Novel foamy virus comprising three plasmids, and use thereof

Abstract

The present invention relates to a novel foamy virus comprising three plasmids, and use thereof. In order to prepare a novel foamy virus vector of the present invention, a wild-type foamy virus genome has been used to produce three plasmids containing: an expression cassette of a 3'UTR deletion-CAS (Gag / Pol deletion)-internal promoter–target gene-PRE structure; a Gag / Pol expression cassette; and an Env expression cassette, and a foamy virus vector has been produced through transfection using the produced plasmids. In addition, it has been identified that gene delivery efficiency increases using P2A sequence and codon optimization in the Gag / Pol plasmid.

Description

Novel foamivirus containing three plasmids and its use

[0001] The present invention relates to a novel foamy virus comprising three plasmids and its use.

[0002] With the advancement of science and technology, recently developed treatment methods for diseases are generally divided into three areas: chemotherapy, which uses chemicals to control disease-causing or body-deficient substances; cellular therapy, which involves injecting or transplanting therapeutic cells; and gene therapy, which introduces therapeutic genes or corrects defective genes. The concept of gene therapy was established in 1972 and first attempted in 1989. Research has steadily advanced, and in 2017, the first treatment method was approved by the FDA for a blind patient with RPE65 gene deficiency. Over 3,000 treatments have been attempted over the past 30 years, with more than half of these in Phase 1 clinical trials. Gene therapy is primarily attempted using vector DNA based on well-known viruses, such as retroviruses or adenoviruses, which have strong infectivity and transmissibility.

[0003] Although the virus-based vectors currently being studied are said to be inactivated, the viruses used cause diseases such as cancer in the human body, leaving the possibility of producing persistently infectious viruses when introduced into the body. This poses long-term safety concerns. Furthermore, unintended gene introduction can occur. While numerous gene therapies using existing vectors have been attempted, very few have actually received FDA approval. Furthermore, most vectors currently being tested are limited to infecting specific cells, lacking broad infectivity, which is a limitation of the technology. To address these issues, foamyviruses, a retrovirus known to be harmless to humans, are attracting attention as candidates for new-generation vectors.

[0004] Accordingly, the present inventors completed the present invention by producing a novel foamy virus vector using a 3-plasmid system.

[0005] The object of the present invention is to provide (1) a sequence-deleted Gag gene and a sequence-deleted Pol gene;

[0006] internal promoter (IPro);

[0007] target gene; and

[0008] A first plasmid for delivery of a target gene, comprising a structure of a PRE (Posttranscriptional Regulatory Element) for enhancing expression of the target gene;

[0009] (2) a second plasmid comprising a Gag gene operably linked to a universal promoter; and a Pol gene; and

[0010] (3) A kit for producing a foamy virus is provided, comprising a third plasmid comprising an Env gene operably linked to a universal promoter.

[0011] Another object of the present invention is to provide a step of culturing cells;

[0012] A step of transfecting the cultured cells with a plasmid containing an expression cassette of SEQ ID NO: 3; a plasmid containing an expression cassette of SEQ ID NO: 4 or SEQ ID NO: 6; and a plasmid containing an expression cassette of SEQ ID NO: 5; and

[0013] A method for producing a foamy virus is provided, comprising the step of obtaining a virus from the transduced cell.

[0014] Another object of the present invention is to provide a foamy virus vector manufactured by the above method.

[0015] Another object of the present invention is to provide a method for introducing a target gene using a foamy virus, comprising the step of infecting a target cell with the foamy virus vector.

[0016] To achieve the above object, the present invention provides (1) a sequence-deleted Gag gene and a sequence-deleted Pol gene;

[0017] internal promoter (IPro);

[0018] target gene; and

[0019] A first plasmid for delivery of a target gene, comprising a structure of a PRE (Posttranscriptional Regulatory Element) for enhancing expression of the target gene;

[0020] (2) a second plasmid comprising a Gag gene operably linked to a universal promoter; and a Pol gene; and

[0021] (3) A kit for producing a foamy virus is provided, comprising a third plasmid comprising an Env gene operably linked to a universal promoter.

