Novel polynucleotide encoding FAF1 protein and FAF1 protein variant

Abstract

An aspect of the present invention relates to a novel polynucleotide encoding FAF1 protein, and an FAF1 protein variant or a polynucleotide encoding same. The polynucleotide exhibits improved stability compared to wild-type FAF1 polynucleotide, and the FAF1 protein variant can exhibit excellent anticancer efficacy and apoptosis-inducing ability compared to wild-type FAF1 protein.

Description

Novel polynucleotides encoding FAF1 protein and FAF1 protein variants

[0001] The present invention relates to a novel polynucleotide encoding a wild-type FAF1 (Fas-associated factor 1) protein, a FAF1 protein variant or a polynucleotide encoding the same, and uses thereof.

[0002] In addition, the present invention relates to a novel polynucleotide encoding a wild-type FAF1 (Fas-associated factor 1) protein, a FAF1 protein variant, or a carrier comprising a polynucleotide encoding the same, and its use.

[0003] Cancer is one of the leading causes of death worldwide. Meanwhile, various genetic mechanisms and the tumor microenvironment are known to be involved in cancer proliferation and metastasis, and active research is underway on proteins that suppress cancer proliferation and metastasis. While various anticancer drugs are currently being developed and marketed, a complete cure remains difficult due to individual patient characteristics and drug resistance.

[0004] Meanwhile, gene therapy is actively being developed to not only directly administer therapeutic proteins to the target organism, but also to enable them to be expressed within target cells to achieve the intended therapeutic effect. In this regard, while improving the tumor-targeting ability of the gene delivery system itself is crucial in gene therapy development, the stability and / or anticancer efficacy of the polynucleotide loaded onto the delivery system are also crucial considerations.

[0005] FAF1 is not only a protein associated with the Fas pathway, which is the most powerful receptor for cell death, but also suppresses cancer proliferation and metastasis through various mechanisms such as β-Catenin inhibition, NF-kB inhibition, TGF-β signaling, and Aurora-A inhibition, and it has been reported that FAF1 expression levels are reduced in various tumors including lung cancer, colon cancer, liver cancer, prostate cancer, brain cancer, and breast cancer (XIE, Feng, et al., FAF1 phosphorylation by AKT accumulates TGF-β type II receptor and drives breast cancer metastasis, Nature communications, 2017, 8.1: 1-16 and L Zhang et al. Fas-associated Factor 1 Is a Scaffold Protein That Promotes β-Transducin Repeat-containing Protein (β-TrCP)-mediated β-Catenin Ubiquitination and Degradation. J Biol Chem. 2012 Aug 31; 287(36): Furthermore, Korean Patent No. 0692416 discloses that FAF1 fragments have angiogenic, tube formation inhibitory, and cell proliferation inhibitory activities as tumor suppressors.

[0006] However, it is not known whether gene therapies containing the FAF1 protein or delivery vehicles such as nanoparticles containing FAF1 polynucleotides can kill cancer cells.

[0007] Furthermore, in order to develop and commercialize a gene therapy product related to the FAF1 protein, it is necessary to be able to manufacture the FAF1 protein at a high yield. Therefore, the production capacity of the FAF1 protein is increased and / or the development of a polynucleotide with improved stability is required to increase the production yield.

[0008] In addition, there is a need for the development of mutant proteins that have improved or at least similar anticancer effects compared to the wild-type FAF1 protein and polynucleotides with excellent anticancer efficacy that can be incorporated into anticancer gene therapy.

[0009] The technical task of the present invention is to provide a novel polynucleotide that increases the production yield of FAF1 protein and / or exhibits an increased anticancer effect compared to a wild-type FAF1 polynucleotide when delivered into the body.

[0010] Another technical task of the present invention is to provide a polynucleotide with improved stability and a use thereof so as to increase the production yield of FAF1 protein.

[0011] Another technical task of the present invention is to provide a carrier comprising a polynucleotide encoding FAF1 protein and a use thereof.

[0012] Another technical task of the present invention is to provide a FAF1 protein mutant having increased anticancer and cell death effects compared to the wild-type FAF1 protein.

[0013] Another technical task of the present invention is to provide a FAF1 protein variant and its use.

[0014] [1] One aspect of the present invention relates to a polynucleotide encoding a FAF1 protein, wherein the polynucleotide has 80% or more sequence homology with the nucleotide sequence of SEQ ID NO: 3. The polynucleotide may be a polynucleotide having 93% or more sequence homology with the nucleotide sequence of SEQ ID NO: 3, and the polynucleotide may be composed of the nucleotide sequence of SEQ ID NO: 3.

[0015] [2] Another aspect of the present invention relates to a FAF1 protein variant having 80% or more sequence homology with the FAF1 protein consisting of the amino acid sequence of SEQ ID NO: 5. The FAF1 protein variant may be a FAF1 protein variant having 90% or more sequence homology with the FAF1 protein consisting of the amino acid sequence of SEQ ID NO: 5.

[0016] [3] In the above [2], the FAF1 protein variant may be characterized by having a mutation selected from the group consisting of the following (1) to (21):

[0017] (1) Substitution of serine (S), amino acid positions 289 and 291 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, with aspartic acid (D);

[0018] (2) Substitution of serine (S), the 582nd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, with alanine (A);

[0019] (3) Serine (S), which is the 289th and 291st amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with aspartic acid (D), and serine (S), which is the 582nd amino acid, is substituted with alanine (A);

[0020] (4) Lysine (K), which is the 139th, 143rd, and 146th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0021] (5) Lysine (K), the 157th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R); (6) Lysine (K), the 163rd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0022] (7) Lysine (K), the 221st amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0023] (8) Lysine (K), the 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R);

[0024] (9) Lysine (K), which is the 163rd, 221st, and 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0025] (10) Aspartic acid (D), which is the 149th and 152nd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A);

[0026] (11) Aspartic acid (D), which is the 292nd, 295th, and 298th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A);

[0027] (12) Aspartic acid (D), which is the 494th and 497th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A);

[0028] (13) Aspartic acid (D), which is an amino acid at positions 494 and 497 from the N-terminus of the FAF1 protein consisting of an amino acid sequence of sequence number 5, is substituted with alanine (A), and aspartic acid (D), which is an amino acid at positions 292, 295, and 298, is substituted with alanine (A);

[0029] (14) The 289th and 291st amino acids of the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, serine (S), are substituted with aspartic acid (D), and the 292nd, 295th and 298th amino acids, aspartic acid (D), are substituted with alanine (A);

[0030] (15) Aspartic acid (D), which is the 494th and 497th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with alanine (A), and serine (S), which is the 582nd amino acid, is substituted with alanine (A);

[0031] (16) From the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, the 289th and 291st amino acids, serine (S), are substituted with aspartic acid (D), the 444th amino acid, lysine (K), is substituted with arginine (R), and the 582nd amino acid, serine (S), is substituted with alanine (A);

[0032] (17) Lysine (K), the 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R), and aspartic acid (D), the 494th and 497th amino acids, is substituted with alanine (A);

[0033] (18) Deletion of amino acids 1 to 87 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5;

[0034] (19) Deletion of amino acids 1 to 139 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5;

[0035] (20) Deletion of amino acids 1 to 286 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5; and

[0036] (21) Amino acids 1 to 286 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5 are deleted, and the 444th amino acid, lysine (K), is replaced with arginine (R).

[0037] [4] In the above [3], the FAF1 protein variant may include a polypeptide consisting of an amino acid sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.

[0038] [5] In the above [3], the FAF1 protein variant can be encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46.

[0039] [6] One aspect of the present invention relates to a carrier comprising a polynucleotide encoding a FAF1 protein having an amino acid sequence of SEQ ID NO: 5; the polynucleotide of [1]; any one of the FAF1 protein variants of [2] to [5]; or any one of the FAF1 protein variants of [2] to [5]. The polynucleotide encoding the FAF1 protein having an amino acid sequence of SEQ ID NO: 5 may be composed of a nucleotide sequence of SEQ ID NO: 1.

[0040] [7] In the above [6], the delivery vehicle may be one selected from the group consisting of a viral vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a bacterial artificial chromosomes (BACs), a yeast artificial chromosomes (YACs), a liposome, an extracellular vesicle, a nanoparticle, a peptide-based vector, and a polymer-based vector.

[0041] [8] In the above [7], the extracellular vesicle may be one selected from the group consisting of exosomes, microvesicles, ectosomes, oncosomes, and prostasomes.

[0042] [9] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition may contain as an active ingredient the polynucleotide of [1]; the FAF1 protein variant of any one of [2] to [5]; the polynucleotide encoding the FAF1 protein variant of any one of [2] to [5]; or the carrier of any one of [6] to [8].

[0043]

[0010] In the above [9], the cancer may be selected from the group consisting of leukemia, lymphoma, myeloma, melanoma, sarcoma, brain tumor, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancer, glioblastoma, ocular cancer, vaginal cancer, vulvar cancer, cervical cancer, endometrial carcinoma, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, basal cancer, salivary gland cancer, pharyngeal cancer, skin cancer, kidney cancer, Wilms' tumor, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, and urethral cancer.

[0044]

[0011] Another aspect of the present invention is a composition for inducing cell death, wherein the composition may contain as an active ingredient the polynucleotide of [1]; the FAF1 protein variant of any one of [2] to [5]; the polynucleotide encoding the FAF1 protein variant of any one of [2] to [5]; or the transporter of any one of [6] to [8].

[0045]

[0012] In the above

[0011] , the cell may be selected from the group consisting of HEK 293 cells, SW480 cells, HeLa cells, Hep3B cells, MIA-PaCa-2 cells, A549 cells, and MDA-MB-231 cells.

