Novel intron fragments

Abstract

To provide novel intron fragments capable of increasing the expression amounts of foreign genes.SOLUTION: An intron fragment of the invention can increase gene expression to an equal or higher level than the level achieved by a full-length intron while maintaining the ability to increase gene expression even when combined with various types of promoters and splicing donor. Particularly, the intron fragment can load a larger foreign gene when used in a size restricted genetic information transfer system such as adeno-associated virus (AAV) and rhabdovirus. Thus, the use of the intron fragment is expected to extend the range of therapeutic genes.SELECTED DRAWING: Figure 6A

Description

Detailed Description of the Invention

[0001] [Technical Field] The present disclosure relates to novel intron fragments (ie, untranslated nucleic acid sequences) that can increase the expression level of foreign genes.

[0002] [Background technology] Eukaryotic pre-mRNAs are composed of exons containing actual genetic information and introns located between exons, with poly(A) sequences at the 3'-end. Introns affect mRNA alternative splicing and regulate protein production (Huh, G.S., et al., "Regulation of Alternative Pre-mRNA Splicing by a Novel Repeated Hexanucleotide Element," Genes Dev 8(13):1561-74 (Jul. 1994); Parenteau, J., et al., "Introns Within Ribosomal Protein Genes Regulate the Production and Function of Yeast Ribosomes," Cell 147(2):320-31 (Oct. 2011)). Furthermore, it has been reported that foreign genes (transgenes) containing introns are transcribed 10 to 100 times more efficiently in mice than those without introns, further affecting their survival (Brinster, R.L., et al., “Introns Increase Transcriptional Efficiency in Transgenic Mice,” Proc. Natl. Acad. Sci. USA. 85(3):836-40 (Feb. 1988); Parenteau, J., et al., “Introns Are Mediators of Cell Response to Starvation,” Nature 565(7741):612-617 (Jan. 2019)). The increase in protein production due to introns is called intron-mediated enhancement (IME), but the introns used for this IME effect are usually quite long, limiting its practical use.For example, adeno-associated viruses (AAVs), which have recently attracted attention as vehicles for gene therapy, can only insert genetic information up to 4.7 kbp long, so the range of genes that can be transferred is very limited if long introns are used. Therefore, it would be highly desirable to find and apply short intron fragments to AAV vectors while maintaining the ability to increase protein expression.

[0003] Summary of the Invention [Problem to be solved by the invention] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Korean Patent Application No. 10-2020-0084038, filed on July 8, 2020, and U.S. Patent Application No. 17 / 365,884, filed on July 1, 2021, the entire texts of which are incorporated herein by reference.

[0004] Provided herein are isolated polynucleotides comprising an untranslated nucleic acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 874 to 924 of SEQ ID NO: 1, wherein the untranslated nucleic acid sequence does not comprise SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 57. In some embodiments, the untranslated nucleic acid sequence consists of the nucleotide sequence set forth in SEQ ID NO: 57.

[0005] Provided herein are isolated polynucleotides comprising an untranslated nucleic acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 852-924 of SEQ ID NO: 1, wherein the untranslated nucleic acid sequence does not comprise SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the untranslated nucleic acid sequence consists of the nucleotide sequence set forth in SEQ ID NO: 3.

[0006] Provided herein are isolated polynucleotides comprising an untranslated nucleic acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 830 to 924 of SEQ ID NO: 1, wherein the untranslated nucleic acid sequence does not comprise SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the untranslated nucleic acid sequence consists of the nucleotide sequence set forth in SEQ ID NO: 2.

[0007] In some embodiments, the polynucleotides described herein further comprise at least 1, at least 2, at least 3, at least 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at the 5' end of the untranslated nucleic acid sequence (the "5' region"). In some embodiments, the polynucleotides comprise one or more contiguous or non-contiguous nucleotides corresponding to positions 1-873 of SEQ ID NO:1 in the 5' region of the untranslated nucleic acid sequence. In some embodiments, the polynucleotides described herein further comprise at least 1, at least 2, at least 3, at least 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at the 3' end of the untranslated nucleic acid sequence (the "3' region").In some embodiments, the untranslated nucleic acid sequence of a polynucleotide described herein is selected from the group consisting of: (i) nucleotides 871 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:58), (ii) nucleotides 861 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:59), (iii) nucleotides 852 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:60), (iv) nucleotides 851 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:61), (v) nucleotides 830 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:2), (vi) nucleotides 821 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:63), (vii) nucleotides 811 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:64), (viii) nucleotides 808 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:65), (ix) nucleotides 801 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:66), and (x) nucleotides 751 to 924 of SEQ ID NO:1. nucleotides at positions 721 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:67), (xi) nucleotides at positions 721 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:68), (xii) nucleotides at positions 701 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:69), (xiii) nucleotides at positions 651 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:70), (xiv) nucleotides at positions 601 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:71), (xv) nucleotides at positions 570 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:72), (xvi) nucleotides at positions 551 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:73), or (xvii) nucleotides at positions 501 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:74).

[0008] In some embodiments, the polynucleotides described herein further comprise an exogenous gene, which in some embodiments is capable of being translated into a polypeptide.

[0009] In some embodiments, the untranslated nucleic acid sequence of the polynucleotides described herein can increase expression of the exogenous gene upon translation compared to baseline expression, where baseline expression includes expression of the exogenous gene upon translation in the absence of the untranslated nucleic acid sequence and / or in the presence of a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence can increase expression of the exogenous gene by at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold over baseline expression.

[0010] In some embodiments, the polynucleotides described herein further comprise a promoter, such as a cytomegalovirus (CMV) promoter, an EF-1α promoter, a β-actin promoter, a GAPDH promoter, an HSP70 promoter, a GRP78 promoter, an eIF4a promoter, an AAT promoter, a TTR promoter, a GFAP promoter, an SV40 promoter, a SYN1 promoter, a GRK promoter, a Rho promoter, or any combination thereof.

[0011] In some embodiments, the promoter is an EF-1α promoter. In some embodiments, the EF-1α promoter comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the CMV promoter comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the promoter comprises a β-actin promoter. In some embodiments, the β-actin promoter comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:8.

[0012] In some embodiments, the polynucleotides described herein further comprise an enhancer. In some embodiments, the enhancer comprises a cytomegalovirus (CMV) enhancer, an SV40 early enhancer, an adenovirus 5 E1A enhancer, an HBV enhancer-1 regulatory region (Eh-1), an HPV-16 or -18 E6 / 7 long control region (LCR), an HIV-1 long terminal repeat (LTR), or any combination thereof. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the CMV enhancer comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:4.

[0013] In some embodiments, the polynucleotides described herein further comprise a splicing donor sequence. In some embodiments, the splicing donor sequence is linked upstream of the untranslated nucleic acid sequence. In some embodiments, the splicing donor sequence comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:10.

[0014] In some embodiments, the polynucleotides described herein further comprise an EF-1α exon 2 (E2) nucleotide sequence, which has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:11.

[0015] In some embodiments, the polynucleotides described herein further comprise a cytomegalovirus (CMV) exon 1 (E1) sequence, an EF-1α E1 sequence, a β-actin E1 sequence, or any combination thereof. In some embodiments, the CMV E1 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, the EF-1α E1 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the β-actin E1 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0016] In some embodiments, the polynucleotides described herein further comprise at least one target sequence of an immune cell-specific microRNA (miRNA). In some embodiments, the miRNA comprises miR142-3p, miR142-5p, or both. In some embodiments, the target sequence of the miRNA can be an antisense oligonucleotide, an antagomir, a short hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule, a ribozyme, a peptide nucleic acid (PNA) oligonucleotide, a locked nucleic acid (LNA), or a ribozyme. In some embodiments, the miRNA target sequence comprises a nucleotide sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 17.

[0017] In some embodiments, the polynucleotides described herein comprise at least two target sequences, at least three target sequences, at least four target sequences, at least five target sequences, at least six target sequences, at least seven target sequences, at least eight target sequences, a target sequence, at least nine target sequences, or at least ten target sequences. In some embodiments, two or more of the target sequences are the same. In some embodiments, the target sequences are different from one another.

[0018] In some embodiments, the polynucleotides described herein comprise a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:18.

[0019] In some embodiments, the polynucleotides described herein further comprise one or more polyadenylation (pA) sequences, which in some embodiments have at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a nucleotide sequence set forth in any of SEQ ID NOs: 19-22.

[0020] When the polynucleotide comprises a foreign gene, in some embodiments, the foreign gene encodes a wild-type polypeptide or any variant thereof, a fusion protein, an antibody or antigen-binding fragment thereof, an RNA-based molecule, or any combination thereof. In some embodiments, the foreign gene comprises a nucleotide sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 23. In some embodiments, the foreign gene encodes a fusion protein. In some embodiments, the fusion protein comprises a vascular endothelial growth factor ("VEGF") inhibitor. In some embodiments, the VEGF inhibitor comprises aflibercept. When the foreign gene is an RNA-based molecule, in some embodiments, the RNA-based molecule comprises an miRNA, shRNA, siRNA, ribozyme, or any combination thereof.

[0021] In some aspects, the polynucleotides described herein are recombinant expression constructs.

[0022] Also provided herein is a polynucleotide comprising (i) a foreign gene and (ii) a regulatory element operably linked to the foreign gene, wherein the regulatory element is (in a 5' to 3' direction): (1) a CMV enhancer as set forth in SEQ ID NO:4; (2) a promoter selected from the CMV promoter sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, the EF-1α promoter sequence as set forth in SEQ ID NO:7, or the chicken β-actin promoter sequence as set forth in SEQ ID NO:8; (3) a CMV enhancer as set forth in SEQ ID NO:12. (4) an exon 1 (E1) sequence selected from the group consisting of the EF-1α E1 sequence set forth in SEQ ID NO:13, the EF-1α E1 sequence set forth in SEQ ID NO:14 or SEQ ID NO:15, the chicken β-actin E1 sequence set forth in SEQ ID NO:9 or SEQ ID NO:10; (5) a non-translated nucleic acid sequence comprising, consisting essentially of, or consisting of the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:57; and (6) a polynucleotide comprising the EF-1α E2 sequence set forth in SEQ ID NO:11.

[0023] The present specification provides a vector comprising any of the polynucleotides of the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises an adenovirus (e.g., a genetically engineered adenovirus), an adeno-associated virus (AAV), a lentivirus, an SV40 virus, a polyomavirus, an Epstein-Barr virus, a papillomavirus, a herpes simplex virus (HSV), a vaccinia virus, a poliovirus, a baculovirus, a retrovirus, a box virus, or any combination thereof. In some embodiments, the viral vector is an AAV.

[0024] In some embodiments, the viral vectors described herein are for use in gene therapy. In some embodiments, the viral vectors are for use in expressing a polypeptide encoded by a foreign gene of a polynucleotide described herein. In some embodiments, the foreign gene comprises the nucleotide sequence set forth in SEQ ID NO:23.

[0025] The present specification provides a cell comprising any of the polynucleotides or vectors described herein.

[0026] The present disclosure also provides methods for producing recombinant viral particles, comprising transducing cells with a construct comprising any of the vectors described herein and the rep and cap genes, and in some embodiments, the method further comprises isolating the recombinant viral particles produced.

[0027] The present specification provides a recombinant viral particle produced by the method. The present specification also provides a recombinant viral particle comprising (a) a capsid protein and (b) any of the vectors described herein. In some embodiments, the recombinant viral particle is an adeno-associated virus (AVV). In some embodiments, the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrhlO. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV5. In some embodiments, the AAV serotype is AAV9.

[0028] The present disclosure further provides a pharmaceutical composition comprising: (a) any polynucleotide, vector, cell, or recombinant viral particle described herein; and (b) a pharmaceutically acceptable excipient.

[0029] The present specification further provides a pharmaceutical composition comprising a recombinant adeno-associated virus particle and a pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus particle comprises (a) an AAV8 type capsid protein and (b) (i) a foreign gene comprising the nucleotide sequence set forth in SEQ ID NO:23, and (ii) a polynucleotide operably linked to the foreign gene (in a 5' to 3' direction) comprising: (1) a cytomegalovirus (CMV) enhancer sequence set forth in SEQ ID NO:4; (2) a chicken β-actin promoter sequence set forth in SEQ ID NO:8; (3) a chicken β-actin exon 1 (E1) sequence set forth in SEQ ID NO:15; (4) a chicken β-actin intron splicing donor sequence set forth in SEQ ID NO:10; (5) a non-translated nucleic acid sequence comprising, consisting essentially of, or consisting of the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:57; and (6) an EF-1α exon 2 (E2) sequence set forth in SEQ ID NO:11.

[0030] The present specification provides a pharmaceutical composition for preventing or treating an ophthalmic disease, comprising a recombinant adeno-associated virus particle and a pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus particle comprises (a) an AAV8 type capsid protein and (b) (i) a foreign gene comprising the nucleotide sequence set forth in SEQ ID NO:23, and (ii) a polynucleotide operably linked to the foreign gene (in a 5' to 3' direction) comprising: (1) a cytomegalovirus (CMV) enhancer sequence set forth in SEQ ID NO:4; (2) a chicken β-actin promoter sequence set forth in SEQ ID NO:8; (3) a chicken β-actin exon 1 (E1) sequence set forth in SEQ ID NO:15; (4) a chicken β-actin intron splicing donor sequence set forth in SEQ ID NO:10; (5) a non-translated nucleic acid sequence that comprises, consists essentially of, or consists of the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:57; and (6) an EF-1α exon 2 (E2) sequence set forth in SEQ ID NO:11.

[0031] In some embodiments, the ophthalmic disease comprises diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, or a combination thereof. In some embodiments, the macular degeneration comprises age-related macular degeneration (AMD).

[0032] Provided herein are methods for increasing expression of an exogenous gene in a cell, the method comprising contacting the cell with any of the polynucleotides, vectors, or recombinant viral particles described herein. In some embodiments, the contacting occurs in vivo. In some embodiments, the method comprises administering the polynucleotide, vector, or recombinant viral particle to a subject prior to the contacting. In some embodiments, the contacting occurs ex vivo. In some embodiments, expression of the exogenous gene is increased by at least about 1 fold, at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, or at least about 10 fold compared to corresponding expression in a reference cell, wherein the reference cell is contacted with a polynucleotide, vector, or recombinant viral particle that lacks the untranslated nucleic acid sequence or that includes the nucleotide sequence set forth in SEQ ID NO:1.

[0033] Provided herein are methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject any of the polynucleotides, vectors, or recombinant viral particles described herein. In some embodiments, the disease or disorder comprises an ophthalmic disease. In some embodiments, the ophthalmic disease comprises diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, or a combination thereof. In some embodiments, the macular degeneration comprises age-related macular degeneration (AMD). In some embodiments, the method comprises administering an additional therapeutic agent to the subject.

[0034] [Mode] First mode An elongation factor-1 alpha (EF-1α) intron fragment for use in expressing a foreign gene, comprising a truncated sequence of consecutive or non-consecutive nucleotides among the nucleotides in the EF-1α intron sequence shown in SEQ ID NO: 1, wherein (a) the EF-1α intron fragment essentially comprises nucleotides at positions 874 to 924 in the sequence shown in SEQ ID NO: 1, and (b) the EF-1α intron fragment increases foreign gene expression when present in an expression construct compared to an intron fragment consisting only of nucleotides at positions 874 to 924 in the sequence shown in SEQ ID NO: 1.

[0035] Second mode In a first embodiment, the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 873 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0036] Third Modality In a second aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 870 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0037] Fourth modality In a third aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 860 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0038] Fifth modality In a fourth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 851 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0039] 6th modality In a fifth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 850 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0040] 7th modality In a sixth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 829 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0041] Eighth modality In a seventh aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 820 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0042] The 9th modality In an eighth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 810 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0043] 10th modality In a ninth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 800 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0044] 11th modality In a tenth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 750 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0045] 12th modality In an eleventh aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 720 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0046] 13th modality In a twelfth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 700 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0047] 14th modality In a thirteenth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 650 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0048] 15th modality In a fourteenth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 600 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0049] 16th modality In a 15th aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 569 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0050] 17th modality In a sixteenth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 550 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0051] 18th modality In a seventeenth aspect, the EF-1α intron fragment, wherein the deletion of consecutive or non-consecutive nucleotides includes deletion of consecutive nucleotides among the nucleotides at positions 1 to 500 in the sequence shown in SEQ ID NO: 1, deletion of non-consecutive nucleotides that remain undeleted between the deleted nucleotides, or insertion of different types of nucleotides at the positions of the deleted consecutive or non-consecutive nucleotides.

[0052] 19th modality In a seventh embodiment, the EF-1α intron fragment comprises the nucleotide sequence set forth in SEQ ID NO:2.

[0053] 20th modality In a fifth embodiment, the EF-1α intron fragment comprises the nucleotide sequence set forth in SEQ ID NO:3.

[0054] 21st modality A recombinant expression construct for expressing a foreign gene, comprising (a) a foreign gene and (b) regulatory elements operably linked to the foreign gene, the regulatory elements including an enhancer, a promoter and an EF-1α intron fragment according to a first aspect, wherein the foreign gene is transcribed and translated from a host cell.

[0055] 22nd modality In a twenty-first embodiment, the recombinant expression construct, wherein the enhancer is a cytomegalovirus (CMV) enhancer.

[0056] 23rd modality In a twenty-second embodiment, the recombinant expression construct, wherein the cytomegalovirus (CMV) enhancer comprises the nucleotide sequence set forth in SEQ ID NO:4.

[0057] 24th modality In a twenty-first embodiment, the recombinant expression construct is one in which the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, an EF-1α promoter, and a β-actin promoter.

[0058] 25th modality In a twenty-fourth aspect, the recombinant expression construct, wherein the CMV promoter comprises the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0059] 26th modality In a twenty-fourth embodiment, the EF-1α promoter comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0060] Sequence No. 7 27th modality In a twenty-fourth embodiment, the recombinant expression construct wherein the β-actin promoter comprises the nucleotide sequence set forth in SEQ ID NO:8.

[0061] 28th modality In a twenty-first embodiment, the recombinant expression construct comprises a splicing donor sequence linked upstream of the EF-1α intron fragment.

[0062] 29th modality In a twenty-eighth embodiment, the recombinant expression construct comprises the splicing donor sequence as set forth in SEQ ID NO:9 or SEQ ID NO:10.

[0063] The 30th mode In a twenty-first embodiment, the recombinant expression construct comprises an EF-1α exon 2 (E2) sequence.

[0064] The 31st mode In a thirtieth embodiment, the recombinant expression construct wherein the EF-1α E2 sequence comprises the nucleotide sequence set forth in SEQ ID NO:11.

[0065] The 32nd modality In a twenty-first embodiment, the recombinant expression construct comprises a cytomegalovirus (CMV), EF-1α, or β-actin exon 1 (E1) sequence.

[0066] The 33rd modality In a thirty-second embodiment, the recombinant expression construct wherein the CMV E1 sequence comprises the nucleotide sequence set forth in SEQ ID NO:12.

[0067] 34th modality In a thirty-second embodiment, the recombinant expression construct wherein the EF-1α E1 sequence comprises the nucleotide sequence set forth in SEQ ID NO:13.

[0068] The 35th modality In a thirty-second embodiment, the recombinant expression construct comprises the β-actin E1 sequence set forth in SEQ ID NO:14 or SEQ ID NO:15.

