Improved recombinant polyadenylated signal sequences and their use

Abstract

This invention relates to an improved recombinant polyadenylated signal sequence and its use.

Description

Technical Field

[0001] Field of the Invention The present invention relates to an improved recombinant polyadenylation signal sequence and its use.

Background Art

[0002] Background of the Invention In research, disease modeling, drug discovery, therapeutic gene expression, and biopharmaceutical production, generating stable high-level recombinant protein-expressing cell lines is essential for cell engineering applications. Achieving robust recombinant protein expression in a desired host cell is determined by the optimization of expression vectors using various enhancers, promoters, introns, polyA, and regulatory sequences. Various approaches have been reported for developing and optimizing the gene elements used in expression vectors to achieve a desired level of transgene expression, which include assembling blocks of natural or newly designed functional elements (Cao et al., 2021; McFarland et al., 2006; Patel et al., 2021; Schlabach et al., 2010), but little consideration has been given to creating high-level multi-gene expression vectors for predictably co-expressing recombinant genes from the same vector. Most efforts to optimize expression vectors have focused on identifying the optimal sequences of a specific recombinant protein and vector, so the extent to which these gene elements can be transferred to other applications may be limited. Furthermore, taking one optimized gene element and replicating it into multiple expression units on a multi-gene expression vector is often not ideal because replicating regulatory sequences or introducing extended regions of repetitive sequences by reusing the same gene element creates a risk of recombination events occurring during vector replication and integration into the target host cell (Bzymek et al., 2001; Finn et al., 1989).

[0003] In mammalian cells, transcription termination and polyadenylation are essential for the efficient protein expression of endogenous genes, as well as recombinant genes expressed from engineered vectors. Specifically, the poly-A tail attached to protein-coding transcripts assists mRNA nuclear export, translation, and stability by protecting the transcript from enzymatic degradation in the cytoplasm. More commonly used polyadenylation signal sequences in vector development include those of bovine growth hormone (BGH) (Goodwin et al., 1992), human growth hormone (hGH) (Pfarr et al., 1986), monkey virus 40 (SV40) (Hans et al., 2000), and rabbit β-globin (RbG) (Lanoix et al., 1988). However, due to significant differences in size and sequence composition, the selection of polyadenylation signal sequences can greatly affect the characteristics of the expression vector. Larger sizes reduce transfection and integration efficiency during cell manipulation, and in the case of viral vector manipulation, the size of the gene cargo may exceed the packaging capacity of the viral vector. Differences in the sequence composition and functional elements within polyadenylation signal sequences can lead to heterogeneous expression levels due to variations in their respective uses and expression levels in different host cells, as is the case with SV40 and RbG, which have been proposed to be more efficient polyadenylation signal sequences compared to others due to the presence of additional upstream and downstream functional elements that contribute to termination and polyadenylation (Gil et al., 1987; Shek et al., 1992). Furthermore, recent data suggest that even slight variations in sequence composition can contribute to differences in transcription termination processes and protein expression within the same host cell and across different host cells (Omelina et al., 2022).

[0004] Therefore, further improvements to the polyadenylation signal sequence are still needed. [Overview of the Initiative]

[0005] This specification provides a repertoire of robust transcription termination and polyadenylation (Poly-A) signal sequences for advanced vector development. Based on minimal core sequences of RbG polyadenylation signal sequences, the inventors have generated a set of rationally designed recombinant polyadenylation signal sequences (Levitt et al., 1989). Due to their small size and clear functional element composition, these recombinant polyadenylation signal sequences are particularly useful for generating multiplex gene expression vectors for manipulating mammalian cells. Furthermore, the sequence composition is specifically designed to accommodate Gibson DNA assembly cloning to facilitate the generation of multiplex gene expression vectors and to reduce the risk of DNA recombination events with low sequence identity. The resulting recombinant polyadenylation signal sequences of the present invention support significantly higher expression levels than known short polyadenylation signal sequences such as core RbG Poly-A sequences (see, e.g., Figure 5) and levels comparable to larger, commonly used polyadenylation signal sequences (see, e.g., Figure 6). Furthermore, the inventors demonstrated that the recombinant polyadenylation signal sequences of the present invention can minimize the influence of upstream 3'UTR sequence configurations (see, e.g., Figure 7) and support robust expression in combination with constituent promoters of varying intensities (see, e.g., Figure 8). In summary, the inventors provide a novel and improved set of recombinant polyadenylation signal sequences that can facilitate the development of advanced multi-gene expression vectors and cell models for research, disease modeling, drug discovery, therapeutic gene expression, and biopharmaceutical manufacturing. Further advantageous effects in specific exemplary use scenarios are described below.

[0006] In one embodiment, a recombinant transcription unit is provided comprising a nucleotide sequence encoding a polypeptide, the nucleotide sequence being operably ligated to a recombinant polyadenylation signal sequence having a sequence length of less than 100 nucleotides, and eukaryotic cells transformed with the recombinant nucleic acid comprising the recombinant transcription unit are capable of expressing the polypeptide at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid comprising the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to the sequence of the recombinant nucleic acid without regard to the recombinant polyadenylation signal sequence with respect to sequence identity.

[0007] In some embodiments, the recombinant polyadenylation signal sequence comprises or consists of nucleotide sequences having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs: 12.

[0008] In some embodiments, a recombinant transcription unit is provided comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, the recombinant polyadenylation signal sequence comprising or comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0009] In some embodiments, the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12.

[0010] In one embodiment, (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence Recombinant nucleic acids, including Recombinant nucleic acids are provided in which the first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%.

[0011] In some embodiments, recombinant nucleic acids are (c) Further comprising a third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence, The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence individually have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% with the third recombinant polyadenylation signal sequence.

[0012] In some embodiments, the first, second, and, if present, third recombinant polyadenylated signal sequences have a sequence length of less than 100 nucleotides.

[0013] In some embodiments, the first, second, and, if present, third recombinant polyadenylation signal sequences are unable to participate in DNA strand exchange for the formation of recombinant intermediates.

[0014] In some embodiments, recombination events between a nucleic acid containing a first recombinant polyadenylation signal sequence and a nucleic acid containing a second recombinant polyadenylation signal sequence are reduced or prevented.

[0015] In some embodiments, recombination events between a nucleic acid containing a first recombinant polyadenylation signal sequence and a nucleic acid containing a third recombinant polyadenylation signal sequence are reduced or prevented, and / or recombination events between a nucleic acid containing a second recombinant polyadenylation signal sequence and a nucleic acid containing a third recombinant polyadenylation signal sequence are reduced or prevented.

[0016] In some embodiments, the first polypeptide, the second polypeptide, and, if present, the third polypeptide, are expressed in eukaryotic cells.

[0017] In some embodiments, the first recombinant transcription unit is a recombinant transcription unit as described herein, the second recombinant transcription unit is a recombinant transcription unit as described herein, and, if present, the third recombinant transcription unit is a recombinant transcription unit as described herein.

[0018] In some embodiments, recombinant nucleic acids as described herein are provided, where, (a) The first recombinant transcription unit further comprises a first promoter operably ligated to a nucleotide sequence encoding the first polypeptide, (b) The second recombinant transcription unit further comprises a second promoter operably ligated to a nucleotide sequence encoding the second polypeptide, The first and second promoters have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%.

[0019] In some embodiments, recombinant nucleic acids as described herein are provided, which recombinant nucleic acids (c) If present, the first recombinant transcription unit further comprises a first promoter operably ligated to a nucleotide sequence encoding the first polypeptide, The first promoter and the second promoter have less than 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or 60% sequence identity with the third promoter.

[0020] In some embodiments, recombinant nucleic acids as described hereinabove are provided, where (i) the first promoter, the second promoter, and, if present, the third promoter are active in eukaryotic cells, (ii) the first promoter drives the expression of the first polypeptide, (iii) the second promoter drives the expression of the second polypeptide, (iv) the third promoter drives the expression of the third polypeptide, and / or (v) the first promoter, the second promoter, and, if present, the third promoter each drive the expression of the first, second, and, if present, third polypeptides, respectively.

[0021] In some embodiments, recombinant nucleic acids as described hereinabove are provided, wherein the first, second, and, if present, third promoters are each individually selected from the group consisting of the hPGK1 promoter, the CMV promoter, and the hEF1α promoter.

[0022] In some embodiments, the recombinant nucleic acid comprises at least one vector.

