g., replication, transcription, translation, or the like) from being expressed.
The certain factor may be a gene associated with the self-replication of a virus.
The certain function may be a self-replication function of a virus.
The virus has a property of delivery of its own gene to another host in order to replicate and proliferate in the host. Such a virus may serve as a vector, which removes pathogenicity and delivers a treatable gene, using the property.
The cell line is a cell line having a virus-producing ability, which is derived from one or a combination of two or more selected from a retrovirus, an adenovirus, an adeno-associated virus (AAV), a lentivirus, a herpes simplex virus (HSV), and the like, but the present invention is not limited thereto.
The cell line may include normal cells, cancer cells, stem cells, immune cells, and the like, but the present invention is not limited thereto. Preferably, the cell line may be a normal cell line.
The cell line may include a human-derived cell line, a mouse-derived cell line, a pig-derived cell line, a monkey-derived cell line, a sheep-derived cell line, a plant-derived cell line, and the like, but the present invention is not limited thereto. Preferably, the cell line may be a human-derived cell line.
The cell line may include i) a first cell line including at least one envelope gene; and ii) a second cell line transfected with the product obtained from the first cell line. The second cell line includes two or more envelope genes. Preferably, the second cell line includes all the envelope genes and envelope gene subtypes.
The product obtained from the first cell line may be a virus expressing at least one envelope gene.
The envelope gene may include one or more selected from E1, E2, E3, and E4 genes, and the like, but the present invention is not limited thereto. More particularly, the envelope gene may be an E1 gene. Preferably, the envelope gene may be an adenoviral E1 gene.
Some of the adenoviral E1, E2 and E4 genes are essential for the viral growth, and the E3 gene is not essential for the viral growth but has information on proteins evading an immune system of host cells.
The envelope gene may also include envelope gene subtypes.
The envelope gene subtypes may include one or more selected from E1A, E1B, E2A, E2B, E3A, E3B, E4A, E4B, and the like, but the present invention is not limited thereto. More particularly, the envelope gene subtypes may be E1A and E1B. Preferably, the envelope gene subtypes may be adenoviral E1A and E1B.
The E1B gene includes a nucleotide sequence (E1B19K) encoding E1B 19K and a nucleotide sequence (E1B55K) encoding E1B 55K, and inhibits an apoptosis signaling pathway.
It should be understood that the envelope genes described hereinafter are interpreted to also include the envelope gene subtypes.
The first cell line may include one or more selected from the envelope genes E1, E2, E3, E4, E1A, E1B, E2A, E2B, E3A, E3B, E4A, E4B, and the like, or a combination thereof, but the present invention is not limited thereto.
According to one embodiment, for example, the first cell line may include one or more genes selected from the envelope genes E1, E1A, and E1B.
According to one embodiment, the first cell line may be a cell line independently including each of the envelope genes E1, E1A, and E1B.
According to one embodiment, the first cell line may include E1; and E1A or E1B.
According to one embodiment, the first cell line may include E1, E1A, and E1B.
The product obtained from the first cell line may be a product obtained in the form of a virus by inserting the envelope gene into a vector. In this case, a non-viral vector or a viral vector may be used as the vector. More particularly, the vector may be a viral vector, preferably a retroviral vector.
According to one embodiment, the first cell line may be a product obtained from the first cell line by inserting each of the E1, E1A and E1B genes into a retroviral vector, thereby obtaining retroviruses including the E1, E1A and E1B genes, respectively.
According to one embodiment, the first cell line may be a product obtained from the first cell line by inserting the E1 and E1A or/and E1B gene into retroviral vectors, thereby obtaining retroviruses including the E1 and E1A or/and E1B genes.
The retroviral vector may be a non-replicating recombinant retroviral vector having no autoreplication capability.
According to one embodiment, the retroviral vector may include a foreign gene as a gene delivery vector.
Examples of the foreign gene include any genes to be expressed in target cells, for example, nucleic acids encoding polypeptides (enzymes, growth factors, cytokines, receptors, structural proteins, and the like), antisense RNA, ribozymes, decoys, RNAs causing RNA interference, and the like, but the present invention is not particularly limited thereto.
Also, a promoter, an enhancer, a terminator or other transcription regulatory elements may be inserted into the nucleic acids in order to control the expression of the foreign gene as described above. Also, the retroviral vector may have a proper marker gene (for example, a drug-resistant gene, a gene encoding a fluorescence protein, a gene encoding an enzyme capable of functioning as a reporter such as β-galactosidase, luciferase, or the like) enabling the selection of the gene-introduced cells.
The drug-resistant gene may be an antibiotic-resistant gene. According to one example of the present invention, the drug-resistant gene may be a gene resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline, but the present invention is not limited thereto.
According to one exemplary embodiment, the promoter may include a murine leukemia virus (MLV) long terminal repeat (LTR) promoter, a cytomegalovirus (CMV) promoter, an adenovirus early promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, an HSV tk promoter, a Rous sarcoma virus (RSV) promoter, an elongation factor-1 alpha (EF1 alpha) promoter, a metallothionein promoter, a beta-actin promoter, a promoter of a human IL-2 gene, a promoter of a human interferon gene, a promoter of a human interleukin-4 gene, a promoter of a human lymphotoxin gene, a promoter of a human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, a human phosphoglycerate kinase (PGK) promoter, or a mouse phosphoglycerate kinase (PGK) promoter, but the present invention is not limited thereto.