[0069] The 36th modality In a twenty-first embodiment, the recombinant expression construct further comprises one or more target sequences for immune cell-specific microRNAs (miRNAs).

[0070] The 37th modality In a thirty-sixth embodiment, the recombinant expression construct is one in which the miRNA is miR142-3p or miR142-5.

[0071] The 38th modality In a 36th aspect, the recombinant expression construct is one in which the target sequence for the miRNA is selected from the group consisting of antisense oligonucleotides, antagomir, short hairpin RNA (shRNA) molecules, short interfering RNA (siRNA) molecules, ribozymes, peptide nucleic acid (PNA) oligonucleotides, and locked nucleic acid (LNA) oligonucleotides, each having a sequence complementary to the full length or a portion of the sequence of miR142-3p or miR142-5p.

[0072] The 39th modality In a thirty-eighth embodiment, the recombinant expression construct comprises a target sequence for miR142-3p comprising the nucleotide sequence set forth in SEQ ID NO:16.

[0073] 40th modality In a 38th embodiment, the number of target sequences of the miRNA is 2 to 6.

[0074] 41st modality In a fortieth embodiment, the recombinant expression construct comprises a target sequence for miR142-3p comprising the nucleotide sequence set forth in SEQ ID NO:17.

[0075] 42nd modality In a twenty-first embodiment, the recombinant expression construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence.

[0076] The 43rd modality In a forty-second embodiment, the recombinant expression construct comprises the WPRE sequence set forth in SEQ ID NO:18.

[0077] 44th modality In a twenty-first embodiment, the recombinant expression construct further comprises one or more polyadenylation (pA) sequences.

[0078] The 45th modality In a 44th embodiment, the recombinant expression construct wherein the polyadenylation sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 19-22.

[0079] 46th modality In a 21st aspect, the regulatory elements comprise: (1) a CMV enhancer sequence shown in SEQ ID NO: 4; (2) a promoter sequence selected from the CMV promoter sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, the EF-1α promoter sequence shown in SEQ ID NO: 7, or the chicken β-actin promoter sequence shown in SEQ ID NO: 8; (3) a CMV E1 sequence shown in SEQ ID NO: 12, the EF-1α E1 sequence shown in SEQ ID NO: 13, or the chicken β-actin E1 sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; (4) a splicing donor sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10; (5) the EF-1α intron fragment sequence described in claim 1; and (6) an EF-1α E2 sequence shown in SEQ ID NO: 11.

[0080] The 47th modality In a twenty-first embodiment, the recombinant expression construct comprises the exogenous gene comprising the nucleotide sequence set forth in SEQ ID NO:23.

[0081] The 48th modality A host cell transfected with a recombinant expression construct according to aspect 21.

[0082] The 49th modality A method for producing a recombinant virus comprising transducing a host cell with a recombinant expression construct according to the 21st aspect and a construct comprising the rep and cap genes.

[0083] 50th modality A recombinant virus comprising (a) a capsid protein and (b) a recombinant expression construct according to the 21st aspect.

[0084] 51st modality In a 50th embodiment, the recombinant virus is an adeno-associated virus (AVV).

[0085] 52nd modality In a fifty-first embodiment, the adeno-associated virus is of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrhlO.

[0086] The 53rd modality A pharmaceutical composition comprising a recombinant expression construct according to the 21st aspect or a recombinant virus according to the 50th aspect.

[0087] 54th modality In a fifty-third embodiment, the pharmaceutical composition is used to prevent or treat an ophthalmic disease.

[0088] The 55th modality In a fifty-fourth embodiment, the pharmaceutical composition is characterized in that the ophthalmological disease is selected from the group consisting of diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, and macular swelling.

[0089] 56th modality Therapeutic use of a recombinant expression construct according to the 21st aspect or a recombinant virus according to the 50th aspect.

[0090] The 57th modality 1. A pharmaceutical composition for preventing or treating an ophthalmic disease, comprising a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus comprises (a) an AAV8 type capsid protein and (b) a foreign gene comprising the nucleotide sequence set forth in SEQ ID NO:23, and the foreign gene is operably linked to the foreign gene and comprises: (1) a cytomegalovirus (CMV) enhancer sequence set forth in SEQ ID NO:4; (2) a chicken β-actin promoter sequence set forth in SEQ ID NO:8; (3) a chicken β-actin exon 1 (E1) sequence set forth in SEQ ID NO:15; (4) a chicken β-actin intron splicing donor sequence set forth in SEQ ID NO:10; (5) an EF-1α intron fragment in which consecutive or non-consecutive nucleotides among the nucleotides at positions 1 to 829 in the sequence set forth in SEQ ID NO:1 are deleted and in which nucleotides at positions 830 to 924 in the sequence set forth in SEQ ID NO:1 are essentially present; and (6) an EF-1α exon 2 (E2) sequence set forth in SEQ ID NO:11.

[0091] [Means for solving the problem] The present disclosure relates generally to non-translated nucleic acid sequences (also referred to herein as "introns") and the use of such sequences for foreign gene expression. As described herein, Applicant has determined that certain fragments of the EF-1α intron sequence exhibit superior ability to increase foreign gene expression. As shown herein, in some aspects, these EF-1α intron fragments increase foreign gene expression at levels comparable to or greater than the full-length EF-1α intron. Further aspects of the present disclosure are provided throughout this application.

[0092] To facilitate understanding of the disclosure set forth herein, a number of terms and phrases are defined. Additional definitions are set forth throughout the detailed description.

[0093] I. Definition Throughout this disclosure, the terms "one" or "any" entity refer to one or more of that entity. For example, "a polypeptide" is understood to refer to one or more polypeptides. Thus, the terms "one" (or "any"), "one or more," and "at least one" can be used interchangeably herein.

[0094] Furthermore, "and / or" as used herein should be considered a specific disclosure of each of two particular features or components together with or alone the remaining other features or components. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include each of the following aspects: A, B and C; A, B or C; A or B; B or C; A and B; B and C; A (single); B (single); and C (single).

[0095] The term "at least" before a number or series of numbers is understood to include the number following the term "at least" and all subsequent numbers or integers that can be clearly and logically included in the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a series of numbers or ranges, it is understood that "at least" can modify each number in the series or range. "At least" is not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%, regardless of the number of significant digits).

[0096] When an aspect is described herein using the term "comprising," it is understood that other similar aspects described in terms of "comprising" and / or "essentially" are also provided.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, "Concise Dictionary of Biomedicine and Molecular Biology," Juo, Pei-Show, 2nd ed., 2002, CRC Press; "Dictionary of Cell and Molecular Biology," 3rd ed., 1999, Academic Press; and "Oxford Dictionary of Biochemistry and Molecular Biology," Revised, 2000, Oxford University Press, provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0098] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) recognized format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, amino acid sequences are written left to right in amino to carboxyl orientation. The headings provided herein are not limitations on the various aspects of the disclosure, which may be read by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0099] As used herein, the term "about" is used to mean approximately, roughly, in the region of, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it extends the boundaries above and below the numerical values ​​set forth to modify that range. In general, the term "about" can modify the numerical value above and below the specified value by, for example, a variance of 10% above or below (higher or lower).

[0100] The term "adeno-associated virus" (AAV) as used herein includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and phylogenetic branches disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), and other currently known or later discovered AAVs. See, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV" includes derivatives of known AAVs. In some embodiments, "AAV" includes modified or engineered AAVs.

[0101] The terms "administration," "administering," and grammatical variations thereof refer to the introduction of a composition (e.g., a polynucleotide comprising a foreign gene and untranslated nucleic acid sequence described herein) into a subject via a pharmaceutically acceptable route. The composition may be introduced into a subject by any appropriate route, including intratumoral, oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intraspinal, periocular, or topical administration. Administration includes self-administration and administration by another. A composition or formulation will exert its intended function via an appropriate route of administration. For example, if the appropriate route is intravenous, the composition is administered by introducing the composition or formulation into the subject's vein.

[0102] As used herein, a "CEE" construct comprises a CMV enhancer, an EF-1α promoter, and an EF-1α intron fragment. A "CE" construct comprises a CMV enhancer and an EF-1α promoter, but does not comprise an intron fragment (e.g., does not comprise an EF-1α intron fragment). A "CAE" construct comprises a CMV enhancer, a chicken β-actin promoter, and an EF-1α intron fragment. As used herein, a "CAG" construct comprises a CMV enhancer, a chicken β-actin promoter, and a chicken β-actin / rabbit β-globin hybrid intron fragment. A "CA" construct comprises a CMV enhancer and a chicken β-actin promoter, but does not comprise an intron fragment (e.g., does not comprise an EF-1α intron fragment). See Figures 4A, 5A, and 5C.

[0103] As used herein, the term "conserved" refers to each nucleotide or amino acid residue of a polynucleotide or polypeptide sequence that appears unchanged in the same position in two or more sequences being compared. A relatively conserved nucleotide or amino acid is one that is more conserved among related sequences than a nucleotide or amino acid that appears elsewhere in the sequence.

[0104] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, or at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, regions, or features thereof.

[0105] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each comprising a nucleobase sequence) related to each other by Watson-Crick base-pairing rules, or between an oligomer and a target gene. For example, the nucleobase sequence "TGA(5'→3')" is complementary to the nucleobase sequence "ACT(3'→5')." Complementarity may be "partial" if fewer than all nucleobases of a given nucleobase sequence match another nucleobase sequence according to the base-pairing rules. For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Thus, in certain embodiments, the term "complementary" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity or complementarity with a target nucleic acid sequence. Alternatively, to continue by way of illustration, there may be "perfect" or "100%" complementarity between a given nucleobase sequence and another nucleobase sequence. In some embodiments, the degree of complementarity between nucleobase sequences significantly affects the efficiency and strength of intersequence hybridization.

[0106] As used herein, the phrase "contiguous or non-contiguous nucleotides are deleted" is intended to include the contiguous or non-contiguous nucleotide sequence remaining as a result of deletion compared to the wild-type (or original) sequence, but is not intended to include any deletion process. The term can include "deletion of contiguous nucleotides," "deletion of non-contiguous nucleotides remaining undeleted between the deleted nucleotides," and "insertion of a different type of nucleotide at the position of the deleted contiguous or non-contiguous nucleotides." For example, in the case of the nucleotide sequence "ATGCCGTC," deletion of contiguous nucleotides includes deletion of one or more contiguous nucleotides, such as "A-_-_-_-CGTC," deletion of non-contiguous nucleotides means that one or more nucleotides remain undeleted between the deleted nucleotides, such as "A-_-G-_-CGT-_," and insertion of a different type of nucleotide at the position of the deleted contiguous or non-contiguous nucleotides means that one or more nucleotides different from the original nucleotides are inserted at the position of the deleted nucleotides, such as "AAG-_-CGTG."

[0107] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence relates to a sequence subsequent to the start of transcription. For example, the translation initiation codon of a gene is located downstream of the transcription start site.

[0108] As used herein, the term "enhancer" refers to a segment of DNA that contains a sequence that can provide enhanced transcription, and in some cases can act independently of its orientation relative to other regulatory sequences. Enhancers can act in concert with, or in addition to, a promoter and / or other enhancer elements.

[0109] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a complex, e.g., a polynucleotide comprising a foreign gene and untranslated nucleic acid sequence as described herein.

[0110] The term "exon" refers to a nucleic acid sequence that appears in the mature form of an RNA molecule after removal of a given portion of a protein-coding nucleic acid or any portion of a preprocessed (or precursor) RNA by splicing. The mature RNA molecule may be messenger RNA (mRNA) or a functional form of non-coding RNA such as rRNA or tRNA.

[0111] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, e.g., an RNA or a polypeptide. This includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA) and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be a nucleic acid, such as an RNA produced by transcription of a gene. As used herein, a gene product can be a nucleic acid or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids resulting from post-transcriptional modification, e.g., polyadenylation or splicing, or polypeptides resulting from post-translational modification, e.g., phosphorylation, methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0112] The term "identity," as used herein, refers to the conservation of all monomers between polymeric molecules, e.g., polynucleotide molecules. The term "identical" without any additional modifiers, e.g., polynucleotide A is identical to polynucleotide B, means that the polynucleotide sequences are 100% identical (100% sequence identity). Describing two sequences as being, e.g., "70% identical," is the same as describing them as having, e.g., "70% sequence identity."

[0113] For example, calculation of percent identity of two polypeptide or polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second polypeptide or polynucleotide sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of an aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids, or in the case of polynucleotides, bases at corresponding amino acid positions are then compared.

[0114] When a particular position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each of the gaps. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm.

[0115] Suitable software programs that can be used to align different sequences (e.g., polynucleotide sequences) are available from several sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). bl2seq performs comparisons between two sequences using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and can be obtained from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0116] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (Clustal W, Clustal X, or Clustal Omega), MUSCLE, and the like.

[0117] Different regions within a single polynucleotide or polypeptide target sequence that are aligned with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identity values ​​are rounded / truncate to the nearest first decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are rounded to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded to 80.2. Note also that length values ​​are always integers.

[0118] In certain embodiments, the percent identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID=100×(Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in an alignment of the first and second sequences (when aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0119] Those skilled in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons that are driven entirely by primary sequence data. Furthermore, it will be understood that sequence alignments can be performed by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. Suitable programs for integrating heterogeneous data to achieve multiple sequence alignments are available at www.tcoffee.org, as well as, for example, T-Coffee, also available from EBI. Furthermore, it will be understood that the final alignment used to calculate percent sequence identity can be organized automatically or manually.

[0120] As used herein, the term "intron" refers to a segment of DNA within a gene (an intervening sequence) that does not code for part of the protein produced by the gene and is spliced ​​out of the mRNA transcribed from the gene before it is exported from the cell nucleus. "Intron sequence" refers to the nucleic acid sequence of an intron. Such sequences are also referred to herein as "non-translated nucleic acid sequences." Thus, an intron is a region of DNA sequence that is transcribed along with the coding sequence (exon) but is removed during the formation of the mature mRNA.

[0121] As used herein, the term "intron fragment" refers to a fragment derived from the full-length EF-1α intron A sequence (i.e., the first intron of EF-1α, e.g., as set forth in SEQ ID NO: 1). The fragment excludes the full-length EF-1α intron. In some embodiments, the "intron fragment" comprises the minimum number of nucleotides or structures necessary to achieve an expression level greater than that achieved by a corresponding construct lacking all nucleotides of EF-1α intron A. Thus, the intron fragment (also referred to herein as a "non-translated nucleic acid sequence") of the present disclosure is not particularly limited, as long as it comprises a fragment of the EF-1α intron and is capable of increasing expression of an exogenous gene. As demonstrated herein, in some embodiments, an intron fragment (i.e., a non-translated nucleic acid sequence) can increase transcription of an exogenous gene, thereby increasing expression of the exogenous gene. Thus, in some embodiments, the intron fragments described herein can be non-translated regulatory elements.

[0122] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably to refer to the state of a preparation of a desired composition of the present disclosure, e.g., a polynucleotide comprising an exogenous gene and untranslated nucleic acid sequences, that has undergone one or more purification procedures. In some aspects, isolation or purification, as used herein, is a process of removing or partially removing (e.g., fractionating) a composition of the present disclosure, e.g., a polynucleotide described herein, from a sample containing contaminants.

[0123] In some embodiments, the isolated composition has no detectable undesired activity or, alternatively, an undesired activity at or below an acceptable level or amount. In other embodiments, the isolated composition has an amount and / or concentration of a desired composition of the present disclosure at or above an acceptable amount and / or concentration and / or activity. In other embodiments, the isolated composition is enriched relative to the starting material from which it is derived. This enrichment may be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% relative to the starting material.

[0124] In some embodiments, the isolated preparation is substantially free of residual biological products. In some embodiments, the isolated preparation is 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, or at least about 90% free of any biological contaminants. Residual biological products may include abiotic materials (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolites.

[0125] As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence may be directly attached or juxtaposed to the second amino acid or polynucleotide sequence, or an intervening sequence may covalently link the first sequence to the second sequence. The term "linked" not only refers to the fusion of the first polynucleotide sequence to the second polynucleotide sequence at the 5'-end or 3'-end, but also includes the insertion of the entire first polynucleotide sequence (or second polynucleotide sequence) at any two nucleotides within the second polynucleotide sequence (or first polynucleotide sequence), respectively. The first polynucleotide sequence can be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker can be, for example, a polynucleotide.

[0126] As used herein, the term "macular degeneration" refers to any of a number of disorders and conditions in which the macula degenerates or loses functional activity. The degeneration or loss of functional activity can occur as a result of, for example, apoptosis, decreased cell proliferation, loss of normal biological function, or a combination thereof. Macular degeneration can lead to and / or manifest as changes in the structural integrity of the cells and / or extracellular matrix of the macula, changes in normal cellular and / or extracellular matrix organization, and / or loss of function of macular cells. The cells can be any cell type normally present in or near the macula, including RPE cells, photoreceptors, and / or capillary endothelial cells. While age-related macular degeneration is the most common type of macular degeneration, the term "macular degeneration" does not necessarily exclude macular degeneration in non-elderly patients. Non-limiting examples of macular degeneration include: age-related macular degeneration (wet or dry); Best macular dystrophy, Sorsby fundus dystrophy, Malattia Leventinese, Doyne honeycomb retinal dystrophy, Stargardt disease (also called Stargardt macular dystrophy, juvenile macular degeneration, or fundus flavimaculatus), and pigment epithelial detachment-associated macular degeneration.

[0127] As used herein, the term "age-related macular degeneration" (AMD) refers to a retinopathy that generally affects older adults and is associated with loss of central vision due to damage to the central portion of the retina (i.e., the macula). AMD is generally characterized by the gradual accumulation or aggregation of yellow, insoluble extracellular deposits called drusen (accumulation of extracellular proteins and lipids, such as amyloid beta) within the macula (primarily between the retinal pigment epithelium (RPE) and the underlying choroid). The accumulation or aggregation of these deposits within the macula can gradually deteriorate the macula and damage central vision. As used herein, the term "macula" refers to the central part of the retina responsible for central, high-resolution color vision.

[0128] The pathogenesis of age-related macular degeneration (AMD) is unknown, although several theories have been proposed, including oxidative stress, mitochondrial dysfunction, and inflammatory processes. An imbalance between the production and degradation of damaged cellular components leads to the accumulation of harmful products, such as intracellular lipofuscin and extracellular drusen. Early atrophy is distinguished by areas of retinal pigment epithelium (RPE) thinning or depigmentation, which precedes geographic atrophy in the early stages of AMD. In advanced stages of AMD, RPE atrophy (geographic atrophy) and / or the development of new blood vessels (neovascularization) leads to photoreceptor death and loss of central vision. In dry (non-exudative) AMD, cellular debris called drusen accumulates between the retina and choroid, causing retinal atrophy and scarring. In the more severe form of wet (exudative) AMD, blood vessels grow in the choroid behind the retina (neovascularization), leaking exudate and fluid, potentially causing bleeding.