[0022] In addition, the present invention comprises a step of culturing cells;

[0023] A step of transfecting the cultured cells with a plasmid containing an expression cassette of SEQ ID NO: 3; a plasmid containing an expression cassette of SEQ ID NO: 4 or SEQ ID NO: 6; and a plasmid containing an expression cassette of SEQ ID NO: 5; and

[0024] A method for producing a foamy virus is provided, comprising the step of obtaining a virus from the transduced cell.

[0025] In addition, the present invention provides a foamy virus vector prepared by the above method.

[0026] In addition, the present invention provides a method for introducing a target gene using a foamy virus, including a step of infecting a target cell with the foamy virus vector.

[0027] The present invention provides a novel formivirus vector, comprising three plasmids comprising an expression cassette having a 3'UTR deletion-CAS (Gag / Pol deletion)-internal promoter-target gene-PRE structure from a wild-type formivirus genome; a Gag / Pol expression cassette and an Env expression cassette; and a formivirus vector was produced by transfection using the produced plasmids. In addition, since the gene transfer ability is increased by optimizing the P2A sequence and codons in the Gag / Pol plasmid, it can be usefully utilized in related industries.

[0028] Figure 1 is a diagram showing a plasmid map encoding the foamy virus vector of the present invention.

[0029] Figure 2 is a schematic diagram of a plasmid in a 3-plasmid system for producing a foamy virus vector of the present invention.

[0030] Figure 3 is a schematic diagram of a process for producing the foamy virus vector of the present invention.

[0031] Figure 4 is a schematic diagram of the pCMV-Gag-P2A-Pol plasmid optimized for producing the foamy virus vector of the present invention.

[0032] Figure 5 is a diagram comparing the transmissibility of a foamy virus vector produced using the 3-plasmid system of the present invention with that of a foamy virus vector produced using the 2-plasmid system.

[0033] A: Gag protein size comparison

[0034] B: Confirmation of fluorescence expression according to gene transferability

[0035] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0036] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0037] The present invention relates to (1) a sequence-deleted Gag gene and a sequence-deleted Pol gene;

[0038] internal promoter (IPro);

[0039] target gene; and

[0040] A first plasmid for delivery of a target gene, comprising a structure of a PRE (Posttranscriptional Regulatory Element) for enhancing expression of the target gene;

[0041] (2) a second plasmid comprising a Gag gene operably linked to a universal promoter; and a Pol gene; and

[0042] (3) A kit for producing a foamy virus is provided, comprising a third plasmid comprising an Env gene operably linked to a universal promoter.

[0043] In the present invention, a plasmid or vector refers to a genetic construct containing a base sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence for regulating the termination of transcription and translation. The vector of the present invention is not particularly limited as long as it can replicate in a cell, and any vector known in the art may be used, for example, a plasmid, a cosmid, a phage particle, or a viral vector. When the vector is operably linked to a gene encoding a target polypeptide to be expressed, it can be used as an expression vector of the target polypeptide capable of expressing the target polypeptide with high efficiency in a suitable host cell, and the recombinant vector can be expressed in the host cell. The host cell may preferably be a eukaryotic cell, and depending on the type of host cell, expression control sequences such as promoters, terminators, enhancers, sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose.

[0044] In the present invention, the foreign gene that can be inserted can be inserted into a cloning site.

[0045] Variants of each base sequence described herein are included within the scope of the present invention. Specifically, it refers to a sequence that has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with each base sequence, and exhibits substantially the same physiological activity as the base sequence of the present invention. The "% of sequence homology" for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences.

[0046] According to one embodiment of the present invention, the Gag gene with the deleted sequence may be one in which the 2090th to 3817th base sequence of the wild type foamy virus sequence of SEQ ID NO: 1 is deleted.

[0047] According to one embodiment of the present invention, the Pol gene with the deleted sequence may be one in which the base sequence from position 3765 to position 5271 of the wild type foamy virus sequence of SEQ ID NO: 1 is deleted.

[0048] The "Gag gene" of the present invention is a group-specific antigen gene, which encodes the core protein of the virus and encodes the protein that constitutes the inner shell of the virus.