According to one exemplary embodiment, the promoter may be an elongation factor-1 alpha (EF1α) promoter.
The recombinant retroviral vector (hereinafter also referred to as a recombinant retrovirus) used in the present invention may be a retroviral vector known in the art.
For example, an MFG vector, an α-SGC vector (pamphlet of International Publication No. 92/07943), retroviral vectors such as pBabe [Nucleic Acids Research, Vol. 18, pp. 3587-3596 (1990)], pLXIN (Clontech Laboratories, Inc.), pDON-AI(Takara-Bio Inc.), a lentiviral vector [a human immunodeficiency virus (HIV)-derived vector, a simian immunodeficiency virus (SIV)-derived vector, or the like], a pBApo-EF1αNeo DNA (TaKaRa Code 3243) or pBApo-EF1αPur DNA (TaKaRa Code 3244) vector, or a modified vector thereof may be used.
According to one exemplary embodiment of the present invention, the retroviral vector may be an MLV-derived vector from which gag, pol and env genes are removed.
According to one embodiment, a retroviral vector including each of nucleic acid sequences encoding adenoviral E1, E1A, E1B proteins may be constructed.
According to one embodiment, the retroviral vector may further include an elongation factor-1 alpha (EF1a) promoter.
According to one embodiment, the retroviral vector may further include an LTR sequence.
According to one embodiment, the retroviral vector may further include an IRES neo sequence.
The obtaining of the product in the form of a virus may include packaging genes of a vector when a virus-producing cell line is transfected with the vector together with a packaging plasmid.
The transfection of the virus-producing cell line with the vector may be performed using various methods known in the related art.
According to one exemplary embodiment, the vector may be imported into cells using methods such as microinjection; calcium phosphate precipitation; electroporation; liposome-mediated transfection; DEAE-dextran treatment; and gene bombardment, but the present invention is not limited thereto.
The packaging plasmid has no cis-acting sequence essential for most of reverse transcription and integration, and is designed to express only gag, pol, and env viral proteins.
According to one exemplary embodiment of the present invention, the retrovirus packaging cell line transfected with the retroviral vector includes various cells.
Packaging cell lines known in the art, for example, PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 or GP+envAm-12 (U.S. Pat. No. 5,278,056), Psi-Crip [Proc. Natl. Acad. Sci. USA., Vol. 85, pp. 6460-6464 (1988)], and the like may be used as the retrovirus packaging cell line as described above, but the present invention is not limited thereto.
According to one exemplary embodiment of the present invention, retrovirus-producing cells may be prepared by introducing a packaging plasmid (a retrovirus packaging kit; disclosed in Korean Patent No. 10-1362111; TAKARA-BIO Inc., and the like) loaded with genes necessary for production of retroviral particles into 293 cells or 293T cells.
According to one exemplary embodiment of the present invention, the retrovirus packaging cell line may be a human-derived cell line. According to a preferred exemplary embodiment of the present invention, the retrovirus packaging cell line may be an L132 cell line.
The first cell line may produce a virus expressing the envelope gene. Preferably, the first cell line may produce a retrovirus.
The retrovirus is characterized in that the retrovirus is effective for transfecting the adenovirus-producing cell line because the retrovirus may serve to stably insert an adenoviral E1 gene and also has a high titer.
According to the present invention, a cell line is transfected with the prepared recombinant retrovirus to obtain a cell line expressing the adenoviral E1 gene.
The second cell line may be a cell line infected with a virus produced by the first cell line. More particularly, the second cell line may be a cell line obtained by transfecting an adenovirus-producing cell line with the first cell line, followed by transfection with an adenoviral vector. In this case, the second cell line may include two or more envelope genes.
Hereinafter, types of envelope genes that may include the second cell line are described by way of example, but the present invention is not limited thereto.
According to one embodiment, when the first cell line independently includes each of E1, E1A, and E1B, the second cell line may be a cell line obtained by transfecting an adenovirus-producing cell line with two or more of the viruses obtained from the first cell line in a simultaneous or sequentially.
According to one embodiment, the second cell line may be a second cell line infected with the E1 product of the first cell line and the E1A product of the first cell line. Also, the second cell line may be a cell line infected with the E1 product of the first cell line and the E1B product of the first cell line. Also, the second cell line may be a cell line infected with the E1 product of the first cell line, the E1A product of the first cell line, and the E1B product of the first cell line.
According to one embodiment, the second cell line may be a second cell line which is first transfected with the E1 product of the first cell line, followed by infection with the E1A product of the first cell line. Also, the second cell line may be a second cell line which is first transfected with the E1 product of the first cell line, followed by infection with the E1B product of the first cell line. Also, the second cell line may be a cell line which is first transfected with the E1 product of the first cell line, followed by transfection with the E1A product of the first cell line and the E1B product of the first cell line.
That is, the second cell line may be a cell line which is transfected with the products of the first cell line in a desired order or combination. Preferably, the second cell line may be a second cell line which is first transfected with the E1 product of the first cell line, followed by infection with the E1A or E1B product of the first cell line.