[0129] Depending on the extent of drusen present, AMD can be classified into three major stages: (i) early, (ii) intermediate, and (iii) advanced or late. Early AMD is characterized by the presence of several small (e.g., less than about 63 microns in diameter) drusen or several intermediate-sized (e.g., about 63-124 microns in diameter) drusen. In the early stage, patients experience no vision loss and no obvious symptoms. The intermediate stage is characterized by the presence of many intermediate-sized drusen or one or more large (e.g., greater than about 125 microns in diameter) drusen. At this stage, some patients may begin to experience blurred spots in the central field of vision. Advanced or late AMD is characterized by damage to large areas of retinal tissue, resulting in a central blind spot and eventual loss of central vision. Based on the type of damage (e.g., the presence or absence of neovascularization), advanced or late stage AMD can be further divided into two subtypes: (i) geographic atrophy (also called dry AMD) and (ii) wet AMD (also called neovascular or exudative AMD).

[0130] There are two major forms of AMD: (i) dry AMD and (ii) wet AMD. Unless otherwise specified, the term "age-related macular degeneration" includes both dry AMD and wet AMD. As used herein, the term "age-related macular degeneration" includes all types of age-related macular degeneration, regardless of cause, and any and all symptoms of age-related macular degeneration. Non-limiting examples of symptoms associated with macular degeneration (e.g., age-related macular degeneration) include: loss of central vision, distortion, decreased contrast sensitivity, blurred vision, difficulty adapting to low light, sudden onset and rapid worsening of symptoms, and decreased color vision. In some aspects, macular degeneration (e.g., age-related macular degeneration) can cause macular edema (i.e., swelling of the macula due to the collection of fluid and protein deposits above or below the macula).

[0131] As used herein, the term "dry AMD" (also referred to as atrophic age-related macular degeneration or non-exudative AMD) refers to any form of AMD that is not wet (neovascular) AMD. This includes early and intermediate forms of AMD, as well as an advanced form of dry AMD known as geographic atrophy. Patients with dry AMD tend to have minimal symptoms in the early stages, and loss of visual function occurs more frequently when symptoms progress to geographic atrophy.

[0132] As used herein, the term "wet AMD" (also known as neovascular age-related macular degeneration or exudative AMD) refers to a retinal condition characterized by the presence of retinal neovascularization and is the most advanced form of AMD. In wet AMD, blood vessels grow from the choriocapillaris and, in some cases, the underlying retinal pigment epithelium (choroidal neovascularization or angiogenesis) through defects in Bruch's membrane. The organization of fluid or hemorrhagic exudates from these vessels can cause the formation of fibrovascular scars in the macular region, along with concomitant degeneration of the neural retina, detachment and rupture of the retinal pigment epithelium, vitreous hemorrhage, and permanent damage to central vision.

[0133] The terms "miRNA," "miR," and "microRNA" are used interchangeably and refer to microRNA molecules found in eukaryotes that are involved in RNA-based gene regulation. This term is used to refer to single-stranded RNA molecules processed from precursors. In some embodiments, the term "antisense oligomer" may be used to describe the microRNA molecules of the present disclosure. Names of miRNAs and their sequences relevant to the present disclosure are provided herein. MicroRNAs down-regulate target gene expression by recognizing and binding to target mRNAs through imperfect base pairing, resulting in destabilization or translational inhibition of the target mRNA. Conversely, miRNA targeting via molecules containing miRNA-binding sites (usually molecules containing sequences complementary to the seed region of the miRNA) can reduce or inhibit miRNA-induced translational inhibition, manifesting as up-regulation of target genes.

[0134] "Nucleic acid," "nucleic acid molecule," "nucleotide sequence," "polynucleotide," and grammatical variations thereof are used interchangeably and refer to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented herein in a 5' to 3' direction. Polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are represented herein by single-letter coding, such as adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0135] As used herein, the terms "operably linked" or "operably linked" mean that the linked DNA sequences are located contiguously so as to perform their desired function. For example, if a particular promoter helps initiate transcription of a coding sequence (e.g., a foreign gene), such a promoter can be operably linked with a coding region. The promoter and coding region do not necessarily have to be located contiguously, so long as such a functional relationship is maintained.

[0136] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," and grammatical variations thereof include all formulations approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause the production of undesirable physiological effects sufficient to prevent administration of the composition to a subject and that does not eliminate the biological activity and properties of the administered conjugate. Included are generally safe, non-toxic, and preferred excipients and carriers that are useful in the preparation of pharmaceutical compositions.

[0137] As used herein, the term "pharmaceutical composition" refers to one or more of the compositions described herein (e.g., polynucleotides, vectors, cells and / or recombinant viruses) mixed with or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients.

[0138] As used herein, the terms "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of regulating the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. A promoter may be derived entirely from a native gene, be composed of different elements derived from different promoters found in nature, or may include synthetic DNA segments. Those skilled in the art will appreciate that different promoters can direct the expression of genes in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters that direct the expression of most genes in most host cell types are commonly referred to as "constitutive promoters." Promoters that direct the expression of genes in specific cell types are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that direct the expression of genes at specific developmental or cell differentiation stages are commonly referred to as "developmental-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced to express genes upon exposure or treatment of cells with promoter-inducing agents, biological molecules, chemicals, ligands, light, etc. are commonly referred to as "inducible promoters" or "regulatable promoters." Furthermore, it is recognized that because in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0139] The promoter sequence typically is bounded at its 3' end by a transcription initiation site and extends upstream (5') to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence, a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1) will be found, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. In some embodiments, promoters that can be used in the present disclosure include tissue-specific promoters.

[0140] As used herein, the term "gene regulatory region" or "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences) or downstream (3' non-coding sequences) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences will usually be located 3' to the coding sequence.

[0141] In some embodiments, polynucleotides described herein (including, e.g., exogenous genes and untranslated nucleic acid sequences) can include a promoter and / or other expression (e.g., transcription) regulatory elements operably associated with one or more coding regions. In operably associated relationships, a coding region for a gene product is associated with one or more regulatory regions such that expression of the gene product is under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in transcription of an mRNA encoding the gene product encoded by the coding region, and the nature of the link between the promoter and the coding region does not interfere with the promoter's ability to direct expression of the gene product or the ability of the DNA template to be transcribed. Other expression regulatory elements outside of promoters, e.g., enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct expression of a gene product.

[0142] As used herein, the terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably and refer to any mammalian subject to which any of the compositions described herein (e.g., polynucleotides, recombinant expression constructs, cells, pharmaceutical compositions, or recombinant viruses) is administered. Non-limiting examples include humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), particularly humans, in need of diagnosis, treatment, or therapy. The methods described herein are applicable to both human therapy and veterinary applications.

[0143] As used herein, the term "subject in need" includes subjects such as mammalian subjects who would benefit from the administration of the compositions described herein.

[0144] As used herein, the term "therapeutically effective amount" refers to the amount of a reagent or pharmaceutical complex comprising a composition of the present disclosure (e.g., a polynucleotide comprising an exogenous gene and an untranslated nucleic acid sequence) sufficient to achieve a desired therapeutic, pharmacological, and / or physiological effect in a subject in need thereof. A therapeutically effective amount may be a "prophylactically effective amount," as prevention may be considered treatment.

[0145] As used herein, the term "foreign gene" refers to at least one polynucleotide or polynucleotide region encoded by a recombinant expression construct, or a polynucleotide or facilitating or regulatory nucleic acid encoding an expression product, polypeptide, or multi-polypeptide, of said polynucleotide or polynucleotide region. In some embodiments, the foreign gene may be heterologous to (i.e., not naturally expressed in) the cell into which it is inserted (or transduced).

[0146] As used herein, the terms "treat," "treatment," or "treating" refer to, for example, reducing the severity of a disease or condition, reducing the duration of a disease, ameliorating or eliminating one or more symptoms associated with a disease or condition, or providing a beneficial effect to a subject with a disease or condition without necessarily curing the disease or condition. The terms also include preventing or preventing a disease, condition, or a symptom thereof.

[0147] The term "upstream" refers to a nucleotide sequence that is located 5' relative to a reference nucleotide sequence.

[0148] As used herein, the term "vector" or "construct" refers to any vehicle into which a nucleic acid or gene can be inserted, e.g., a delivery vehicle that can be introduced into a cell where a nucleic acid sequence can be inserted and replicated. The nucleic acid sequence that can be inserted into a vector can be exogenous or heterologous. The nucleic acid sequence can be a foreign gene. Examples of constructs include, but are not limited to, plasmids, cosmids, and viruses (e.g., AAV). Those skilled in the art can generate such vectors or constructs using standard recombinant techniques (e.g., 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). As used herein, the term "expression vector" or "expression construct" refers to a vector or construct that contains a nucleotide sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression constructs can contain a variety of regulatory elements. Along with regulatory sequences that control transcription and translation, vectors and expression vectors can also contain nucleotide sequences that serve other functions as well.

[0149] Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells containing the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that integrate and express other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialarphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

[0150] II. Polynucleotides II.A. Non-translated Nucleic Acid Sequences The present disclosure relates to polynucleotides comprising untranslated nucleic acid sequences that, upon translation, can increase expression of a foreign gene. Specifically, the present disclosure provides elongation factor-1α (EF-1α) intron sequences that are shorter than the full-length EF-1α intron.

[0151] Elongation factor 1α (EF-1α) is a gene located on chromosome 6 (nucleotides 73,489,308 to 73,525,587 of GenBank accession number NC_000006.12; negative strand orientation). The EF-1α gene contains eight exons and seven introns and encodes the eukaryotic elongation factor 1A (also known as eEF1A1 and eEF1A) protein, which plays an important role in mRNA translation (e.g., transporting aminoacyl-tRNA to the A site of the ribosome as the ternary complex eEF1A1-GTP-aa-tRNA). See Scaggiante et al., Atlas Genet Cytogenet Oncol Haematol 19(4):256-265 (March 2015). The nucleotide sequence of the full-length EF-1α intron is set forth in SEQ ID NO:1 (924 nucleotides long). As described herein, the non-translated nucleic acid sequences of the present disclosure (i.e., EF-1α intron fragment sequences) offer distinct advantages over the full-length EF-1α intron (or other introns known in the art). For example, in some embodiments, the non-translated nucleic acid sequences described herein can increase foreign gene expression to a greater extent than the full-length EF-1α intron. Furthermore, because the non-translated nucleic acid sequences of the present disclosure are shorter than their full-length counterparts, in some embodiments they can be used in combination with larger foreign genes. For example, AAV capsids (such as those described herein) can accommodate up to approximately 4.7 kb of nucleic acid. Thus, in some embodiments, larger foreign genes and / or additional cis-elements can be integrated with the EF-1α intron fragments (i.e., non-translated nucleic acid sequences) described herein to further increase gene expression.

[0152] In some embodiments, the untranslated nucleic acid sequence described herein (i.e., the EF-1α intron fragment) includes, but does not include, nucleotides 874-924 of the sequence set forth in SEQ ID NO:1. In some embodiments, the untranslated nucleic acid sequence consists essentially of nucleotides 874-924 of SEQ ID NO:1. In some embodiments, the untranslated nucleic acid sequence consists of nucleotides 874-924 of SEQ ID NO:1. Such an EF-1α intron fragment is also referred to herein as a "T3.2 fragment" and is set forth in SEQ ID NO:57. In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 874-924 of SEQ ID NO:1, but the untranslated nucleic acid sequence does not include SEQ ID NO:1.

[0153] In some embodiments, the untranslated nucleic acid sequence described herein (i.e., the EF-1α intron fragment) comprises, but does not include, nucleotides 852-924 of SEQ ID NO:1. In some embodiments, the untranslated nucleic acid sequence of the present disclosure essentially begins with nucleotides 852-924 of SEQ ID NO:1. In some embodiments, the untranslated nucleic acid sequence consists of nucleotides 852-924 of SEQ ID NO:1. Such an EF-1α intron fragment is also referred to herein as a "T3.1.2 fragment" and is set forth in SEQ ID NO:3. In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 852-924 of SEQ ID NO:1, but the untranslated nucleic acid sequence does not include SEQ ID NO:1.

[0154] In some embodiments, the untranslated nucleic acid sequence described herein (i.e., the EF-1α intron fragment) comprises, but does not include, nucleotides 830-924 of SEQ ID NO:1. In some embodiments, the untranslated nucleic acid sequence of the present disclosure essentially begins with nucleotides 830-924 of SEQ ID NO:1. In some embodiments, the untranslated nucleic acid sequence consists of nucleotides 830-924 of SEQ ID NO:1. Such an EF-1α intron fragment is also referred to herein as a "T3.1.1 fragment" and is set forth in SEQ ID NO:2. In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 830-924 of SEQ ID NO:1, but the untranslated nucleic acid sequence does not include SEQ ID NO:1.

[0155] As is apparent from the present disclosure, in addition to the nucleotides described above (i.e., positions 874-924, 852-924, and 830-924 of SEQ ID NO:1, i.e., SEQ ID NO:57, SEQ ID NO:3, and SEQ ID NO:2, respectively), in some embodiments, the untranslated nucleic acid sequence of the present disclosure can further include contiguous or non-contiguous nucleotides of the EF-1α intron sequence in the 5' region, as shown in SEQ ID NO:1. For example, in some embodiments, the contiguous or non-contiguous nucleotides added to the 5' region can be added anywhere upstream of nucleotides 874-924, 852-924, or 830-924 of SEQ ID NO:1. In some embodiments, the contiguous or non-contiguous nucleotides added to the 5' region are derived from the full-length EF-1α intron sequence, i.e., SEQ ID NO:1. In some embodiments, the contiguous or non-contiguous nucleotides added to the 5' and / or 3' regions are derived from any sequence heterologous to the full-length EF-1α intron sequence, i.e., SEQ ID NO:1.

[0156] Thus, in some embodiments, the untranslated nucleic acid sequences described herein do not further include any contiguous or non-contiguous nucleotides derived from nucleotide positions 1 to 873 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 874 to 924 of SEQ ID NO:1; "T3.2 fragment"; SEQ ID NO:57).

[0157] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-870 in the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 871-924 of SEQ ID NO:1; i.e., SEQ ID NO:58). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-870 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-870 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-870 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-870 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0158] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-860 of SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 861-924 of SEQ ID NO:1; i.e., SEQ ID NO:59). In some embodiments, such untranslated nucleic acid sequences can contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-860 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-860 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-860 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences can include one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-860 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0159] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-851 in the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 852-924 of SEQ ID NO:1; i.e., SEQ ID NO:60). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-851 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-851 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-851 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-851 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0160] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-850 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 851-924 of SEQ ID NO:1; i.e., SEQ ID NO:61). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-850 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-850 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-850 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-850 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0161] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-829 of SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 830-924 of SEQ ID NO:1; i.e., SEQ ID NO:2). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-829 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-829 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-829 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-829 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0162] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-820 of the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 821-924 of SEQ ID NO:1; i.e., SEQ ID NO:63). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-820 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-820 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-820 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-820 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0163] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-810 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 811-924 of SEQ ID NO:1; i.e., SEQ ID NO:64). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-810 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-810 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-810 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-810 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0164] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-807 in the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 808-924 of SEQ ID NO:1; i.e., SEQ ID NO:65). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-807 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-807 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-807 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-807 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0165] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-800 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 801-924 of SEQ ID NO:1; i.e., SEQ ID NO:66). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-800 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-800 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-800 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-800 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0166] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-750 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 751-924 of SEQ ID NO:1; i.e., SEQ ID NO:67). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-750 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-750 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-750 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-750 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0167] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-720 of SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 721-924 of SEQ ID NO:1; i.e., SEQ ID NO:68). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-720 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-720 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-720 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-720 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0168] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-700 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 701-924 of SEQ ID NO:1; i.e., SEQ ID NO:69). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-700 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-700 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-700 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-700 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0169] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-650 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 651-924 of SEQ ID NO:1; i.e., SEQ ID NO:70). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-650 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-650 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-650 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-650 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0170] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-600 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 601-924 of SEQ ID NO:1; i.e., SEQ ID NO:71). In some embodiments, such untranslated nucleic acid sequences can contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-600 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-600 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-600 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences can include one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-600 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0171] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-569 of SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 570-924 of SEQ ID NO:1; i.e., SEQ ID NO:72). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-569 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-569 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-569 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-569 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0172] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-550 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 551-924 of SEQ ID NO:1; i.e., SEQ ID NO:73). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-550 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-550 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-550 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-550 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0173] In some embodiments, the untranslated nucleic acid sequences described herein do not contain any contiguous or non-contiguous nucleotides derived from nucleotides 1-500 within the sequence set forth in SEQ ID NO:1 (resulting in an EF-1α intron fragment comprising, consisting essentially of, or consisting of nucleotides 501-924 of SEQ ID NO:1; i.e., SEQ ID NO:74). In some embodiments, such untranslated nucleic acid sequences may contain one or more contiguous or non-contiguous nucleotides derived from within nucleotide positions 1-500 of SEQ ID NO:1, but the one or more contiguous or non-contiguous nucleotides do not span the entire length of nucleotide positions 1-500 of SEQ ID NO:1 (e.g., shorter than nucleotides 1-500 of SEQ ID NO:1). In some embodiments, such untranslated nucleic acid sequences may contain one or more different types of nucleotides (i.e., nucleotides not derived from nucleotide positions 1-500 of SEQ ID NO:1) at the 5' and / or 3' ends of the untranslated nucleic acid sequence.

[0174] In some embodiments, the untranslated nucleic acid sequences described herein include (i) nucleotides 871 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:58), (ii) nucleotides 861 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:59), (iii) nucleotides 852 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:60), (iv) nucleotides 851 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:61), (v) nucleotides 830 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:2), (vi) nucleotides 821 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:63), (vii) nucleotides 811 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:64), (viii) nucleotides 808 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:65), (ix) nucleotides 801 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:66), (x) nucleotides 751 to 924 of SEQ ID NO:1. (xiv) nucleotides 601 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO: 71), (xv) nucleotides 570 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO: 72), (xvi) nucleotides 551 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO: 73), or (xvii) nucleotides 501 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO: 74).

[0175] In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 871-924 of SEQ ID NO:1 (i.e., SEQ ID NO:58). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 861-924 of SEQ ID NO:1 (i.e., SEQ ID NO:59). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 852-924 of SEQ ID NO:1 (i.e., SEQ ID NO:60). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 851-924 of SEQ ID NO:1 (i.e., SEQ ID NO:61). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 830-924 of SEQ ID NO:1 (i.e., SEQ ID NO:2). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 821-924 of SEQ ID NO:1 (i.e., SEQ ID NO:63).In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 811 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 64). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 808 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 65). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 801-924 of SEQ ID NO:1 (i.e., SEQ ID NO:66). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 751-924 of SEQ ID NO:1 (i.e., SEQ ID NO:67). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 721-924 of SEQ ID NO:1 (i.e., the sequence of SEQ ID NO:68). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 701-924 of SEQ ID NO:1 (i.e., SEQ ID NO:69).In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 651-924 of SEQ ID NO:1 (i.e., SEQ ID NO:70). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 601-924 of SEQ ID NO:1 (i.e., SEQ ID NO:71). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 570 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 72). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 551 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 73). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to nucleotides 501 to 924 of SEQ ID NO:1 (i.e., SEQ ID NO:74).

[0176] As is evident from the disclosure above, in some embodiments, a polynucleotide comprising an untranslated nucleic acid sequence described herein further comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at the 5' end of the untranslated nucleic acid sequence (the "5' region"). In some embodiments, the polynucleotides described herein further comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at the 3' end of the untranslated nucleic acid sequence (the "3' region"). In some embodiments, a polynucleotide comprising an untranslated nucleic acid sequence further comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at both the 5' end ("5' region") and the 3' end ("3' region") of the untranslated nucleic acid sequence.