[0049] The "Pol gene" of the present invention is a gene of DNA polymerase, encodes a reverse transcriptase, and encodes an enzyme that reverse transcribes viral RNA into double-stranded DNA, and encodes an integrase, which is an enzyme that inserts viral DNA into a host gene.

[0050] According to one embodiment of the present invention, the first plasmid may include an expression cassette having a structure of sequence-deleted 3'UTR - sequence-deleted Gag gene - sequence-deleted Pol gene; and internal promoter - target gene - PRE - sequence-deleted 3'UTR, and the expression cassette may include a base sequence of SEQ ID NO: 2 or a base sequence of SEQ ID NO: 3.

[0051] According to one embodiment of the present invention, the 3'UTR with the sequence deleted may be a sequence in which the 11,974th to 12,622nd base sequence of the wild type foamy virus of sequence number 1 (GenBank Acc. No. Y07725) is deleted.

[0052] According to one embodiment of the present invention, the second plasmid may include an expression cassette of a codon-optimized Gag gene encoding a core protein and a codon-optimized Pol gene encoding a reverse transcriptase, and the expression cassette may include a base sequence of SEQ ID NO: 4.

[0053] According to one embodiment of the present invention, the second plasmid may include a codon-optimized expression cassette of the Gag gene - 2A peptide P2A-Pol gene structure, and the expression cassette may include a base sequence of SEQ ID NO: 6.

[0054] According to one embodiment of the present invention, the third plasmid may include a codon-optimized Env expression cassette encoding an envelope protein, and the expression cassette may include a base sequence of SEQ ID NO: 5.

[0055] The "Env gene" of the present invention encodes a protein that forms the viral envelope. Expression of the Env gene enables the virus to target and attach to specific cell types and penetrate the cell membrane.

[0056] In addition, the present invention comprises a step of culturing cells;

[0057] A step of transfecting the cultured cells with a plasmid containing an expression cassette of SEQ ID NO: 3; a plasmid containing an expression cassette of SEQ ID NO: 4 or SEQ ID NO: 6; and a plasmid containing an expression cassette of SEQ ID NO: 5; and

[0058] A method for producing a foamy virus is provided, comprising the step of obtaining a virus from the transduced cell.

[0059] In addition, the present invention provides a foamy virus vector prepared by the above method.

[0060] In addition, the present invention provides a method for introducing a target gene using a foamy virus, including a step of infecting a target cell with the foamy virus vector.

[0061] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0062] To produce a novel foamivirus vector of the present invention, a foamivirus vector was produced using a 3-plasmid system. A CMV promoter was inserted in front of the 5' end of the viral gene (SEQ ID NO: 1). Then, for plasmid replication, a bacterial origin (ori) and an origin for animal cells (SV40ori) were inserted. Then, for plasmid screening, an antibiotic resistance gene, ampicillin R or Kanamycin R, was inserted, and a portion other than the CAS (cis-acting sequence) required for virus packaging was removed. Then, to improve safety, the 3'UTR, which is the self-promoter, was removed, the foamivirus Env gene region was removed, and two BsmBI sites were inserted as cloning sites for insertion of external genes, thereby producing the pcHFV-CAS-Transgene plasmid (SEQ ID NO: 2 or SEQ ID NO: 3). In addition, the CAS portions Gag and Pol were linked to the universal promoter pCMV to construct the pcGag / pol plasmid (SEQ ID NO: 4), and in order to structurally include the Env gene region removed in the plasmid construction into the foamy virus vector, EnV was linked to the universal promoter pCMV to construct the pcEnv plasmid (SEQ ID NO: 5).

[0063] To produce a non-integrating foamy virus vector, the gene insertion function was eliminated through N129V and D186A mutations of Pol (integrase).

[0064] In addition, in order to produce a cassette of the inserted transgene, a sequence overlapping with the restriction enzyme site BsmBI at both ends was inserted, and an internal promoter was inserted in front of the 5' end of the transgene (target gene). After that, a PRE (Posttranscriptional Regulatory Element) was inserted behind the 3' end of the transgene to improve protein expression, and the transgene was produced by codon optimization (SEQ ID NO: 3). The plasmid map produced in the present invention is shown in Fig. 1, and the three-plasmid system is shown in Fig. 2.