According to one embodiment of the present invention, the second cell line is a cell line obtained by transfecting an adenovirus-producing cell line with an E1 retrovirus.
According to another embodiment of the present invention, the second cell line is a cell line obtained by first transfecting an adenovirus-producing cell line with an E1 retrovirus, followed by further transfection with an E1A retrovirus.
The transfection is as described above.
The second cell line may further include an adenoviral vector.
The adenoviral vector may further include an enhancer, a terminator or other transcription regulatory elements for controlling the expression of a foreign gene. According to another exemplary embodiment of the present invention, the adenoviral vector may further include a marker gene. The marker gene may be a drug-resistant gene, a gene encoding a fluorescence protein, a gene encoding an enzyme capable of functioning as a reporter such as β-galactosidase or luciferase, and the like, but the present invention is not limited thereto.
A cell line expressing an adenoviral E1 protein may be transfected with the adenoviral vector of the present invention. A method of introducing the adenoviral vector may be performed using various methods known in the related art.
According to one exemplary embodiment of the present invention, the vector may be imported into cells using methods such as microinjection; calcium phosphate precipitation; electroporation; liposome-mediated transfection; DEAE-dextran treatment; and gene bombardment, but the present invention is not limited thereto.
The adenoviral vector of the present invention may enable the production of an RNA transcript from the adenoviral vector, and the transcript may be packaged into an adenovirus, which is then excreted into a medium.
Therefore, the adenovirus having a limited autoreplication capability according to the present invention may be obtained by collecting and concentrating the medium.
According to one exemplary embodiment of the present invention, the adenovirus obtained by collection and concentration of the medium may further undergo a purification process for clinical applications, which may be carried out using methods known in the art.
According to one exemplary embodiment of the present invention, the purification of the adenovirus may be performed using methods such as differential centrifugation, density-gradient centrifugation, column chromatography, expanded chromatography, virus precipitation, contaminant modification, and cell component breakdown using an enzyme, but the present invention is not limited thereto.
The first cell line and the second cell line are cell lines having different virus-producing abilities. Particularly, the first cell line may be a cell line having a retrovirus-producing ability, and the second cell line may be a cell line having an adenovirus-producing ability.
The retrovirus is considered to be a vector most useful for stably inserting a therapeutic gene into an RNA virus. However, the retrovirus may be inserted into only dividing cells, and the therapeutic gene may not be imported into non-dividing cells. Also, the retrovirus has drawbacks in that a replication competent retrovirus (RCR) showing partial replicability may be generated, and the retrovirus may be inactivated by serum complements.
The adenovirus is in the form of double-stranded DNA, and thus shows a high expression level in both dividing and non-dividing cells. Also, a recombinant virus having good stability and a high titer may be produced. However, the adenoviruses may be isolated from various organisms spanning from algae to humans, and may be divided into six subgenes, depending on their structural, biochemical, and immunological characteristics. Ad2 and Ad5 have been widely used as the adenoviral vector used for gene therapy so far. However, when there is a replication competent adenovirus (RCA), a gene delivery vector should be used to minimize production of the RCA because the RCA may cause severe side effects in an immunodeficient patient.
The adenovirus type 5 has a replication origin essential for virus replication, and includes E1, E2, E3, and E4 genes, and genes encoding a viral capsid protein.
The envelope gene and the envelope gene subtypes are genes associated with the replication competent adenovirus (RCA).
The RCA refers to an adenovirus in which a viral vector included in the first cell line and the second cell line is self-replicating when the first cell line and the second cell line are applied as a delivery tool for gene therapy. In this case, when the RCA is produced, side effects may be caused in patients deficient in an immune response.
The cell line may be a cell line having a low RCA-producing probability and an improved adenovirus-producing ability.
According to some embodiments of the present invention, the result of RCA reactivity experiments on the cell lines having an adenovirus-producing ability, which are obtained by transfecting an adenovirus cell line with the E1 and E1A or/and E1B retroviruses, shows that the RCA reactivity of the adenovirus cell line may be confirmed by formation of an E1 band, as shown in
The adenovirus produced by the cell line for producing an adenovirus having a limited autoreplication capability according to the present invention may not have an E1 region therein, and thus may have no autoreplication capability, thereby reducing an immune response caused by the autoreplication capability of the adenovirus when the adenovirus is used as a gene therapeutic agent, and the like.
Also, the cell line for producing an adenovirus having a limited autoreplication capability according to the present invention has a characteristic of not producing the replication competent adenovirus (RCA) because the cell line of the present invention does not have the same sequence as the adenovirus-producing vector, thereby producing an adenovirus that can be used for gene therapy, and the like.
The cell line according to the present invention is a cell line for producing an adenovirus having a limited autoreplication capability, which has an enhanced adenovirus-producing ability.
The cell line may be a cell line producing the adenovirus. Preferably, the cell line may be a cell line producing the adenoviral E1 gene.
The cell line producing the E1 gene may be screened by measuring cell viability.