[0023] In some embodiments, the recombinant nucleic acid comprises a first vector comprising a first recombinant transcription unit and a second vector comprising a second recombinant transcription unit, and, if a third recombinant transcription unit is present, a third vector comprising the third recombinant transcription unit.

[0024] In some embodiments, at least one vector comprises a selectable marker operably linked to the first, second, or, if present, third recombinant transcription unit, respectively.

[0025] In some embodiments, the selectable marker is selected from the group consisting of a hygromycin selectable marker, a neomycin selectable marker, a G418 selectable marker, dihydrofolate reductase (DHFR), thymidine kinase, glutamine synthetase, asparagine synthetase, tryptophan synthetase, histidinol dehydrogenase, and a nucleic acid conferring resistance to puromycin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.

[0026] In some embodiments, the first, second, and / or, if present, the third vector comprises a bacterial origin of replication, particularly the pUC19 origin of replication.

[0027] In some embodiments, there are provided host cells comprising a recombinant transcription unit as described herein and / or a recombinant nucleic acid as described herein.

[0028] In some embodiments, the host cell is a eukaryotic host cell.

[0029] In some embodiments, the host cell is selected from the group consisting of CHO, BHK, HEK, and Sp2 / 0.

[0030] In some embodiments, there is provided a recombinant viral vector comprising a vector genome, the vector genome comprising, in order from 5' to 3': (i) a 5' ITR sequence, (ii) a promoter sequence, (iii) a sequence encoding a polypeptide, (iv) a recombinant polyadenylation signal sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and (v) a 3' ITR sequence and comprising.

[0031] In some embodiments, the recombinant polyadenylated signal sequence is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12.

[0032] In some embodiments, the recombinant viral vector is selected from the group consisting of retroviral vectors, adenovirus vectors, helper-dependent adenovirus vectors, hybrid adenovirus vectors, herpes simplex virus vectors, lentivirus vectors, poxvirus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, human cytomegalovirus vectors, lentivirus vectors, adenovirus vectors or adeno-associated virus (AAV) vectors, or recombinant variants derived therefrom.

[0033] In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) vector.

[0034] In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, AAV3B, AAV-2i8 capsids, or mutant capsids derived therefrom.

[0035] In some embodiments, a method for producing a target polypeptide is provided, the method comprising the following steps: (a) a step of providing host cells as described herein, (b) A step of incubating host cells under conditions suitable for polypeptide expression, (c) A step of recovering the target polypeptide from the cell culture and Includes.

[0036] In some embodiments, a method for producing a target polypeptide is provided, the method comprising the following steps: (a) A step of providing a host cell comprising recombinant nucleic acid as described herein, wherein the polypeptide of interest is a first polypeptide, and a second polypeptide and, if present, a third polypeptide are required for the production of the polypeptide of interest or improve the production of the polypeptide of interest. (b) Incubating host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide; (c) A step of recovering the target polypeptide from the cell culture, Optional, (d) A step of formulating the recovered target polypeptide for therapeutic use. Includes.

[0037] In some embodiments, a method for producing recombinant adeno-associated virus (rAAV) vectors is provided, which involves the following steps: (a) A step of providing a host cell comprising recombinant nucleic acid as described herein, wherein a first polynucleotide sequence encodes a therapeutic payload, a second nucleotide sequence encodes viral vector rep and cap proteins, and a third nucleotide sequence encodes E4, E2a and VA proteins, (b) A step of incubating host cells under conditions suitable for the preparation of recombinant rAAV vectors, (c) A step of recovering the viral vector from the cell culture, Optional, (d) A step of formulating the recovered target polypeptide for therapeutic use. Includes.

[0038] In some embodiments, the methods previously described herein are provided, and the host cells are selected from the group consisting of CHO cells, BHK cells, HEK cells, and Sp2 / 0 cells.

[0039] In some embodiments, the methods previously described herein are provided, and the rAAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, AAV3B, AAV-2i8 vectors, or vector variants derived therefrom.

[0040] In some embodiments, the use of recombinant transcription units for recombinant production of the target polypeptide is provided, the recombinant transcription units being as previously defined herein.

[0041] In some embodiments, the use of recombinant nucleic acids for recombinant production of the target polypeptide is provided, the recombinant nucleic acids being as defined herein above. [Brief explanation of the drawing]

[0042] [Figure 1] This is a schematic diagram of a reporter plasmid used to test recombinant polyadenylation signal sequences, consisting of a constitutive promoter (Prom.), enhanced green fluorescent protein (EGFP), a P2A self-cleaving peptide sequence, NanoLuc luciferase (Nluc), a PEST proteolytic signal, and a 3' untranslated region (3'UTR), with the recombinant polyadenylation signal sequence of interest following downstream. [Figure 2A] Schematic diagram of a reporter plasmid containing either BGH or a 2x sNRP-1 polyadenylated signal sequence. [Figure 2B] Transient studies of each reporter construct in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. The values ​​represent the mean ± standard deviation of n=16 biologically independent samples, normalized to the mean Nluc relative luminescence (RLU) of reporter plasmid samples encoding BGH polyA (normalized luminescence; %). [Figure 3]A schematic diagram of the 95nt recombinant polyadenylation signal sequence design presented herein. The sequence consists of (i) a 46nt heterogeneous U-rich upstream sequence element (USE) region designed to include a unique primer annealing site with a Tm of 70–72°C that fits into a Gibson assembly, (ii) a polyadenylation signal (PAS), (iii) a variable spacer region containing two 15–20nt cytosine-adenine (CA) mRNA cleavage sites downstream of the PAS, and (iv) two GU / U-rich downstream sequence element (DSE; DSE1 and DSE2) regions. [Figure 4A] Schematic diagram of a reporter plasmid containing a novel recombinant polyadenylated signal sequence. [Figure 4B] Transient testing of each reporter construct in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of all samples (normalized luminescence; %). [Figure 5A] This is a schematic diagram of a reporter plasmid containing either the rabbit β-globulin polyadenylation signal sequence defined by Levitt et al., or one of three selected recombinant polyadenylation signal sequences (Poly-A-2.3, -3.3, -4.3). [Figure 5B] Transient testing of each reporter construct in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of poly(A) encoding the reporter plasmid sample (normalized luminescence; %). [Figure 6A] Schematic diagram of a reporter plasmid containing either hGH, BGH, SV40, or one of three selected recombinant polyadenylated signal sequences (poly-A-2.3, -3.3, -4.3). [Figure 6B]Transient testing of each reporter construct in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of BGH polyA encoding the reporter plasmid sample (normalized luminescence; %). [Figure 7A] Schematic diagram of a reporter plasmid containing one of three selected recombinant polyadenylation signal sequences (Poly-A-2.3, -3.3, -4.3) combined with three different novel 3'UTR sequences (3'UTR1, 2, 3). [Figure 7B] Transient testing of each reporter construct in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of all samples (normalized luminescence; %). [Figure 8A] Schematic diagram of a reporter plasmid containing one of three selected recombinant polyadenylation signal sequences (Poly-A-2.3, -3.3, -4.3) in combination with three constitutive promoters of different strengths (hPGK1, CMV, hEF1α). [Figure 8B] Transient tests of (8B)hPGK1-, (8C)CMV-, and (8D)hEF1α-driven reporter constructs in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of all samples tested with the corresponding promoter (normalized luminescence; %). [Figure 8C]Transient tests of (8B)hPGK1-, (8C)CMV-, and (8D)hEF1α-driven reporter constructs in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of all samples tested with the corresponding promoter (normalized luminescence; %). [Figure 8D] Transient tests of (8B)hPGK1-, (8C)CMV-, and (8D)hEF1α-driven reporter constructs in HEK293T, evaluated in terms of Nluc expression levels 24 hours after transfection. Bars represent the mean ± standard deviation of n=16 independent replicas normalized to the mean Nluc relative luminescence (RLU) of all samples tested with the corresponding promoter (normalized luminescence; %). [Modes for carrying out the invention]