[0046]

[0013] One aspect of the present invention relates to a kit for preventing or treating cancer, wherein the kit may include an instruction manual and a pharmaceutical composition of any one of [9] or

[0010] .

[0047]

[0014] Another aspect of the present invention relates to a method for preventing or treating cancer, comprising administering to a subject a therapeutically effective amount of the polynucleotide of [1]; the FAF1 protein variant of any one of [2] to [5]; the polynucleotide encoding the FAF1 protein variant of any one of [2] to [5]; or the carrier of any one of [6] to [8].

[0048]

[0015] Another aspect of the present invention relates to the use of a therapeutically effective amount of the polynucleotide of [1]; the FAF1 protein variant of any one of [2] to [5]; the polynucleotide encoding the FAF1 protein variant of any one of [2] to [5]; or the carrier of any one of [6] to [8] for the prevention or treatment of cancer.

[0049]

[0016] Another aspect of the present invention relates to the use of a therapeutically effective amount of the polynucleotide of [1]; the FAF1 protein variant of any one of [2] to [5]; the polynucleotide encoding the FAF1 protein variant of any one of [2] to [5]; or the carrier of any one of [6] to [8] for the manufacture of a medicament for the prevention or treatment of cancer.

[0050] The FAF1 polynucleotide having 80% or more sequence homology to the nucleotide sequence of SEQ ID NO: 3 of the present invention exhibits a superior yield of FAF1 protein production and / or anticancer effect compared to the wild-type FAF1 polynucleotide. Furthermore, the FAF1 protein variant of the present invention exhibits an increased anticancer effect compared to the wild-type FAF1 protein.

[0051] In addition, the FAF1 protein variant of the present invention or the polynucleotide encoding the same exhibits a superior anticancer effect compared to wild-type FAF1 or the polynucleotide thereof.

[0052] Furthermore, a delivery vehicle (e.g., lipid nanoparticle) comprising the FAF1 polynucleotide or FAF1 protein variant of the present invention exhibits excellent cancer cell killing effects.

[0053] Figure 1 shows the GC content of wild-type FAF1 and three candidate FAF1 polynucleotides and the sequence homology between individual polynucleotides.

[0054] Figure 2 shows the results of Western blot detection of FAF1 protein produced in cells into which four types of in vitro transcripts (IVT) were introduced.

[0055] Figure 3 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ② FAF1 IVT treatment. *: p-value <0.05, **: p-value <0.01

[0056] Figure 4 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ①, ②, and ③ FAF1 IVT treatment in HeLa cells.

[0057] Figure 5 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ①, ②, and ③ FAF1 IVT treatment in Hep3B cells.

[0058] Figure 6 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ①, ②, and ③ FAF1 IVT treatment in MIA-PaCa-2 cells.

[0059] Figure 7 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ①, ②, and ③ FAF1 IVT treatment in A549 cells.

[0060] Figure 8 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ①, ②, and ③ FAF1 IVT treatment in MDA-MB-231 cells.

[0061] Figure 9 shows the results of analyzing the apoptosis induction ability according to wild-type FAF1 IVT and candidate ①, ②, and ③ FAF1 IVT treatment in SW480 cells.

[0062] Figure 10 illustrates the results of observing changes in tumor volume according to treatment with wild-type or candidate ② FAF1 mRNA-LNP in a CT26 allograft tumor model.

[0063] Figure 11 is a schematic diagram of a plasmid vector DNA constructed for transduction / protein expression of a FAF1 protein mutant.

[0064] Figure 12 shows the results of a Western blot detecting FAF1 protein expressed by cells transduced with plasmid DNA encoding a FAF1 protein variant.

[0065] Figure 13 shows the results of comparing the apoptosis induction ability upon transduction of a wild-type FAF1 polynucleotide in HEK293 cells with the apoptosis induction ability upon transduction of a polynucleotide encoding a FAF1 protein mutant.

[0066] Figure 14 shows the results of comparing the apoptosis induction ability upon transduction of a wild-type FAF1 polynucleotide in SW480 cells with the apoptosis induction ability upon transduction of a polynucleotide encoding a FAF1 protein mutant.

[0067] Figure 15 shows the results of observing changes in tumor volume and weight by vehicle, wild-type FAF1 protein, and three FAF1 protein mutants (Ca01, Ca02, Ca03) in the SW480 xenograft tumor model. *: p-value <0.05

[0068] Figure 16 illustrates the results of comparing the apoptosis induction ability upon transduction of a wild-type FAF1 polynucleotide in HeLa cells with the apoptosis induction ability upon transduction of a polynucleotide encoding a FAF1 protein mutant.

[0069] Figure 17 illustrates the results of comparing the apoptosis induction ability upon transduction of a wild-type FAF1 polynucleotide in Hep3B cells with the apoptosis induction ability upon transduction of a polynucleotide encoding a FAF1 protein mutant.

[0070] Figure 18 illustrates the results of comparing the apoptosis induction ability upon transduction of wild-type FAF1 polynucleotide in MIA-PaCa-2 cells with the apoptosis induction ability upon transduction of polynucleotide encoding FAF1 protein mutants.

[0071] Figure 19 illustrates the results of comparing the apoptosis induction ability upon transduction of a wild-type FAF1 polynucleotide in A549 cells with the apoptosis induction ability upon transduction of a polynucleotide encoding a FAF1 protein variant.

[0072] Figure 20 illustrates the results of comparing the apoptosis induction ability upon transduction of a wild-type FAF1 polynucleotide in MDA-MB-231 cells with the apoptosis induction ability upon transduction of a polynucleotide encoding a FAF1 protein mutant.

[0073] Figure 21 shows the results of measuring the protein expression levels in AAV expressing the FAF1 wild-type protein and AAV expressing the FAF1 protein mutant (Tr03) using Western blot.

[0074] Figure 22 shows the results of comparing the cell death effect when treated with AAV expressing the wild-type FAF1 protein and the cell death effect when treated with AAV expressing the FAF1 protein mutant (Tr03). *: p-value <0.05, **: p-value <0.01

[0075] definition

[0076] The term "FAF1" stands for Fas-associated factor 1, which mediates necrosis through JNK-dependent mitochondrial dysfunction and participates in cell proliferation by negatively regulating Aurora-A to inhibit the G2 / M phase of the cell cycle. In addition, FAF1 regulates protein degradation by binding to ubiquitinated proteins and VCP (Valosin Containing Protein) and participating in the ubiquitin-proteasome pathway, and unnecessary FAF1 is ubiquitinated through Parkin and degraded through the proteasome pathway. FAF1 activates multiple pathways and participates in various biochemical processes, including apoptosis, inflammation, cell proliferation, and protein homeostasis. In particular, FAF1 is a tumor suppressor that induces cell death by forming the Fas death-inducing signaling complex (Fas-DISC; death-inducing signaling complex), plays a tumor suppressive role by suppressing NF-κB, and also suppresses tumor metastasis through TGF-β signaling.

[0077] In the present specification, the wild-type FAF1 protein is encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence of SEQ ID NO: 5.

[0078] The term “polynucleotide” refers to a strand of DNA or RNA, which is a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds.

[0079] The term "protein variant" encompasses a polypeptide having an amino acid sequence that differs from the original amino acid sequence by substituting one or more amino acids in the polypeptide. Such variants may retain the function or properties of the original polypeptide, or may exhibit increased functions or properties. This includes "truncated forms" of the polypeptide, in which some amino acids are deleted, but the function or properties of the polypeptide are retained, or even exhibit increased functions or properties.

[0080] The term "transduction" means the transfer or introduction of an exogenous polynucleotide into a host cell, and is used herein to include "transfection" or "infection" or "transformation".

[0081] The term "transporter" refers to any delivery vehicle capable of introducing a nucleic acid or gene into a cell, for example, a delivery vehicle capable of introducing a nucleic acid sequence into a cell in which the nucleic acid sequence can be inserted and replicated, or a delivery vehicle capable of delivering a polypeptide to a target cell. The delivery vehicle capable of introducing the nucleic acid or gene into a cell may be any material known in the art to function as a vector without limitation. The vector may include, but is not limited to, plasmids, cosmid vectors, bacteriophage vectors, and viral vectors. Vectors that can be used as the above vectors can be produced by manipulating plasmids (such as pCMV6, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), bacteriophages (such as λgt4λB, λCharon, and M13), or viruses (such as AVV, Poxvirus, CMV, and SV40) that are frequently used in the art. In addition, the above vectors can be introduced into cells using well-known techniques for introducing polynucleotides into cells. Those skilled in the art can construct the vector by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0082] Additionally, liposomes; extracellular vesicles such as exosomes, microvesicles, ectosomes, oncosomes, and prostasomes; nanoparticles such as lipid nanoparticles; peptide-based vectors and polymer-based vectors can be used as the above-described carriers.

[0083] The term "apoptosis" refers to the death of cells due to changes in cell morphology and internal biochemical properties, and includes apoptotic cell death, autophagic cell death, and necrotic cell death.

[0084] The term "cancer" refers to a cellular disorder characterized by uncontrolled or dysregulated cell proliferation, decreased cell differentiation, an inappropriate ability to invade surrounding tissues, and / or the ability to establish new growth at heterotopic sites. Furthermore, tumors include, but are not limited to, solid tumors and blood-borne tumors, and further include diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels, as well as primary and metastatic tumors.

[0085] The terms “cancer prevention” or “cancer treatment” refer to any action that improves or beneficially alters the symptoms of cancer.

[0086] Additionally, the term “subject” herein refers to an animal, preferably a human.