The cell line producing the adenoviral E1 gene may include various cells. According to one exemplary embodiment of the present invention, the cell line producing the adenoviral E1 gene may include carcinomic human alveolar basal epithelial cells (A549), human embryonic retinoblasts (HER), human cervical cancer cells (HeLa), Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK), mouse embryonic fibroblast cells (3T3), myeloma cells, rat pheochromocytoma cells (PC12), human amniocytes, human dermal fibroblasts (WS1), human lung fibroblasts (MRC5), human lung fibroblast cells (WI38), or human lung cells (L132), but the present invention is not limited thereto.
According to one specific embodiment of the present invention, the cell line may be a cell line having an adenovirus-producing ability, which is obtained by introducing an E1 gene into the L132 cell line.
According to another specific embodiment of the present invention, the cell line may be a cell line having an adenovirus-producing ability, which is obtained by introducing an E1 gene into the L132 cell line to improve stability, followed by introduction of an E1A or/and E1B gene.
Therefore, the cell line of the present invention may provide the adenoviral E1 gene with high stability to cross-complement an E1-deleted adenovirus, and thus may provide a cell line capable of producing an adenovirus with high efficiency.
II. Method of Producing Cell Line
Another aspect of the present invention is directed to a method of producing a cell line.
One exemplary embodiment of the method of producing a cell line may include a first step of obtaining a first cell line and second step of preparing a second cell line.
The method of producing a cell line is a method of transfecting a cell line with the product obtained from the first cell line to prepare the second cell line.
Hereinafter, the respective steps will be described.
The first step is to obtain a first cell line.
To obtain a first cell line including at least one envelope gene, the first step may include inserting the envelope gene; and producing an intermediate for producing a second cell line.
The inserting of the envelope gene includes inserting a gene sequence into an expression construct (vector) expressing the envelope gene. One or more selected from a lentiviral vector, an adenoviral vector, a retroviral vector, and the like may be used as the expression construct, but the present invention is not limited thereto. Preferably, the expression construct may be a retroviral vector.
The retroviral vector is as described above.
The insertion of the envelope gene into the expression construct will be described below with reference to examples, but the present invention is not limited thereto.
According to one exemplary embodiment, two or more genes selected from E1, E1A, and E1B may be inserted to be prepare: a retroviral vector including the E1 and E1A genes, a retroviral vector including the E1 and E1B genes, a retroviral vector including the E1A and E1B genes, and a retroviral vector including the E1, E1A and E1B genes. According to one exemplary embodiment, all three of the E1, E1A, E1B genes may be inserted to prepare a retroviral vector including the E1, E1A and E1B genes.
The order of insertion of the two or three genes may vary depending on a purpose.
Preferably, the E1, E1A, and E1B genes may be inserted to prepare an E1 retroviral vector, an E1A retroviral vector, and an E1B retroviral vector, respectively.
According to some embodiments of the present invention, the insertion of the E1 gene into the retroviral vector may be confirmed by determining whether the gene is inserted into the vector by means of PCR, as shown in
According to some embodiments of the present invention, the insertion of the E1A or E1B gene into the retroviral vector may be confirmed by determining whether the gene is inserted into the vector by means of PCR, as shown in
The producing of the intermediate includes introducing the vector into which the envelope gene is inserted into proper cells to obtain the intermediate in the form of a virus. More particularly, a retrovirus-producing packaging cell line is transfected with the envelope gene-inserted retroviral vector together with a packaging plasmid to harvest a retrovirus.
When the retrovirus-producing cell line is transfected with the retroviral vector together with the packaging plasmid, genes of the retroviral vector may be packaged to produce the retrovirus. The transfection method is as described above.
The retrovirus-producing cell line is transfected with the retroviral vector together with the packaging plasmid, and cultured under conditions enabling the retrovirus production. The transfected cells are cultured for a predetermined time until the transfected cells form colonies to screen clones of the cell line. The screening of the clones may be performed using all methods known in the related art, such as a method of culturing a cell line, into which the retrovirus is introduced, in a medium, adding a select agent (for example, an antibiotic) to the medium, and collecting and sorting the cells resistant to the select agent or screening the cells resistant to the select agent as clones.
According to one embodiment, the retrovirus may be produced by transducing cells with a retroviral vector and gag-pol- and env-expressing plasmids at the same time, and concentrating and purifying only a supernatant of the cells after a predetermined time.
According to some embodiments of the present invention, the retrovirus is characterized in that the retrovirus is effective for transfecting the adenovirus-producing cell line because the retrovirus may serve to stably insert the adenoviral E1 gene and also has a high titer.
According to one exemplary embodiment of the present invention, the retrovirus including a nucleic acid sequence encoding the adenoviral E1 protein is prepared to prepare a cell line expressing the E1 protein for producing an adenovirus having a limited autoreplication capability, which is used for gene therapy.
When the adenovirus-producing cell line is infected with the produced retrovirus, only a structure of the retroviral vector may be inserted into a chromosome of the cell line, thereby inducing expression of the adenoviral E1 protein which is encoded by a foreign gene.
The second step is to obtain a second cell line.
To obtain a second cell line including two or more envelope genes, the second step may include transfecting proper cells with the product of the first step to obtain the second cell line.
As described above, the second cell line is a cell line including two or more envelope genes. According to one exemplary embodiment, the second cell line is an adenovirus-producing cell line including one envelope gene and one envelope subtype gene.