[0043] References Bzymek et al. (2001). Instability of repetitive DNA sequences: the role of replication in multiple mechanisms. Proceedings of the National Academy of Sciences, 98(15), 8319-8325. Batt et al. (1995). Characterization of the polyomavirus late polyadenylation signal.Molecular and Cellular Biology,15:4783-4790 Cao et al. (2021). High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies.Nature communications,12(1),4138. Coleら(1985)。Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.Molecular and Cellular Biology.5:2104-2113 Finnら(1989)。Homologous plasmid recombination is elevated in immortally transformed cells.Molecular and Cellular biology,9(9),4009-4017。 Gilら(1987)。Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit β-globin mRNA 3′ end formation.Cell,49(3),399-406。 Gilら(1984)。A sequence downstream of AAUAAA is required for rabbit β-globin mRNA 3′-end formation.Nature,312:473-474 Gimmiら(1989)。Alterations in the pre-mRNA topology of the bovine growth hormone polyadenylation region decrease poly(A)site efficiency.Nucleic Acid Research,17(17):6983-98 Goodwinら(1992)。The 3’-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation.Journal of Biological Chemistry,267(23),16330-16334。 Hansら(2000)。Functionally significant secondary structure of the simian virus 40 late polyadenylation signal.Molecular and Cellurar Biology,20(8),2926-2932。 Lanoixら(1988)。A rabbit beta‐globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA.EMBO journal,7(8)、2515-2522。 Levittら(1989)。Definition of an efficient synthetic poly(A)site.Genes&Development,3(7),1019-1025。 McFarlandら(2006)。Evaluation of a novel short polyadenylation signal as an alternative to the SV40 polyadenylation signal.Plasmid,56(1),62-67。 Murthyら(1995)。The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3’-end formation.Genes&Development 9:2672-2683 Omelinaら(2022)。Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells.International Journal of Molecular Sciences,23(24),15485 Patelら(2021)。Control of multigene expression stoichiometry in mammalian cells using synthetic promoters.ACS Synthetic Biology,10(5),1155-1165 Pfarrら(1986)。Differential effects of polyadenylation regions on gene expression in mammalian cells.DNA,5(2),115-122 Schekら(1992)。Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses.Molecular and Cellular Biology,12(12),5386-5393 Schlabachら(2010).Synthetic design of strong promoters.Proceedings of the National Academy of Sciences,107(6),2538-2543 Takagakiら(1997)。RNA recognition by the human polyadenylation factor CstF.Molecular and Cellular Biology 17:3907-3914 Takagaki et al. (1992). The human 64 kDa polyadenylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs.Proceedings of the National Academy of Sciences 1992;89:1403-1407

[0044] Detailed description of the invention The inventors have generated improved polyadenylation signal sequences that, when incorporated into a transcription unit (also called a transcription cassette), induce potent expression of the target polypeptide. The novel sequences are short, making them advantageous for many applications, such as the incorporation of recombinant polyadenylation signal sequences into size-constrained transcription units (e.g., recombinant adeno-associated virus vectors). Furthermore, the inventors have generated multiple recombinant polyadenylation signal sequences that share low sequence homology, i.e., the sequence identity of multiple sequences prevents recombination events. In eukaryotic cells, recombination events can occur when sequences with high sequence identity are in close proximity to each other. For example, this can happen when sequences with high sequence identity are incorporated into the same genomic locus, or when multiple plasmid vectors sharing sequences with high homology are transfected into cells.

[0045] As shown in the examples, short polyadenylation signal sequences known in the art (e.g., the 2x sNRP-1 signal sequence described by McFarland et al., SEQ ID NO: 14, 49 nucleotides long) cannot achieve an expression level of the target polypeptide comparable to that of longer polyadenylation signal sequences known in the art (e.g., the BGH sequence, SEQ ID NO: 16, 208 nucleotides long). This is shown, for example, in Figure 2B.

[0046] In contrast, the novel recombinant polyadenylation signal sequences of the present invention (SEQ ID NOs: 1-12) yield comparable or even stronger expression levels compared to longer polyadenylation signal sequences known in the art (e.g., hGH, BGH, and SV40 sequences, SEQ ID NOs: 15-17, 122-477 nucleotide lengths, etc.). This is illustrated, for example, in Figures 4B, 5B, and 6B.

[0047] Accordingly, novel and improved recombinant polyadenylation signal sequences are provided herein. Furthermore, recombinant transcription units comprising the improved recombinant polyadenylation signal sequences according to the present invention are provided. These transcription units can result in potent expression of a nucleotide sequence encoding a polypeptide of interest and operably linked to the improved recombinant polyadenylation signal sequences according to the present invention.

[0048] In one embodiment, a recombinant nucleic acid is provided which comprises a recombinant transcription unit containing a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence.

[0049] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used synonymously to mean multiple “nucleotides” (i.e., molecules containing a sugar (e.g., ribose or deoxyribose) bonded to an interchangeable organic base that is either a phosphate group and a substituted pyrimidine (e.g., cytosine (C), thymine (T), or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G))). In this specification, these terms refer to oligoribonucleotides and oligodeoxyribonucleotides. These terms also include polynucleosides (i.e., polynucleotides without phosphates) and any other organic base-containing polymers. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomes or cDNA), but may also be produced by synthesis (e.g., by oligonucleotide synthesis).

[0050] In this specification, “recombinant” nucleic acids refer to nucleic acids that do not exist in nature. Recombinant nucleic acids are sometimes also called “synthetic” nucleic acids. Similarly, recombinant transcription units and recombinant polyadenylation signal sequences refer to transcription units and polyadenylation signal sequences that do not exist in nature, for example, containing or consisting of recombinant nucleic acids. Recombinant nucleic acids may contain or consist of polynucleotide sequences that are not and / or do not encode in the genome of naturally occurring organisms (e.g., wild-type organisms). Recombinant nucleic acids may contain or consist of polynucleotide sequences that are not contained in the polynucleotide sequences of (RNA) transcripts produced by naturally occurring organisms. Recombinant nucleic acids may be produced using recombinant nucleic acid technologies. Recombinant nucleic acid technologies include technologies for constructing and manipulating nucleotide sequences of nucleic acids, and include molecular cloning.

[0051] The term "transcription unit" refers to a DNA sequence that codes for a single RNA molecule (e.g., an mRNA molecule). A transcription unit contains a sequence of nucleotides necessary for transcription. For example, a transcription unit typically includes a promoter, a polynucleotide sequence that codes for the target protein, and a terminator sequence (such as the 3' untranslated region, also called the 3'-UTR).

[0052] The term "operably ligated" refers to a situation where the nucleic acid encoding the recombinant polypeptide of interest and a regulatory nucleic acid sequence (e.g., a polyadenylation signal, promoter, and / or enhancer) are covalently linked to one or more regulatory nucleic acid sequences so as to place the expression of the nucleic acid encoding the polypeptide of interest under the influence or control of one or more regulatory nucleic acid sequences (thereby forming a transcription unit, or expression cassette). Thus, the (regulatory) sequence is operably ligated to a selected nucleic acid sequence if the regulatory sequence is capable of transcribing the nucleic acid sequence. The resulting one or more transcripts can then be translated into one or more desired polypeptides of interest.

[0053] The term "polyadenylation signal sequence" refers to a sequence that terminates the transcription of a transcription unit, ensuring that the nucleic acid sequence encoding the polypeptide is properly transcribed and translated. Polyadenylation signals are recognized by RNA cleavage complexes that result in RNA cleavage and polyadenylation catalyzed by polyadenylation polymerases.

[0054] An example of a naturally occurring eukaryotic polyadenylation signal is the rabbit β-globin poly(A) signal, which has been characterized as potent in the literature (Gil and Proudfoot, Cell 49:399-406 (1987); Gil and Proudfoot, Nature 312:473-474 (1984)). One of its main features is the structure of its downstream elements, which contain both UG-rich and U-rich domains. Other polyadenylation signal sequences include synthetic polyA, HSV thymidine kinase polyA (see Cole, CN and TPStacy, MoI. Cell. Biol. 5:2104-2113 (1985)); human α-globin polyA, SV40 polyA (see Schek, N, Cooke, C, and JC Alwine, MoI. Cell Biol. 12:5386-5393 (1992)); human β-globin polyA (see Gil, A., and NJProudfoot, Cell 49:399-406 (1987)); polyomavirus polyA (see Batt, DB and GG Carmichael, MoI. Cell. Biol. 15:4783-4790 (1995)); bovine growth hormone polyA (see Gimmi, ER, Reff, ME, and ICDeckman, Nucleic Acid). Res. (1989) is one example.