[0087] Polynucleotide encoding FAF1 protein with improved stability

[0088] One aspect of the present invention relates to a polynucleotide encoding a FAF1 protein, which has an excellent apoptosis-inducing ability and comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology, particularly at least 93% or 98% sequence homology, with the nucleotide sequence of SEQ ID NO: 3. The polynucleotide may have a GC content of at least 50%, and may preferably be a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 3.

[0089] The polynucleotide with improved stability encoding the FAF1 protein may be DNA or RNA, and if the polynucleotide is RNA, cytosine may be substituted with 5-methylcytosine and / or uracil may be substituted with N1-methylpsuedouracil and / or the 5' end of the FAF1 polynucleotide with improved stability may be capped. In this case, the 5' end capping may be performed using a known method known to be suitable for capping mRNA.

[0090] When the polynucleotide encoding the FAF1 protein with improved stability is transduced into cancer cells or cells for mass production of proteins via a vector, it can achieve an FAF1 protein production amount that is increased by 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more compared to when the wild-type FAF1 polynucleotide (SEQ ID NO: 1) is introduced.

[0091] As the above vector, viruses, plasmids (including episomes), cosmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), liposomes, extracellular vesicles (exosomes, microvesicles, ectosomes, oncosomes, prostasomes, etc.), nanoparticles such as lipid nanoparticles, peptide-based vectors, or polymer-based vectors can be used.

[0092] The FAF1 polynucleotide with improved stability, when transduced or delivered into cancer cells via a carrier, is more effective in inducing apoptosis of cancer cells than when transduced with a wild-type FAF1 polynucleotide. The FAF1 polynucleotide with improved stability, when transduced or delivered into cancer cells via a carrier, can induce apoptosis in cancer cells that is 1.5 times, 2 times, or 2.5 times more effective than when transduced with a wild-type FAF1 polynucleotide at the same dose. The novel FAF1 polynucleotide can induce apoptosis of cancer cells to the same extent when transduced with a carrier, directly delivered to cancer cells, or administered to a subject having cancer at a dose that is 1 / 2, 1 / 3, 1 / 4, or 1 / 5 of the dose of the wild-type FAF1 polynucleotide. The carrier is as described below.

[0093] FAF1 protein mutants

[0094] One aspect of the present invention relates to a FAF1 protein variant having a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a FAF1 protein having an amino acid sequence of SEQ ID NO: 5.

[0095] The above FAF1 protein variant may be characterized by having a mutation selected from the group consisting of the following (1) to (21):

[0096] (1) Substitution of serine (S), amino acid positions 289 and 291 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, with aspartic acid (D);

[0097] (2) Substitution of serine (S), the 582nd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, with alanine (A);

[0098] (3) Serine (S), which is the 289th and 291st amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with aspartic acid (D), and serine (S), which is the 582nd amino acid, is substituted with alanine (A);

[0099] (4) Lysine (K), which is the 139th, 143rd, and 146th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0100] (5) Lysine (K), the 157th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R);

[0101] (6) Lysine (K), the 163rd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R);

[0102] (7) Lysine (K), the 221st amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0103] (8) Lysine (K), the 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R);

[0104] (9) Lysine (K), which is the 163rd, 221st, and 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R);

[0105] (10) Aspartic acid (D), which is the 149th and 152nd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A);

[0106] (11) Aspartic acid (D), which is the 292nd, 295th, and 298th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A);

[0107] (12) Aspartic acid (D), which is the 494th and 497th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A);

[0108] (13) Aspartic acid (D), which is an amino acid at positions 494 and 497 from the N-terminus of the FAF1 protein consisting of an amino acid sequence of sequence number 5, is substituted with alanine (A), and aspartic acid (D), which is an amino acid at positions 292, 295, and 298, is substituted with alanine (A);

[0109] (14) The 289th and 291st amino acids of the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, serine (S), are substituted with aspartic acid (D), and the 292nd, 295th and 298th amino acids, aspartic acid (D), are substituted with alanine (A);

[0110] (15) Aspartic acid (D), which is the 494th and 497th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with alanine (A), and serine (S), which is the 582nd amino acid, is substituted with alanine (A);

[0111] (16) From the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, the 289th and 291st amino acids, serine (S), are substituted with aspartic acid (D), the 444th amino acid, lysine (K), is substituted with arginine (R), and the 582nd amino acid, serine (S), is substituted with alanine (A);

[0112] (17) Lysine (K), the 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R), and aspartic acid (D), the 494th and 497th amino acids, is substituted with alanine (A);

[0113] (18) Deletion of amino acids 1 to 87 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5;

[0114] (19) Deletion of amino acids 1 to 139 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5;

[0115] (20) Deletion of amino acids 1 to 286 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5; and

[0116] (21) Amino acids 1 to 286 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5 are deleted, and the 444th amino acid, lysine (K), is replaced with arginine (R).

[0117] The above FAF1 protein variant may comprise a polypeptide consisting of an amino acid sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.

[0118] The above FAF1 protein variant can be encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46.

[0119] The above FAF1 protein variant exhibits a superior apoptosis-inducing effect in cancer cells compared to the wild-type protein consisting of the amino acid sequence of SEQ ID NO: 5. The polynucleotide encoding the above FAF1 protein variant can exhibit a superior apoptosis-inducing effect when transduced into cancer cells via a delivery vehicle, directly delivered to cancer cells, or administered to a subject having cancer compared to the wild-type FAF1 polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1. The delivery vehicle is as described below.

[0120] carrier

[0121] One aspect of the present invention relates to a polynucleotide encoding a FAF1 protein having an amino acid sequence of SEQ ID NO: 5, a polynucleotide encoding the aforementioned FAF1 protein having improved stability, a variant of the aforementioned FAF1 protein, or a carrier comprising a polynucleotide encoding the same.

[0122] The above delivery system can be used to transduce or deliver into cells a polynucleotide encoding a FAF1 protein having an amino acid sequence of SEQ ID NO: 5, a polynucleotide encoding the FAF1 protein with improved stability, or a polynucleotide encoding a variant of the FAF1 protein described above, into cancer cells of a subject, or to deliver the FAF1 protein variant described above into cells.

[0123] The above delivery vehicle may be one selected from the group consisting of viral vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), liposomes, extracellular vesicle nanoparticles, peptide-based vectors, and polymer-based vectors.

[0124] The above extracellular vesicles may be one selected from the group consisting of exosomes, microvesicles, ectosomes, oncosomes, and prostasomes.

[0125] As the above viral vector, a retrovirus, lentivirus, adenovirus (AV), adeno-associated virus (AAV), herpes simplex virus vector (HSV), or poxvirus vector can be used.

[0126] The above cosmid vector is a type of hybrid plasmid containing a cosmid lambda phage. Any cosmid vector suitable for delivering or transducing a polynucleotide into a cell may be used without limitation as a delivery vehicle of the present invention.

[0127] In addition, any cosmid vector, plasmid vector (including episome and fosmid vectors) and bacteriophage vector known in the technical field to which the present invention pertains may be used without limitation as a delivery vehicle of the present invention.

[0128] The bacterial artificial chromosome is known to be capable of delivering a DNA fragment larger than a plasmid vector (about 150 to 350 kbp) into a cell, and the yeast artificial chromosome is a chromosome obtained by genetically manipulating DNA obtained from Saccharomyces cerevisiae and then linking it to a bacterial plasmid, and is known to be capable of inserting a DNA fragment of about 100 to 1000 kb. Any material known as the artificial chromosome can be used without limitation as the delivery vehicle of the present invention.

[0129] The above liposome is a carrier composed of a phospholipid bilayer and can contain both hydrophobic and hydrophilic drugs. Any biocompatible material capable of performing liposome function can be used without limitation as a carrier of the present invention.

[0130] The above extracellular vesicles are small membrane-structured particles secreted from cells that contain various bioactive substances such as proteins, lipids, and nucleic acids, and can protect the internal contents from the external environment and deliver them to specific cells.

[0131] The above exosomes are nano-sized (approximately 20 to 1,000 nm) vesicles surrounded by a lipid bilayer that cells secrete outside the cell for information exchange.

[0132] The above microvesicles are vesicles of approximately 200 to 1,000 nm in size, containing various proteins and lipids, formed by direct outward budding of the cell membrane.

[0133] The above ectosomes are microvesicle-like vesicles that are released by budding directly from the plasma membrane for intercellular signal transmission.

[0134] The above oncosome is an extracellular vesicle secreted by cancer cells that is involved in the growth and metastasis of cancer cells.

[0135] The above prostasomes are extracellular vesicles secreted from prostate epithelial cells.

[0136] The above nanoparticles are various particles used to deliver medicines or proteins to a target site, and refer to particles, devices, or systems with a size of less than a micron (mainly 3 to 200 nm) and can be manufactured from various materials such as metals, ceramics, polymers, fats, and proteins. Examples of nanoparticles include, but are not limited to, polymer nanogels, lipid nanoparticles, and micelles.

[0137] Polymer nanogels are nanoparticles obtained by cross-linking hydrophilic polymer chains, or hydrophilic or amphiphilic polymer chains, with a size of tens to hundreds of nanometers, forming three-dimensional cross-links. Polymer nanogels can encapsulate drugs or proteins through hydrogen bonding, salt bonding, or hydrophobic interactions (Duan, Qiu-Yi, et al. "Nanogels: Synthesis, properties, and recent biomedical applications." Progress in Materials Science, 2023, 1, 1, 101167, Adv. Drug Delivery Reviews 2012, 64:9;836-851).