The second step includes transfecting an adenovirus-producing cell with the retrovirus produced from the first cell line, followed by transfection with an adenoviral vector.
The transfection method is as described above.
According to one embodiment, the second cell line may be made by preparing a retrovirus including a nucleic acid sequence of one of E1, E1A, and E1B genes; obtaining each of the constructed retroviral vectors as the retrovirus; and transfecting a cell line for producing an adenovirus with the retrovirus.
According to one embodiment, the second cell line may be made by constructing retroviral vectors including nucleic acid sequences of each of E1, E1A and E1B genes; obtaining each of the constructed retroviral vectors as the retrovirus; and transfecting a cell line for producing an adenovirus with the E1 retrovirus, the E1A retrovirus, and the E1B retrovirus.
According to one embodiment, the second cell line may be made by constructing each of retroviral vectors including nucleic acid sequences of E1, E1A and E1B genes;
obtaining each of the constructed retroviral vectors as the retrovirus; and transfecting a cell line for producing an adenovirus with the E1 retrovirus, the E1A retrovirus, and the E1B retrovirus.
According to one embodiment, the second cell line may be made by constructing retroviral vectors including nucleic acid sequences of two of E1, E1A, and E1B genes; obtaining each of the constructed retroviral vectors as the retrovirus; transfecting a cell line for producing an adenovirus with the retrovirus; and further infecting the cell line for producing an adenovirus, which has been transfected with the retrovirus including the two nucleic acid sequences, with the retrovirus including the other one nucleic acid sequence.
According to one embodiment, the second cell line may be made by constructing retroviral vectors including nucleic acid sequences of each of E1, E1A and E1B genes; obtaining each of the constructed retroviral vectors as the retrovirus; transfecting a cell line for producing an adenovirus with the E1 retrovirus; and further infecting the cell line for producing an adenovirus, which has been transfected with the E1 retrovirus, with the E1A and/or E1B retrovirus.
According to one embodiment of the present invention, the adenovirus-producing efficiency of the cell line producing the adenoviral E1 gene may be confirmed by means of real-time PCR by measuring a titer of the cell line to determine how much the cell line produces the adenovirus, as shown in
According to another embodiment of the present invention, it can be seen that the adenovirus-producing cell line transfected with the E1 retrovirus followed by transfection with the E1A or/and E1B retrovirus has a higher amount of produced adenovirus, compared to the adenovirus-producing cell line transfected with the E1 retrovirus, as shown in
According to another embodiment of the present invention, it can be seen that the adenovirus-producing cell line transfected with the E1 retrovirus followed by transfection with the E1A or/and E1B retrovirus has no RCA reaction, as shown in
When the method of producing a cell line described above is used, a cell line capable of producing the adenovirus having a limited autoreplication capability may be prepared. In this case, the cell line capable of producing the adenovirus having a limited autoreplication capability may be a cell line derived from human cells, but the present invention is not limited thereto. Preferably, the cell line derived from human cells may be an L132 cell line.
Additionally, the adenovirus having a limited autoreplication capability according to the present invention may be obtained by collecting and concentrating the medium as described above.
According to one exemplary embodiment of the present invention, the adenovirus obtained by collection and concentration of the medium may further undergo a purification process for clinical applications, which may be carried out using methods known in the art.
According to one exemplary embodiment of the present invention, the purification of the adenovirus may be performed using methods such as differential centrifugation, density-gradient centrifugation, column chromatography, expanded chromatography, virus precipitation, contaminant modification, and cell component breakdown using an enzyme, but the present invention is not limited thereto.
The adenovirus produced from the cell line of the present invention has a limited autoreplication capability. The limited autoreplication capability of the adenovirus produced from the cell line of the present invention may be confirmed by amplifying an adenoviral genome present in a culture supernatant of the cell line of the present invention using a polymerase chain reaction (PCR) to analyze whether an E1 region exists in the adenoviral genome.
The adenovirus produced from the cell line for producing an adenovirus having a limited autoreplication capability according to the present invention may not have an E1 region, and thus may have no autoreplication capability, thereby reducing an immune response caused by the autoreplication capability of the adenovirus when the adenovirus is used as a gene therapeutic agent, and the like.
Also, because the cell line for producing an adenovirus having a limited autoreplication capability according to the present invention does not have the same sequence as the adenovirus-producing vector, the cell line is characterized in that no replication competent adenovirus (RCA) is produced, thereby producing the adenovirus capable of being used for gene therapy, and the like.
The cell line prepared by the method of the present invention is a cell line for producing an adenovirus having a limited autoreplication capability, which has an improved adenovirus-producing ability.
III. Use of Cell Line
Packaging System for Producing AAV
The adenovirus produced from the adenovirus-producing cell line of the present invention may be used to produce an adeno-associated virus.
The adeno-associated virus (AAV) of the present invention has an ability to effectively infect various cells (muscle, brain, lung, retina, liver, ear, heart, blood vessel, and the like) and is inserted into a chromosome of a target cell to stably express a therapeutic protein for a long period of time.
According to one exemplary embodiment of the present invention, the adenovirus obtained by the cell line for producing an adenovirus having a limited autoreplication capability; a cell line including a nucleic acid sequence encoding a rep protein of an adeno-associated virus (AAV) and a nucleic acid sequence encoding a cap protein of the AAV; and a packaging system for producing the AAV, which includes an AAV vector, may be provided.
