Novel foamy virus comprising two plasmids and use thereof

Abstract

The present invention relates to a novel foamy virus comprising two plasmids and a use thereof. To produce a novel foamy virus vector in the present invention, two plasmids comprising an expression cassette having the structure of 3' UTR deletion-Gag / Pol-internal promoter-target gene-PRE and the an Env expression cassette were constructed from the wild-type foamy virus genome, and foamy virus vector was prepared by transfection using the constructed plasmids. In addition, it was confirmed that the produced virus delivers a target gene into cells and enables stable expression of the delivered gene.

Description

Novel foamivirus containing two plasmids and its use

[0001] The present invention relates to a novel foamy virus comprising two 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 well-known virus-based vector DNA, 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 two-plasmid system.

[0005] The object of the present invention is to provide a method for producing a nucleic acid molecule comprising: (1) an internal promoter (IPro);

[0006] target gene; and

[0007] 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;

[0008] (2) A kit for producing a foamy virus is provided, comprising a second plasmid comprising an Env gene operably linked to a universal promoter.

[0009] Another object of the present invention is to provide a step of culturing cells;

[0010] A step of transfecting the cultured cells with a plasmid containing an expression cassette of sequence number 3 and a plasmid containing an expression cassette of sequence number 4; and

[0011] A method for producing a foamy virus is provided, comprising the step of obtaining a virus from the transduced cell.

[0012] Another object of the present invention is to provide a foamy virus vector manufactured by the above method.

[0013] 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.

[0014] To achieve the above purpose, the present invention comprises: (1) an internal promoter (IPro);

[0015] target gene; and

[0016] 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;

[0017] (2) A kit for producing a foamy virus is provided, comprising a second plasmid comprising an Env gene operably linked to a universal promoter.

[0018] In addition, the present invention comprises a step of culturing cells;

[0019] A step of transfecting the cultured cells with a plasmid containing an expression cassette of sequence number 3 and a plasmid containing an expression cassette of sequence number 4; and

[0020] A method for producing a foamy virus is provided, comprising the step of obtaining a virus from the transduced cell.

[0021] In addition, the present invention provides a foamy virus vector prepared by the above method.

[0022] 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.

[0023] The present invention provides a novel formivirus vector. Two plasmids were constructed from the wild-type formivirus genome, each containing an expression cassette of the 3'UTR deletion-Gag / Pol-internal promoter-target gene-PRE structure and an Env expression cassette. The constructed plasmids were then used for transfection to construct a formivirus vector. In addition, it was confirmed that the constructed virus could deliver the transfer gene into cells, and since the transfer gene could be stably expressed, it can be usefully utilized in related industries.

[0024] Figure 1 is a diagram showing a plasmid map encoding the foamy virus vector of the present invention.

[0025] Figure 2 is a schematic diagram of a plasmid in a two-plasmid system for producing a foamy virus vector of the present invention.

[0026] Figure 3 is a schematic diagram of a process for producing the foamy virus vector of the present invention.

[0027] Figure 4 is a diagram confirming the gene transfer ability of the foamy virus vector of the present invention using the GFP gene.

[0028] A: Confirmation of fluorescence expression according to 2-plasmid concentration

[0029] B: Quantification of fluorescence expression according to 2-plasmid concentration

[0030] C: Confirmation of fluorescence expression following long-term culture of gene-transferred cells

[0031] 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.

[0032] 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.

[0033] The present invention comprises (1) an internal promoter (IPro);

[0034] target gene; and

[0035] 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;

[0036] (2) A kit for producing a foamy virus is provided, comprising a second plasmid comprising an Env gene operably linked to a universal promoter.

[0037] 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.

[0038] In the present invention, the foreign gene that can be inserted can be inserted into a cloning site.

[0039] 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.

[0040] According to one embodiment of the present invention, the first plasmid may include a Gag gene encoding a sequence-deleted 3'UTR-core protein - a Pol gene encoding a reverse transcriptase; and an expression cassette having an internal promoter - target gene - PRE - sequence-deleted 3'UTR structure, and the expression cassette may include a base sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0041] 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.

[0042] The "Pol gene" of the present invention is a gene of DNA polymerase, which encodes a reverse transcriptase enzyme and encodes an enzyme that reverse transcribes viral RNA into double-stranded DNA.

[0043] 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.

[0044] According to one embodiment of the present invention, the second 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: 4.

[0045] 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.