[0138] Lipid nanoparticles refer to particulate drug delivery vehicles composed of lipids capable of encapsulating poorly soluble substances. The lipid nanoparticles are composed of ionizable cationic lipids capable of neutralizing the negative charge of mRNA, phospholipids and cholesterol that maintain the lipid bilayer structure of the lipid nanoparticles and allow for good permeation of cell membranes, and polyethylene glycol (PEG) lipid components that enhance the stability of the lipid nanoparticles in the body. Any lipid nanoparticle that allows polynucleotides such as mRNA to be delivered into cells without degradation may be used without limitation as the delivery vehicle of the present invention.

[0139] Micelles are materials based on amphiphilic block polymers that self-assemble in a water-soluble state to form a core / shell (outer) structure. Any known micelle that allows polynucleotides to be delivered into cells without degradation may be used without limitation as a delivery vehicle of the present invention.

[0140] The above peptide-based vector is a cationic peptide rich in basic amino acids (lysine, arginine, histidine), and any peptide vector known to be useful for cell delivery of genes or nucleic acids can be used without limitation as a delivery vehicle of the present invention. Examples of the above peptide-based vector include, but are not limited to, poly-L-lysines (PLLs), arginine-rich peptides, and poly-L-ornithine (PLO).

[0141] The polymer-based vectors are polymer-based nanoparticles studied for drug and gene delivery, and include, but are not limited to, polyethyleneimine (PEI) and its derivatives, polymethacrylate, β-cyclodextrin, chitosan, dextran, polyamidoamine dendrimers (PAMAM), and polypropylene diamine dendrimers.

[0142] Pharmaceutical compositions, compositions for inducing cell death, methods of treatment, uses and kits for the manufacture of pharmaceuticals

[0143] One aspect of the present invention relates to a pharmaceutical use of the FAF1 polynucleotide with improved stability described above, the FAF1 protein variant described above or a polynucleotide encoding the same or the carrier described above.

[0144] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, which may include the FAF1 polynucleotide with improved stability described above, the FAF1 protein variant described above or a polynucleotide encoding the same, or the carrier described above as an active ingredient.

[0145] In another aspect of the present invention, the cancer is (1) leukemia including but not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, myelodysplastic syndrome, preleukemia, and chronic myelomonocytic leukemia (CMML); (2) chronic leukemia including but not limited to chronic myelocytic (granular) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; (3) lymphoma including but not limited to Hodgkin's disease and non-Hodgkin's disease; (4) multiple myeloma including but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; (5) Bone and connective tissue sarcomas, including but not limited to osteosarcoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, schwannoma, rhabdomyosarcoma, and synovial sarcoma; (6) brain tumors, including but not limited to glioma, glioblastoma, astrocytic, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pinealoma, pineoblastoma, oligodendroglioma, and primary brain lymphoma; (7) breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary carcinoma, Paget's disease, and inflammatory breast cancer; (8) adrenal cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; (9) thyroid cancer, including but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; (10) pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;(11) pituitary cancers including, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; (12) ocular cancers including, but not limited to, ocular melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; (13) vaginal cancers including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; (14) vulvar cancers including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; (15) cervical cancers including, but not limited to, squamous cell carcinoma and adenocarcinoma; (16) endometrial carcinoma; (17) uterine cancers including, but not limited to, uterine sarcoma; (18) ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; (19) esophageal cancers including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; (20) gastric cancers including but not limited to adenocarcinoma, mycosis (polypoid), ulcerative, superficial spreading, diffuse expansile, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; (21) colon cancer; (22) rectal cancer; (23) liver cancers including but not limited to hepatocellular carcinoma and hepatoblastoma; (24) gallbladder cancers including but not limited to adenocarcinoma; (25) cholangiocarcinomas including but not limited to papillary, nodular, and diffuse; (26) lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid cystic carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; (27) testicular cancer, including but not limited to germ cell tumor, seminoma, anaplastic, classical (typical), spermatocyte, nonseminomatous, embryonal carcinoma, teratoma carcinoma, and choriocarcinoma (yolk sac tumor); (28) prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; (29) penile cancer;(30) oral cancers including but not limited to laryngeal, pharyngeal, nasopharyngeal, oropharyngeal, and squamous cell carcinomas; (31) basal cell carcinomas; (32) salivary gland cancers including but not limited to adenocarcinomas, mucoepidermoid carcinomas, and adenoid cystic carcinomas; (33) pharyngeal cancers including but not limited to squamous cell carcinomas and verrucous; (34) skin cancers including but not limited to basal cell carcinomas, squamous cell carcinomas, and melanomas, superficial spreading melanomas, nodular melanomas, lentigo malignant melanomas, and acral lentiginous melanomas; (35) kidney cancers including but not limited to renal cell carcinomas, adenocarcinomas, renal cell carcinomas, fibrosarcomas, and transitional cell carcinomas (renal pelvis and / or ureter); (36) Wilms' tumors; (37) bladder cancers including but not limited to transitional cell carcinomas, squamous cell carcinomas, adenocarcinomas, and carcinosarcoma; (38) Head and neck cancer (including cancer of the mouth, nose, pharynx, larynx, paranasal sinuses or salivary glands, and head and neck squamous cell carcinoma); (39) Hepatocellular carcinoma;And (40) other cancers including, but not limited to, appendicitis, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), malignant peripheral nerve sheath tumor (MPNST), and urethral cancer (see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America). For reference, colon cancer or rectal cancer is a type of colorectal cancer, so colorectal cancer is also classified as a cancer to be treated. Meanwhile, the tumor may be a metastatic tumor, an unresectable tumor, or a locally advanced tumor.;

[0146] The cancer may be a solid cancer or a blood cancer, and the cancer may be selected from the group consisting of leukemia, lymphoma, myeloma, melanoma, sarcoma, brain tumor, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancer, glioblastoma, ocular cancer, vaginal cancer, vulvar cancer, cervical cancer, endometrial carcinoma, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, basal cancer, salivary gland cancer, pharyngeal cancer, skin cancer, kidney cancer, Wilms' tumor, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, and urethral cancer.

[0147] Another aspect of the present invention is a composition for inducing cell death, which may include the FAF1 polynucleotide with improved stability described above, the FAF1 protein variant described above or a polynucleotide encoding the same, or the carrier described above as an active ingredient.

[0148] In another aspect of the present invention, the cell may be selected from the group consisting of HEK 293 cells, SW480 cells, HeLa cells, Hep3B cells, MIA-PaCa-2 cells, A549 cells, and MDA-MB-231 cells.

[0149] The pharmaceutical composition may include a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier is determined in part by the specific composition to be administered, as well as the specific method used to administer the composition. The pharmaceutical composition is generally formulated aseptically and in full compliance with the Good Manufacturing Practice (GMP) regulations of regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the Korea Food and Drug Administration (KFDA).

[0150] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art.

[0151] The pharmaceutical composition may be administered intratumorally, parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, intramuscularly, or as an inhalant or eye drop.

[0152] One aspect of the present invention relates to a kit for preventing or treating cancer, comprising an instruction manual and the pharmaceutical composition described above.

[0153] Another aspect of the present invention relates to a method for preventing or treating cancer, comprising administering to a subject a therapeutically effective amount of the FAF1 polynucleotide with improved stability described above; the FAF1 protein variant described above; a polynucleotide encoding the same; or the carrier described above.

[0154] Another aspect of the present invention relates to the use of a therapeutically effective amount of a FAF1 polynucleotide having improved stability as described above; a FAF1 protein variant as described above; a polynucleotide encoding the same; or a carrier as described above for the prevention or treatment of cancer.

[0155] Another aspect of the present invention relates to the use of a therapeutically effective amount of the FAF1 polynucleotide having improved stability as described above; the FAF1 protein variant as described above; the polynucleotide encoding the same; or the carrier as described above for the manufacture of a medicament for the prevention or treatment of cancer.

[0156] The composition of the pharmaceutical composition among the terms or elements mentioned in the above method, use and kit is understood to be the same as mentioned in the description of the pharmaceutical composition and combination above.

[0157] Hereinafter, the present invention will be described in detail with examples and the like to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples.

[0158] Example 1. Preparation of novel FAF1 polynucleotide

[0159] Example 1.1. Preparation of a novel polynucleotide capable of encoding FAF1 protein

[0160] A group of novel FAF1 nucleotides that can code for the FAF1 protein were identified by Genscript's GenSmart TMUsing the tool, three candidate polynucleotides with GC contents of 50% or more were selected as a group of mRNA optimization candidate sequences. The base sequences of the three selected candidate polynucleotides (respectively, candidate ①, candidate ②, and candidate ③) were synthesized by the PCR (Polymerase Chain Reaction) method. The characteristics of the wild-type FAF1 polynucleotide and the three candidate polynucleotides are shown in Table 1, the sequences of each polynucleotide are shown in Table 2, and the sequence similarity between these polynucleotides is illustrated in Figure 1.