[0177] In some embodiments, the polynucleotide comprises one or more consecutive or non-consecutive nucleotides corresponding to positions 1 to 873 of SEQ ID NO:1 in the 5' region of the untranslated nucleic acid sequence.

[0178] II.B. Foreign Genes In some embodiments, polynucleotides described herein that include a non-translated nucleic acid sequence further include a foreign gene. Thus, in some embodiments, polynucleotides described herein include a foreign gene and a non-translated nucleic acid sequence, wherein the non-translated nucleic acid sequence includes, consists essentially of, or consists of nucleotides 874 to 924 of SEQ ID NO:1, but does not include SEQ ID NO:1 (e.g., SEQ ID NO:57). In some embodiments, polynucleotides described herein include a foreign gene and a non-translated nucleic acid sequence, wherein the non-translated nucleic acid sequence includes, consists essentially of, or consists of nucleotides 852 to 924 of SEQ ID NO:1, but does not include SEQ ID NO:1 (e.g., SEQ ID NO:3). In some embodiments, polynucleotides described herein include a foreign gene and a non-translated nucleic acid sequence, wherein the non-translated nucleic acid sequence includes, consists essentially of, or consists of nucleotides 830 to 924 of SEQ ID NO:1, but does not include SEQ ID NO:1 (e.g., SEQ ID NO:2).

[0179] The exogenous genes useful in the present disclosure are not particularly limited, as long as the exogenous genes can be translated into a polypeptide when transduced into a cell. Therefore, any suitable exogenous gene of interest can be used in the non-translated nucleic acid sequences of the present disclosure. In some embodiments, the exogenous gene encodes a polypeptide (or any variant thereof), a fusion protein, an antibody or an antigen-binding fragment thereof, an RNA-based molecule (e.g., miRNA, shRNA, ribozyme, siRNA), or any combination thereof.

[0180] In some embodiments, the exogenous gene encodes a protein useful for treating a disease or disorder, such as those described herein. In some embodiments, the exogenous gene encodes a peptide for treating a specific disease intended for sustained expression in the subject's or patient's body. In some embodiments, the exogenous gene has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 23. In some embodiments, the exogenous gene encodes a fusion protein, wherein the fusion protein is an inhibitor of vascular endothelial growth factor ("VEGF"). In some embodiments, VEGF inhibitors include uprebercept (EYLEA® and ZALTRAP®).

[0181] II.C. Adjustment factors In some embodiments, the polynucleotides described herein further comprise a regulatory element. Thus, in some embodiments, the polynucleotide comprises (1) a regulatory element, (2) a non-translated nucleic acid sequence described herein, and (3) an exogenous gene.

[0182] As used herein, the term "regulatory element" refers to a nucleic acid sequence that regulates (e.g., increases or decreases) expression of an operably linked nucleic acid. Regulatory elements useful in the present disclosure include enhancers (e.g., CMV enhancers), promoters (e.g., CMV promoters, EF-1α promoters, or β-actin promoters), exons (e.g., exon 1 or exon 2), splicin donor sequences, acceptor sequences, or combinations thereof. In some embodiments, the regulatory element may include a sequence for transcription termination (e.g., polyA), a sequence for stable expression of a foreign gene (e.g., a WPRE sequence), a sequence for reducing the development of foreign gene-specific immunity (e.g., an miRNA target sequence), or a combination thereof.

[0183] II.C.1. Enhancers In some embodiments, the regulatory element is an enhancer. Thus, in some embodiments, the polynucleotides described herein include (in no particular order): (1) an enhancer, (2) an untranslated nucleic acid sequence, and (3) an exogenous gene. In some embodiments, the polynucleotides described herein include (in 5' to 3' direction): (1) an enhancer, (2) an untranslated nucleic acid sequence, and (3) an exogenous gene.

[0184] Any suitable enhancer known in the art can be used in the present disclosure. Non-limiting examples of suitable enhancers include the cytomegalovirus (CMV) enhancer, the SV40 early enhancer, the adenovirus 5 E1A enhancer, the HBV enhancer-1 regulatory region (Eh-1), the HPV-16 or -18 E6 / 7 long regulatory region (LCR), the HIV-1 long terminal repeat (LTR), or any combination thereof. In some embodiments, the enhancer is a cytomegalovirus (CMV) enhancer. In some embodiments, the CMV enhancer comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:4. In some embodiments, the cytomegalovirus (CMV) enhancer comprises the nucleotide sequence set forth in SEQ ID NO:4.

[0185] II.C.2. Promoter In some embodiments, the regulatory element is a promoter. Thus, in some embodiments, the polynucleotides described herein include (in no particular order): (1) a promoter, (2) a non-translated nucleic acid sequence, and (3) a foreign gene. In some embodiments, the polynucleotide includes (in 5' to 3' direction): (1) a promoter, (2) a non-translated nucleic acid sequence, and (3) a foreign gene. In some embodiments, the regulatory element includes both an enhancer and a promoter. In such embodiments, the polynucleotide can include (in no particular order): (1) an enhancer, (2) a promoter, (3) a non-translated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the polynucleotide includes (in 5' to 3' direction): (1) an enhancer, (2) a promoter, (3) a non-translated nucleic acid sequence, and (4) a foreign gene. Any suitable promoter known in the art can be used in the present disclosure.

[0186] In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter, an EF-1α promoter, a β-actin promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a 70-kDa heat shock protein (HSP70) promoter, a 78-kDa glucose-regulated protein (GRP78) promoter, a eukaryotic initiation factor-4A (eIF4a) promoter, an alpha-1-antitrypsin (AAT) promoter, a transthyretin (TTR) promoter, a glial fibrillary acidic protein (GFAP) promoter, an early promoter of simian phorolar virus 40 (SV40) promoter, a synapsin I (SYN1) promoter, a G protein-coupled receptor kinase (GRK) promoter, a rhodopsin (Rho) promoter, or a combination thereof.

[0187] In some embodiments, a promoter useful in the present disclosure is a CMV promoter. Thus, in some embodiments, a polynucleotide comprises (1) an enhancer (e.g., a CMV enhancer), (2) a CMV promoter, (3) a non-translated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the CMV promoter comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the CMV promoter comprises the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0188] In some embodiments, the promoter that can be used in the present disclosure is the EF-1α promoter. In some embodiments, the polynucleotide comprises (1) an enhancer (e.g., a CMV enhancer), (2) the EF-1α promoter, (3) a non-translated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the EF-1α promoter comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:7. In some embodiments, the EF-1α promoter comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0189] In some embodiments, the promoter is a β-actin promoter. Thus, in some embodiments, the polynucleotides described herein include (1) an enhancer (e.g., a CMV enhancer), (2) a β-actin promoter, (3) a non-translated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the β-actin promoter includes a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:8. In some embodiments, the β-actin promoter is a chicken β-actin promoter as set forth in SEQ ID NO:8.

[0190] As shown herein (see FIG. 4A), in some embodiments, a polynucleotide described herein (e.g., including an exogenous gene and a non-translated nucleic acid sequence) can contain multiple promoters. For example, in some embodiments, a polynucleotide includes a combination of a CMV promoter, an EF-1α promoter, and / or a β-actin promoter. In some embodiments, a polynucleotide includes both a CMV promoter and an EF-1α promoter. In such embodiments, the CMV promoter can be a portion of a full-length CMV promoter, such as the sequence set forth in SEQ ID NO:5 (i.e., the first 31 nucleotides from the 5' end of SEQ ID NO:6).

[0191] II.C.3. Splicing donor sequence In some embodiments, the polynucleotides described herein (i.e., including untranslated nucleic acid sequences) comprise a splice donor sequence. As used herein, the term "splice donor sequence" or "splice donor site" refers to a guanine-thymine (GT)-rich domain present at the 5' end of an intron (e.g., the EF-1α intron) (implying the boundary between the intron and the exon). As provided herein, such sequences can be targeted to generate untranslated nucleic acid sequences of the present disclosure. In some embodiments, the splice donor sequence is linked upstream of an EF-1α intron fragment (i.e., the untranslated nucleic acid sequence). For example, in some embodiments, the polynucleotides described herein comprise (in no particular order): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) a splice donor sequence, (4) an untranslated nucleic acid sequence, and (5) an exogenous gene. In some embodiments, the polynucleotide comprises (from 5' to 3'): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) a splicing donor sequence, (4) a non-translated nucleic acid sequence, and (5) an exogenous gene.

[0192] In some embodiments, splicing donor sequences useful in the present disclosure have at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the splicing donor sequence comprises the nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:10.

[0193] II.C.4. Exon Sequence As provided herein, in some embodiments, the polynucleotides of the present disclosure further comprise one or more exon sequences. For example, in some embodiments, the polynucleotides described herein comprise an EF-1α exon 2 (E2) sequence. Thus, in some embodiments, the polynucleotides described herein comprise the following features (in no particular order): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) a splice donor sequence, (4) a non-translated nucleic acid sequence, (5) an EF-1α E2 sequence, and (6) an exogenous gene. In some embodiments, such a polynucleotide comprises (from 5' to 3'): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) a splicing donor sequence, (4) a non-translated nucleic acid sequence, (5) an EF-1α E2 sequence, and (6) a foreign gene. In some embodiments, the EF-1α E2 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:11. In some embodiments, the EF-1α E2 sequence comprises the nucleotide sequence set forth in SEQ ID NO:11.

[0194] In some embodiments, one or more exon sequences that can be included in the polynucleotides described herein include cytomegalovirus (CMV), EF-1α, or β-actin exon 1 (E1) sequences. In some embodiments, the polynucleotides described herein can include both E1 and E2 sequences. For example, in some embodiments, the polynucleotides include (in no particular order): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, and (7) a foreign gene. In some embodiments, the polynucleotide comprises (from 5' to 3'): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or β-actin), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α promoter, and / or β-actin), (4) a promoter (e.g., a CMV E1 sequence, an EF-1α promoter, and / or β-actin), (5) a promoter (e.g., a CMV E1 sequence, an EF-1α promoter, and / or β-actin), (6) a promoter (e.g., a CMV E1 sequence, an EF-1α promoter, and / or β-actin), (7) a promoter (e.g., a CMV E1 sequence, an EF-1α promoter, and / or β-actin), (8) a promoter (e.g E1 sequence and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, and (7) a foreign gene.

[0195] In some embodiments, the E1 sequence that can be used in the present disclosure is a CMV E1 sequence. In some embodiments, the CMV E1 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 12. In some embodiments, the CMV E1 sequence comprises the nucleotide sequence set forth in SEQ ID NO: 12.

[0196] In some embodiments, the E1 sequence is an EF-1α E1 sequence. In some embodiments, the EF-1α E1 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13. In some embodiments, the EF-1α E1 sequence comprises the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the E1 sequence is a β-actin E1 sequence. In some embodiments, the β-actin E1 sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the β-actin E1 sequence comprises the nucleotide sequence set forth in SEQ ID NO:14 or SEQ ID NO:15.

[0197] II.C.5. miRNA Target Sequences As described herein, in some embodiments, the regulatory elements of the polynucleotides described herein include one or more target sequences for immune cell-specific microRNAs (miRNAs) ("miRNA target sequences"). Accordingly, in some embodiments, the polynucleotides described herein include the following features (in no particular order): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) an exogenous gene, and (8) more than one miRNA target sequence. In some embodiments, the polynucleotide comprises (from 5' to 3'): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) an exogenous gene, and (8) one or more miRNA target sequences.

[0198] In some embodiments, the polynucleotides described herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more miRNA target sequences. In some embodiments, the number of target sequences for miRNAs that may be included in the polynucleotides described herein is about 2 to about 6 (e.g., 2 to 6). In some embodiments, the multiple miRNA target sequences are the same. In some embodiments, one or more of the multiple miRNA target sequences are different from each other.

[0199] As is apparent from the present disclosure, the inclusion of one or more miRNA target sequences can improve the specificity of the polynucleotides described herein. For example, if inhibition of the foreign gene is desired in a specific cell type (e.g., immune cells), a target sequence for an immune cell-specific miRNA can be used to suppress the expression of the foreign gene in the immune cells. As a result, the development of foreign gene-specific immunity by the immune cells can be blocked. Therefore, by using different miRNA target sequences, the expression of the foreign gene can be regulated in different cells / tissues.

[0200] Any suitable miRNA target sequence known in the art can be used in the present disclosure. In some embodiments, the miRNA target sequence is specific to miR142-3p or miR142-5p. In some embodiments, the target sequence for the miRNA is selected from antisense oligonucleotides, antagomirs, short hairpin RNA (shRNA) molecules, short interfering RNA (siRNA) molecules, ribozymes, peptide nucleic acid (PNA) oligonucleotides, locked nucleic acid (LNA) oligonucleotides, or combinations thereof, each having a sequence complementary to the entire or partial sequence of miR142-3p or miR142-5p.

[0201] In some embodiments, the miRNA target sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 16. In some embodiments, the miRNA target sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 17. In some embodiments, the miRNA target sequence comprises the nucleotide sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 17.

[0202] II.C.6.WPRE Sequence In some embodiments, the polynucleotides described herein (i.e., including untranslated nucleic acid sequences) further comprise a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence. Thus, in some embodiments, the polynucleotides described herein comprise (in no particular order): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) an exogenous gene, (8) one or more miRNA target sequences, and (9) a WPRE sequence. In some embodiments, the polynucleotides described herein include (from 5' to 3'): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) an exogenous gene, (8) one or more miRNA target sequences, and (9) a WPRE sequence.

[0203] In some embodiments, the WPRE sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 18. In some embodiments, the WPRE sequence comprises the nucleotide sequence set forth in SEQ ID NO: 18.

[0204] II.C.7. Polyadenylation Sequences In some embodiments, the polynucleotides described herein (i.e., including non-translated nucleic acid sequences) further comprise one or more polyadenylation (pA) sequences. Thus, in some embodiments, the polynucleotides comprise (in no particular order): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) an exogenous gene, (8) one or more miRNA target sequences, (9) a WPRE sequence, and (10) one or more pA sequences. In some embodiments, the polynucleotide includes (from 5' to 3'): (1) an enhancer (e.g., a CMV enhancer), (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin), (3) an E1 sequence (e.g., a CMV E1 sequence, an EF-1α E1 sequence, and / or a β-actin E1 sequence), (4) a splicing donor sequence, (5) a non-translated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) an exogenous gene, (8) one or more miRNA target sequences, (9) a WPRE sequence, and (10) one or more pA sequences.

[0205] Any suitable pA sequence known in the art can be used in the present disclosure. In some embodiments, examples of polyadenylation sequences include, but are not limited to, human growth hormone (hGH) pA sequence, bovine growth hormone (bGH) pA sequence, simian hormonal virus 40 (SV40) early pA sequence, and SV40 late pA sequence.

[0206] In some embodiments, the pA sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 19. In some embodiments, the pA sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 20. In some embodiments, the pA sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21. In some embodiments, the pA sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 22. In some embodiments, the polyadenylation sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 19-22.

[0207] As noted above, in some embodiments, the polynucleotides described herein include (i) an exogenous gene (e.g., SEQ ID NO: 23) and (ii) a regulatory element operably linked to the exogenous gene, the regulatory element including (from 5' to 3'): (1) the CMV enhancer sequence set forth in SEQ ID NO: 4; (2) the CMV promoter sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, the EF-1α promoter sequence set forth in SEQ ID NO: 7, or the chicken β-actin promoter sequence set forth in SEQ ID NO: 8; (3) the CMV E1 sequence set forth in SEQ ID NO: 12, the EF-1α E1 sequence set forth in SEQ ID NO: 13, or the chicken β-actin E1 sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15; (4) the splicing donor sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10; (5) an EF-1α intron fragment sequence (i.e., an untranslated nucleic acid sequence) comprising, consisting essentially of, or consisting of the nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 57; and (6) the EF-1α E2 sequence set forth in SEQ ID NO: 11.

[0208] III. Vector In some aspects, the present disclosure provides vectors (e.g., expression vectors) containing any of the polynucleotides described herein (e.g., containing a foreign gene and a non-translated nucleic acid sequence). As described herein, such vectors are useful for recombinant expression in host cells and cells targeted for therapeutic intervention. In some aspects, vectors (e.g., containing a foreign gene and a non-translated nucleic acid sequence) useful for delivering the polynucleotides described herein include viral vectors. Examples of viruses that can be used as vectors in the present disclosure include, but are not limited to, retroviruses, herpes simplex viruses, lentiviruses, box viruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, adeno-associated viruses (AAVs), baculoviruses, and combinations thereof. In some aspects, vectors that can be used in the present disclosure include non-viral vectors. Non-limiting examples of such vectors include plasmids, cosmids, yeast artificial chromosomes (YACs), bacteriophages, and combinations thereof.

[0209] III.A. Adeno-associated virus (AAV) In some embodiments, polynucleotides described herein (e.g., including foreign genes and non-translated nucleic acid sequences) are delivered to cells, for example, using AAV. Adeno-associated virus (AAV), a single-stranded DNA virus, is a helper-dependent human parvovirus. The AAV genome is approximately 4.7 kbp in size and consists of an N-terminus encoding the rep gene involved in viral replication and viral gene expression, a C-terminus encoding the cap gene encoding the viral capsid protein, and inverted repeat repeats (ITRs) with approximately 145 bases inserted at each end. The 145-bp ITRs have a T-shaped structure and function as origins of replication during viral replication and as primary packaging signals. The ITRs are the only cis-acting sequences required for constructing recombinant AAV (rAAV) constructs. The IRT has enhancer activity in the presence of Rep protein but has very weak activity in its absence. When cloning a foreign gene into a recombinant AAV construct, these characteristics must be taken into consideration when constructing an expression construct by appropriately configuring enhancers, promoters, pAs, etc. (RJ Samulski and N Muzycka, Annu. Rev. Virolo. 2014.1:427-451). Four proteins are translated from the rep gene. These proteins are classified according to their molecular weight as rep78, rep68, rep52, and rep40, and play important roles in AAV DNA replication. Four proteins are translated from the cap gene. Of these, VP1, VP2, and VP3 are structural proteins that make up AAV particles, and assembly-activating proteins (AAPs) promote the assembly of AAV particles by these structural proteins. For efficient replication, adeno-associated virus requires some proteins and RNA derived from a helper virus such as adenovirus or herpes simplex virus (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0210] AAVs possess unique characteristics that make them attractive as vectors for delivering foreign DNA into cells. AAV infection of cells in culture is generally noncytopathic, and natural infection in humans and other animals is asymptomatic and symptomless. Furthermore, AAVs can infect many different mammalian cell types and potentially target many different tissues in vivo. AAVs also promote a weaker immune response compared to other forms of gene delivery, and have additional advantages that make them a particularly attractive viral system for gene delivery, including persistent expression based on non-integrating, episomal vector DNA in both dividing and quiescent cells. Furthermore, AAVs can withstand conditions used to inactivate adenovirus (56°C–65°C for several hours), making cold storage of rAAV-based vaccines less critical.