[0065] <Comparative Example 1> Formyvirus vector produced using 2 plasmid systems

[0066] In order to confirm the gene transfer ability of the foamivirus vector using the 3 plasmid system of the present invention, the 2 plasmid system was used to produce the foamivirus vector, and two plasmids were produced. The CMV promoter was inserted in front of the 5' end of the viral gene (SEQ ID NO: 1). Then, for plasmid replication, the bacterial origin (ori) and the animal cell origin (SV40ori) were inserted. Then, for plasmid screening, the antibiotic resistance gene, ampicillin R, was inserted, and the 3'UTR, which is the self-promoter, was removed to improve safety. Then, the foamivirus Env gene region was removed, and two BsmBI sites were inserted as cloning sites for insertion of external genes, thereby producing the pcHFV-GP plasmid.

[0067] Additionally, a pcEnv plasmid was constructed to structurally include the Env gene region removed in the above plasmid construction into the foamy virus vector.

[0068] To produce a non-integrating foamy virus vector, the gene insertion function was eliminated through N129V and D186A mutations of Pol (integrase).

[0069] In addition, in order to construct a cassette of the inserted transgene, a sequence overlapping with the restriction enzyme site BsmBI was inserted at both ends, and an internal promoter was inserted in front of the 5' end of the transgene (target gene, EGFP). After that, a PRE (PosttranscriptionalRegulatoryElement) was inserted behind the 3' end of the transgene to improve protein expression, thereby constructing the pcHFV-GP-EGFP plasmid.

[0070] <Example 2> Production of a novel formic virus vector

[0071] To produce the novel foamy virus vector of the present invention, the 293T cell line was used. Specifically, the 293T cell line was seeded at an appropriate cell density in a cell culture dish and cultured until cell confluency reached 70%. The cells were then co-transfected with the pcHFV-CAS-Transgene plasmid, the pcGag / pol plasmid, and the pcEnv plasmid, and cultured for 2 to 3 days. After the culture was completed, the produced virus was harvested, filtered, stored at -80°C, and concentrated as needed. The produced virus was quantified using qPCR. The specific production process of the foamy virus vector is shown in Figure 3.

[0072] <Example 3> Optimization of a novel foamy virus vector and verification of its transduction ability.

[0073] <3-1> Plasmid optimization and Gag protein size analysis

[0074] In order to optimize the novel foamy virus vector of the present invention, the structure of the plasmid was changed. Specifically, in the pcGag / pol plasmid of the present invention (SEQ ID NO: 4), the P2A sequence of the 2A peptide system was inserted between the Gag gene and the Pol gene to express Gag and Pol at the same ratio, and pCMV-Gag-P2A-Pol (SEQ ID NO: 6), which is optimized for expression in human cells, was constructed through human codon optimization. The constructed pCMV-Gag-P2A-Pol plasmid is schematically illustrated in Fig. 4. Thereafter, in order to confirm whether the insertion of the P2A sequence increases the size of the Gag protein, the size of the Gag protein expressed using pcHFV-GP-EGFP in the 2-plasmid system constructed in Comparative Example 1 and the pCMV-Gag-P2A-Pol plasmid of the present invention was analyzed by Western blotting.

[0075] As a result, as shown in Fig. 5A, it was confirmed that the size of the protein expressed in the P2A and codon-optimized pCMV-Gag-P2A-Pol plasmids among the 3-plasmid systems of the present invention was increased compared to the protein expressed in the 2-plasmid system, thereby confirming that the gene transfer ability can be increased by increasing the size of the Gag core protein.

[0076] <3-2> Confirmation of increased gene transfer ability by pCMV-Gag-P2A-Pol plasmid

[0077] It was confirmed whether the novel foamivirus vector produced by the 3-plasmid system of the present invention has increased gene transfer ability compared to the foamivirus vector produced by the 2-plasmid system. Specifically, while producing the foamivirus vector in Example 2, the pcGag / pol plasmid (SEQ ID NO: 4) was replaced with pcCMV-Gag-P2A-Pol (SEQ ID NO: 6), and EGFP was used as the transfer gene (pcHFV-CAS-EGFP). The foamivirus vector of the 2-plasmid system of Comparative Example 1 was also produced using the 293T cell line using the same method (pcGFV-GP-EGFP). Afterwards, each produced foamivirus vector was infected into HT1080 cells, which are target cells, and cultured for 2 days, and the expression of each fluorescence was confirmed.