[0161] Sequence number GC content (%) Free energy (Kcal / mol) Wild type FAF1 SEQ ID NO: 144.7-548.1 Candidate ① SEQ ID NO: 256.7-675.1 Candidate ② SEQ ID NO: 355-658.9 Candidate ③ SEQ ID NO: 450.13-596.9

[0162]

[0163] SEQ ID NO: 1TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:2TCAGCTCCAGATACGGCGATTGCCACCCCGTGTTCTTCATCGGCAGCCTGGAGGCCGCCTTCCAGGAAGCCTTTTACGTGAAGGCCAGAGATAGAAAGCTGCTGGCCATCTACCTGCACCACGACGAGAGCGTGTTGACGAACGTGTTCTGCAGCCAGATGCTGTGCGCCGAGTCAATCGTGTCCTACCTGAGCCAGAATTTTATCACATGGGCCTGGGACCTGACAAAGGACTCTAATAGAGCCAGATTCCTGACCATGTGTAACCGGCATTTCGGCAGCGTGGTGGCTCAAACAATCCGCACCCAGAAAACCGACCAGTTTCCACTGTTCCTCATCATCATGGGCAAGCGGAGCAGCAATGAGGTGCTGAACGTGATCCAGGGCAACACCACCGTTGACGAACTGATGATGAGACTGATGGCTGCCATGGAAATCTTCACCGCCCAACAACAGGAGGACATCAAGGACGAGGATGAGCGGGAAGCACGAGAAAACGTCAAAAGAGAACAGGACGAGGCCTACAGACTGAGCCTGGAGGCCGATCGGGCCAAGAGAGAGGCCCACGAGCGCGAAATGGCCGAACAATTCAGACTGGAACAGATCAGAAAGGAGCAAGAGGAAGAACGGGAAGCCATCAGGCTGAGTCTGGAGCAAGCCCTGCCACCTGAGCCCAAGGAAGAGAACGCCGAACCTGTATCCAAGCTGAGAATCAGAACCCCTTCTGGCGAGTTCCTGGAACGGCGGTTCCTGGCCAGCAACAAGCTGCAGATTGTGTTCGACTTTGTCGCCTCCAAAGGCTTCCCATGGGATGAGTATAAGCTGCTGTCCACCTTCCCCAGACGGGATGTGACCCAGCTGGATCCTAACAAATCTCTGTTGGAGGTGAAGCTGTTCCCTCAGGAGACACTCTTTCTGGAAGCTAAAGAGTGASEQ ID NO:3TCAGCTCCAGATACGGCGATTGCCACCCCGTGTTCTTCATCGGCAGCCTGGAGGCCGCCTTCCAGGAAGCCTTTTACGTGAAGGCCAGAGATAGAAAGCTGCTGGCCATCTACCTGCACCACGACGAGAGCGTGTTGACGAACGTGTTCTGCAGCCAGATGCTGTGCGCCGAGTCAATCGTGTCCTACCTGAGCCAGAATTTTATCACATGGGCCTGGGACCTGACAAAGGACTCTAATAGAGCCAGATTCCTGACCATGTGTAACCGGCATTTCGGCAGCGTGGTGGCTCAAACAATCCGCACCCAGAAAACCGACCAGTTTCCACTGTTCCTCATCATCATGGGCAAGCGGAGCAGCAATGAGGTGCTGAACGTGATCCAGGGCAACACCACCGTTGACGAACTGATGATGAGACTGATGGCTGCCATGGAAATCTTCACCGCCCAACAACAGGAGGACATCAAGGACGAGGATGAGCGGGAAGCACGAGAAAACGTCAAAAGAGAACAGGACGAGGCCTACAGACTGAGCCTGGAGGCCGATCGGGCCAAGAGAGAGGCCCACGAGCGCGAAATGGCCGAACAATTCAGACTGGAACAGATCAGAAAGGAGCAAGAGGAAGAACGGGAAGCCATCAGGCTGAGTCTGGAGCAAGCCCTGCCACCTGAGCCCAAGGAAGAGAACGCCGAACCTGTATCCAAGCTGAGAATCAGAACCCCTTCTGGCGAGTTCCTGGAACGGCGGTTCCTGGCCAGCAACAAGCTGCAGATTGTGTTCGACTTTGTCGCCTCCAAAGGCTTCCCATGGGATGAGTATAAGCTGCTGTCCACCTTCCCCAGACGGGATGTGACCCAGCTGGATCCTAACAAATCTCTGTTGGAGGTGAAGCTGTTCCCTCAGGAGACACTCTTTCTGGAAGCTAAAGAGTGASEQ ID NO:4TCAGCTCCAGATACGGCGATTGCCACCCCGTGTTCTTCATCGGCAGCCTGGAGGCCGCCTTCCAGGAAGCCTTTTACGTGAAGGCCAGAGATAGAAAGCTGCTGGCCATCTACCTGCACCACGACGAGAGCGTGTTGACGAACGTGTTCTGCAGCCAGATGCTGTGCGCCGAGTCAATCGTGTCCTACCTGAGCCAGAATTTTATCACATGGGCCTGGGACCTGACAAAGGACTCTAATAGAGCCAGATTCCTGACCATGTGTAACCGGCATTTCGGCAGCGTGGTGGCTCAAACAATCCGCACCCAGAAAACCGACCAGTTTCCACTGTTCCTCATCATCATGGGCAAGCGGAGCAGCAATGAGGTGCTGAACGTGATCCAGGGCAACACCACCGTTGACGAACTGATGATGAGACTGATGGCTGCCATGGAAATCTTCACCGCCCAACAACAGGAGGACATCAAGGACGAGGATGAGCGGGAAGCACGAGAAAACGTCAAAAGAGAACAGGACGAGGCCTACAGACTGAGCCTGGAGGCCGATCGGGCCAAGAGAGAGGCCCACGAGCGCGAAATGGCCGAACAATTCAGACTGGAACAGATCAGAAAGGAGCAAGAGGAAGAACGGGAAGCCATCAGGCTGAGTCTGGAGCAAGCCCTGCCACCTGAGCCCAAGGAAGAGAACGCCGAACCTGTATCCAAGCTGAGAATCAGAACCCCTTCTGGCGAGTTCCTGGAACGGCGGTTCCTGGCCAGCAACAAGCTGCAGATTGTGTTCGACTTTGTCGCCTCCAAAGGCTTCCCATGGGATGAGTATAAGCTGCTGTCCACCTTCCCCAGACGGGATGTGACCCAGCTGGATCCTAACAAATCTCTGTTGGAGGTGAAGCTGTTCCCTCAGGAGACACTCTTTCTGGAAGCTAAAGAGTGA

[0164]

[0165] The polynucleotides of the above candidates ①, ②, and ③ differ in sequence by approximately 25% from the wild-type FAF1 polynucleotide. In addition, the polynucleotides of the above candidates ①, ②, and ③ have a GC content that is 5 to 10% higher than the wild-type FAF1 polynucleotide.

[0166] Example 1.2. Preparation of in vitro transcript (IVT) for transduction of novel FAF1 polynucleotides.

[0167] mRNA corresponding to FAF1 polynucleotides of SEQ ID NOs: 1 to 4 described in Table 2 was manufactured by requesting ST Pharm. In the sequences of SEQ ID NOs: 1 to 4, thymidine was substituted with N1-methylpseudouridine, and naked mRNA was manufactured by applying ST Pharm's unique capping technology, SmartCap® (https: / www.stpharm.co.kr / ko / cdmo / mrna). The manufactured naked mRNA was purified using affinity chromatography and tangential flow filtration (TFF) (yield: 80%), and used as the wild-type FAF1 IVT, the IVT of candidate ①, the IVT of candidate ②, and the IVT of candidate ③.

[0168] Example 2. Efficacy of novel polynucleotides

[0169] Example 2.1. FAF1 protein expression ability

[0170] The human colon cancer cell line SW480 was maintained by subculturing every 3 to 4 days in RPMI-1640 medium containing serum and antibiotics (5% CO2, 37°C). 5 × 10 of the SW480 cells were seeded in a 6-well scale cell culture dish. 5Inoculated with 1.0 μg of IVT (i.e., wild-type FAF1 IVT, IVT of candidate ①, IVT of candidate ②, and IVT of candidate ③) prepared in Example 1.2 above and 0.4, 1.0, or 2.5 μl of messenger Max Lipofectamine® (Thermo Fisher Sci.) were added to Opti-MEM TM (Thermo Fisher Sci.) and reacted. IVT transduction was performed by treating SW480 cells 24 hours after inoculation with the reaction solution.

[0171] Protein samples were isolated from SW480 cells 48 hours after transduction, and FAF1 protein expression levels were analyzed by Western blot. The results of the FAF1 protein expression analysis are shown in Figure 2.

[0172] As confirmed in Figure 2, the IVT of candidate ② showed the best FAF1 protein expression level compared to the wild-type FAF1 IVT.

[0173] Example 2.2. Apoptosis induction (in vitro)

[0174] The human colon cancer cell line SW480 was maintained in RPMI-1640 medium containing serum and antibiotics at 37°C with 5% CO2, with subculture every 3 to 4 days. 5 × 10 SW480 cells were seeded in a 6-well scale cell culture dish. 5 The dog was inoculated into the well.

[0175] In the above Example 2.1, since the IVT of candidate ② showed superior protein expression efficiency compared to the wild-type FAF1 IVT, the IVT of candidate ② was selected as the experimental group, and the wild-type FAF1 IVT was selected as the control group. Transduction was performed by treating SW480 cells with the experimental and control FAF1 IVTs 24 hours after inoculation.

[0176] Specifically, 1.0 μg of wild-type FAF1 IVT prepared in Example 1.2 and 3.0 μl of messenger Max Lipofectamine® (Thermo Fisher Sci.) and Opti-MEM TM (Thermo Fisher Sci.) were mixed and reacted, and the reaction solution was treated on SW480 cells. IVT (1.0 μg, 0.5 μg, or 0.25 μg) of candidate ② prepared in Example 1.2 and messenger Max Lipofectamine® (3.0 μl, 1.5 μl, or 0.75 μl) were mixed in a 1:3 ratio and mixed in Opti-MEM TM (Thermo Fisher Sci.) and then treated with the reaction solution to SW480 cells. After culturing the transduced cells for 48 hours, they were suspended, stained with propidium iodide (PI), and Guava® easyCyte TM Cell death was analyzed using a Flow Cytometer (CYTEK®).