[0211] The types or serotypes of adeno-associated virus that can be used in the present disclosure include AAVrh.10 (AAVrh10), AAV-DJ (AAVDJ), AAV-DJ8 (AAVDJ8), AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV'130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.4 4、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R 4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu. 17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AA Vhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu. u.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.12、AAVrh.13、AAVr h.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAV rh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、A AVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh8R A586R mutant、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.14、AAVhEr1.18、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、 AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAVhEr2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM10-2, AAV Shuffle100-1, AAV Shuffle100-3, AAV Shuffle100-7, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle10-8, AAV Shuffle100-2, AAV. SM10-1, AAV SM10-8, AAV SM100-3, AAV SM100-10, BP61 AAV, BP62 AAV, BP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10 and Japanese AAV10 serotypes, including but not limited to these.

[0212] In some embodiments, the adeno-associated virus serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrhlO. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV5. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9.

[0213] III.B. Non-AAV Vectors The present specification also provides non-AAV vectors that include the polynucleotides described herein (eg, including foreign genes and untranslated nucleic acid sequences).

[0214] In some embodiments, the vector can be a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage, or eukaryotic viral DNA. In addition to the AAV vector, numerous other vector backbones useful for protein expression and known in the art can be used. These vectors include, but are not limited to, adenovirus, retrovirus, boxvirus, baculovirus, herpesvirus, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), and Moloney rat leukemia virus. Furthermore, one class of vectors contains DNA elements derived from viruses such as bovine papillomavirus, polyomavirus, baculovirus, retrovirus, or Semliki Forest virus. Such vectors are commercially available or can be assembled from the described sequences by methods well known in the art.

[0215] It will be apparent to those skilled in the art that certain disclosures relating to AAV vectors provided herein are equally applicable to non-AAV vectors, and thus, unless otherwise specified, the term "vector" includes both AAV and non-AAV vectors.

[0216] IV. Cell In some aspects, the present specification provides cells comprising any of the polynucleotides described herein (e.g., including untranslated nucleic acid sequences). For example, in some aspects, the cells described herein are transduced, transfected, or transformed with a recombinant expression construct comprising a foreign gene and untranslated nucleic acid sequences for expression of the foreign gene.

[0217] Without being bound by any theory, in some embodiments, the cells described herein (e.g., transduced with a polynucleotide comprising an untranslated nucleic acid sequence) are useful for producing proteins, such as those encoded by the exogenous genes described herein (e.g., VEGF inhibitors). As described herein, in some embodiments, the untranslated nucleic acid sequences described herein (i.e., EF-1α intron fragments) can enhance expression of the protein encoded by the exogenous gene (the "encoded protein") in a cell. Thus, in some embodiments, the cells described herein (e.g., transduced with a polynucleotide comprising an exogenous gene and an untranslated nucleic acid sequence of the present disclosure) result in greater expression of the encoded protein compared to a reference cell. In some embodiments, the reference cell is transduced with the polynucleotide of interest but lacks the untranslated nucleic acid sequence.

[0218] In some embodiments, the cells described herein are capable of producing the protein encoded by the exogenous gene in vitro. In certain embodiments, the cells described herein are capable of producing the encoded protein in vivo (e.g., in a subject administered a polynucleotide described herein). In some embodiments, the cells described herein are capable of producing the encoded protein both in vitro and in vivo.

[0219] In some embodiments, cells that can be used to produce proteins encoded by foreign genes (e.g., in vitro) include host cells. As used herein, the term "host cell" is intended to include the cells of any organism that can be transduced with the expression construct (e.g., a vector) to replicate the expression construct or express the gene encoded by the expression construct. Such cells include eukaryotic and prokaryotic cells. As used herein, the term "transduction" is intended to include transfection and transformation. The host cell can be transduced, transfected, or transformed with the expression construct. This process refers to the delivery or introduction of an exogenous nucleic acid molecule into the host cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, a transgenic mammalian cell, and a plant cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0220] In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is Sf9. In some embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used in the present disclosure include HEK293, HeLa, ARPE-19, RPE-1, HepG2, Hep3B, Huh-7, C8D1a, Neuro2A, CHO, MES13, BHK-21, COS7, COP5, A549, MCF-7, HC70, HCC1428, BT-549, PC3, LNCaP, Capan-1, Panc-1, MIAPaCa-2, SW480, HCT166, LoVo, A172, MKN-45, MKN-74, Kato-III, NCI-N87, HT-144, SK-MEL-2, SH-SY5Y, C6, HT-22, PC-12, NIH3T3 cells, and combinations thereof.

[0221] In some embodiments, cells that can be used to produce (e.g., in vivo) proteins encoded by exogenous genes described herein include human cells. In some embodiments, the human cells are cells of a subject administered a nucleic acid molecule described herein. In certain embodiments, the human cells are derived from a donor (e.g., a healthy human subject).

[0222] V. Pharmaceutical Compositions The various nucleic acid molecules, cells, and vectors (also referred to herein as "active compounds") disclosed herein can be incorporated into pharmaceutical compositions suitable for administration. Accordingly, in some aspects, the present disclosure relates to such pharmaceutical compositions.

[0223] In some aspects, the present specification discloses pharmaceutical compositions comprising (a) any of the polynucleotides described herein (e.g., including exogenous genes and untranslated nucleic acid sequences) and (b) one or more pharmaceutically acceptable carriers. In some aspects, the present specification discloses pharmaceutical compositions comprising (a) a vector described herein (e.g., rAAV) and (b) one or more pharmaceutically acceptable carriers. In some aspects, the present specification discloses pharmaceutical compositions comprising (a) a cell described herein and (b) one or more pharmaceutically acceptable carriers.

[0224] In some aspects, the pharmaceutical compositions described herein comprise a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus comprises (a) an AAV8 capsid protein and (b) a polynucleotide comprising (i) a foreign gene comprising the nucleotide sequence set forth in SEQ ID NO:23, and (ii) a regulatory element operably linked to the foreign gene, wherein the regulatory element comprises (in a 5' to 3' direction): (1) a CMV enhancer sequence set forth in SEQ ID NO:4; (2) a chicken β-actin promoter sequence set forth in SEQ ID NO:8; (3) a chicken β-actin exon 1 (E1) sequence set forth in SEQ ID NO:15; (4) a chicken β-actin intron splicing donor sequence set forth in SEQ ID NO:10; (5) a non-translated nucleic acid sequence comprising, consisting essentially of, or consisting of the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:57; and (6) an EF-1α exon 2 (E2) sequence set forth in SEQ ID NO:11.

[0225] The pharmaceutically acceptable carriers that can be used in the present disclosure are those that are commonly used in formulations. Examples of the pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical compositions of the present disclosure may further comprise one or more additives selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0226] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of suitable parenteral routes of administration include intravenous injection, transdermal injection, subcutaneous injection, intramuscular injection, intravitreal injection, subretinal injection, choroidal space injection, ophthalmic injection, intraventricular injection, intraspinal injection, intraamniotic injection, intra-arterial injection, intra-articular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisternal injection, intracoronary injection, intracranial injection, intradural injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection, intraperitoneal injection, intrathoracic injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection, topical injection, intraperitoneal injection, and combinations thereof.

[0227] In some embodiments, the pharmaceutical composition is administered at a daily dose of 0.0001 to 100 mg / kg.

[0228] The pharmaceutical compositions of the present disclosure can be formulated with one or more pharmaceutically acceptable carriers and / or excipients. The pharmaceutical compositions can be provided in unit dosage form or dispensed in multi-dose containers. The formulations can be in the form of solutions, suspensions, or emulsions in oily or aqueous media, or in the form of tablets, powders, granules, tablets, or capsules. The formulations can further include dispersants or stabilizers.

[0229] VI. Kit This specification also discloses kits that include one or more polynucleotides (e.g., including exogenous genes and untranslated nucleic acid sequences) disclosed herein, one or more vectors (e.g., rAAV) disclosed herein, one or more cells disclosed herein, any pharmaceutical composition disclosed herein, or any combination thereof. In some embodiments, the kits also include instructions for administering any of the foregoing or any combination thereof to a subject in need thereof.

[0230] As used herein, the terms "kit" and "system" are intended to refer, in some aspects, to at least one or more polynucleotides disclosed herein, one or more vectors disclosed herein (e.g., rAAV), one or more host cells disclosed herein, any pharmaceutical composition disclosed herein, or any combination thereof, in combination with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packaging such as commercial packaging, instructions for use, etc.).

[0231] VII. Uses and Methods VII.A. Production Methods The present specification also discloses methods for producing a polypeptide encoded by an exogenous gene. In some embodiments, such methods comprise culturing a cell described herein (e.g., transduced with a polynucleotide comprising an exogenous gene and an untranslated nucleic acid molecule) under appropriate conditions and recovering the encoded protein. In particular embodiments, the method for producing a polypeptide encoded by an exogenous gene comprises administering a polynucleotide (e.g., comprising an exogenous gene and an untranslated nucleic acid molecule) of the present disclosure to a subject in need thereof, thereby producing the encoded polypeptide in the subject. Further disclosure regarding such in vivo methods for producing polypeptides is provided elsewhere in this disclosure (see, e.g., therapeutic uses).

[0232] In some aspects, the present disclosure provides methods for producing a recombinant adeno-associated virus (rAAV) comprising a polynucleotide (e.g., including a foreign gene and a non-translated nucleic acid sequence) described herein. In some aspects, the method for producing such a recombinant AAV comprises culturing cells transfected with an AAV vector described herein under conditions for producing the recombinant AAV. In some aspects, the method further comprises recovering the recombinant AAV from the cell culture supernatant.

[0233] In some embodiments, the recombinant adeno-associated virus vector can be produced using (i) an AAV construct containing the foreign gene and a non-translated nucleic acid sequence (see, e.g., FIG. 6C), (ii) a construct containing the rep and cap genes, and (iii) a helper construct for transducing the foreign gene into a host cell. In this embodiment, the helper construct can contain an E2A gene that promotes AAV genome replication and gene transcription, an E4 gene that enables AAV mRNA to move from the nucleus to the cytoplasm, and a VA region that generates two VA RNAs that play a role in regulating translation.

[0234] In some embodiments, the three constructs described above can be replaced by two constructs for transducing host cells. In such embodiments, an AAV construct comprises a foreign gene and a non-translated nucleic acid sequence, and a separate construct comprises the rep and cap genes, E2A gene, E4 gene, and VA region. Additional methods for producing the AAV particles described herein are generally known in the art. See, for example, Clement et al., Mol Ther Methods Clin Dev 3:16002 (March 2016); Clark, Kidney, J. Med., 1999, 14 ... See Int.61:S9-15 (January 2002); and Xiao et al., J Virol 72(3):2224-32 (March 1998).

[0235] VII.B. Therapeutic uses The nucleic acid molecules (e.g., comprising exogenous genes and untranslated nucleic acid sequences), vectors and recombinant viruses (e.g., rAAV) comprising such nucleic acid molecules, and the methods described herein have numerous in vitro and in vivo utilities. For example, the polynucleotides, e.g., vectors, e.g., AAV vectors, described herein can be administered to cells in culture, in vitro, or ex vivo, or to human subjects, e.g., in vivo, to treat disease. Thus, in some aspects, the present disclosure provides therapeutic uses of any of the polynucleotides (e.g., comprising exogenous genes and untranslated nucleic acid sequences) described herein, the recombinant expression constructs described herein, the cells described herein, the pharmaceutical compositions described herein, or the recombinant viruses described herein.

[0236] In some aspects, the present specification discloses a method of expressing an exogenous gene in a subject in need thereof, comprising administering to the subject a polynucleotide (e.g., comprising an exogenous gene and a non-translated nucleic acid sequence) disclosed herein, a vector disclosed herein, a recombinant virus (e.g., rAAV) disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein, wherein expression of the exogenous gene in the subject is increased following said administration.

[0237] As described herein, the non-translated nucleic acid sequences of the present disclosure can increase expression of an exogenous gene when the exogenous gene is translated. Thus, in some aspects, the present disclosure provides a method for increasing expression of an exogenous gene in a cell, the method comprising contacting the cell with any of the polynucleotides, vectors, or recombinant viruses (e.g., rAAV) disclosed herein. The contacting can be performed in vitro or in vivo. When the contacting proceeds in vivo, the method can further comprise administering any of the polynucleotides, vectors, or recombinant viruses to a subject prior to the contacting.

[0238] In some embodiments, expression of the exogenous gene after the contacting is increased by at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold or more compared to baseline expression. In some embodiments, the baseline expression is expression of the exogenous gene in the cell prior to contacting. In some embodiments, the baseline expression is expression of the exogenous gene in the cell that is not contacted with a polynucleotide, vector, or recombinant virus described herein (e.g., lacking a non-translated nucleic acid sequence or comprising the nucleotide sequence set forth in SEQ ID NO: 1).

[0239] Yet another aspect of the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of any of the polynucleotides, vectors, cells, recombinant viruses, or pharmaceutical compositions described above. As is apparent from the present disclosure, the compositions described herein (e.g., polynucleotides, recombinant expression constructs, cells, pharmaceutical compositions, or recombinant viruses) can be used to treat any disease in a subject, for example, by modifying an exogenous gene.

[0240] Diseases that can be prevented, ameliorated, or treated by the present disclosure are not limited and include all diseases that require reduced drug administration. Non-limiting examples of such diseases include ophthalmic diseases. In some embodiments, the ophthalmic disease is selected from diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, or a combination thereof.

[0241] In some embodiments, the ophthalmic disease treatable by the present disclosure includes macular degeneration. In some embodiments, the macular degeneration includes age-related macular degeneration (AMD). Age-related macular degeneration can be divided into dry (atrophic) macular degeneration and wet (neovascular or exudative) macular degeneration. Age-related macular degeneration can be divided into early AMD, intermediate AMD, and late or advanced AMD (map atrophy). In some embodiments, the ophthalmic disease treatable by the present disclosure includes diabetic retinopathy. In some embodiments, the diabetic retinopathy is nonproliferative diabetic retinopathy (NPDR). In some embodiments, the diabetic retinopathy is proliferative diabetic retinopathy (PDR). In some embodiments, the diabetic retinopathy is diabetic macular disease. In some embodiments, the diabetic retinopathy is diabetic macular edema. In some embodiments, the diabetic retinopathy is any retinopathy associated with ischemic damage in the retina. Unless otherwise stated, the present disclosure can be used to treat any form of AMD and / or diabetic retinopathy.

[0242] Yet another aspect of the present disclosure provides a gene therapy agent or method for treating a disease that can achieve sustained expression of an exogenous gene.

[0243] Using the viral delivery systems described herein, it is possible to administer the compositions described herein at intervals of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year or more. In some embodiments, the intervals are about 2 to about 3 months. In some embodiments, the intervals are about 6 months. In some embodiments, the intervals are about 1 year. In some embodiments, the intervals are at least about 1 year. That is, using the viral delivery systems described herein can significantly reduce the frequency of administration of the composition, thereby avoiding the discomfort caused to physicians, patients, or subjects by repeated administration of the composition. Depending on the patient's symptoms or needs, the composition can be initially administered at least 2 to 3 times at intervals of 1 to 2 weeks, and then once every 2 to 3 months, every 6 months, or once every 1 year or more.

[0244] The present disclosure will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that the scope of the present disclosure is not limited to these examples, according to the gist of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS These and / or other aspects and advantages of the present disclosure will be apparent and more readily understood from the following description of said aspects with reference to the accompanying drawings, in which: FIG. 1 shows a cleavage map of the pAAV-eGFP expression construct described herein.

[0245] Figures 2A and 2B show increased expression of eGFP in cells transduced with an expression construct containing the full-length EF-1α intron together with the EF-1α promoter ("CEE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron ("CE"). In Figure 2A, an ITR-deleted animal cell expression construct was used. In Figure 2B, an ITR-containing pAAV expression construct was used. In both Figures 2A and 2B, eGFP expression is shown in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows:

[0246] Figures 3A, 3B, and 3C show eGFP expression in cells transduced with an expression construct containing the full-length EF-1α intron with various promoters ("CCE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron. In Figure 3A, a combination of the CMV enhancer and CMV promoter was inserted into an animal cell expression construct (i.e., ITR-deleted) and used to transduce the cells. In Figure 3B, a combination of the CMV enhancer and CMV promoter was inserted into a pAAV expression construct and used to transduce cells. In Figure 3C, the cells were transduced with an animal cell expression construct containing a combination of the CMV enhancer and chicken β-actin promoter. Figures 3A, 3B, and 3C, respectively, show eGFP expression in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows:

[0247] Figures 4A and 4B show the effect of increasing or decreasing various EF-1α intron fragment sequences (i.e., non-translated nucleic acid sequences described herein) on gene expression. Figure 4A shows a schematic of a series of CEE constructs produced by sequentially deleting EF-1α intron sequences. Each construct shown contained: (1) a CMV enhancer (380 base pairs); (2) a promoter containing a portion of the CMV promoter (the first 31 base pairs from the 5' end) and the EF-1α promoter (201 base pairs); and (3) EF-1α exon 1 (E1) sequence (29 base pairs). The control "CE" construct contained the EF-1 alpha intron sequence but no additional components. Other constructs further contained the following additional components: (4) a splice donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1 alpha intron sequence (i.e., SEQ ID NO:1); (5) an EF-1 alpha intron sequence; and (6) an EF-1 alpha exon 2 (E2) sequence (9 base pairs). The EF-1 alpha intron sequence may be the full-length sequence (924 base pairs) ("CEE-FL") or: (ii) nucleotides 570 to 924 of SEQ ID NO:1 (355 base pairs) ("CEE-T2"); (iii) nucleotides 721 to 924 of SEQ ID NO:1 (204 base pairs) ("CEE-T3"); (iv) nucleotides 808 to 924 of SEQ ID NO:1 (117 base pairs) ("CEE-T3.1"); (v) nucleotides 808 to 924 of SEQ ID NO:1 (117 base pairs) ("CEE-T3.1"); (vi) nucleotides 830-924 (95 base pairs) of SEQ ID NO:1 ("CEE-T3.1.1"); (vi) nucleotides 852-924 (73 base pairs) of SEQ ID NO:1 ("CEE-T3.1.2"); (vii) nucleotides 874-924 (51 base pairs) of SEQ ID NO:1 ("CEE-T3.2"); and (viii) nucleotides 896-924 (29 base pairs) of SEQ ID NO:1 ("CEE-T4"). The full length of each construct is provided on the right. Figure 4B shows the effect of different EF-1α intron sequences (full or deleted) on eGFP (i.e., exogenous gene) expression in five different cell lines (HeLa, Hep3B, Huh-7, ARPE-19, and RPE-1). ARPE-19 and RPE-1 cells were derived from the retina.Huh-7 and Hep3B cells were derived from the liver. HeLa cells were derived from the cervix. eGFP expression was shown as the expression rate (%) observed in cells transduced with a foreign gene using the CEE-FL construct. "ns" = not significant. "**" = p<0.01 and "***" = p<0.001.