[0055] Additional polyadenylation sites can be identified or constructed using methods known in the art. The minimal polyadenylation site consists of AAUAAA and a second recognition sequence located approximately 30 nucleotides downstream, which is typically a G / U-rich sequence. In this specification, the sequences are presented as DNA rather than RNA to facilitate the preparation of DNA suitable for incorporation into expression vectors. When presented as DNA, the polyadenylation site consists of AATAAA, for example, accompanied by a G / T-rich region downstream. Both sequences must be present to form an effective polyadenylation site. The purpose of these sites is to recruit specific RNA-binding proteins to RNA. AAUAAA binds to a cleavage and polyadenylation-specific factor (CPSF; Murthy KG, and Manley JL (1995), Genes Dev 9:2672-2683), while a second site, often a G / U sequence, binds to a cleavage-stimulating factor (CstF; Takagaki Y. and Manley JL (1997), MoI Cell Biol 17:3907-3914). CstF is composed of several proteins, but the protein involved in RNA binding is CstF-64, a member of the ribonucleoprotein domain family of proteins (Takagaki et al. (1992), Proc Natl Acad Sci USA 89:1403-1407).

[0056] While not strictly theoretical, polyadenylated signal sequences are understood to belong to the 3' regulatory elements, which are DNA sequences located in the 3' untranslated region (UTR) of mRNA transcripts downstream of the coding region. Other 3' regulatory elements include AU-rich elements (AREs) and microRNA (miRNA) binding sites. Although 3' regulatory elements are not translated into proteins, they play a crucial role in regulating gene expression, including influencing mRNA transcript stability, localization, and translation, ultimately affecting the expression (level) of protein-coding genes.

[0057] This specification provides improved recombinant polyadenylation signal sequences having several enhanced properties. The recombinant polyadenylation signal sequences of the present invention result in improved expression of the target protein (encoded by a nucleotide sequence operably linked to the recombinant polyadenylation signal sequence). Furthermore, the recombinant polyadenylation signal sequences of the present invention are shorter than effective polyadenylation signal sequences known in the art, compared to polyadenylation signal sequences selected from the group consisting of, for example, rabbit β-globin poly(A) signal, HSV thymidine kinase polyA, human α-globin polyA, SV40 polyA, human β-globin polyA, polyomavirus polyA, and bovine growth hormone polyA.

[0058] In one embodiment, the recombinant polyadenylation signal sequence disclosed herein contains 100, 99, 98, 97, or fewer than 96 nucleotides. In one embodiment, the recombinant polyadenylation signal sequence disclosed herein has a sequence length of 100, 99, 98, 97, or fewer than 96 nucleotides. In one embodiment, the recombinant polyadenylation signal sequence disclosed herein has a sequence length of 25-100, 30-100, 35-100, 40-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 85-100, 90-100 nucleotides, or 95-100 nucleotides. In one embodiment, the recombinant polyadenylation signal sequence disclosed herein is recognized by RNA polymerase, where the RNA polymerase releases an RNA molecule. In one embodiment, the RNA polymerase is a eukaryotic RNA polymerase. In one embodiment, a eukaryotic cell comprising a transcription unit comprising a nucleotide sequence encoding a polypeptide is capable of expressing the polypeptide if the nucleotide sequence is operably ligated to a recombinant polyadenylation signal sequence as disclosed herein. In one embodiment, the recombinant polyadenylation signal sequence results in the termination (termination of transcription) and polyadenylation of the mRNA transcript of the transcription unit in the eukaryotic cell. Thus, the recombinant polyadenylation signal sequence initiates the termination of transcription of the transcription unit.

[0059] While not strictly theoretical, shorter regulatory elements are advantageous in many applications of recombinant transcription units. For example, viral vectors may have size limitations. Recombinant adeno-associated viruses (rAAVs) are limited to AAV transgenes of less than 5 kilobases, and the transgene must contain the coding sequence of the gene of interest, along with the promoter sequence, enhancer, and polyadenylation signal. Shorter regulatory elements leave more space for the coding sequence.

[0060] The term "expression" refers to the process by which information from nucleic acids is used to synthesize functional polynucleotides that enable the production of (gene) products, such as the protein of interest. Expression can include transcription, RNA splicing, translation, and post-translational modification. Regulation of expression controls the timing, location, and quantity of a given expression product (such as the protein of interest) present in the cell.

[0061] The term "termination" refers to the process in which RNA polymerase stops adding nucleotides to the growing RNA chain and releases the RNA molecule. "Polyadenylation" refers to the process in which a chain of adenine nucleotides, also called the poly(A) tail, is added to the 3' end of the newly synthesized RNA molecule. Polyadenylation is catalyzed by poly(A) polymerase and occurs after the RNA molecule is cleaved at a specific site downstream of the coding region (polyadenylation signal). The terms termination and polyadenylation refer to both processes that occur sequentially to produce a mature mRNA transcript.

[0062] An "mRNA transcript," also known as "messenger RNA" or simply "mRNA," refers to a type of RNA molecule used as a template for transporting genetic information from DNA in the cell nucleus to ribosomes and for synthesizing proteins. In the transcription process, the DNA sequence of a protein-coding gene is used as a template to produce a complementary RNA molecule. This RNA molecule is then processed and modified to form a mature mRNA transcript. Modifications for forming a mature mRNA transcript include, for example, 5' capping, splicing, and polyadenylation.

[0063] In one embodiment, a recombinant transcription unit is provided comprising a nucleotide sequence encoding a polypeptide, the nucleotide sequence being operably linked to a recombinant polyadenylation signal sequence having a sequence length of less than 100 nucleotides. In one embodiment, eukaryotic cells transformed with the recombinant nucleic acid comprising the recombinant transcription unit are capable of expressing the polypeptide at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid comprising the recombinant polyadenylation signal sequence of SEQ ID NO: 2. In one embodiment, the nucleotide sequence of the reference nucleic acid is identical to the sequence of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity.

[0064] The term "expression level" refers to the quantitative determination of the level at which a particular open reading frame (e.g., contained in a transcription unit) is expressed by a cell. Expression levels can be determined by detecting products of open reading frames (e.g., proteins) by methods known in the art, such as Western blotting. However, it may often be simpler to detect one of the protein precursors, such as mRNA, and infer gene expression levels from these measurements. mRNA levels can be quantitatively measured by methods known in the art, such as Northern blotting, RT-qPCR, or hybridization microarrays. In one embodiment, mRNA expression levels are determined by RT-qPCR. Another method for determining expression levels is the use of a reporter gene (also known as a reporter). This is a gene that, when operably ligated to a regulatory sequence, can be readily identified and measured, for example, by fluorescence or luminescence. Such reporter genes are well known in the art and are described herein.

[0065] For example, Example 2 describes a recombinant transcription unit containing the luminescent protein NanoLuc luciferase. The expression level of luciferase is known in the art and can be determined by the method shown in Example 1.3.

[0066] In one embodiment, the expression level handed down by a recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, is determined by: (a) Provide a nucleotide sequence of a recombinant transcription unit, and generate a reporter transcription unit by replacing the nucleotide sequence encoding the polypeptide with the nucleotide sequence of SEQ ID NO: 19 encoding luciferase, (b) Transfect HEK293T cells with a reporter plasmid containing the reporter transcription unit, and culture the HEK293T cells under conditions suitable for the expression of the reporter transcription unit. (c) Measure the level of luciferase 24 hours after transfection.

[0067] In one embodiment, the expression level is determined for the recombinant nucleic acid as described above to obtain a first expression level, then the expression level is determined for the reference nucleic acid as described above to obtain a second expression level, and then the first expression level and the second expression level are compared to determine whether the first expression level is the same as or higher than the second expression level.

[0068] In this specification, “reference nucleic acid” means a nucleic acid that is similar to or identical to the recombinant nucleic acid of interest (such as a recombinant nucleic acid containing the recombinant transcription unit of the present invention), except for the sequence element of interest. The reference nucleic acid can be used to compare or evaluate the functionality of the recombinant nucleic acid of interest (e.g., the resulting expression level) against a specific reference (e.g., a nucleic acid containing a recombinant polyadenylation signal sequence consisting of the nucleotide sequence of SEQ ID NO: 2). In some embodiments, the nucleotide sequence of the reference nucleic acid is identical to the nucleotide sequence of the recombinant nucleic acid of interest, except for the sequence element of interest, such as the recombinant polyadenylation signal sequence of the present invention (without considering sequence identity). In some embodiments, the nucleotide sequence of the recombinant polyadenylation signal sequence is not considered in determining sequence identity. For example, the nucleotide sequence of the recombinant polyadenylation signal sequence can be omitted (deleted) from the recombinant nucleic acid of interest and the reference nucleic acid for sequence comparison.