[0177] The degree of cell death according to the IVT transduction amount of candidate ② was analyzed using the cell death rate according to wild-type FAF1 IVT transduction as a standard (1.0), and the results of the analysis are presented in the box-and-whisker graph of Fig. 3.

[0178] As confirmed in Fig. 3, the IVT of candidate ② induced increased cell death even when using only 1 / 4 the dose compared to the wild-type FAF1 IVT of sequence number 1, and was found to induce approximately 2-fold increased cell death at the same dose.

[0179] Example 2.3. Apoptosis induction ability (in vitro)

[0180] To analyze whether IVT of candidates ①, ②, and ③ also have apoptotic activity in other cancer cells, human cervical cancer cell line HeLa, human hepatoma cell line Hep3B, human pancreatic cancer cell line MIA PaCa-2, human lung cancer cell line A549, human breast cancer cell line MDA-MB-231, and human colon cancer cell line SW480 were maintained by subculturing every 3 to 4 days in a culture medium containing serum and antibiotics at 5% CO2 and 37°C. HeLa, Hep3B, and MIA PaCa-2 were cultured in DMEM, and A549, MDA-MB-231, and SW480 were cultured in RPMI-1640.

[0181] 0.5 μg of wild-type FAF1 IVT prepared in Example 1.2 above, 1.5 μl of messenger Max Lipofectamine® (Thermo Fisher Sci.), and Opti-MEM TM (Thermo Fisher Sci.) were mixed and reacted, and the reaction solution was treated on HeLa, Hep3B, MIA PaCa-2, A549, MDA-MB-231, and SW480 cells. IVT (0.5 μg) of candidates ①, ②, and ③ prepared in Example 1.2 and messenger Max Lipofectamine® 1.5 μl were mixed in a 1:3 ratio, and Opti-MEM TM (Thermo Fisher Sci.) and the reaction solution was treated with HeLa, Hep3B, MIA PaCa-2, A549, MDA-MB-231, and SW480 cells. After culturing the transduced cells for 48 hours, they were suspended, stained with Propidium iodide (PI), and labeled with Guava® easyCyte. TM Cell death was analyzed using a Flow Cytometer (CYTEK®), and the analysis results are shown in Figures 4 to 9.

[0182] Specifically, in HeLa cells, only candidate ③ induced a 1.11-fold increase in apoptosis (Fig. 4); in Hep3B cells, candidate ② induced a 1.18-fold increase in apoptosis, and candidate ③ induced a 1.22-fold increase in apoptosis (Fig. 5); in MIA-PaCa-2 cells, candidate ① induced a 1.38-fold increase in apoptosis, candidate ② induced a 1.21-fold increase in apoptosis, and candidate ③ induced a 2.57-fold increase in apoptosis (Fig. 6); in A549 cells, candidate ③ induced a 1.75-fold increase in apoptosis (Fig. 7); in MDA-MB-231 cells, candidate ① induced a 1.21-fold increase in apoptosis, candidate ② induced a 1.19-fold increase in apoptosis, and candidate ③ induced a 1.44-fold increase in apoptosis (Fig. 8); In SW480 cells, candidate ① induced cell death by 1.212-fold, candidate ② by 1.298-fold, and candidate ③ by 1.777-fold (Fig. 9).

[0183] IVT of candidates ①, ②, and ③ was shown to induce increased apoptosis compared to the same dose of wild-type FAF1 IVT of sequence number 1 in different cancer types, and candidate ③ induced increased apoptosis in the most cancer types.

[0184] Example 3. Anticancer efficacy of nanoparticles containing FAF1 mRNA (in vivo)

[0185] <FAF1 mRNA-LNP의 제조>

[0186] Lipid nanoparticles (LNPs) containing wild-type FAF1 mRNA and LNPs containing mRNA of candidate ② were manufactured by requesting Estifarm.

[0187] Wild-type FAF1 mRNA and candidate ② mRNA were prepared according to the mRNA preparation method described in Example 1.2. The prepared mRNA was encapsulated with STLNP®, an LNP from ST Pharm, to prepare FAF1 mRNA-LNP (final concentration: 0.5 mg / ml). STLNP® from ST Pharm is known to contain helper lipids, ionizable lipids, cholesterol, and PEG (see https: / / www.stpharm.co.kr / ko / cdmo / mrna). As controls, STLNP without mRNA encapsulation (hereinafter referred to as empty LNP) and LNP dilution buffer (LDB) were used, respectively. The characteristics of the prepared wild-type FAF1 mRNA-LNP and FAF1 mRNA-LNP encapsulating candidate ② mRNA are summarized in Table 3.

[0188] Control (empty LNP) Wild type FAF1 mRNA-LNP Candidate ② mRNA-encapsulated FAF1 mRNA-LNP Appearance White suspension White suspension White suspension RNA concentration X 0.5256 g / L 0.5434 g / L RNA encapsulation X 96.55% 96.64% Particle z-diameter (d, nm) 100.4 116.7 117.7 nm Particle PDI 0.03 3 0.08 (0.07567) 0.07 (0.06564)

[0189]

[0190] <Production of tumor model animals>

[0191] Mouse colon cancer cell line CT26 was maintained in RPMI-1640 medium containing serum and antibiotics at 37°C with 5% CO2, with 3- to 4-day subculture cycles. 5 × 10 cells were cultured in 150 μl of PBS. 5 An allograft tumor model was created by mixing CT26 cells from dogs and transplanting them into the right dorsal side of 6-week-old male BALB / c mice using a 26G syringe.

[0192] Efficacy Experiment and Results

[0193] Tumor volume (shortened) 6 days after CT26 cell transplantation 2 × long axis × 0.5) is 65 mm on average 3 From the time point reached, 0.1 mg / kg (mpk) of LDB, empty LNP, and IVT-LNP (wild-type FAF1 mRNA-LNP, FAF1 mRNA-LNP encapsulating mRNA of candidate ②) were administered intratumorally to tumor model animals at 2-day intervals for a total of 6 times using a 31G syringe with a total dose of 50 μl, and the tumor volume was measured 3 times a week.

[0194] At the end of the experiment, 41 days after tumor cell transplantation, the tumor tissue weight was measured, and the measurement results are shown in Figure 10.

[0195] Specifically, both wild-type FAF1 mRNA-LNPs and FAF1 mRNA-LNPs encapsulating mRNA of candidate ② exhibited superior tumor growth inhibition effects compared to the control group (Fig. 10). Furthermore, candidate ③, which demonstrated superior anticancer efficacy compared to candidate ② in the in vitro experiment of Example 2.3, is expected to exhibit even superior tumor growth inhibition effects.

[0196] Example 4. Preparation of protein variants of FAF1

[0197] Example 4.1. Production of FAF1 protein mutants

[0198] We identified candidate mutation sites and deletion sites expected to improve the stability and function of the wild-type FAF1 protein. A total of 21 FAF1 protein variants were designed using point mutations, truncations, and combination mutations of two or more mutations. The characteristics and sequence numbers of the FAF1 protein variants are listed in Table 4, and the base and amino acid sequences for each variant are listed in Table 5.

[0199]

[0200]

[0201] SEQ ID NO: 6TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:7MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVDDDDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:8TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCGCTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:9MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPAGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:10TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCGCTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:11MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVDDDDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPAGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:12TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:13MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSRMLLRGWRTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:14TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:15MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLRSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:16TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:17MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPRNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:18TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:19MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVRRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:20TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAGAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:21MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQRTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:22TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAGAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:23MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPRNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVRRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQRTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:24TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGCTATAAAGGCCGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:25MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGAVEASTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:26TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:27MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSAGDAFEAATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:28TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGCTATAAAGGCCGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:29MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEAIKAEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:30TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGCTATAAAGGCCGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:31MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSAGDAFEAATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEAIKAEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:32TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:33MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVDDDAGDAFEAATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:34TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGCTATAAAGGCCGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCGCTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:35MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEAIKAEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPAGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:36TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAGAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCGCTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:37MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVDDDDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQRTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPAGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO:38TTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAGAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGCTATAAAGGCCGAGGATGAACGTGAAGCCAGAGAAAATGTGAAGAGAGAGCAAGATGAGGCCTATCGCCTTTCACTTGAGGCTGACAGAGCAAAGAGGGAAGCTCACGAGAGAGAGATGGCAGAACAGTTTCGTTTGGAGCAGATTCGCAAAGAACAAGAAGAGGAACGTGAGGCCATCCGGCTGTCCTTAGAGCAAGCCCTGCCTCCTGAGCCAAAGGAAGAAAATGCTGAGCCTGTGAGCAAACTGCGGATCCGGACCCCCAGTGGCGAGTTCTTGGAGCGGCGTTTCCTGGCCAGCAACAAGCTCCAGATTGTCTTTGATTTTGTAGCTTCCAAAGGATTTCCATGGGATGAGTACAAGTTACTGAGCACCTTTCCTAGGAGAGACGTAACTCAACTGGACCCAAATAAATCATTATTGGAGGTAAAGTTGTTCCCTCAAGAAACCCTTTTCCTTGAAGCAAAAGAGTAASEQ ID NO:39MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQRTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEAIKAEDEREARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVFDFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKESEQ ID NO: 40GCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAASEQ ID NO:41MPSRQIVERQPRMLDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDESEQ ID NO: 42TCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAASEQ ID NO: 43MLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITHREVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDESEQ ID NO:44ATGGTTAGTGATAGCGATGGAGATGACTTTGAAGATGCTACAGAATTTGGGGTGGATGATGGAGAAGTATTTGGCATGGCGTCATCTGCCTTGAGAAAATCTCCAATGATGCCAGAAAACGCAGAAAATGAAGGAGATGCCTTATTACAATTTACAGCAGAGTTTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAAAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAASEQ ID NO: 45MVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDESEQ ID NO:46ATGGTTAGTGATAGCGATGGAGATGACTTTGAAGATGCTACAGAATTTGGGGTGGATGATGGAGAAGTATTTGGCATGGCGTCATCTGCCTTGAGAAAATCTCCAATGATGCCAGAAAACGCAGAAAATGAAGGAGATGCCTTATTACAATTTACAGCAGAGTTTTCTTCAAGATATGGTGATTGCCATCCTGTATTTTTTATTGGCTCATTAGAAGCTGCTTTTCAAGAGGCCTTCTATGTGAAAGCCCGAGATAGAAAGCTTCTTGCTATCTACCTCCACCATGATGAAAGTGTGTTAACCAACGTGTTCTGCTCACAAATGCTTTGTGCTGAATCCATTGTTTCTTATCTGAGTCAAAATTTTATAACCTGGGCTTGGGATCTGACAAAGGACTCCAACAGAGCAAGATTTCTCACTATGTGCAATAGACACTTTGGCAGTGTTGTGGCACAAACCATTCGGACTCAAAGAACGGATCAGTTTCCGCTTTTCCTGATTATTATGGGAAAGCGATCATCTAATGAAGTGTTGAATGTGATACAAGGGAACACAACAGTAGATGAGTTAATGATGAGACTCATGGCTGCAATGGAGATCTTCACAGCCCAACAACAGGAAGATATAAAGGACGAGGATGAASEQ ID NO: 47MVSDSDGDDFEDATEFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESIVSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQRTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