[0248] Figures 5A, 5B, 5C, and 5D show the ability of EF-1α intron fragments T3.1.1 (i.e., nucleotides 830-924 (95 base pairs) of SEQ ID NO:1) and T3.1.2 (i.e., nucleotides 852-924 (73 base pairs) of SEQ ID NO:1) to increase expression of foreign genes. Figure 5A shows a schematic representation of a series of CAE constructs containing EF-1α intron fragments T3.1.1 and T3.1.2, along with the following additional components: (1) a CMV enhancer ( (3) chicken β-actin promoter (279 base pairs); (3) chicken β-actin exon 1 (E1) sequence (32 base pairs); (4) EF-1α exon 1 (E1) sequence (29 base pairs); (5) splicing donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1α intron sequence (i.e., SEQ ID NO: 1); (6) EF-1α exon 2 (E2) sequence (9 base pairs). The control "CA" construct contained the CMV enhancer, chicken β The control "CAE-FL" construct contained only the EF-1α intron sequence and the β-actin promoter. The full length of the constructs is provided on the left. Figure 5B shows eGFP (i.e., foreign gene) expression in HeLa (left graph) and ARPE-19 (right graph) cells transduced with the CAE-T3.1.1 and CAE-T3.1.2 constructs. Gene expression was significantly enhanced by the CA constructs (i.e., the EF-1α intron sequence). The results are shown as percentage expression in cells transduced with the EF-1α intron fragment T3.1.1 or T3.1.2 and the chicken β-actin intron fragment. Figure 5C shows a schematic diagram of a series of hybrid intron CA constructs (i.e., CA-T3.1.1 and CA-T3.1.2 constructs) containing both the EF-1α intron fragment T3.1.1 or T3.1.2 and the chicken β-actin intron fragment. The two constructs further contain: (1) a CMV enhancer; (2) a chicken β-actin promoter; (3) a chicken β-actin E1 sequence; and (4) an EF-1α E2 sequence.The control "CAG-FL" construct contained: (1) a CMV enhancer (380 base pairs); (2) a chicken β-actin promoter (279 base pairs); (3) a chicken β-actin E1 sequence (93 base pairs); (4) a chimeric intron (containing chicken β-actin and rabbit β-globin introns) (924 base pairs); and (5) a rabbit β-globin exon 3 (E3) sequence (48 base pairs). The "CA" construct was the same as that described in Figure 5A. Figure 5D shows the expression of eGFP in HeLa (left graph) and Hep3B (right graph) cells transduced with constructs CA-T3.1.1 and CA-T3.1.2. Gene expression is shown as the percentage of expression in the corresponding cells transduced with the CA construct (i.e., lacking the EF-1α intron sequence).

[0249] Figures 6A, 6B, and 6C show the effect of EF-1α intron fragments T3.1.1 and T3.1.2 on increasing foreign gene (i.e., apprebercept) expression when delivering genes using AAV. Figure 6A shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vitro gene transfer. Figure 6B shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vivo gene transfer. Figure 6C shows the cleavage map of the pAAV-CA-T3.1.1 vector containing apprebercept as the foreign gene. [Mode for Carrying Out the Invention] Materials and Methods Example 1. Preparation of a pAAV-eGFP construct that does not contain an enhancer-promoter-intron sequence Example 1-1. Insertion of the bGH polyadenylation signal sequence Using the pcDNA5 / FRT / TO construct (Invitrogen, USA, Cat. No. V6520-20) as a template, polymerase chain reaction (PCR) was performed using oligos #001 and #002 to obtain the bovine growth hormone (bGH) polyadenylation signal A sequence (polyA). Next, the human growth hormone (hGH) polyA was removed using the BglII / BstEII sites of the pAAV-MCS-mu promoter plasmid (Cellbiolabs, USA, Cat. No. VPK-411) and inserted into the bGH polyA.

[0250] Example 1-2. Insertion of eGFP The human codon-optimized eGFP gene from the pUCIDT-KAN-eGFP construct (GeneArt, Germany) was obtained and cloned into the BamHI / HindIII sites of the pAAV-bGH construct prepared in Example 1-1.

[0251] Example 1-3. Insertion of WPRE sequence The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence was obtained from the pUC57-WPRE construct (GenScript, USA) and cloned into the HindIII / BglII sites of the pAAV-eGFP-bGH construct prepared in Example 1-2.

[0252] Example 1-4. Insertion of four copies of miRNA142-3p target sequence Oligos #003 and #004, as well as #005 and #006, were annealed to prepare two DNA fragments, each containing two copies of the miRNA142-3p target sequence. The two short DNA fragments were cloned into the HindIII / SalI sites of the pAAV-eGFP-WPRE-bGH construct prepared in Examples 1-3, resulting in the insertion of a total of four copies of the miRNA142-3p target sequence. This resulted in the construction of the pAAV-eGFP construct, which does not contain an enhancer-promoter-intron sequence (Figure 1).

[0253] Example 2. Preparation of constructs containing various types of enhancer-promoter-intron sequences Example 2-1. Preparation of the CEE series (CEE-FL, CEE-T2, -T3, -T3, -T3.1, -T3.1.1, -T3.1.2, -T3.2, -T4) and constructs containing CE sequences Example 2-1-1. Preparation of a construct containing the CEE-FL (full-length) base sequence A DNA fragment CEE-FL (CMV enhancer (SEQ ID NO: 4)-31 bp CMV promoter (SEQ ID NO: 5)-EF-1α promoter (SEQ ID NO: 7)-29 bp EF-1α exon 1 (SEQ ID NO: 13)-EF-1α intron (SEQ ID NO: 1)-9 bp EF-1α exon 2 (SEQ ID NO: 11)) was obtained from the pMK-RQ3_PEM construct (GeneArt, Germany), and cloned into the EcoRI / BamHI sites of the construct prepared in Example 1.

[0254] Example 2-1-2. Preparation of a construct containing the CEE-T2 base sequence PCR was performed using the constructs prepared in Example 2-1-1 as templates using the oligo #007 / 008 combination and the #009 / 010 combination, and the two DNA fragments were ligated using Gibson Assembly (registered trademark) (NEB, USA, Cat No. E2611) to prepare a construct containing the CEE-T2 sequence.

[0255] Example 2-1-3. Preparation of a construct containing the CEE-T3 base sequence PCR was performed using the constructs prepared in Example 2-1-1 as templates and the oligo #011 / 008 combination and #012 / 010 combination, and the two DNA fragments were ligated via Gibson Assembly to prepare a construct having the CEE-T3 sequence.

[0256] Example 2-1-4. Preparation of a construct containing the CEE-T3.1 base sequence PCR was performed using the combination of oligos #013 / 008 and #014 / 010 and the construct produced in Example 2-1-1 as a template, and the two DNA fragments were ligated via Gibson Assembly to produce a construct having the CEE-T3.1 sequence.

[0257] Example 2-1-5. Preparation of a construct containing the CEE-T3.2 base sequence PCR was performed using the combination of oligos #015 / 008 and #016 / 010 and the construct produced in Example 2-1-1 as a template, and the two DNA fragments were ligated via Gibson Assembly to produce a construct having the CEE-T3.2 sequence.

[0258] Example 2-1-6. Preparation of a construct containing the CEE-T4 base sequence PCR was performed using the combination of oligos #017 / 008 and #018 / 010 and the construct produced in Example 2-1-1 as a template, and the two DNA fragments were ligated via Gibson Assembly to produce a construct having the CEE-T4 sequence.

[0259] Example 2-1-7. Preparation of a construct containing the CEE-T3.1.1 base sequence The CEE-T3.1.1 nucleotide sequence from the pUC57-T3.1.1 construct (GenScript, USA) was secured and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-1-1.

[0260] Example 2-1-8. Preparation of a construct containing the CEE-T3.1.2 base sequence The CEE-T3.1.2 nucleotide sequence from the pUC57-T3.1.2 construct (GenScript, USA) was secured and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-1-1.

[0261] Example 2-1-9. Preparation of a construct containing a CE base sequence A CE fragment was isolated by PCR from the construct prepared in Example 2-1-1 using oligos #019 and #020, and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-1-1.

[0262] Example 2-2. Preparation of constructs containing CCE-FL and CC sequences Example 2-2-1. Preparation of a construct containing the CCE-FL sequence A DNA fragment having the sequence CCE-FL (CMV enhancer (SEQ ID NO: 4)-CMV promoter (SEQ ID NO: 6)-CMV exon 1 30 bp (SEQ ID NO: 12)-EF-1α exon 1 29 bp (SEQ ID NO: 13)-EF-1α intron (SEQ ID NO: 1)-EF-1α exon 2 9 bp (SEQ ID NO: 11)) was obtained from the pMK-RQ4_PME construct (GeneArt, Germany) and cloned into the EcoRI / BamHI sites of the pAAV-eGFP construct obtained in Example 1.

[0263] Example 2-2-2. Preparation of a construct containing a CC sequence A CC fragment was isolated by PCR from the construct prepared in Example 2-2-1 using oligos #019 and #021, and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-2-1.

[0264] Example 2-3. Preparation of constructs containing CAG-FL, CA-T3.1.1, CA-T3.1.2, and CA sequences Example 2-3-1. Preparation of a construct containing the CAG-FL sequence A CAG fragment was obtained from the pCAG-Neo construct (Wako Pure Chemical Industries, Ltd., Japan, Cat No. 163-25601) and cloned into the SnaBI / BamHI site of the construct obtained in Example 2-1.

[0265] Example 2-3-2. Preparation of a construct containing a CA sequence The CA fragment was secured from the pUC57-CA construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-3-1.

[0266] Example 2-3-3. Preparation of a construct containing the CA-T3.1.1 sequence A T3.1.1 fragment was obtained by PCR from the construct obtained in Example 2-1-7 using oligos #022 and #023, and this was cloned into the AfeI / BamHI site of the construct prepared in Example 2-3-1.

[0267] Example 2-3-4. Preparation of a construct containing the CA-T3.1.2 sequence The T3.1.2 fragment obtained by annealing oligos #024 and #025 was cloned into the AfeI / BamHI sites of the construct prepared in Example 2-3-1.

[0268] Examples 2-4. Preparation of constructs containing CAE-FL, CAE-T3.1.1, and CAE-T3.1.2 sequences Example 2-4-1. Preparation of a construct containing the CAE-FL sequence The CAE (CMV enhancer (SEQ ID NO: 4)-chicken β-actin promoter (SEQ ID NO: 8)-32 bp chicken β-actin exon 1 (SEQ ID NO: 14)-29 bp EF-1α exon 1 (SEQ ID NO: 13)-924 bp EF-1α intron (SEQ ID NO: 1)-9 bp EF-1α exon 2 (SEQ ID NO: 11)) fragment was obtained from the pUC57-CAE construct (GenScript, USA) and cloned into the EcoRI / BamHI sites of the construct prepared in Example 1.

[0269] Example 2-4-2. Preparation of a construct containing the CAE-T3.1.1 sequence The CAE-T3.1.1 fragment was isolated from the construct prepared in Example 2-1-7 using oligos #026 and #027 and cloned into the KpnI / BamHI site of the construct prepared in Example 2-1-7.

[0270] Example 2-4-3. Preparation of a construct containing the CAE-T3.1.2 sequence The CAE-T3.1.2 fragment was isolated from the construct prepared in Example 2-1-8 using oligos #026 and #027, and cloned into the KpnI / BamHI site of the construct prepared in Example 2-1-8.

[0271] Example 2-5. Preparation of pAAV construct containing the upreversecept gene The apribercept gene DNA fragment obtained by PCR from the pcDNA3.1(+)-IgG-apribercept construct (GenScript, USA) using oligos #028 and #029 was cloned into the BamHI / HindIII sites of the constructs prepared in Examples 2-3-1, 2-3-3, 2-3-4, 2-4-1, 2-4-2, and 2-4-3, respectively.

[0272] Example 2-6. Preparation of an animal cell expression construct lacking ITR sequences Example 2-6-1. Preparation of pcDNA3.1(+)-eGFP construct The construct prepared in Example 1 was digested with BamHI / AfeI to obtain the eGFP gene fragment, which was then cloned into the BamHI / EcoRV sites of the pcDNA3.1(+) construct.

[0273] Example 2-6-2. Preparation of an animal cell expression construct containing a CEE sequence The construct prepared in Example 2-1-1 was digested with NdeI / BamHI and secured, and then cloned into the NdeI / BamHI site of the construct prepared in Example 2-6-1.

[0274] Example 2-6-3. Preparation of animal cell expression constructs containing CE sequences The construct prepared in Example 2-1-9 was cleaved with NdeI / BamHI to obtain a CE fragment, which was then cloned into the NdeI / BamHI site of the construct prepared in Example 2-6-1.

[0275] Example 2-6-4. Preparation of animal cell expression constructs containing CCE sequences The construct prepared in Example 2-2-1 was cleaved with NdeI / BamHI to obtain the CCE fragment, which was then cloned into the NdeI / BamHI site of the construct prepared in Example 2-6-1.

[0276] Example 2-6-5. Preparation of animal cell expression constructs containing CC sequences The construct prepared in Example 2-2-2 was cleaved with NdeI / BamHI to obtain a CC fragment, which was then cloned into the NdeI / BamHI site of the construct prepared in Example 2-6-1.

[0277] Example 2-6-6. Preparation of animal cell expression constructs containing CAE sequences The construct prepared in Example 2-4-1 was cleaved with NdeI / BamHI to obtain the CAE fragment, which was then cloned into the NdeI / BamHI site of the construct prepared in Example 2-6-1.

[0278] Example 2-6-7. Preparation of animal cell expression constructs containing CA sequences The construct prepared in Example 2-3-2 was cleaved with NdeI / BamHI to obtain the CA fragment, which was then cloned into the NdeI / BamHI site of the construct prepared in Example 2-6-1.

[0279] Example 2-7. Confirmation of base sequence All constructs obtained by cloning were sequenced by DNA sequencing (MACROGEN, Korea or BIONEER, Korea).

[0280] Example 3. Cell culture HEK293 and HeLa cell lines were cultured in MEM medium (Gibco, USA, Cat. No. 42360-032), ARPE-19 cell lines in DMEM / F12 medium (Gibco, USA, Cat. No. 11330-032), and RPE-1 and Hep3B cell lines in DMEM medium (Gibco, USA, Cat. No. 10569-010) under humidified conditions of 5% CO2 and 37°C. All media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA, Cat. No. 16000-044) and 1% penicillin-streptomycin (Gibco, USA, Cat. No. 15140-163). The Expi293 cell line was cultured in wet culture in Expi293 medium (Gibco, USA, Cat. No. A14351-01) supplemented with 1% penicillin-streptomycin under conditions of 8% CO and 37°C with shaking at 250 rpm.

[0281] Example 4. Transduction Example 4-1. Transduction of adherent cells For transduction, each cell line was washed twice with DPBS (Gibco, USA, Cat No. 14190-250) and then removed from the culture dish with trypsin-EDTA (Gibco, USA, Cat No. 25200-114). The cells were then seeded into 12-well plates at 80% saturation. After 24 hours of culture, the cells were transduced with the respective plasmid DNA using Lipofectamine 3000 (Thermo Fisher Scientific, USA, Cat No. L300075).

[0282] Example 4-2. Transduction of suspension cells for AAV production For AAV production, 6 x 10 cells were cultured in a 1 L Ellenmeyer culture flask. 8The cells were inoculated into 220 ml of expi293 medium. After approximately 3–4 hours of incubation for stabilization, 3.73 pmoles each of pHelper plasmid DNA, pUC-RC2 plasmid DNA, or pUC-RC8 plasmid DNA, and the AAV construct plasmid DNA containing the foreign gene were dissolved in 10 ml of Opti-MEM (Gibco, USA, Cat. No. 51985-034). Polyethyleneamine (PEI, Polyscience, USA, Cat. No. 23966-1) equivalent to twice the total amount of DNA was then diluted with 10 ml of Opti-MEM, and the two solutions were immediately mixed to prepare the transfection solution. After incubation at room temperature for 30 minutes, a total of 20 ml of the transfection solution was added to a culture flask.

[0283] Example 5. Purification of AAV Example 5-1. Purification of AAV2 72 hours after transduction in Example 4-2, the cell culture medium was collected and centrifuged to remove the medium and collect the cells. The cell pellet was washed with DPBS, and the cells were resuspended in 16 ml of DPBS. This was subjected to three freeze / thaw cycles to lyse the cells, which were then centrifuged to collect the supernatant containing AAV2. The supernatant and AAVanced TM The mixture was mixed with Concentration Reagent (System Bioscience, USA, Cat No. AAV110A-1) at a 4:1 ratio, stirred at 4°C for 16 hours, then centrifuged to remove the supernatant and collect the AAV2-containing pellet. The pellet was washed with 500 μl of Opti-MEM, and after removing all of the supernatant, it was finally resuspended in 400 μl of ice-cold DPBS to obtain AAV.

[0284] Example 5-2. Purification of AAV8 72 hours after transduction in Example 4-2, the cell culture medium was collected. Cell debris was removed using a 0.45 μm filter, and AAV was isolated using anion exchange and affinity chromatography.

[0285] Example 6. AAV titration qPCR (Bio-Rad, USA, CFX96) was performed to determine the titer of AAV2 purified in Example 5. ANAV was treated with DNaseI in DNaseI reaction buffer (New England Biolab, USA, M0303S) at 37°C for 1 hour. The DNaseI-treated sample was then treated with proteinase K (Invitrogen, USA, Cat No. AM2548) at 55°C for 30 minutes, and then reacted at 95°C for 15 minutes to inactivate the proteinase K. The prepared sample was used as a template for qPCR, and the AAV construct (7.4 x 10) was used to generate a standard curve. 8 -7.4×10 4 , 10-fold dilution), Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2-RSS, ATCC, USA, Cat No. VR-1616), or Recombinant Adeno-associated Virus 8 Reference Standard Stock (rAAV8-RSS, ATCC, USA, Cat No. VR-1816) were used as positive controls. qPCR for titer determination was performed using 2x SsoAdvanced Universal Probe Supermix (Bio-Rad, USA, Cat No. VR-1616). The primers used were AAV2-ITR-specific primers (#030, #031) and probe (#032, FAM-CACT CCCTCTCTGCGCGCTCG-BHQ1). Each qPCR cycle consisted of denaturation at 95°C for 10 minutes, incubation at 95°C for 30 seconds, and incubation at 60°C for 1 minute, repeated 40 times. Standard curves and quantification were analyzed using Bio-Rad CFX Maestro 1.1 software (Bio-Rad, USA).

[0286] Example 7. Transduction with AAV2 in vitro For AAV2 transduction, HEK293 cells were cultured in 24-well culture plates at 4 x 10 cells per well. 5After 24 hours, each well was treated with MG132 (Sigma-Aldrich, USA, Cat No. M7449) at a concentration of 5 μM for 8 hours. AAV2 was then transduced at a multiplicity of infection (MOI) of 25,000 and cultured for 72 hours.

[0287] Example 8. Transduction with AAV8 in vivo After perforating the lower part of the eyeball with a 31G needle, the needle from the IO kit was inserted and stopped when the tip of the needle touched the wall of the eyeball. The success of the subretinal injection was determined by checking the formation of a retinal bleb on OCT immediately after administration. After 4 weeks, the eyeball was removed and Halt was performed. TM 200 μl of RIPA solution (Thermo Fisher Scientific, USA, Cat. No. 89900) supplemented with Protease Inhibitor Cocktail 100x (Thermo Fisher Scientific, USA, Cat. No. 78429) was added, and the eyeballs were crushed using an Axygen tissue grinder (Axygen, Cat. No. 14-222-358). After incubation at 4°C for 1 hour, the tissue was centrifuged at 13,000 g for 15 minutes at 4°C, and the supernatant was collected and analyzed.