According to one exemplary embodiment of the present invention, the nucleic acid sequence encoding the rep protein of the adeno-associated virus (AAV) and the nucleic acid sequence encoding the cap protein of the AAV may be introduced into the cell line using methods known in the art.
In this case, a method of introducing the nucleic acid sequence into the AAV-producing cell line may include methods such as calcium phosphate precipitation, lipofection, DEAE dextran treatment, polyethylene imine treatment, electroporation, and the like, but the present invention is not limited thereto.
Also, the introduction of the nucleic acid into the AAV-producing cell line may be performed using a vector or a plasmid, and the vector may include a retroviral vector, a lentiviral vector, an adenoviral vector, and the like, but the present invention is not limited thereto.
According to one exemplary embodiment of the present invention, the AAV vector may be constructed using methods known in the related art.
According to one exemplary embodiment of the present invention, the vector may be imported into the cells using methods such as microinjection; calcium phosphate precipitation; electroporation; liposome-mediated transfection; DEAE-dextran treatment; and gene bombardment, but the present invention is not limited thereto.
The AAV obtained by the aforementioned packaging system for producing AAV may be used for gene therapy because the AAV shows efficient infection ability and is stably inserted into a target cell.
Gene Therapeutic Agent
A gene therapeutic agent may be prepared using the adenoviruses produced by the prepared cell line for producing an adenovirus having a limited autoreplication capability and the packaging system for producing an adenovirus having a limited autoreplication capability.
The gene therapy of the present invention is a method of treating a congenital or acquired genetic disorder, which is difficult to treat using conventional methods, using a genetic engineering method.
According to one exemplary embodiment of the present invention, a therapeutic method of administering a genetic material such as DNA and RNA into the human body to express a therapeutic protein or inhibit expression of a certain protein in order to treat and prevent a chronic disease such as congenital or acquired genetic defects, viral diseases, cancers, or cardiovascular diseases may be provided as the gene therapy.
According to one exemplary embodiment of the present invention, the adenovirus may include a target gene necessary for gene therapy to deliver the target gene in vivo.
According to another exemplary embodiment of the present invention, the adenovirus may deliver the target gene into cells collected from a patient.
According to still another exemplary embodiment of the present invention, the adenovirus may include a target gene instead of the E1 gene.
According to one exemplary embodiment of the present invention, the adenovirus may include a target gene instead of the E1 gene.
When the adenovirus obtained from the cell line for producing an adenovirus having a limited autoreplication capability according to the present invention is used for gene therapy, the adenovirus has advantages in that the adenovirus may reduce an immune response due to the limited autoreplication capability, and has no risk of RCA production.
Antibody-Producing Cell Line
The cell line for producing an adenovirus having a limited autoreplication capability according to the present invention may be used to produce an antibody.
For production of a therapeutic antibody using the cell line, one important step is to screen a cell line having high productivity.
The adenoviral E1 gene of the present invention may be endowed with high productivity by means of immortalization, that is, persistent cell division.
Because the adenoviral E1A gene of the present invention is associated with the permanent cell division and the E1B gene has an ability to inhibit cell death (i.e., apoptosis), when a recombinant cell line is constructed by transfecting a cell line with an E1 gene to express the E1 gene, followed by further transfection with an E1A or E1B gene to express the E1A or E1B gene as in the present invention, it is possible to obtain a cell line having high productivity.
In the present invention, an antibody is a specific protein molecule directed against an antigenic site. For the purpose of the present invention, the antibody is produced by the cell line of the present invention, and is an antibody that specifically binds to an antigen. In this case, the term “antibody” includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.
According to one exemplary embodiment of the present invention, the polyclonal antibody using the cell line may be produced using methods widely used in the related art. In this case, the polyclonal antibody may be prepared from any animal hosts such as a goat, a rabbit, a sheep, a monkey, a horse, a pig, cattle, a dog, and the like, but the present invention is not limited thereto.
According to one exemplary embodiment of the present invention, the monoclonal antibody using the cell line may be prepared using a hybridoma method widely known in the related art (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or a phage antibody library technique (Clackson et al., Nature, 352: 624-628, 1991; Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).
According to one exemplary embodiment of the present invention, the antibody produced by the cell line may be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.
The produced antibody includes a complete antibody form having two full-length light chains and two full-length heavy chain as well as functional fragments of the antibody molecule. The functional fragments of the antibody molecule refers to fragments that retain at least an antigen-binding function, and include Fab, F(ab′), F(ab′) 2, Fv, and the like.
According to one exemplary embodiment of the present invention, a human cell line may be used as an antibody-producing cell line, but the present invention is not limited thereto.
According to one exemplary embodiment of the present invention, a CHO cell line may be used as an antibody- and protein-producing cell line.
Because the antibody-producing cell line of the present invention does not have a morphological difference in glycosylation, the antibody-producing cell line has a characteristic of very low immunogenicity. Therefore, the antibody produced by the cell line may be effectively used as a therapeutic agent.
Production of Vaccine
The adenovirus-producing cell line of the present invention may be used as a vaccine-producing cell line.