[0069] In one embodiment, eukaryotic cells transformed (individually) with recombinant nucleic acids containing recombinant transcription units are able to express polypeptides at the same or higher expression levels compared to eukaryotic cells of the same type transformed with a reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity. In one embodiment, the expression level is determined by (individually) incorporating each of the recombinant polyadenylation signal sequences into a recombinant transcription unit containing the nucleotide sequence of SEQ ID NO: 19 (encoding NanoLuc luciferase) and the recombinant polyadenylation signal sequence of interest in a 5'-3' order, (individually) transfecting HEK293T cells with a reporter plasmid containing the recombinant transcription unit, culturing the HEK293T cells under conditions suitable for the expression of the recombinant transcription unit, and measuring the level of luciferase 24 hours after transfection.

[0070] In one embodiment, the recombinant polyadenylation signal sequence includes or consists of nucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs: 12.

[0071] A particular aspect of the recombinant polyadenylation signal sequences according to the present invention is that they are functional in eukaryotic cells. For example, the recombinant polyadenylation signal sequences provided herein are recognized by RNA cleavage complexes. In some embodiments, the RNA cleavage complex is a eukaryotic RNA cleavage complex. In some embodiments, the recombinant polyadenylation signal sequence includes a TG-rich domain and a T-rich domain. In some embodiments, the recombinant polyadenylation signal sequence includes the nucleotide sequence AATAAA (SEQ ID NO: 18). In some embodiments, the recombinant polyadenylation signal sequence includes a G / T-rich sequence located approximately 30 nucleotides downstream of the nucleotide sequence AATAAA (SEQ ID NO: 18). In some embodiments, the recombinant polyadenylation signal sequence, when present within an RNA molecule, has the ability to bind to a cleavage-polyadenylation-specific factor (CPSF). In some embodiments, the recombinant polyadenylation signal sequence, when present within an RNA molecule, has the ability to bind to a cleavage-stimulating factor (CstF). In some embodiments, at least one polyadenylation signal sequence results in termination and polyadenylation of an mRNA transcript operably linked to at least one polyadenylation signal sequence.

[0072] In some embodiments, a recombinant transcription unit is provided comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, the recombinant polyadenylation signal sequence comprising or comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0073] In a preferred embodiment, the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12.

[0074] In some embodiments, recombinant nucleic acids comprising at least one polyadenylation signal sequence described herein are provided. When a recombinant nucleic acid (such as one or more plasmids) requires multiple regulatory elements (such as polyadenylation signal sequences), a common obstacle known in the art is that identical sequences in close proximity can trigger a recombination event. Therefore, it is advantageous to reduce or eliminate such recombination events. One aspect of the present invention is a plurality of novel short recombinant polyadenylation signal sequences having (sharing) low sequence homology (low sequence identity) with respect to each other. Furthermore, the recombinant polyadenylation signal sequences result in potent expression of a nucleotide sequence encoding a polypeptide of interest operably ligated to the recombinant polyadenylation signal sequences.

[0075] In some embodiments, the recombinant nucleic acid comprises two or more recombinant polyadenylation signal sequences described herein. In some embodiments, the recombinant nucleic acid comprises two or more recombinant polyadenylation signal sequences described herein. In some embodiments, the recombinant nucleic acid comprises three recombinant polyadenylation signal sequences described herein. In preferred embodiments, the recombinant nucleic acid comprises the polyadenylation signal sequences of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12. In certain such embodiments, the recombinant nucleic acid comprises three individual polyadenylation signal sequences, the first polyadenylation signal sequence comprising the nucleotide sequence of SEQ ID NO: 6, the second polyadenylation signal sequence comprising the nucleotide sequence of SEQ ID NO: 9, and the third polyadenylation signal sequence comprising the nucleotide sequence of SEQ ID NO: 12.

[0076] In one embodiment, (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, and (b) comprising a second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably ligated to a second recombinant polyadenylation signal sequence, Recombinant nucleic acids are provided in which the first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%.

[0077] In one embodiment, recombinant nucleic acid is (c) Further comprising a third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence, The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence individually have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% with the third recombinant polyadenylation signal sequence.

[0078] In one embodiment, recombinant nucleic acid is (c) Further comprising a third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence, The first recombinant polyadenylation signal sequence has 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% sequence identity with the third recombinant polyadenylation signal sequence.

[0079] In one embodiment, recombinant nucleic acid is (c) Further comprising a third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence, The second recombinant polyadenylation signal sequence has 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% sequence identity with the third recombinant polyadenylation signal sequence.

[0080] In some embodiments, the first, second, and, if present, third recombinant polyadenylation signal sequences have a sequence length of less than 100, 99, 98, 97, or 96 nucleotides.

[0081] The term “sequence identity percentage (%)” is defined as the percentage of nucleotides in the target sequence that are identical to nucleotides in the candidate sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage. Alignment can be performed using various methods well known in the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0082] In some embodiments, the sequence identity percentage (%) of the first and second recombinant polyadenylated signal sequences is determined by: (a) Align the sequences of the first and second recombinant polyadenylated signal sequences using Align-2 software and standard settings. (b) Determine the percentage of nucleotides in the first recombinant polyadenylation signal sequence that are identical to the nucleotides in the second recombinant polyadenylation signal sequence to obtain the sequence identity percentage (%).

[0083] In some embodiments, the sequence identity percentage (%) between the first and second recombinant polyadenylation signal sequences and the third recombinant polyadenylation signal sequence is determined individually by: (a) Align the sequences of the first and third recombinant polyadenylated signal sequences using Align-2 software and standard settings. (b) Determine the proportion of nucleotides in the first recombinant polyadenylation signal sequence that are identical to the nucleotides in the third recombinant polyadenylation signal sequence, and obtain the sequence identity percentage (%) of the first and third recombinant polyadenylation signal sequences. (c) Align the sequences of the second and third recombinant polyadenylated signal sequences using Align-2 software and standard settings. (d) Determine the proportion of nucleotides in the second recombinant polyadenylation signal sequence that are identical to the nucleotides in the third recombinant polyadenylation signal sequence, and obtain the sequence identity percentage (%) of the second and third recombinant polyadenylation signal sequences.

[0084] "Recombination events" between nucleic acids (e.g., plasmids) can occur through various mechanisms, including homologous recombination and site-directed recombination. Such events can result in the transfer of genetic material from one plasmid to another, or the integration of a plasmid into a chromosome. The resulting plasmids / chromosomes may have different genetic content and confer different functions to cells. In the context of the present invention, such recombination events are undesirable, and the inventors aimed to provide novel and improved sequences for reducing or inhibiting recombination events. Accordingly, in some embodiments, recombination events between nucleic acids containing a first recombinant polyadenylation signal sequence and nucleic acids containing a second recombinant polyadenylation signal sequence (and a third recombinant polyadenylation signal sequence, if present) are reduced or prevented.

[0085] The inventors have created novel and improved recombinant polyadenylation signal sequences that reduce / inhibit / prevent recombination events. The novel and improved sequences provided are short and share a low degree of sequence identity. In some embodiments, the first, second, and, if present, third polyadenylation signal sequences are unable to participate in DNA strand exchange to form a recombinant intermediate. "DNA strand exchange" is a crucial step in the process of cross-recombination. Two DNA molecules are cleaved at corresponding sites, exchanging segments of their strands with each other, and then rejoined to form two new hybrid DNA molecules. DNA strand exchange involves the formation of a heteroduplex structure in which the single-stranded ends of the cleaved polynucleotide molecules penetrate each other's double helix, forming a base-pairing region (Holiday junction) between the two molecules. This "recombination intermediate" allows the DNA strands to cross with each other, facilitating the exchange of DNA segments between the two molecules. Subsequently, the Holiday junction can be degraded by strand cleavage, leading to the formation of the hybrid DNA molecule.

[0086] For a Holliday junction to form during homologous recombination, a considerable degree of sequence homology is required between the two DNA molecules involved in the exchange. Specifically, the homologous sequences must be long enough to form a stable heteroduplex DNA structure and possess a sufficiently high degree of similarity. While not theoretically bound, the minimum length and degree of homology required to form a Holliday junction can vary depending on the DNA molecules involved, as well as the specific enzymes and cofactors involved. Generally, it is believed that at least 100–200 base pairs of consecutive homologous DNA sequences are necessary to form a stable Holliday junction.