[0046] In addition, the present invention comprises a step of culturing cells;

[0047] A step of transfecting the cultured cells with a plasmid containing an expression cassette of sequence number 3 and a plasmid containing an expression cassette of sequence number 4; and

[0048] A method for producing a foamy virus is provided, comprising the step of obtaining a virus from the transduced cell.

[0049] According to one embodiment of the present invention, the plasmid of sequence number 3 and the plasmid of sequence number 4 may be introduced by mixing at a ratio of 10 to 50: 0.5 to 1.5, and preferably, may be introduced by mixing at a ratio of 35 to 45: 1, but is not limited thereto.

[0050] In addition, the present invention provides a foamy virus vector prepared by the above method.

[0051] 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.

[0052] 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.

[0053] <Example 1> Two-plasmid system for the production of a novel formic virus vector

[0054] To construct a novel foamivirus vector of the present invention, a foamivirus vector was constructed using a two-plasmid method. 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 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 constructing the pcHFV-GP plasmid (SEQ ID NO: 2).

[0055] In addition, in order to structurally include the Env gene region removed in the above plasmid construction into the foamy virus vector, a pcEnv plasmid (SEQ ID NO: 4) was constructed by linking Env to the pCMV promoter, which is a universal promoter.

[0056] To produce a non-integrating foamy virus vector, the gene insertion function was eliminated through N129V and D186A mutations of Pol (integrase).

[0057] 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 codon optimized to produce the pcHFV-CAS-pEF-1a-EGFP-WPRE plasmid (SEQ ID NO: 3). The plasmid map produced in the present invention is shown in Fig. 1, and the two plasmid systems are shown in Fig. 2.

[0058] <Example 2> Production of a novel formic virus vector

[0059] 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 pcHFV-GP-Transgene plasmid and pcEnv were co-transfected, and cultured for 2 to 3 days. After completion of the culture, 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.

[0060] <Example 3> Transduction using a novel foamy virus vector

[0061] Gene transfer of the novel foamy virus vector of the present invention was confirmed. Specifically, the GFP gene was transduced as a transgene into target cells, HT1080, using the foamy virus vector produced in Example 2, and cultured for 2 days. In addition, while producing the foamy virus in Example 2, the ratio of the pcHFV-GP-EGFP plasmid and the helper Env plasmid (pcEnv) was adjusted to compare the gene transfer ability. Thereafter, the expression of GFP in the cells was confirmed using a fluorescence microscope. In addition, the transduced cells were cultured for an additional 4 weeks to confirm whether the delivered gene was expressed long-term and maintained in stability.

[0062] As a result, as shown in Figures 4A and 4B, it was confirmed that fluorescence expression significantly increased when pcHFV-GP-EGFP and helper Env were mixed at a ratio of 40:1. In addition, it was confirmed that the transduced transgene expressed fluorescence even after 4 weeks of cell culture, confirming that the foamy virus vector of the present invention can stably express the transgene for a long period of time.

[0063] Therefore, in order to produce a novel foamivirus vector, the present invention constructed two plasmids from the wild-type foamivirus genome, each containing an expression cassette of the 3'UTR deletion-Gag / Pol-internal promoter-destination gene-PRE structure and an Env expression cassette, and constructed a foamivirus vector by transfection using the constructed plasmids. In addition, it was confirmed that the constructed virus could deliver the transfer gene into cells, and it was confirmed that the transfer gene could be stably expressed.

Claims

1. (1) 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 kit for producing a foamy virus comprising a second plasmid comprising an Env gene operably linked to a universal promoter.

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

3. In paragraph 2, A kit wherein the above expression cassette comprises a base sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

4. In paragraph 2, The above sequence-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.

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

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

7. Step of culturing cells; A step of transfecting the cultured cells with a plasmid containing an expression cassette of sequence number 3 and a plasmid containing an expression cassette of sequence number 4; and A method for producing a foamy virus, comprising the step of obtaining a virus from the transduced cell.

8. In paragraph 7, A method wherein a plasmid including an expression cassette of the above sequence number 3 and a plasmid including an expression cassette of the above sequence number 4 are introduced by mixing them at a ratio of 10 to 50: 0.5 to 1.

5.

9. A foamy virus vector manufactured by the method of Article 7.

10. 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 9.

11. In paragraph 10, A method wherein the above target gene is selected from a group consisting of genes associated with single-gene diseases and reporter genes.

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