Like in the antibody-producing cell line, high productivity is also important in the vaccine-producing cell line.
Therefore, in the present invention, a recombinant cell line may be constructed by transfecting a cell line with an E1 gene to express the E1 gene, followed by further transfection with an E1A or E1B gene to express the E1A or E1B gene, thereby obtaining a cell line having high productivity.
According to one exemplary embodiment of the present invention, a chicken egg or canine cells may be used to produce an influenza vaccine.
The vaccine produced according to the present invention may be properly formulated with a pharmaceutically available carrier. Therefore, the vaccine may be used to prepare a medical or pharmaceutical composition, and may be formulated into a solution for parenteral administration or a freeze-dried powder. The powder may be reconstructed before use by adding a suitable diluent or other pharmaceutically available carriers thereto. A liquid formulation may be a buffer solution, an isotonic solution, or an aqueous solution. The powder may be sprayed in an anhydrous form. Examples of the suitable diluent generally include isotonic saline, a standard 5% dextrose solution in water, or a buffered sodium or ammonium acetate solution. The formulation is particularly suitable for parenteral administration, and may also be used for oral administration or may be contained in a metered dose inhaler or a sprayer. Excipients, for example, polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride, sodium citrate, and the like may be preferably added.
The vaccine produced by the cell line of the present invention may be prepared for oral administration. A pharmaceutically available solid or liquid carrier may be added to improve or stabilize a composition. The solid carrier includes starch, lactose, calcium sulfate dihydrate, white clay, magnesium stearate or stearic acid, talc, pectin, acacia, agar, or gelatin. The liquid carrier includes syrup, peanut oil, olive oil, saline, or water. Also, the carrier may include a delayed release material (for example, glyceryl monostearate or glyceryl distearate) alone or in combination with a wax. An amount of the solid carrier may vary, and may preferably be in a range of approximately 20 mg to approximately 1 g per unit dose. When the liquid carrier is used, the formulation may be in the form of a syrup, an elixir, an emulsion, or an aqueous or non-aqueous suspension.
The vaccine produced by the cell line of the present invention may be formulated into forms including a medically available drug or a biological agent.
Also, the vaccine produced by the cell line of the present invention may be administered together with other drugs or biological agents useful for treatment of a targeted disease or symptoms thereof.
When the cell line according to the present invention is used to produce a vaccine, the cell line has low immunogenicity, thereby producing a safe vaccine.
Hereinafter, the present invention will be described in further detail with reference to examples thereof. However, it should be understood that the following examples are given for the purpose of illustration of the present invention only, and are not intended to limit the scope of the present invention, as will be apparent to those skilled in the art.
1-1: Cloning of E1, E1A and E1B genes into retroviral vector PCR was performed using an E1 gene (2,962 bp) of adenovirus type 5 as a template and an E1A gene (998 bp) and an E1B gene (1,808 bp) as specific primers to obtain an E1A band of approximately 1 Kb and an E1B band of approximately 1.8 Kb.
A gene was cloned into a pGEM-T easy vector, and then sequenced to confirm DNA sequences of the E1A and E1B genes (see SEQ ID NO: 1 shown in
Referring
In the clones whose DNA sequences were identified in the various clones, each of the E1, E1A, E1B, pro-E1, pro-E1A, and pro-E1B genes was cloned into a retroviral vector by means of enzyme digestion.
1-2: Production of Retrovirus
A packaging cell line expressing a gag-pol protein was co-transfected with the retroviral vector into which the gene was inserted together with a plasmid expressing a surface protein (an envelope), and then cultured for 3 days to obtain a supernatant. The supernatant was concentrated and purified using a centrifuge to produce the constructed retrovirus.
1-3: Confirmation of Retrovirus Production
To measure a titer using real-time PCR, a standard solution was serially diluted from 109 to 105 and used in consideration of the concentration and size of the vector plasmid DNA. For evaluation of optimal multiplicity of infection (MOD, cells were transfected with a control virus expressing GFP at a density of 1, 10, and 100, and an expression level of GFP was observed using a fluorescence microscope. From these results, it can be seen that the retrovirus was produced.
1-4: Confirmation of Introduction of E1, E1A and E1B Genes
To verify whether the E1 gene of Example 1-1 was expressed, mRNA expression was confirmed using RT-PCR. Introduction of the E1 gene was checked by measuring the E1A gene in which the shapes of mRNA and gDNA were distinguished by PCR because introns were included in the E1 gene.
The E1 gene was inserted into a retroviral vector, and HT1080 cells were transfected with the retroviral vector using JetPEI (Polyplus, France). Then, two different sets of primers (E1A-1 and E1A-2) specific to the E1A were used to measure an expression level of mRNA.