[0087] In some embodiments, recombination events between a nucleic acid containing a first polyadenylation signal sequence and a nucleic acid containing a second polyadenylation signal sequence are reduced or prevented. In some embodiments, recombination events between a nucleic acid containing a first polyadenylation signal sequence and a nucleic acid containing a third polyadenylation signal sequence are reduced or prevented. In some embodiments, recombination events between a nucleic acid containing a second polyadenylation signal sequence and a nucleic acid containing a third polyadenylation signal sequence are reduced or prevented.

[0088] Recombination events can be detected by methods known in the art. For example, a recombination event can be detected by Sanger sequencing of the relevant PCR amplicon, followed by sequence alignment (e.g., using CLUSTALW), and identification of the recombination event (e.g., using a recombination detection program). In some embodiments, a recombination event is detected by Sanger sequencing of a PCR amplicon of a nucleic acid containing a recombinant polyadenylation signal sequence provided herein, followed by alignment of the PCR amplicon using standard settings of CLUSTALW, and identification of the recombination event using standard settings of a recombination detection program 5. In preferred embodiments, no recombination event is detected.

[0089] The recombinant polyadenylation signal sequences of the present invention are useful for a variety of applications. Polyadenylation signal sequences are necessary for efficient protein expression. Therefore, in some embodiments, the recombinant transcription units according to the present invention are capable of driving the expression of a nucleotide sequence encoding a target polypeptide. In some embodiments, the nucleotide sequence encoding a target polypeptide is operably ligated to the recombinant transcription unit. In some embodiments, the nucleotide sequence encoding a target polypeptide is operably ligated to the recombinant polyadenylation signal sequence described herein.

[0090] In some embodiments, the first and second, and if present, the third recombinant transcription units are active in eukaryotic cells. In some embodiments, the first and second, and if present, the third polypeptides are expressed by eukaryotic cells. In some embodiments, eukaryotic cells are incubated under conditions suitable for the expression of the first, second, and if present, the third polypeptides. In some embodiments, eukaryotic cells are cultured under conditions suitable for the expression of the first, second, and if present, the third polypeptides. In some embodiments, a method for producing one (or more) polypeptides is provided, comprising the step of culturing host cells containing at least one recombinant transcription unit described herein under conditions suitable for the expression of one (or more) polypeptides.

[0091] Protein expression can be measured by assays readily available in the art, such as those described in the examples provided below.

[0092] The terms plasmid, construct, and vector are used throughout this specification. In this specification, “plasmid” refers to a circular supercoiled DNA molecule assembled from various nucleic acid molecules encoding regulatory sequences, open reading frames, cloning sites, stop codons, spacer regions, or other sequences selected for structural or functional regions, and used as a vector to express genes within a vertebrate host. Furthermore, in this specification, “plasmid” refers to a molecule capable of replicating in bacterial strains. In this specification, “construct” refers to a specific vector or plasmid having a particular arrangement of genes and regulatory elements. A nucleic acid sequence can be “exogenous,” meaning it is foreign to the cell into which the vector is introduced; “heterogeneous,” meaning it originates from a different genetic source; or “homological,” meaning the sequence is structurally related to a sequence within the cell but is located in a host cell nucleic acid where that sequence is not normally found. Methods for constructing the vectors of the present invention or modifying plasmids are well known in the art through standard recombination techniques, such as those described, for example, in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1989) and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, New York, (1995), both of which are incorporated herein by reference.

[0093] The term “vector” is used to refer to a carrier nucleic acid molecule that can be inserted to introduce a specified nucleic acid molecule encoding one or more antigens into a cell capable of expressing it. Examples of vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). The term “expression vector” refers to a vector containing a nucleic acid sequence encoding at least a portion of a transcriptable gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of expressed interfering RNA (eiRNA), small interfering RNA (siRNA), antisense molecules, or ribozymes. Expression vectors may contain a variety of “regulatory sequences.” Regulatory sequences are nucleic acid sequences necessary for the transcription and, possibly translation, of a coding sequence operably linked in a particular host organism. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, as described below.

[0094] Apart from recombinant polyadenylation signal sequences, it is naturally understood that, in order to prevent recombination events within nucleic acids containing multiple transcription units, further elements of the transcription units may also form recombinant intermediates that can cause recombination events. Therefore, it is preferable that different recombinant transcription units do not contain elements that share high sequence homology. It will be further understood that nucleotide sequences encoding a polypeptide of interest typically do not share high sequence homology. However, in situations where closely related genes encoding a polypeptide of interest are contained in different transcription units contained in the nucleic acids provided herein, it is preferable that the nucleotide sequences encoding different polypeptides have 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% sequence identity. Further elements of a transcription unit that can form recombinant intermediates are promoters contained within the transcription unit. In some embodiments, the nucleic acid contains a first promoter and a second promoter. In some embodiments, the first promoter and the second promoter are not the same promoter. In some embodiments, the nucleic acid further contains a third promoter. In some embodiments, the third promoter is not the same promoter as the first and / or second promoter.

[0095] In one embodiment, recombinant nucleic acids previously described herein are provided, where (a) The first recombinant transcription unit further comprises a first promoter operably ligated to a nucleotide sequence encoding the first polypeptide, (b) The second recombinant transcription unit further comprises a second promoter operably ligated to a nucleotide sequence encoding the second polypeptide, The first and second promoters have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%.

[0096] In one embodiment, recombinant nucleic acid is (c) If present, the first recombinant transcription unit further comprises a first promoter operably ligated to a nucleotide sequence encoding the first polypeptide, The first and second promoters have sequence identity with the third promoter of 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or less than 60%.

[0097] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 6, and (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 9, Optional, Eukaryotic cells transformed with recombinant nucleic acid containing the first recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity. Eukaryotic cells transformed with recombinant nucleic acid containing a second recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity.

[0098] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 6, and (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 12. Optional, Eukaryotic cells transformed with recombinant nucleic acid containing the first recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity. Eukaryotic cells transformed with recombinant nucleic acid containing a second recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity.

[0099] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 9, and (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 12. Optional, Eukaryotic cells transformed with recombinant nucleic acid containing the first recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity. Eukaryotic cells transformed with recombinant nucleic acid containing a second recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity.

[0100] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 6, and (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably ligated to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having SEQ ID NO: 9.

[0101] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 6, and (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having SEQ ID NO: 12.

[0102] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 9, and (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having SEQ ID NO: 12.

[0103] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 6. (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 9, and (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence, wherein the third recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO: 12. Optional, Eukaryotic cells transformed with recombinant nucleic acid containing the first recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity. Eukaryotic cells transformed with recombinant nucleic acid containing a second recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity. Eukaryotic cells transformed with recombinant nucleic acids containing a third recombinant transcription unit are capable of expressing polypeptides at the same or higher expression levels compared to eukaryotic cells transformed with reference nucleic acids containing the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid when the recombinant polyadenylation signal sequence is not considered for sequence identity.

[0104] In one embodiment, recombinant nucleic acids comprising the following are provided: (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 6, (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 9, and (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence, wherein the third recombinant polyadenylation signal sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 12.

[0105] In some embodiments, the first, second, and, if present, third promoters are active in eukaryotic cells. In some embodiments, the first, second, and, if present, third promoters are capable of driving the expression of one or more polypeptides of interest in eukaryotic cells. The expression of the polypeptides of interest can be measured by assays readily available in the art, such as those described in the examples provided below. In some embodiments, the first promoter drives the expression of the first polypeptide. In some embodiments, the second promoter drives the expression of the second polypeptide. In some embodiments, the third promoter drives the expression of the third polypeptide. In some embodiments, the first promoter is capable of driving the expression of the first polypeptide, the second promoter is capable of driving the expression of the second polypeptide, and, if present, the third promoter is capable of driving the expression of the third polypeptide.

[0106] The term "promoter" refers to a polynucleotide sequence that controls the transcription of a gene / structural gene or nucleic acid sequence to which it is operably ligated. Promoters contain signals for RNA polymerase binding and transcription initiation. The promoter used functions within the cell to which the expression of the selected structural gene is intended. Numerous promoters, including constitutive, inductive, and repressive promoters, originating from a diverse range of different sources, are well known in the art (identified in databases such as GenBank) and are available as cloned polynucleotides or within cloned polynucleotides (e.g., from contract organizations such as ATCC and other commercial or individual sources).