[0078] As a result, as shown in Fig. 5B, it was confirmed that the fluorescence expression of pcHFV-CAS-EGFP produced by the 3-plasmid system of the present invention was increased compared to pcGFV-GP-EGFP produced by the 2-plasmid system, thereby confirming that the gene transfer ability of the foamy virus vector produced by the 3-plasmid system of the present invention was increased.

[0079] Therefore, the present invention provides a novel formivirus vector, comprising three plasmids comprising an expression cassette of the 3'UTR deletion-CAS (Gag / Pol deletion)-internal promoter-destination gene-PRE structure from the wild-type formivirus genome; a Gag / Pol expression cassette and an Env expression cassette; and a formivirus vector was produced by transfection using the produced plasmids. In addition, it was confirmed that gene transfer ability was increased by optimizing the P2A sequence and codon in the Gag / Pol plasmid.

Claims

1. (1) Gag gene with sequence deletion and Pol gene with sequence deletion; internal promoter (IPro); target gene; and A first plasmid for delivery of a target gene, comprising a structure of a PRE (Posttranscriptional Regulatory Element) for enhancing expression of the target gene; (2) a second plasmid comprising a Gag gene operably linked to a universal promoter; and a Pol gene; and (3) A kit for producing a foamy virus comprising a third plasmid comprising an Env gene operably linked to a universal promoter.

2. In paragraph 1, The above sequence-deleted Gag gene is a kit in which the base sequence from position 2090 to position 3817 of the wild-type formivirus sequence of sequence number 1 is deleted.

3. In paragraph 1, The above sequence-deleted Pol gene is a kit in which the base sequence from position 3765 to position 5271 of the wild-type foamy virus sequence of sequence number 1 is deleted.

4. In paragraph 1, A kit, wherein the first plasmid comprises an expression cassette of a sequence-deleted 3'UTR-sequence-deleted Gag gene-sequence-deleted Pol gene; and an internal promoter-target gene-PRE-sequence-deleted 3'UTR structure.

5. In paragraph 4, A kit wherein the above expression cassette comprises the base sequence of sequence number 2 or sequence number 3.

6. In paragraph 4, The above sequenced deleted 3`UTR is a kit in which the base sequence from position 11,664 to position 12,878 of the wild type foamy virus sequence of sequence number 1 is deleted.

7. In paragraph 1, A kit wherein the second plasmid comprises an expression cassette of a codon-optimized Gag gene encoding a core protein and a codon-optimized Pol gene encoding a reverse transcriptase.

8. In paragraph 7, A kit wherein the above expression cassette comprises the base sequence of sequence number 4.

9. In paragraph 1, A kit wherein the second plasmid comprises a codon-optimized expression cassette of the Gag gene - 2A peptide P2A-Pol gene structure.

10. In paragraph 9, A kit wherein the above expression cassette comprises the base sequence of SEQ ID NO:

6.

11. In paragraph 1, A kit wherein the third plasmid comprises a codon-optimized Env expression cassette encoding an envelope protein.

12. In paragraph 11, A kit wherein the above expression cassette comprises the base sequence of SEQ ID NO:

5.

13. Step of culturing cells; A step of transfecting the cultured cells with a plasmid containing an expression cassette of SEQ ID NO: 3; a plasmid containing an expression cassette of SEQ ID NO: 4 or SEQ ID NO: 6; and a plasmid containing an expression cassette of SEQ ID NO: 5; and A method for producing a foamy virus, comprising the step of obtaining a virus from the transduced cell.

14. A foamy virus vector manufactured by the method of Article 13.

15. A method for introducing a target gene using a foamy virus, comprising the step of infecting a target cell with the foamy virus vector of Article 14.

16. In paragraph 15, A method wherein the target gene is a single-gene disease-related gene or a reporter gene.

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