[0288] Example 9. Measurement of GFP gene expression intensity by flow cytometry eGFP expression was measured by flow cytometry (Beckman Coulter, USA, CytoFlex). The eGFP measurement value was calculated by correcting the transduction efficiency for the red fluorescence measurement of the co-transfected pCMV-dsRed (Clontech, Japan, Cat. No. 632416) construct. 72 hours after transduction, cells were washed with DPBS and detached with trypsin. Cells were collected by centrifugation at 1500 rpm for 5 minutes and resuspended in 500 μl of DPBS supplemented with 2% FBS. In flow cytometry, single-cell regions were distinguished using an FSC vs. SSC plot, and FL1-A (green) and FL2-A (red) were measured within them. Using samples transfected with a single fluorescent vector (pEGFP-C1, Clontech, Japan, Cat. No. 632416), we measured the red fluorescence of the co-transfected pCMV-dsRed construct (Clontech, Japan, Cat. No. 632416). Complementation of pCMV-DsRed-Expression2 and pCMV-DsRed-Expression2 was performed and reflected in the measurement results. All flow cytometry analysis results were analyzed using FlowJo software 10.5.3 (Becton Dickinson & Company, USA).

[0289] Example 10. Measurement of expression level of Apribercept by ELISA ELISA was performed to quantify the concentration of secreted protein. 48 hours after AAV transduction, the cell culture medium was collected and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was used for ELISA. Uprivercept ELISA (Eagle Bioscience, USA, Cat. No. IG-AA115) was performed according to the protocol provided by the manufacturer. ELISA results were measured using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, USA) and analyzed using SkanIt software (Thermo Fisher Scientific, USA). Experiments using cell lines were performed in duplicate, and animal experiments were performed once per eye.

[0290] Example 11. Statistical analysis method All experiments using cell lines, except for ELISA, were performed in triplicate. Results were analyzed using GraphPad Prism software 8.1.1 (GraphPad Software, Inc., USA) with Student's t-test for comparisons between two groups and one-way ANOVA for comparisons between three or more groups. Animal experiments were analyzed with the Wilcoxon matched-pairs signed rank test. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0291] [Table 1]

[0292] Oligonucleotide sequences used in this disclosure Experimental results 1. Increased expression of eGFP gene by EF-1α intron in combination with EF-1α promoter We tested the effect of the EF-1α intron on eGFP gene expression regulated by the combination of the cytomegalovirus (CMV) enhancer and the human elongation factor 1α (EF-1α) promoter. First, we compared the effect of the EF-1α intron on eGFP gene expression regulated by the combination of the cytomegalovirus (CMV) enhancer and the human elongation factor 1α (EF-1α) promoter with an ITR-free animal cell expression construct. The EF-1α intron increased eGFP expression in all cell lines tested (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) by 459.6%, 276.3%, 181.8%, 163.4%, 471.1%, and 494.3% (Figure 2A). Similarly, the EF-1α intron also increased eGFP expression in the ITR-containing pAAV construct by 224.0%, 167.7%, 218.7%, 229.5%, 202.5%, and 260.5% (Figure 2B). These results demonstrate that the EF-1α intron in combination with the EF-1α promoter can increase gene expression in various forms of exogenous gene expression constructs.

[0293] 2. Increased expression of eGFP gene by EF-1α intron in combination with various promoters In addition to the EF-1α promoter, we also examined whether the EF-1α intron increased gene expression when used in combination with other promoters. First, we examined the effect of the EF-1α intron on eGFP gene expression induced by the cytomegalovirus (CMV) enhancer in combination with the CMV promoter. First, we compared the expression of the eGFP gene with an animal cell expression construct that does not contain an ITR. We confirmed that eGFP expression was increased by the EF-1α intron in all cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) by 284.3%, 464.0%, 217.5%, 180.4%, 405.7%, and 370.6% (Figure 3A).

[0294] Similarly, in pAAV constructs containing ITRs, the EF-1α intron increased eGFP expression (241.4%, 494.8%, 266.8%, 185.5%, 415.5%, and 367.8%), regulated by the combination of the cytomegalovirus (CMV) enhancer and the CMV promoter (Figure 3B).

[0295] Next, we examined the effect of the EF-1α intron on eGFP gene expression regulated by the combination of the CMV enhancer and chicken β-actin promoter. Comparison of animal cell expression constructs confirmed that the EF-1α intron increased eGFP expression (415.2%, 396.7%, 233.9%, 217.9%, 353.7%, and 297.7%) in all cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) (Figure 3C). These results suggest that the EF-1α intron can increase gene expression not only when used with the EF-1α promoter, but also when combined with various promoters.

[0296] 3. Determination of the minimum length of the EF-1α intron fragment that increases gene expression 3-A. Construction of a series of CEE constructs with sequential deletions of EF-1α intron sequences The CEE-FL construct was designed to contain the CMV enhancer and a portion of the CMV promoter (31 bp at the 5' end), the EF-1α promoter, 29 bp of EF-1α exon 1, 924 bp of the EF-1α intron, and 9 bp of EF-1α exon 2. The core sequence related to the intron splicing function contained a 5' splicing donor and a 3' splicing acceptor containing a branch point site (BPS). To identify the minimum length of the EF-1α intron sequence that could increase gene expression, the 5' splicing donor sequence in the intron was preserved, and then the intron sequences were sequentially deleted. Specifically, a CEE construct was prepared in the same manner as in Example 2-1, containing up to 19 bp of the 5' end of the EF-1α intron, which is presumed to be the consensus splicing donor sequence for the EF-1α intron, but lacking subsequent deletions of 659 bp (T2), 720 bp (T3), 807 bp (T3.1), 829 bp (T3.1.1), 851 bp (T3.1.2), 873 bp (T3.2), and 895 bp (T4) (Figure 4A).

[0297] 3-B. Effect of increasing or decreasing gene expression by sequential deletion of EF-1α intron sequences We compared eGFP expression from eight constructs, ranging from the full-length EF-1α intron sequence (CEE-FL) to those with sequential deletions, in five animal cell lines. First, in HeLa, Hep3B, and Huh-7 cell lines, eGFP gene expression was maintained from CEE-T2 to CEE-T3.1.2 compared to the full-length EF-1α intron A. However, gene expression rapidly decreased in CEE-T3.2 and CEE-T4. Next, in ARPE-19 and RPE-1 cell lines, eGFP expression was reduced to approximately 50% of that in CEE-FL in CEE-T2, while eGFP expression from CEE-T3 to CEE-T3.1.2 was similar to or slightly higher than that in CEE-FL. However, as with the previous cell lines, expression rapidly decreased in CEE-T3.2 and CEE-T4. These results indicate that the intron A fragments that have the function of increasing gene expression in EF-1α intron A are T2 to T3.1.2, with the shortest being T3.1.2 (or a fragment longer than T3.2) (Fig. 4B).

[0298] 4. Increased gene expression by EF-1α intron fragments T3.1.1 and T3.1.2 4-A. Construction of EF-1α intron fragments T3.1.1 and T3.1.2 combined with the chicken β-actin promoter The CAE-FL construct was designed to contain the CMV enhancer and chicken β-actin promoter, 32 bp of chicken β-actin exon 1, 29 bp of EF-1α exon 1, 924 bp of EF-1α intron A, and 9 bp of EF-1α exon 2. CA is a construct lacking the entire EF-1α intron. The CAE-T3.1.1 construct is identical to CAE-FL except that it contains 19 bp of the 5' end of the EF-1α intron and the T3.1.1 sequence at the 3' end, with an 829-bp deletion. The CAE-T3.1.2 construct is identical to CAE-FL except that it contains 19 bp of the 5' end of the EF-1α intron A and the T3.1.2 sequence at the 3' end, with an 851-bp deletion (Figure 5A).

[0299] 4-B. Increased gene expression by EF-1α intron fragment T3.1.1 or T3.1.2 in combination with the chicken β-actin promoter We compared eGFP expression in two animal cell lines (HeLa and ARPE-19) using three constructs: CA, which contains a complete deletion of all nucleotides in the EF-1α intron, and CAE-T3.1.1 and CAE-T3.1.2, which contain a partial deletion of the EF-1α intron. Compared to the CA construct, the CAE-T3.1.1 and CAE-T3.1.2 constructs increased eGFP expression levels by 330.5% and 243.9% in HeLa and 170.8% and 165.9% in ARPE-19 (Figure 5B). These results demonstrate that the EF-1α intron fragments T3.1.1 and T3.1.2 can increase gene expression even in combination with the chicken β-actin promoter.

[0300] 4-C. Construction of constructs combining EF-1α intron fragments T3.1.1 and T3.1.2 with chicken β-actin splicing donor We constructed CA-T3.1.1 and CA-T3.1.2 constructs, which contain hybrid intronic structures containing a 95-bp (T3.1.1) or 73-bp (T3.1.2) 3'-end fragment of the EF-1α intron and 9-bp of EF-1α exon 2, while maintaining the CMV enhancer, chicken β-actin promoter, 93-bp of chicken β-actin exon 1, and 43-bp of the intron. Chicken β-actin exon 1 and a portion of the chicken β-actin intron are expected to function as splicing donors, while the EF-1α intron A fragment functions as a splicing acceptor. A CA construct without intronic sequences was also tested as a control (Figure 5C).

[0301] 4-D. Increased gene expression by hybridized introns consisting of the splicing donor fragment of chicken β-actin and the 3'-end fragments T3.1.1 and T3.1.2 of EF-1α intron A. After transfection of the CA, CA-T3.1.1, and CA-T3.1.2 constructs into HeLa and Hep3B cell lines, respectively, we compared gene expression levels and confirmed that gene expression was increased in CA-T3.1.1 and CA-T3.1.2 compared to the intronless CA (HeLa: 215.7%, 211.0%, Hep3B: 155.0%, 167.5%). These results suggest that the EF-1α intron fragments T3.1.1 and T3.1.2 can increase gene expression even when combined with splicing donors from other genes (Figure 5D).

[0302] 5. Increased gene expression by T3.1.1 and T3.1.2 fragments when delivering genes using AAV 5-A. Increased gene expression by T3.1.1 and T3.1.2 fragments during in vitro gene delivery using AAV2 To confirm whether the EF-1α intron fragments T3.1.1 and T3.1.2 could efficiently induce gene expression when delivering genes using AAV, we generated AAV2 containing these fragments (CA-T3.1.1, CA-T3.1.2, CAE-T3.1.1, and CAE-T3.1.2) and compared them with AAV2 containing CAG-FL or CAE-FL in the gene expression regulatory region. The constructed AAV2 was designed to express the extracellularly secreted apribercept protein (the signal peptide sequence used was the nucleotide sequence encoding the hIgG signal peptide: SEQ ID NO: 26). MG132-treated HEK293 cells were infected with AAV2 at an MOI of 25,000. After 48 hours, the concentration of apribercept in the cell culture medium was measured by ELISA. We confirmed that AAV2 infection with CA-T3.1.1 or CA-T3.1.2 increased apriorcept expression by 200.1% and 213.1%, respectively, compared with CAG-FL. Furthermore, compared with CAE-FL, AAV2 infection with CAE-T3.1.1 and CAE-T3.1.2 increased apriorcept expression by 118.2% and 166.5%, respectively (Figure 6A). These results suggest that the EF-1α intron A fragments T3.1.1 and T3.1.2 can efficiently increase gene expression when delivered using AAV2, as well as when delivered using plasmid DNA.

[0303] 5-B. Increased gene expression by T3.1.1 and T3.1.2 fragments during in vivo gene transfer using AAV8 To confirm whether the EF-1α intron fragment T3.1.1 can efficiently induce gene expression when delivering genes in vivo using AAV, AAV8 containing this fragment (CA-T3.1.1, CAE-T3.1.1) was produced and compared with AAV8 containing CAG-FL or CAE-FL in the gene expression regulatory region. The AAV8 produced was designed to express the extracellularly secreted Apribercept protein (the signal peptide sequence used was the nucleotide sequence encoding the hIgG signal peptide: SEQ ID NO: 26). After virus production, 1 x 10 virions were injected subretinally into both eyes of eight mice per group. 9 AAV8 was delivered to the eyes of mice with a single dose of AAV8 (vg) and 28 days later, apriorcept expression in each eye was confirmed by ELISA. The CAE-T3.1.1-treated group exhibited 147.6% higher apriorcept expression levels than the CAE-FL group, and the CA-T3.1.1-treated group exhibited 118.7% higher apriorcept expression levels than the CAG-FL group (Figure 6B). These results demonstrate that the EF-1α intron fragment T3.1.1 can efficiently induce gene expression in vivo.

[0304] The cleavage map of the pAAV-CA-T3.1.1 vector into which aprivacept was inserted as the foreign gene is the same as that shown in Figure 6C.

[0305] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes, as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

[0306] Although the present disclosure has been described with reference to the above embodiments, it will be understood by those skilled in the art that various modifications and variations can be made by adding, changing, deleting, or inserting elements without departing from the spirit of the present disclosure as defined in the claims. Such modifications and variations will be understood to be within the scope of the present disclosure. [Brief explanation of the drawings]