[0107] Typically, promoters are located in the 5' non-coding or untranslated region of a gene, adjacent to the transcription start site of a structural gene. Sequence elements within a promoter that function in initiating transcription are often characterized by consensus nucleotide sequences. These elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs), cyclic AMP response elements (CREs), serum response elements (SREs), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE / ATF, AP2, SP1, cAMP response element-binding protein (CREB), and octamer factors. If the promoter is an inducible promoter, the transcription rate increases in response to inducers, such as the CMV promoter with its two subsequent tet operator sites, metallothionein, and heat shock promoters. If the promoter is a constitutively active promoter, the transcription rate is not regulated by inducers. Exemplary eukaryotic promoters identified as potent expression promoters include the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Roussarcoma virus long terminal repeat sequence, the Chinese hamster elongation factor 1α (CHEF-1), human EF-1α, ubiquitin, and the human cytomegalovirus major pre-early promoter (hCMV MIE).

[0108] In some embodiments, the first, second, and, if present, third promoters are individually selected from the group consisting of the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Roussarcoma virus long terminal repeat sequence, Chinese hamster elongation factor 1α (CHEF-1), human EF-1α, ubiquitin, and the human cytomegalovirus major pre-early promoter (hCMV MIE).

[0109] The nucleic acid according to the present invention may be contained in a vector or in multiple vectors.

[0110] Accordingly, this disclosure also provides a vector or a plurality of vectors comprising a nucleic acid or a plurality of nucleic acids according to the present invention. The vector can facilitate the delivery of nucleic acids encoding one or more recombinant transcription units to cells according to this disclosure. The vector may be an expression vector comprising elements necessary for the expression of recombinant polypeptides according to this disclosure. The vector may comprise elements that facilitate the integration of one or more nucleic acids into the genomic DNA of the cell into which the vector is introduced.

[0111] The nucleic acids and vectors provided herein may be provided in purified or isolated forms, i.e., from other nucleic acids or naturally occurring biomaterials.

[0112] A vector can be a vector for expressing nucleic acids in cells (i.e., an expression vector). Such a vector may include a promoter sequence operably ligated to a nucleotide sequence encoding the recombinant polypeptide according to the Disclosure. The vector may also include a stop codon (i.e., the 3' of the nucleotide sequence of the vector relative to the nucleotide sequence encoding one or more recombinant polypeptides) and an expression enhancer. A peptide or polypeptide can be expressed from the vector according to the Disclosure using any suitable vector, promoter, enhancer, and stop codon known in the Art.

[0113] Vectors intended in connection with this disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugated plasmids (e.g., F plasmids), non-conjugated plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, e.g., gamma retroviral vectors (e.g., mouse leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), for example, as described by Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9. Both of these are incorporated herein by reference in their entirety. In some embodiments, the vectors according to this disclosure are lentiviral vectors.

[0114] In some embodiments, the vector may be a eukaryotic vector, i.e., a vector containing the elements necessary for expressing a protein from the vector within a eukaryotic cell. In some embodiments, the vector may be a mammalian vector containing, for example, a cytomegalovirus (CMV) or SV40 promoter that drives protein expression.

[0115] In some embodiments, the first, second, and / or third vectors, if present, include a bacterial origin of replication. In some embodiments, the first vector includes a bacterial origin of replication. In some embodiments, the second vector includes a bacterial origin of replication. In some embodiments, the third vector includes a bacterial origin of replication. A bacterial origin of replication is necessary for replicating vectors such as plasmids within the bacterium. Bacterial origins of replication are known in the art. In some embodiments, the bacterial origin of replication is a pUC origin of replication.

[0116] In some embodiments, the recombinant nucleic acids provided herein include a first vector as described herein, comprising a first recombinant transcription unit as described herein; a second vector as described herein, comprising a second recombinant transcription unit as described herein; and a third vector as described herein, comprising a third recombinant transcription unit, if a third recombinant transcription unit is present. The recombinant nucleic acids may be provided in one or more vials. For example, the first, second, and third vectors, if present, may be provided together in one vial. Alternatively, the first, second, and third vectors, if present, may be provided in separate vials. In some embodiments, the first, second, and third vectors, if present, may be provided in separate vials, but the vials together constitute the recombinant nucleic acids provided herein. In some embodiments, the first, second, and third vectors, if present, may be provided in the same vial.

[0117] In some embodiments, recombinant nucleic acids include at least one selection marker. In some embodiments, vectors as described herein include a selection marker. The term “selection marker” means a nucleic acid that enables cells having it to be specifically selected in the presence of a corresponding selective agent. Typically, a selection marker confers resistance to a drug or compensates for a metabolic or catabolic defect in the cell into which it is introduced. A selection marker can be positive, negative, or bifunctional. A useful positive selection marker is an antibiotic resistance gene, which enables the selection of cells transformed by it in the presence of a corresponding selective agent, such as an antibiotic. Untransformed cells cannot proliferate or survive under selective conditions, i.e., in the presence of the selective agent. A negative selection marker enables the selective removal of cells having that marker. Selective markers used in eukaryotic cells include, for example, structural genes encoding aminoglycoside phosphotransferase (APH), such as hygromycin (hyg), neomycin (neo), and the G418 selective marker, as well as nucleic acids that confer resistance to dihydrofolate reductase (DHFR), thymidine kinase (tk), glutamine synthase (GS), asparagine synthase, tryptophan synthase, tryptophan synthetase (selective agent indole), histidinol dehydrogenase (selective agent histidinol D), and puromycin, bleomycin, phleomycin, chloramphenicol, zeosin, and mycophenolic acid.

[0118] In some embodiments, the selection marker is selected from the group consisting of hygromycin selection markers, neomycin selection markers, G418 selection markers, dihydrofolate reductase (DHFR), thymidine kinase, glutamine synthase, asparagine synthase, tryptophan synthase, histidinol dehydrogenase, and nucleic acids that confer resistance to puromycin, bleomycin, phleomycin, chloramphenicol, zeosin, and mycophenolic acid.

[0119] In some embodiments, recombinant nucleic acids contain at least one bacterial origin of replication. In some embodiments, vectors as described herein contain a bacterial origin of replication. For a vector / plasmid to replicate independently within a bacterial cell, it must have a stretch of DNA that can function as an origin of replication. An origin of replication (also called a replication origin) is a specific sequence from which replication begins. An exemplary origin of replication can be derived from the pUC plasmid cloning vector constructed by Joachim Messing and colleagues (Yanisch-Perron, C.; Vieira, J.; Messing, J. (1985). Gene. 33(1):103-119). In some embodiments, the first, second, and / or third vectors, if present, contain a bacterial origin of replication, particularly the pUC19 origin of replication.

[0120] It will be understood that the nucleic acids of the present invention can be used for recombinant protein production. As previously described herein, the use of novel recombinant polyadenylation signal sequences is advantageous in reducing the risk of recombination events between highly homologous or identical sequence stretches in the context of recombinant polypeptide expression.

[0121] Therefore, cells (e.g., host cells) containing recombinant nucleic acids according to the present invention are further provided.

[0122] In some embodiments, the cells are host cells. The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which, regardless of passage number, include primary transformed cells and their offspring. The offspring may not have nucleic acid content that is exactly the same as that of the parent cell and may contain mutations. In this specification, mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0123] For the recombinant production of the target protein, the nucleic acid encoding the target protein is isolated, inserted into one or more vectors, and further cloned and / or expressed in host cells. Such nucleic acids may be readily isolated and sequenced using conventional procedures, generated by recombinant methods, or obtained by chemical synthesis.

[0124] Suitable host cells for cloning or expressing the target protein include prokaryotic or eukaryotic cells as described herein. For example, (recombinant) polypeptides may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (Also see Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.) After expression, the target protein can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0125] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable hosts for cloning or expressing recombinant polypeptide-encoding vectors, including fungal and yeast strains whose glycosylation pathways are "humanized," resulting in the production of polypeptides with partially or completely human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22(2004) 1409-1414, and Li, H. et al., Nat. Biotech. 24(2006) 210-215.

[0126] Host cells suitable for the expression of (glycosylated) polypeptides can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0127] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0128] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspensions may be useful. Other examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney (HEK) cell line (e.g., 293 or 293T cells described by Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described by Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT060562); TRI cells (e.g., Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68); MRC These include 5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77(1980) 4216-4220), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0129] In some embodiments, the (host) cell is a eukaryotic cell. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the (host) cell is a mammalian (host) cell. In some embodiments, the (host) cell is selected from the group consisting of CHO, BHK, HEK, and Sp2 / 0. In some embodiments, the (host) cell is a CHO K1 cell.

