Expression Cassette for Production of High-Expression and High-Functionality Target Protein and Use Thereof

Patent metadata

Field	Value
Publication number	US-2020299721-A1
Application number	US-201816755453-A
Country	US
Kind code	A1
Filing date	Oct 11, 2018
Priority date	Oct 11, 2017
Publication date	Sep 24, 2020
Grant date	—

Abstract

The present invention relates to an expression cassette for a target protein, comprising a promoter, a polynucleotide coding for the target protein, an intron sequence, and a poly A sequence, an expression vector, and a transformant. The expression cassette for a target protein according to the present invention can simultaneously perform the expression of the intron sequence and the target protein through one transduction and exhibits the effect of inducing the high expression and high functionality of the target protein by regulating the expression of an endogenous gene.

First claim

1 . An expression cassette for production of a target protein, comprising a promoter, a polynucleotide coding for the target protein, an intronic RNA sequence and a poly A sequence, wherein the intronic RNA sequence comprises a splicing donor, a branch and a splicing acceptor.

2 . The expression cassette for production of a target protein of claim 1 , wherein the target protein is an antibody or a fragment thereof.

3 . The expression cassette for production of a target protein of claim 1 , wherein at least one intronic RNA sequence is present on a single target protein expression cassette.

4 . The expression cassette for production of a target protein of claim 1 , wherein the expression cassette for production of a target protein comprises at least one intronic RNA sequence selected from the group consisting of:
a) at least one intronic RNA sequence located between a promoter and a polynucleotide coding for the target protein; b) at least one intronic RNA sequence located between a polynucleotide coding for the target protein and a poly A; and c) at least one intronic RNA sequence located between a promoter and a polynucleotide coding for the target protein and at least one intronic RNA sequence located between a polynucleotide coding for the target protein and a poly A.

5 . The expression cassette for production of a target protein of claim 1 , wherein the intronic RNA sequence further comprises an RNA sequence for target gene expression regulation.

6 . The expression cassette for production of a target protein of claim 5 , wherein the target gene is at least one selected from the group consisting of FUT8 (Alpha-1,6-fucosyltransferase), HDAC5 (Histone Deacetylase 5), LDHA (Lactate dehydrogenase A), CXCR4 (C—X—C chemokine receptor type 4), DHFR (Dihydrofolate reductase), PDK4 (Pyruvate dehydrogenase lipoamide kinase isozyme 4), MAPK3 (Mitogen-activated protein kinase 3), KANK4 (KN Motif And Ankyrin Repeat Domains 4), PDI (Protein disulfide isomerase), CNX (Calnexin), CRT (Calreticulin), elF2alpha (Non-phosphorylatable version of the eukaryotic translation initiation factor 2 alpha), ZFP-TF (Artificial zinc finger protein transcription factor), ATF4 (Activating transcription factor 4), GADD34 (Growth arrest and DNA damage inducible protein 34), mTOR (Mammalian target of rapamycin), BIP (Heat shock 70 kDa protein 5), ATF6C (Activating transcription factor 6C), XBP1 (X-box binding protein 1), BCL2 (B-cell lymphoma 2), BCLxL (BCL2-like 1), Mutated form of BCL-xL (Asp29Asn variant), XIAP (X-linked inhibitor of apoptosis), a mutant form of XIAP (EAX197), AVEN (Apoptosis, caspase inhibitor), C-MYC (Myelocytomatosis oncogene), FAIM (Fas apoptotic inhibitory molecule), 30Kc6 (Apoptosis-inhibiting 30K protein), TERT (Telomerase reverse transcriptase), E1B-19K (Control protein E1B 19K), MDM2 (Murine double-mutant 2), E2F1 (E2F transcription factor 1), HSP27 (Heat shock proteins 27), HSP70 (Heat shock proteins 70), MCL1 (Myeliod cell leukemia 1), AKT1 (RAC-alpha serine/threonineprotein kinase), Beclin-1, ST6GAL (Alpha 2,6 sialyltransferase), GnT-IV (Alpha-1,3-D-mannoside beta 1,4 Nacetylglucosaminyltransferase), GnT-V (alpha 1,6 Dmannoside beta-1,6 Nacetylglucosaminyltransferase), ST3GAL (Alpha 2,3 sialyltransferase), GalT (beta 1,4 galactosyltransferase), CMP-SAT (CMP-sialic acid transporter), CMP-SAS (CMP-sialic acid synthetase), GNE (Mutant uridine diphosphate-N-acetyl glucosamine 2-epimerase), GnT-III (Beta 1,4 Nacetylglucosaminyltransferase III), ManII (Golgi alphamannosidase II), C2GnT (Beta 1,6 Nacetylglucosaminyltransferase), RMD (GDP-6-deoxy-d-lyxo-4-hexulose reductase), VHb ( Vitreoscilla hemoglobin), CPS I (Carbamoyl phosphate synthetase I), OTC (Ornithine transcarbamoylase), PC (Pyruvate carboxylase), GLUT5 (Glucose transporter protein 5), MDH2 (Malate dehydrogenase II), TAUT (Taurine transporter), ALT1 (Alanine aminotransferase 1), XBP1 (X-box binding protein 1), XBP1s (Spliced form of XBP-1), SLY1 (Suppressor of loss of YPT1 protein 1), MUNC18C (syntaxin binding protein 3), CERT (Ceramide transfer protein), Mutant form of CERT (S132A), SNAP-23 (Synaptosome-associated protein of 23 kDa), VAMP8 (Vesicle-associated membrane protein 8), SRP14 (Human signaling receptor protein 14), p21CIP1 (Cyclin-dependent kinase Inhibitor 1A), C/EBP-alpha (CCAAT/enhancer-binding protein alpha), p27KIP1 (Cyclin-dependent kinase inhibitor 1B), CDKL3 (Cyclin-dependent kinase like 3), COX15 (Cytochrome c oxidase subunit), VCP (Valosin-containing protein), BAX (BCL2-associated X protein), BAK (BCL2-antagonist/killer), GS (Glutamine synthetase), MGAT1 (N-acetylglucosaminyltransferase 1), SLC35C1 (GDP-fucose transporter), SLC35A1 (CMPsialic acid transporter), B4GALT1 (Beta 1,4 galactosyltransferase 1), B3GNT2 (Beta 1,3 Nacetylglucosaminyltransferase 2), PAM (Peptidylglycine alphaamidating monooxygenase), Caspase 3, Caspase 7, Caspase 8, Caspase 9, ALG2 (Alpha-1,3/1,6-mannosyltransferase), REQ (Requiem), FADD (Fas(TNFRSF6)-associated via death domain), FAIM (Fas apoptotic inhibitory molecule), NEU2 (Sialidase 2), NEU1 (Sialidases 1), NEU3 (Sialidases 3), GMD (GDP-fucose 4,6-dehydratase), GFT (GDP-fucose transporter), CFL1 (Cofilin), ATR (Ataxia telangiectasia and Rad3 related), ENO1 (Enolase 1) and PDHK (Pyruvate dehydrogenase kinase).