[0307] [Figure 1] 1 shows a cleavage map of the pAAV-eGFP expression construct described herein. [Figure 2A] Figure 2 shows increased expression of eGFP in cells transduced with an expression construct containing the full-length EF-1α intron with the EF-1α promoter ("CEE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron ("CE"). In Figure 2A, an ITR-deleted animal cell expression construct was used. In Figure 2B, an ITR-containing pAAV expression construct was used. In both Figures 2A and 2B, eGFP expression is shown in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows: [Figure 2B] Figure 2 shows increased expression of eGFP in cells transduced with an expression construct containing the full-length EF-1α intron with the EF-1α promoter ("CEE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron ("CE"). In Figure 2A, an ITR-deleted animal cell expression construct was used. In Figure 2B, an ITR-containing pAAV expression construct was used. In both Figures 2A and 2B, eGFP expression is shown in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows: [Figure 3A]Figure 3 shows eGFP expression in cells transduced with an expression construct containing the full-length EF-1α intron with various promoters ("CCE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron. In Figure 3A, a combination of the CMV enhancer and CMV promoter was inserted into an animal cell expression construct (i.e., ITR-deleted) and used to transduce the cells. In Figure 3B, a combination of the CMV enhancer and CMV promoter was inserted into a pAAV expression construct and used to transduce cells. In Figure 3C, the cells were transduced with an animal cell expression construct containing a combination of the CMV enhancer and chicken β-actin promoter. Figures 3A, 3B, and 3C, respectively, show eGFP expression in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows: [Figure 3B] Figure 3 shows eGFP expression in cells transduced with an expression construct containing the full-length EF-1α intron with various promoters ("CCE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron. In Figure 3A, a combination of the CMV enhancer and CMV promoter was inserted into an animal cell expression construct (i.e., ITR-deleted) and used to transduce the cells. In Figure 3B, a combination of the CMV enhancer and CMV promoter was inserted into a pAAV expression construct and used to transduce cells. In Figure 3C, the cells were transduced with an animal cell expression construct containing a combination of the CMV enhancer and chicken β-actin promoter. Figures 3A, 3B, and 3C, respectively, show eGFP expression in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows: [Figure 3C]Figure 3 shows eGFP expression in cells transduced with an expression construct containing the full-length EF-1α intron with various promoters ("CCE-FL"). Control cells were transduced with the expression construct lacking the EF-1α intron. In Figure 3A, a combination of the CMV enhancer and CMV promoter was inserted into an animal cell expression construct (i.e., ITR-deleted) and used to transduce the cells. In Figure 3B, a combination of the CMV enhancer and CMV promoter was inserted into a pAAV expression construct and used to transduce cells. In Figure 3C, the cells were transduced with an animal cell expression construct containing a combination of the CMV enhancer and chicken β-actin promoter. Figures 3A, 3B, and 3C, respectively, show eGFP expression in cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) transduced as follows: [Figure 4A]The effects of increasing or decreasing various EF-1α intron fragment sequences (i.e., non-translated nucleic acid sequences described herein) on gene expression are shown. Figure 4A shows a schematic of a series of CEE constructs produced by sequentially deleting EF-1α intron sequences. Each construct shown contained: (1) a CMV enhancer (380 base pairs); (2) a promoter containing a portion of the CMV promoter (the first 31 base pairs from the 5' end) and the EF-1α promoter (201 base pairs); and (3) EF-1α exon 1 (E1) sequence (29 base pairs). The control "CE" construct contained the EF-1 alpha intron sequence but no additional components. Other constructs further contained the following additional components: (4) a splice donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1 alpha intron sequence (i.e., SEQ ID NO:1); (5) an EF-1 alpha intron sequence; and (6) an EF-1 alpha exon 2 (E2) sequence (9 base pairs). The EF-1 alpha intron sequence may be the full-length sequence (924 base pairs) ("CEE-FL") or: (ii) nucleotides 570 to 924 of SEQ ID NO:1 (355 base pairs) ("CEE-T2"); (iii) nucleotides 721 to 924 of SEQ ID NO:1 (204 base pairs) ("CEE-T3"); (iv) nucleotides 808 to 924 of SEQ ID NO:1 (117 base pairs) ("CEE-T3.1"); (v) nucleotides 808 to 924 of SEQ ID NO:1 (117 base pairs) ("CEE-T3.1"); (vi) nucleotides 830-924 (95 base pairs) of SEQ ID NO:1 ("CEE-T3.1.1"); (vi) nucleotides 852-924 (73 base pairs) of SEQ ID NO:1 ("CEE-T3.1.2"); (vii) nucleotides 874-924 (51 base pairs) of SEQ ID NO:1 ("CEE-T3.2"); and (viii) nucleotides 896-924 (29 base pairs) of SEQ ID NO:1 ("CEE-T4"). The full length of each construct is provided on the right. Figure 4B shows the effect of different EF-1α intron sequences (full or deleted) on eGFP (i.e., exogenous gene) expression in five different cell lines (HeLa, Hep3B, Huh-7, ARPE-19, and RPE-1). ARPE-19 and RPE-1 cells were derived from the retina. Huh-7 and Hep3B cells were derived from the liver.HeLa cells were derived from the cervix. eGFP expression was expressed as the percentage of expression observed in cells transduced with a foreign gene using the CEE-FL construct. "ns" = not significant. "**" = p<0.01 and "***" = p<0.001. [Figure 4B]The effects of increasing or decreasing various EF-1α intron fragment sequences (i.e., non-translated nucleic acid sequences described herein) on gene expression are shown. Figure 4A shows a schematic of a series of CEE constructs produced by sequentially deleting EF-1α intron sequences. Each construct shown contained: (1) a CMV enhancer (380 base pairs); (2) a promoter containing a portion of the CMV promoter (the first 31 base pairs from the 5' end) and the EF-1α promoter (201 base pairs); and (3) EF-1α exon 1 (E1) sequence (29 base pairs). The control "CE" construct contained the EF-1 alpha intron sequence but no additional components. Other constructs further contained the following additional components: (4) a splice donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1 alpha intron sequence (i.e., SEQ ID NO:1); (5) an EF-1 alpha intron sequence; and (6) an EF-1 alpha exon 2 (E2) sequence (9 base pairs). The EF-1 alpha intron sequence may be the full-length sequence (924 base pairs) ("CEE-FL") or: (ii) nucleotides 570 to 924 of SEQ ID NO:1 (355 base pairs) ("CEE-T2"); (iii) nucleotides 721 to 924 of SEQ ID NO:1 (204 base pairs) ("CEE-T3"); (iv) nucleotides 808 to 924 of SEQ ID NO:1 (117 base pairs) ("CEE-T3.1"); (v) nucleotides 808 to 924 of SEQ ID NO:1 (117 base pairs) ("CEE-T3.1"); (vi) nucleotides 830-924 (95 base pairs) of SEQ ID NO:1 ("CEE-T3.1.1"); (vi) nucleotides 852-924 (73 base pairs) of SEQ ID NO:1 ("CEE-T3.1.2"); (vii) nucleotides 874-924 (51 base pairs) of SEQ ID NO:1 ("CEE-T3.2"); and (viii) nucleotides 896-924 (29 base pairs) of SEQ ID NO:1 ("CEE-T4"). The full length of each construct is provided on the right. Figure 4B shows the effect of different EF-1α intron sequences (full or deleted) on eGFP (i.e., exogenous gene) expression in five different cell lines (HeLa, Hep3B, Huh-7, ARPE-19, and RPE-1). ARPE-19 and RPE-1 cells were derived from the retina. Huh-7 and Hep3B cells were derived from the liver.HeLa cells were derived from the cervix. eGFP expression was expressed as the percentage of expression observed in cells transduced with a foreign gene using the CEE-FL construct. "ns" = not significant. "**" = p<0.01 and "***" = p<0.001. [Figure 5A]The ability of EF-1α intron fragments T3.1.1 (i.e., nucleotides 830-924 (95 base pairs) of SEQ ID NO:1) and T3.1.2 (i.e., nucleotides 852-924 (73 base pairs) of SEQ ID NO:1) to increase expression of foreign genes is shown. Figure 5A shows a series of CAE constructs containing EF-1α intron fragments T3.1.1 and T3.1.2, along with the following additional components: (1) CMV enhancer (380 base pairs); (2) CMV enhancer (380 base pairs); (2) chicken β-actin promoter (279 base pairs); (3) chicken β-actin exon 1 (E1) sequence (32 base pairs); (4) EF-1α exon 1 (E1) sequence (29 base pairs); (5) splicing donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1α intron sequence (i.e., SEQ ID NO: 1); (6) EF-1α exon 2 (E2) sequence (9 base pairs). The control group "CA" construct contained the CMV enhancer, chicken β-actin promoter, and EF-1α exon 2 (E2) sequence. The control "CAE-FL" construct contained only the EF-1α intron and chicken β-actin E1 sequences. The full-length constructs are provided on the left. Figure 5B shows eGFP (i.e., foreign gene) expression in HeLa (left graph) and ARPE-19 (right graph) cells transduced with the CAE-T3.1.1 and CAE-T3.1.2 constructs. Gene expression was significantly higher in the CA construct (i.e., lacking the EF-1α intron sequence). The results are shown as percentage expression in the corresponding cells transduced with EF-1α. Figure 5C shows a schematic diagram of a series of hybrid intron CA constructs (i.e., CA-T3.1.1 and CA-T3.1.2 constructs) containing both the EF-1α intron fragment T3.1.1 or T3.1.2 and a chicken β-actin intron fragment. The two constructs further contain: (1) a CMV enhancer; (2) a chicken β-actin promoter; (3) a chicken β-actin E1 sequence; and (4) an EF-1α E2 sequence.The control "CAG-FL" construct contained: (1) a CMV enhancer (380 base pairs); (2) a chicken β-actin promoter (279 base pairs); (3) a chicken β-actin E1 sequence (93 base pairs); (4) a chimeric intron (containing chicken β-actin and rabbit β-globin introns) (924 base pairs); and (5) a rabbit β-globin exon 3 (E3) sequence (48 base pairs). The "CA" construct was the same as that described in Figure 5A. Figure 5D shows the expression of eGFP in HeLa (left graph) and Hep3B (right graph) cells transduced with constructs CA-T3.1.1 and CA-T3.1.2. Gene expression is shown as the percentage of expression in the corresponding cells transduced with the CA construct (i.e., lacking the EF-1α intron sequence). [Figure 5B]The ability of EF-1α intron fragments T3.1.1 (i.e., nucleotides 830-924 (95 base pairs) of SEQ ID NO:1) and T3.1.2 (i.e., nucleotides 852-924 (73 base pairs) of SEQ ID NO:1) to increase expression of foreign genes is shown. Figure 5A shows a series of CAE constructs containing EF-1α intron fragments T3.1.1 and T3.1.2, along with the following additional components: (1) CMV enhancer (380 base pairs); (2) CMV enhancer (380 base pairs); (2) chicken β-actin promoter (279 base pairs); (3) chicken β-actin exon 1 (E1) sequence (32 base pairs); (4) EF-1α exon 1 (E1) sequence (29 base pairs); (5) splicing donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1α intron sequence (i.e., SEQ ID NO: 1); (6) EF-1α exon 2 (E2) sequence (9 base pairs). The control group "CA" construct contained the CMV enhancer, chicken β-actin promoter, and EF-1α exon 2 (E2) sequence. The control "CAE-FL" construct contained only the EF-1α intron and chicken β-actin E1 sequences. The full-length constructs are provided on the left. Figure 5B shows eGFP (i.e., foreign gene) expression in HeLa (left graph) and ARPE-19 (right graph) cells transduced with the CAE-T3.1.1 and CAE-T3.1.2 constructs. Gene expression was significantly higher in the CA construct (i.e., lacking the EF-1α intron sequence). The results are shown as percentage expression in the corresponding cells transduced with EF-1α. Figure 5C shows a schematic diagram of a series of hybrid intron CA constructs (i.e., CA-T3.1.1 and CA-T3.1.2 constructs) containing both the EF-1α intron fragment T3.1.1 or T3.1.2 and a chicken β-actin intron fragment. The two constructs further contain: (1) a CMV enhancer; (2) a chicken β-actin promoter; (3) a chicken β-actin E1 sequence; and (4) an EF-1α E2 sequence.The control "CAG-FL" construct contained: (1) a CMV enhancer (380 base pairs); (2) a chicken β-actin promoter (279 base pairs); (3) a chicken β-actin E1 sequence (93 base pairs); (4) a chimeric intron (containing chicken β-actin and rabbit β-globin introns) (924 base pairs); and (5) a rabbit β-globin exon 3 (E3) sequence (48 base pairs). The "CA" construct was the same as that described in Figure 5A. Figure 5D shows the expression of eGFP in HeLa (left graph) and Hep3B (right graph) cells transduced with constructs CA-T3.1.1 and CA-T3.1.2. Gene expression is shown as the percentage of expression in the corresponding cells transduced with the CA construct (i.e., lacking the EF-1α intron sequence). [Figure 5C]The ability of EF-1α intron fragments T3.1.1 (i.e., nucleotides 830-924 (95 base pairs) of SEQ ID NO:1) and T3.1.2 (i.e., nucleotides 852-924 (73 base pairs) of SEQ ID NO:1) to increase expression of foreign genes is shown. Figure 5A shows a series of CAE constructs containing EF-1α intron fragments T3.1.1 and T3.1.2, along with the following additional components: (1) CMV enhancer (380 base pairs); (2) CMV enhancer (380 base pairs); (2) chicken β-actin promoter (279 base pairs); (3) chicken β-actin exon 1 (E1) sequence (32 base pairs); (4) EF-1α exon 1 (E1) sequence (29 base pairs); (5) splicing donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1α intron sequence (i.e., SEQ ID NO: 1); (6) EF-1α exon 2 (E2) sequence (9 base pairs). The control group "CA" construct contained the CMV enhancer, chicken β-actin promoter, and EF-1α exon 2 (E2) sequence. The control "CAE-FL" construct contained only the EF-1α intron and chicken β-actin E1 sequences. The full-length constructs are provided on the left. Figure 5B shows eGFP (i.e., foreign gene) expression in HeLa (left graph) and ARPE-19 (right graph) cells transduced with the CAE-T3.1.1 and CAE-T3.1.2 constructs. Gene expression was significantly higher in the CA construct (i.e., lacking the EF-1α intron sequence). The results are shown as percentage expression in the corresponding cells transduced with EF-1α. Figure 5C shows a schematic diagram of a series of hybrid intron CA constructs (i.e., CA-T3.1.1 and CA-T3.1.2 constructs) containing both the EF-1α intron fragment T3.1.1 or T3.1.2 and a chicken β-actin intron fragment. The two constructs further contain: (1) a CMV enhancer; (2) a chicken β-actin promoter; (3) a chicken β-actin E1 sequence; and (4) an EF-1α E2 sequence.The control "CAG-FL" construct contained: (1) a CMV enhancer (380 base pairs); (2) a chicken β-actin promoter (279 base pairs); (3) a chicken β-actin E1 sequence (93 base pairs); (4) a chimeric intron (containing chicken β-actin and rabbit β-globin introns) (924 base pairs); and (5) a rabbit β-globin exon 3 (E3) sequence (48 base pairs). The "CA" construct was the same as that described in Figure 5A. Figure 5D shows the expression of eGFP in HeLa (left graph) and Hep3B (right graph) cells transduced with constructs CA-T3.1.1 and CA-T3.1.2. Gene expression is shown as the percentage of expression in the corresponding cells transduced with the CA construct (i.e., lacking the EF-1α intron sequence). [Figure 5D]The ability of EF-1α intron fragments T3.1.1 (i.e., nucleotides 830-924 (95 base pairs) of SEQ ID NO:1) and T3.1.2 (i.e., nucleotides 852-924 (73 base pairs) of SEQ ID NO:1) to increase expression of foreign genes is shown. Figure 5A shows a series of CAE constructs containing EF-1α intron fragments T3.1.1 and T3.1.2, along with the following additional components: (1) CMV enhancer (380 base pairs); (2) CMV enhancer (380 base pairs); (2) chicken β-actin promoter (279 base pairs); (3) chicken β-actin exon 1 (E1) sequence (32 base pairs); (4) EF-1α exon 1 (E1) sequence (29 base pairs); (5) splicing donor sequence consisting of the first 19 base pairs from the 5' end of the full-length EF-1α intron sequence (i.e., SEQ ID NO: 1); (6) EF-1α exon 2 (E2) sequence (9 base pairs). The control group "CA" construct contained the CMV enhancer, chicken β-actin promoter, and EF-1α exon 2 (E2) sequence. The control "CAE-FL" construct contained only the EF-1α intron and chicken β-actin E1 sequences. The full-length constructs are provided on the left. Figure 5B shows eGFP (i.e., foreign gene) expression in HeLa (left graph) and ARPE-19 (right graph) cells transduced with the CAE-T3.1.1 and CAE-T3.1.2 constructs. Gene expression was significantly higher in the CA construct (i.e., lacking the EF-1α intron sequence). The results are shown as percentage expression in the corresponding cells transduced with EF-1α. Figure 5C shows a schematic diagram of a series of hybrid intron CA constructs (i.e., CA-T3.1.1 and CA-T3.1.2 constructs) containing both the EF-1α intron fragment T3.1.1 or T3.1.2 and a chicken β-actin intron fragment. The two constructs further contain: (1) a CMV enhancer; (2) a chicken β-actin promoter; (3) a chicken β-actin E1 sequence; and (4) an EF-1α E2 sequence.The control "CAG-FL" construct contained: (1) a CMV enhancer (380 base pairs); (2) a chicken β-actin promoter (279 base pairs); (3) a chicken β-actin E1 sequence (93 base pairs); (4) a chimeric intron (containing chicken β-actin and rabbit β-globin introns) (924 base pairs); and (5) a rabbit β-globin exon 3 (E3) sequence (48 base pairs). The "CA" construct was the same as that described in Figure 5A. Figure 5D shows the expression of eGFP in HeLa (left graph) and Hep3B (right graph) cells transduced with constructs CA-T3.1.1 and CA-T3.1.2. Gene expression is shown as the percentage of expression in the corresponding cells transduced with the CA construct (i.e., lacking the EF-1α intron sequence). [Figure 6A] Figure 6 shows the effect of EF-1α intron fragments T3.1.1 and T3.1.2 on increasing foreign gene (i.e., apprebercept) expression when delivering genes using AAV. Figure 6A shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vitro gene delivery. Figure 6B shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vivo gene delivery. Figure 6C shows the cleavage map of the pAAV-CA-T3.1.1 vector containing apprebercept as the foreign gene. [Figure 6B] Figure 6 shows the effect of EF-1α intron fragments T3.1.1 and T3.1.2 on increasing foreign gene (i.e., apprebercept) expression when delivering genes using AAV. Figure 6A shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vitro gene delivery. Figure 6B shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vivo gene delivery. Figure 6C shows the cleavage map of the pAAV-CA-T3.1.1 vector containing apprebercept as the foreign gene. [Figure 6C]Figure 6 shows the effect of EF-1α intron fragments T3.1.1 and T3.1.2 on increasing expression of a foreign gene (i.e., apprebercept) when delivering a gene using AAV. Figure 6A shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vitro gene delivery. Figure 6B shows the effect of T3.1.1 and T3.1.2 fragments on increasing gene expression during in vivo gene delivery. Figure 6C shows the cleavage map of the pAAV-CA-T3.1.1 vector into which apprebercept has been inserted as the foreign gene. [Sequence Table Free Text]

[0308] The contents of SEQ ID NO: 11 are as follows: <210> 11 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> EF-1 alpha exon 2 fragment <400> 11 gtgtcgtga 9

Claims

1. An isolated polynucleotide comprising a foreign gene sequence and a non-translated nucleic acid sequence consisting of a splicing donor sequence and an intron fragment sequence, wherein the intron fragment sequence is 51 to 117 nucleotides in length and comprises the nucleotide sequence set forth in SEQ ID NO:

57.

2. The polynucleotide of claim 1, wherein the intron fragment sequence comprises: (i) nucleotides 871 to 924 of SEQ ID NO:1 (SEQ ID NO:58), (ii) nucleotides 861 to 924 of SEQ ID NO:1 (SEQ ID NO:59), (iii) nucleotides 852 to 924 of SEQ ID NO:1 (SEQ ID NO:3), (iv) nucleotides 851 to 924 of SEQ ID NO:1 (SEQ ID NO:61), (v) nucleotides 830 to 924 of SEQ ID NO:1 (SEQ ID NO:2), (vi) nucleotides 821 to 924 of SEQ ID NO:1 (SEQ ID NO:63), (vii) nucleotides 811 to 924 of SEQ ID NO:1 (SEQ ID NO:64), or (viii) nucleotides 808 to 924 of SEQ ID NO:1 (SEQ ID NO:65).

3. The polynucleotide according to claim 2, wherein the intron fragment sequence consists of (i) nucleotides 871 to 924 of SEQ ID NO:1 (SEQ ID NO:58), (ii) nucleotides 861 to 924 of SEQ ID NO:1 (SEQ ID NO:59), (iii) nucleotides 852 to 924 of SEQ ID NO:1 (SEQ ID NO:3), (iv) nucleotides 851 to 924 of SEQ ID NO:1 (SEQ ID NO:61), (v) nucleotides 830 to 924 of SEQ ID NO:1 (SEQ ID NO:2), (vi) nucleotides 821 to 924 of SEQ ID NO:1 (SEQ ID NO:63), (vii) nucleotides 811 to 924 of SEQ ID NO:1 (SEQ ID NO:64), or (viii) nucleotides 808 to 924 of SEQ ID NO:1 (SEQ ID NO:65).

4. 4. The polynucleotide of claim 1, wherein the intron fragment sequence is 54 nucleotides, 64 nucleotides, 73 nucleotides, 74 nucleotides, 95 nucleotides, 104 nucleotides, 114 nucleotides, or 117 nucleotides in length.

5. The polynucleotide of any one of claims 1 to 4, further comprising: (i) a promoter; (ii) an enhancer; (iii) an exon sequence; (iv) a target sequence for a microRNA (miRNA); (v) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence; (vi) a polyadenylation (pA) sequence; or (vii) a combination thereof.

6. (i) the exogenous gene encodes a wild-type polypeptide or any variant thereof, a fusion protein, an antibody or an antigen-binding fragment thereof, an RNA-based molecule, or any combination thereof; (ii) the promoter comprises a cytomegalovirus (CMV) promoter, an EF-1α promoter, a β-actin promoter, a GAPDH promoter, an HSP70 promoter, a GRP78 promoter, an eIF4a promoter, an AAT promoter, a TTR promoter, a GFAP promoter, an SV40 promoter, a SYN1 promoter, a GRK promoter, a Rho promoter, or any combination thereof; (iii) the enhancer comprises a cytomegalovirus (CMV) enhancer, an SV40 early enhancer, an adenovirus 5 E1A enhancer, an HBV enhancer-1 regulatory region (Eh-1), an HPV-16 or -18 E6 / 7 long regulatory region (LCR), an HIV-1 long terminal repeat (LTR), or any combination thereof; (iv) the splicing donor sequence is linked upstream of the intron fragment sequence; (v) the exon sequence comprises an EF-1α exon 2 (E2) nucleotide sequence, a cytomegalovirus (CMV) exon 1 (E1) sequence, an EF-1α E1 sequence, a β-actin E1 sequence, or any combination thereof; (vi) the target sequence for the miRNA comprises an antisense oligonucleotide, an antagomir, a short hairpin RNA (shRNA) molecule, a short interfering RNA (siRNA) molecule, a ribozyme, a peptide nucleic acid (PNA) oligonucleotide, a locked nucleic acid (LNA) oligonucleotide, or any combination thereof; or (vii) any combination of (i) to (vi); The polynucleotide of claim 5, characterized in that

7. 1. A polynucleotide comprising: (i) a foreign gene set forth in SEQ ID NO:23; and (ii) a regulatory element operably linked to the foreign gene, wherein the regulatory element comprises (in a 5' to 3' direction): (1) a CMV enhancer set forth in SEQ ID NO: 4; (2) the chicken β-actin promoter sequence shown in SEQ ID NO: 8; (3) chicken β-actin E1 sequence shown in SEQ ID NO: 15; (4) a splicing donor sequence shown in SEQ ID NO: 10; (5) an intron fragment sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; and (6) EF-1α E2 sequence shown in SEQ ID NO: 11 The polynucleotide of claim 6, comprising:

8. A vector comprising the polynucleotide according to any one of claims 1 to 7.

9. The vector according to claim 8, which is a viral vector.

10. 10. The vector according to claim 8 or 9 for use in gene therapy.

11. 10. The vector according to claim 8 or 9, for use in expressing a polypeptide encoded by the foreign gene.

12. An in vitro cell comprising the polynucleotide of any one of claims 1 to 7 or the vector of claim 8 or 9.

13. A method for producing recombinant viral particles (excluding transduction of cells in the human body) comprising transducing cells with a construct comprising the vector of claim 8 or 9 and the rep and cap genes.

14. A recombinant viral particle comprising (a) a capsid protein and (b) a vector according to claim 8 or 9.

15. 15. The recombinant viral particle according to claim 14, characterized in that it is an adeno-associated virus (AAV).

16. The recombinant viral particle of claim 15, characterized in that the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAVrhlO.

17. (a) a polynucleotide according to any one of claims 1 to 7, a vector according to claim 8 or 9, a cell according to claim 12, or a recombinant viral particle according to claim 14; and (b) a pharmaceutically acceptable excipient A pharmaceutical composition comprising:

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