PCR was performed using HT1080 cells (HT) as the negative control, HEK293 cells (293) as the positive control, and plasmid gE1 in the form of genomic DNA as the control to avoid contamination of the plasmid. The results are shown in
Referring to
A retrovirus-producing packaging cell line was transfected with each of the retroviral vectors into which the E1A and E1B genes of Example 1-1 were cloned, and then cultured to obtain the genes. DNA was extracted from a colony of the cultured cells using a mini-prep method, digested with a restriction enzyme, and then subjected to electrophoresis. The corresponding size was able to be measured to determine whether the E1A and E1B genes were introduced (
Referring to
At least ten normal cell lines were purchased from the American Type Culture Collection (ATCC, USA), and proliferation capacities of the normal cell lines were evaluated by an MTT assay or the like to select six normal cell lines having an excellent proliferation capacity. The six selected cell lines were primarily transfected with each of the two E1 gene-expressing retroviruses prepared in Example 1, and then treated with G418 (Sigma, USA) to select cells into which the E1 gene was inserted.
mRNAs were isolated from the selected cells, and expression levels of E1A, E1B19K, and E1B55K were compared to those of the HEK293 cells. A method of transfecting each of the cell lines with each of the retroviruses expressing the E1, E1A, and E1B genes and selecting the cells based on the expression levels of the E1, E1A, and E1B genes was repeated.
In this case, the expression levels of E1A, E1B19K, and E1B55K were compared by means of real-time PCR and western blotting to select the cell lines having an expression level of 80% or more.
L132 cells were infected with the retrovirus obtained by the method of Example 1, and then transfected with an adenoviral vector to prepare a cell line for producing an adenovirus having a limited autoreplication capability. A titer of the produced adenovirus was measured to check whether the adenovirus was produced in the established cell line.
The results of experiments when the L132 cells were infected only with the E1 retrovirus; and when the L132 cells were infected with the E1 retrovirus followed by further infection with the E1A retrovirus using the method of the present invention were compared. The results are shown in
Referring to
Also, it can be seen that an amount of produced adenovirus in the 11th cell line (#11) was 3.8 times larger when the cell line was further infected with the E1A retrovirus.
Therefore, from these results, it can be seen that the adenovirus-producing ability was able to be enhanced by additionally inserting the E1A gene using the retrovirus according to the present invention.
For RCA inspection, a total of six clones (one for each cell line) were selected from the cell lines, and then passed at least five times to produce the adenoviruses depending on the passages.
When the adenovirus-producing cell line had a cytopathic effect (CPE) after the cell line was infected with the adenoviral vector as described in Examples 1 to 3, the cells were harvested and sonicated to obtain a supernatant. Thereafter, the supernatant was concentrated with CsCl and purified using an ultracentrifuge to obtain the produced adenovirus.
PCR was performed using specific primers to determine whether or not the produced virus had the E1 gene. In this case, HEK293 cells were used as the positive control. From the results of inspection, it was confirmed that the adenovirus produced in the cell line for producing an adenovirus according to the present invention did not have the E1 gene.
5-1: Analysis of E1 and E1A Genes in GT541 Cell Line
The E1 and E1A genes were cloned into the retroviral vector as described in Example 1-1. When the genes were delivered through the retroviral vector, the gene (E1) inserted into the retroviral vector was inserted into the genomic DNA of the host cell. Therefore, to determine whether the E1 and E1A genes were inserted into the GT541 clone (referred to as a cell line in which E1 was introduced into L132 cells), cellular gDNA was extracted (using a Qiagene Tissue kit), and then subjected to PCR using the following corresponding primers (see Tables 1 and 2)
PCR was performed using L132 cells (L132) as the negative control, HEK293A cells (293A) as the positive control, and a cell line (named 1132-2E1-3E1A#6′) in which the E1 and E1A genes were inserted into L132 cells as the experimental group. The results are shown in
As a result, it can be seen that the E1 and E1A genes were introduced and expressed in the L132 cells.
5-2: RCA Test
To check whether an RCA reaction occurred in the cell line of Example 5-1, the cell line was infected with Ad-GFP, and the viruses were harvested. Thereafter, gDNA was extracted from the harvested viruses, and then subjected to a PCR method using the primers corresponding to the E1 gene.
From the corresponding experiment, it was confirmed using the primer sequences (241 bp) for verification of E1 RCA that the RCA reaction occurred (see Table 3)
PCR was performed using viral gDNA (named ‘293A’) extracted after 293A cells were infected with the virus, DNA of 293A cells extracted as the positive control (named ‘293A cells’), and a GT541 cell line (named 1132-2E1-3E1A#6′) as the experimental group. The results are shown in
Referring to
5-3: Measurement of Adenoviral Titer
To determine whether the adenovirus was produced in the cell line of Example 5-1, a titer of the produced adenovirus was measured.
The cell lines were cultured, and infected with Ad-GFP. Then, the cells were harvested, and then stored at −70° C. The next day, the cells were lysed using a sonicator, and centrifuged to obtain a supernatant. Then, gDNA was extracted from the supernatant, and then quantified using real-time PCR.
Referring to
Therefore, from the results, it can be seen that the adenovirus-producing cell line had a high adenovirus-producing ability when the cells were infected with the E1 retrovirus, followed by further infection with the E1A retrovirus, and also that the adenovirus-producing cell line was able to be used as the adenovirus cell line having high stability because the adenovirus-producing cell line had no RCA reaction.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0051829 | Apr 2017 | KR | national |
This application is a continuation application of International Application No. PCT/KR2018/004701 filed on Apr. 23, 2018, which claims priority to Korean Application No. 10-2017-0051829 filed on Apr. 21, 2017. The applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2018/004701 | Apr 2018 | US |
| Child | 16653171 | US |