7 . The expression cassette for production of a target protein of claim 5 , wherein the intronic RNA sequence is any one selected from the group consisting of shRNA (short hairpin RNA), miRNA (micro RNA), stRNA (small temporal RNA), siRNA (small interfering RNA), piRNA (piwi-interacting RNA), snoRNA (small nucleolar RNA), snRNA (small nuclear RNA), exRNA (extracellular RNA), scaRNA (small Cajal body RNA), lncRNA (long noncoding RNA), smRNA (small modulatory dsRNA), and snRNA (small noncoding RNA).

8 . The expression cassette for production of a target protein of claim 7 , wherein the shRNA sequence for target gene expression regulation is at least one selected from the group consisting of intronic FUT8 shRNA represented by SEQ ID NO: 24, intronic HDAC5 (histone deacetylase 5) shRNA represented by any one of SEQ ID NOS: 25 to 27, intronic LDHA (lactate dehydrogenase A) shRNA represented by any one of SEQ ID NOS: 28 to 30, and intronic DHFR (dihydrofolate reductase) shRNA represented by any one of SEQ ID NOS: 31 to 33.

9 . The expression cassette for production of a target protein of claim 7 , wherein the miRNA sequence for target gene regulation is miR483 represented by any one of SEQ ID NOS: 34 to 36.

10 . The expression cassette for production of a target protein of claim 1 , wherein the splicing donor sequence is at least one selected from among SEQ ID NOS: 12 to 15,
the branch sequence is at least one selected from among SEQ ID NOS: 16 to 18, and the splicing acceptor sequence is at least one selected from among SEQ ID NOS: 19 to 22.

11 . The expression cassette for production of a target protein of claim 1 , wherein the expression cassette for production of a target protein has at least one effect selected from the group consisting of inducing the high expression of the target protein, inhibition of lactate production, regulating of histone protein deacetylation, regulating of glucose metabolism, regulating of cell growth, regulating of cell proliferation, inducing the high functionality of the target protein, and decreased fucose content of a target protein, through intron splicing or endogenous gene expression regulation.

12 . A vector comprising the expression cassette for production of a target protein of claim 1 .

13 . A transformant transformed with the vector of claim 12 .

14 . The transformant of claim 13 , wherein the trans

Assignees

• Celltrion Inc

Inventors

• Kim Man Su
• Kim Min Soo
• Cho Jong Moon
• Chang Shin Jae

Classifications

• C07K16/11 Primary

  Paramyxoviridae (F); Pneumoviridae (F), e.g. respiratory syncytial virus [RSV] · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12N15/85 Primary

  for animal cells · CPC title