[0202]

[0203] A plasmid DNA expression vector expressing the nucleotide sequence of the FAF1 protein variant described in Table 5 was constructed based on plasmid DNA into which the wild-type sequence was inserted, using site-directed mutagenesis PCR, standard PCR, SLIC (Sequence and ligation independent Cloning) method, and restriction enzyme reaction. The plasmid DNA expression vector has the structure shown in Fig. 11.

[0204] Example 4.2. FAF1 protein expression level

[0205] Human embryonic kidney-derived cell line HEK-293 was maintained by subculturing every 3 to 4 days in MEM medium containing serum and antibiotics at 37°C and 5% CO2. 5 × 10 cells were seeded in 6-well scale cell culture dishes. 5 HEK-293 cells were inoculated with 1 / well. 24 hours after inoculation, plasmid DNA (2.0 μg) prepared in Example 4.1 was mixed with 3.0 μl of BioT® (Bioland) and treated into HEK-293 cells to transduce DNA. Protein samples were isolated from HEK-293 cells 48 hours after transfection. Protein expression levels were analyzed by Western blot, and the results are shown in Figure 12.

[0206] Example 5. Efficacy of FAF1 protein variants

[0207] Example 5.1. Apoptosis induction (in vitro)

[0208] To analyze the apoptosis-inducing ability of FAF1 protein mutants, human embryonic kidney-derived cell line HEK-293 and human colon cancer cell line SW480 were maintained by subculturing every 3 to 4 days under 5% CO2 and 37°C conditions. HEK293 cell culture medium was MEM culture medium containing serum and antibiotics, and SW480 cell culture medium was RPMI-1640 culture medium containing serum and antibiotics. 2 to 3 × 10 were seeded in a 12-well scale cell culture dish. 5 HEK-293 cells and SW480 cells were each inoculated with 1.0 μg of the plasmid DNA prepared in Example 4.1 and 1.5 μl of BioT® (Bioland) 24 hours after inoculation, the HEK-293 cells or SW480 cells were treated with a mixture to complete transformation. The transformed cells were cultured for 48 hours, suspended, and the cell viability of each group was measured using the Muse® Count & Viability Kit (CYTEK®). The results are shown in Figures 13 and 14 (in Figures 13 and 14, the degree of cell death by expression of each mutant protein was shown using the cell death rate by introduction of the wild-type protein as the standard (1.0). FAF1 protein mutants that have a higher ability to induce cell death in HEK293 or SW480 cells than the wild-type FAF1 protein are listed in Tables 6 and 7 below.

[0209] HEK 293Ph021.205Ph031.235Ub011.234Ub021.321Ub041.386Ub061.441Ca011.223Ca031.382Ca041.218Ph01 Ca021.407Ph02Ca031.273Ph03Ub051.279Ub05Ca031.302Tr011.324Tr021.519Tr031.305Ub05Tr031.297

[0210]

[0211] SW480Ca031.204Ph01Ca021.200Ph03Ub051.239Tr011.287

[0212]

[0213] Specifically, all of the Ph02, Ph03, Ub01, Ub02, Ub04, Ub06, Ca01, Ca03, Ca04, Ph01Ca02, Ph02Ca03 Ph03Ub05, Ub05Ca03, Tr01, Tr02, Tr03, and Ub05Tr03 FAF1 protein mutants were found to have a higher apoptosis-inducing ability in HEK293 cells than the wild-type FAF1 protein, and among them, Tr02 was found to be the most effective (Table 6 and Fig. 13).

[0214] Additionally, Ca03, Ph01Ca02, Ph03Ub05, and Tr01 FAF1 protein mutants were all shown to have a higher apoptosis-inducing ability in SW480 cells than the wild-type FAF1 protein, and among them, Tr01 was shown to be the most effective (Table 7 and Fig. 14).

[0215] Example 5.2. Anticancer efficacy (in vivo)

[0216] Human colon cancer cell line SW480 was maintained by subculturing every 3 to 4 days in RPMI-1640 medium containing serum and antibiotics at 37°C and 5% CO2.

[0217] Each of the wild-type FAF1 Plasmid DNA, FAF1 Ca01 Plasmid DNA, FAF1 Ca02 Plasmid DNA, FAF1 Ca03 Plasmid DNA and vehicle plasmid (VC) containing eGFP prepared in Example 4.1 above were mixed with BioT® and transfected into SW480 cells seeded in a 150 Φ culture dish. 24 hours after the transfection, 5 × 10 SW480 cells were transfected. 6Six-week-old male BALB / c nude mice were transplanted with a 26G syringe into the right dorsal flank with 150 μl (1:1) of a mixture of PBS and Matrigel® (corning). Tumor volume was measured twice a week starting one week after cell transplantation, and tumor tissue volume and weight were measured on the 36th day after the experiment was terminated.

[0218] The results of the analysis of changes in tumor tissue volume and weight are shown in Fig. 15. As shown in Fig. 15, the tumor volume and tumor weight were similar or lower in animals transplanted with SW480 cells transduced with FAF1 Ca01 Plasmid DNA or FAF1 Ca02 Plasmid DNA compared to animals transplanted with SW480 cells without transduction, SW480 cells transduced with wild-type FAF1 Plasmid DNA, or SW480 cells transduced with VC.

[0219] Example 5.3. Apoptosis induction (in vitro)

[0220] To analyze the apoptosis-inducing ability of FAF1 protein variants in other cancer types, human cervical cancer cell line HeLa, human hepatoma cell line Hep3B, human pancreatic cancer cell line MIA PaCa-2, human lung cancer cell line A549, and human breast cancer cell line MDA-MB-231 were maintained in culture medium containing serum and antibiotics at 5% CO2 and 37°C with 3- to 4-day cycles of subculture. HeLa, Hep3B, and MIA PaCa-2 were cultured in DMEM, and A549 and MDA-MB-231 were cultured in RPMI-1640.

[0221] 2 × 10 in a 12-well scale cell culture dish 5 dog / well to 3 × 10 5HeLa cells, Hep3B cells, MIA PaCa-2 cells, A549 cells, or MDA-MB-231 cells were each inoculated per well, and 24 hours after inoculation, a mixture of 1.0 μg of the plasmid DNA prepared in Example 4.1 and 1.5 μl of BioT® (Bioland) was treated to HeLa cells, Hep3B cells, MIA PaCa-2 cells, A549 cells, or MDA-MB-231 cells to complete transformation. The transformed cells were cultured for 48 hours, suspended, and the cell viability of each group was measured using the Muse® Count& Viability Kit (CYTEK®). The results are shown in Tables 8 to 12 and Figures 16 to 20.

[0222] HeLaPh011.767Ub011.315Ub031.320Ub051.269Ub061.680Ca021.457Ca031.239Ca0 41.537Ph01Ca021.238Ph02Ca031.518Ph03Ub051.845Ub05Ca031.695Ub05Tr031.497

[0223]

[0224] Hep3BPh031.403Ub021.183Tr031.197Ub05Tr031.290

[0225]

[0226] MIA-PaCa-2Ub011.191Ub021.249

[0227]

[0228] A549Ph021.275Ub031.187Ub041.348Ca011.164Ph01Ca021.475

[0229]

[0230] MDA-MB-231Ph021.494Ca021.257Ph01Ca021.235Ph02Ca031.182

[0231]

[0232] Specifically, all of the Ph01, Ub01, Ub03, Ub05, Ub06, Ca02, Ca03, Ca04, Ph01Ca02, Ph02Ca03, Ph03Ub05, Ub05Ca03, and Ub05Tr03 FAF1 protein mutants were found to have a higher apoptosis-inducing ability in HeLa cells than the wild-type FAF1 protein, and among them, Ph03Ub05 was found to be the most effective (Table 8 and Figure 16).

[0233] Additionally, the Ph03, Ub02, Tr03, and Ub05Tr03 FAF1 protein mutants were all found to have a higher apoptosis-inducing ability in Hep3B cells than the wild-type FAF1 protein, with Ph03 being the most effective (Table 9 and Figure 17).

[0234] In addition, both Ub01 and Ub02 FAF1 protein mutants were shown to have a higher apoptosis-inducing ability in MIA-PaCa-2 cells than the wild-type FAF1 protein, and among them, Ub02 was shown to be the most effective (Table 10 and Figure 18).

[0235] Regarding the ability to induce A549 cell death, the Ph02, Ub04, Ca01, and Ph01Ca02 FAF1 protein mutants were all found to be higher than the wild-type FAF1 protein, and among them, Ph01Ca02 was found to be the most effective (Table 11 and Fig. 19).

[0236] Regarding the ability to induce MDA-MB-231 cell death, the Ph02, Ca02, and Ph01Ca02 FAF1 protein mutants were all found to be higher than the wild-type FAF1 protein, with Ph02 being the most effective (Table 12 and Fig. 20).

[0237] As a result, when the average apoptosis-inducing ability of each FAF1 protein mutant was compared, Ph01Ca02 was found to be the most effective, with a 20.5% increase in the overall apoptosis-inducing ability compared to the wild-type FAF1 protein.

[0238] Example 6. Preparation of a viral vector containing a FAF1 truncated form mutant and its efficacy

[0239] Example 6.1. Production of viruses containing FAF1 protein variants

[0240] To confirm the efficacy of the FAF1 protein mutant (Tr03) using a viral vector, we commissioned VIROVEK (USA) to produce adeno-associated virus 2 (AAV2) containing the nucleotide sequence of the FAF1 protein mutant (Tr03) and AAV2 containing wild-type FAF1 or GFP. The CMV promoter was used in the preparation of the vector for AAV2 production, and the viruses produced from the viral vector containing the wild-type FAF1 nucleotide sequence, the AAV2-CMV-Tr03 vector containing the nucleotide sequence of the FAF1 protein mutant (Tr03), and the AAV2-GFP vector containing GFP were purified using the final buffer (1x PBS, 0.001% pluronic F-68, and 100 mM sodium citrate).

[0241] Example 6.2. Protein expression following transduction of a viral vector containing a FAF1 protein variant.

[0242] Human embryonic kidney-derived cell line HEK-293 was maintained by subculturing every 3 to 4 days in MEM medium containing serum and antibiotics at 37°C and 5% CO2. 3 × 10 5HEK-293 cells were inoculated with 1 × 10 cells / well. 24 hours after inoculation, 1 × 10 cells / well were inoculated with the prepared GFP-AAV, FAF1 WT-AAV, or Tr03-AAV. 5 HEK-293 cells were transduced at a concentration of vg / cell. Protein samples were isolated from HEK-293 cells 5 days after transduction. Protein expression levels were analyzed by Western blot, and the results are shown in Figure 21.

[0243] Example 6.3. Efficacy of FAF1 protein variants (in vitro)

[0244] Human colon cancer cell line SW480 was maintained in RPMI-1640 medium containing serum and antibiotics at 37°C with 5% CO2, with 3- to 4-day subculture cycles. 2 × 10 5 SW480 cells were seeded with 1 × 10 / well and cultured for 24 hours. Wild-type FAF1-AAV, Tr03-AAV, or GFP-AAV prepared in Example 6.1 were added at a density of 1 × 10 6 SW480 cells were treated at a concentration of 100 vg / cell. Transfected SW480 cells cultured for 6 days after AAV treatment were suspended, stained with propidium iodide (PI), and labeled with Guava® easyCyte TM Analysis was performed using a Flow Cytometer (CYTEK®). The analysis results are shown in Figure 22.

[0245] As confirmed in Figure 22, Tr03-AAV showed superior apoptosis induction ability compared to FAF1-AAV or GFP-AAV. Therefore, the FAF1 protein mutant (Tr03) maintained apoptosis induction ability despite being a mutant of the wild-type FAF1 protein, and even showed superior apoptosis induction ability compared to the wild-type FAF1.

Claims

A polynucleotide encoding the FAF1 protein, which has 80% or more sequence homology with the nucleotide sequence of SEQ ID NO:

3. A FAF1 protein variant having 80% or more sequence homology to the FAF1 protein having an amino acid sequence of sequence number 5. In the second paragraph, a FAF1 protein variant characterized by having a mutation selected from the group consisting of (1) to (21): (1) Substitution of serine (S), amino acid positions 289 and 291 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, with aspartic acid (D); (2) Substitution of serine (S), the 582nd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, with alanine (A); (3) Serine (S), which is the 289th and 291st amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with aspartic acid (D), and serine (S), which is the 582nd amino acid, is substituted with alanine (A); (4) Lysine (K), which is the 139th, 143rd, and 146th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R); (5) Lysine (K), the 157th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R); (6) Lysine (K), the 163rd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R); (7) Lysine (K), the 221st amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R); (8) Lysine (K), the 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with arginine (R); (9) Lysine (K), which is the 163rd, 221st, and 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R); (10) Aspartic acid (D), which is the 149th and 152nd amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A); (11) Aspartic acid (D), which is the 292nd, 295th, and 298th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A); (12) Aspartic acid (D), which is the 494th and 497th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is replaced with alanine (A); (13) Aspartic acid (D), which is an amino acid at positions 494 and 497 from the N-terminus of the FAF1 protein consisting of an amino acid sequence of sequence number 5, is substituted with alanine (A), and aspartic acid (D), which is an amino acid at positions 292, 295, and 298, is substituted with alanine (A); (14) The 289th and 291st amino acids of the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, serine (S), are substituted with aspartic acid (D), and the 292nd, 295th and 298th amino acids, aspartic acid (D), are substituted with alanine (A); (15) Aspartic acid (D), which is the 494th and 497th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with alanine (A), and serine (S), which is the 582nd amino acid, is substituted with alanine (A); (16) From the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, the 289th and 291st amino acids, serine (S), are substituted with aspartic acid (D), the 444th amino acid, lysine (K), is substituted with arginine (R), and the 582nd amino acid, serine (S), is substituted with alanine (A); (17) Lysine (K), the 444th amino acid from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5, is substituted with arginine (R), and aspartic acid (D), the 494th and 497th amino acids, is substituted with alanine (A); (18) Deletion of amino acids 1 to 87 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5; (19) Deletion of amino acids 1 to 139 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5; (20) Deletion of amino acids 1 to 286 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5; and (21) Amino acids 1 to 286 from the N-terminus of the FAF1 protein consisting of the amino acid sequence of sequence number 5 are deleted, and the 444th amino acid, lysine (K), is replaced with arginine (R). In the third paragraph, the FAF1 protein variant comprises a polypeptide consisting of an amino acid sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47. In the third paragraph, the FAF1 protein variant is encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46. A polynucleotide encoding a FAF1 protein consisting of an amino acid sequence of sequence number 5; Polynucleotide of the first paragraph; A FAF1 protein variant according to any one of claims 2 to 5; or A polynucleotide encoding a FAF1 protein variant according to any one of claims 2 to 5. A carrier containing . In claim 6, the carrier is one selected from the group consisting of a viral vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a bacterial artificial chromosomes (BACs), a yeast artificial chromosomes (YACs), a liposome, an extracellular vesicle, a nanoparticle, a peptide-based vector, and a polymer-based vector. In claim 7, the extracellular vesicle is a delivery vehicle selected from the group consisting of exosomes, microvesicles, ectosomes, oncosomes, and prostasomes. As a pharmaceutical composition for preventing or treating cancer, Polynucleotide of the first paragraph; A FAF1 protein variant according to any one of claims 2 to 5; A polynucleotide encoding a FAF1 protein variant of any one of claims 2 to 5; or The carrier of any one of the clauses 6 to 8 A pharmaceutical composition comprising as an active ingredient. In claim 9, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, melanoma, sarcoma, brain tumor, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancer, glioblastoma, ocular cancer, vaginal cancer, vulvar cancer, cervical cancer, endometrial carcinoma, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, basal cancer, salivary gland cancer, pharyngeal cancer, skin cancer, kidney cancer, Wilms' tumor, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, and urethral cancer. As a composition for inducing cell death, Polynucleotide of the first paragraph; A FAF1 protein variant according to any one of claims 2 to 5; A polynucleotide encoding a FAF1 protein variant of any one of claims 2 to 5; or The carrier of any one of the clauses 6 to 8 A composition comprising as an active ingredient. In Article 11, A composition wherein the cells are selected from the group consisting of HEK 293 cells, SW480 cells, HeLa cells, Hep3B cells, MIA-PaCa-2 cells, A549 cells, and MDA-MB-231 cells. As a kit for preventing or treating cancer, Instruction manual; and A kit comprising the pharmaceutical composition of claim 9 or 10. Polynucleotide of the first paragraph; A FAF1 protein variant according to any one of claims 2 to 5; A polynucleotide encoding a FAF1 protein variant of any one of claims 2 to 5; or A method for preventing or treating cancer, comprising administering to a subject a therapeutically effective amount of a carrier according to any one of claims 6 to 8. A therapeutically effective amount for the prevention or treatment of cancer Polynucleotide of the first paragraph; A FAF1 protein variant according to any one of claims 2 to 5; A polynucleotide encoding a FAF1 protein variant of any one of claims 2 to 5; or Use of the carrier according to any one of claims 6 to 8. A therapeutically effective amount of a drug for the prevention or treatment of cancer Polynucleotide of the first paragraph; A FAF1 protein variant according to any one of claims 2 to 5; A polynucleotide encoding a FAF1 protein variant of any one of claims 2 to 5; or Use of the carrier according to any one of claims 6 to 8.

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