[0130] Therefore, in one embodiment, a method for producing a polypeptide is provided, comprising the following steps: (a) A step of providing a host cell containing recombinant nucleic acid previously described herein, (b) A step of incubating host cells under conditions suitable for polypeptide expression, (c) A step of recovering the target polypeptide from the cell culture.

[0131] In one embodiment, a method for producing a polypeptide is provided, comprising the following steps: (a) A step of providing a cell comprising a recombinant nucleic acid previously described herein, wherein the at least one polyadenylation signal sequence is operably linked to a nucleotide sequence encoding a polypeptide, (b) A step of incubating cells under conditions suitable for polypeptide expression, (c) A step of recovering the target polypeptide from the cell culture.

[0132] In one embodiment, a method for producing a target polypeptide is provided, comprising the following steps: (a) A step of providing a host cell comprising a recombinant nucleic acid as previously described herein, wherein the polypeptide of interest is a first polypeptide, and a second polypeptide and, if present, a third polypeptide are required for the production of the polypeptide of interest or improve the production of the polypeptide of interest. (b) Incubating host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide; (c) A step of recovering the target polypeptide from the cell culture, Optional, (d) A step of formulating the recovered polypeptide for therapeutic use.

[0133] Furthermore, the present invention provides for the construction of viral vectors using recombinant polyadenylation signal sequences. The construction of viral vectors using multiple distinct plasmids is a widely used and effective technique that enables the generation of high-quality viral particles for research and clinical applications. However, because multiple plasmids are used, it is necessary to mitigate the possibility of recombination events between highly homologous or identical sequence stretches on different plasmids. The polyadenylation signal sequences according to the present invention are advantageous in this situation. Moreover, viral vector genomes are typically limited in size. Therefore, it is advantageous to incorporate short 5' and 3' regulatory sequences to maximize the sequence length available for the (therapeutic) transgene.

[0134] In some embodiments, recombinant viral vectors containing the recombinant polyadenylated signal sequence described above are provided.

[0135] In some embodiments, recombinant viral vectors are provided that include a capsid and a vector genome package therein. In certain embodiments, viral vectors that may be used in the present invention include, but are not limited to, retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, Epstein-Barr viruses, vaccinia viruses, and human cytomegalovirus vectors (including their recombinant versions). In preferred embodiments, the recombinant viral vector includes a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) that includes an adeno-associated virus (AAV) capsid and a vector genome packaged therein.

[0136] In one embodiment, a recombinant viral vector comprising a vector genome is provided, wherein the vector genome is arranged in the order 5' to 3', (i) 5'ITR sequence, (ii) Promoter sequence, (iii) Sequence encoding polypeptide, (iv) Recombinant polyadenylated signal sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and (v)3'ITR sequence Includes.

[0137] In a preferred embodiment, the recombinant polyadenylated signal sequence is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12.

[0138] The term "recombinant" is used as a modifier for viral vectors (e.g., recombinant AAV (rAAV) vectors) to mean that the composition has been manipulated in a way that does not generally occur in nature (i.e., genetically modified). A specific example of a recombinant AAV vector would be the insertion of nucleic acids (heterogeneous polynucleotides) that are not normally present in the wild-type AAV genome into the viral genome. One example would be cloning a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence into a vector, with or without the presence of the 5', 3', and / or intron regions that are normally associated within the AAV genome. The term "recombinant" is not always used herein in relation to AAV vectors, but despite such omissions, the recombinant form is explicitly included.

[0139] An "rAAV vector" is derived from the wild-type AAV genome by, for example, using molecular methods to remove all or part of the wild-type AAV genome and replacing it with non-natural (heterogeneous) nucleic acids, such as nucleic acids encoding therapeutic proteins or polynucleotide sequences. Typically, for rAAV vectors, one or both of the reverse-end repeat (ITR) sequences of the AAV genome are retained. rAAV is distinguished from the AAV genome because all or part of the AAV genome has been replaced with non-natural sequences with respect to the AAV genome nucleic acids, such as heterogeneous nucleic acids encoding therapeutic proteins or polynucleotide sequences. Therefore, the incorporation of non-natural (heterogeneous) sequences defines the AAV as a "recombinant" AAV vector and is sometimes called an "rAAV vector."

[0140] In some embodiments, eukaryotic cells containing the aforementioned vector genomes are capable of expressing polypeptides. In some embodiments, recombinant polyadenylation signal sequences result in the termination of mRNA transcripts of transcription units (transcription termination) and polyadenylation in eukaryotic cells.

[0141] Recombinant AAV vector sequences can be packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo, and may be referred to herein as “particles.” When a recombinant vector sequence is capsidated or packaged into an AAV particle, that particle may also be referred to as “rAAV,” “rAAV particle,” and / or “rAAV virion.” Such rAAV, rAAV particle, and rAAV virion contain a protein that capsidates or packages the vector genome. A specific example, in the case of AAV, is the capsid protein.

[0142] The "vector genome," sometimes abbreviated as "vg," refers to the portion of a recombinant plasmid sequence that is ultimately packaged or capsidized to form rAAV particles. When a recombinant plasmid is used to construct or manufacture a recombinant AAV vector, the AAV vector genome does not include the "plasmid" portion that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," and while it is crucial for plasmid cloning and amplification—processes necessary for proliferation and recombinant AAV vector production—it is not packaged or capsidized into rAAV particles itself. Therefore, the "vector genome" refers to the nucleic acid packaged or capsidized by rAAV.

[0143] In this specification, the term “serotype” with respect to AAV vectors means a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the absence of cross-reactivity between antibodies against one AAV and antibodies against another. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Antibodies against one AAV may cross-react with one or more other AAV serotypes due to homology in capsid protein sequences.

[0144] In the conventional definition, a serotype refers to a virus of interest that, when tested for neutralizing activity against serums specific to all existing characterized serotypes, does not yield any antibodies that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or more capsid variants are generated, serological differences from currently existing serotypes may or may not exist. Therefore, if a new virus (e.g., AAV) has no serological differences, this new virus (e.g., AAV) becomes a subgroup or variant of the corresponding serotype. Often, serological testing for neutralizing activity has not yet been performed on variant viruses with capsid sequence modifications to determine whether they are other serotypes according to the conventional definition of serotype. Therefore, for convenience and to avoid repetition, the term “serotype” in a broad sense refers to both serologically distinct viruses (e.g., AAV) and serologically non-distinguishable viruses (e.g., AAV) that may fall within a subgroup or variant of a given serotype.

[0145] rAAV viral vectors can contain any virus strain or serotype. For example, but are not limited to, an rAAV vector genome or particle (capsid such as VP1, VP2, and / or VP3) may be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10, AAV3B, or AAV-2i8. Such vectors may be based on the same strain or serotype (or subgroup or variant), or they may be different from one another. For example, but are not limited to, an rAAV plasmid or vector genome or particle (capsid) based on one serotype genome may be identical to one or more capsid proteins that package the vector. Furthermore, an rAAV plasmid or vector genome may be based on a different AAV serotype genome than one or more capsid proteins that package the vector genome, in which case at least one of the three capsid proteins may be a different AAV serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, or a variant thereof. More specifically, an rAAV2 vector genome may contain an AAV2 ITR, but the capsids may originate from a different serotype, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, or a variant thereof. Therefore, rAAV vectors contain gene / protein sequences identical to those characteristic of a particular serotype, as well as "mixed" serotypes, which may also be called "pseudotypes."

[0146] In certain embodiments, the rAAV plasmid or vector genome or particle is based on AAV variants from reptiles or invertebrates, e.g., snake and lizard parvovirus (Penzes et al., 2015, J. Gen. Virol., 96:2769-2779) or insect and shrimp parvovirus (Roekring et al., 2002, Virus Res., 87:79-87).

[0147] In certain embodiments, the recombinant plasmid or vector genome or particle is based on a bokavirus variant. Human bokavirus variants are described, for example, Guido et al., 2016, World J. Gastroenterol., 22:8684-8697.

[0148] In one embodiment, the recombinant AAV (rAAV) vector contains VP1, VP2, and / or VP3 capsid proteins having 70% or more sequence identity to VP1, VP2, and / or VP3 capsid proteins selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8 VP1, VP2, and / or VP3 capsid proteins. In one embodiment, the recombinant AAV (rAAV) vector contains VP1, VP2, and / or VP3 capsid proteins selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8 VP1, VP2, and / or VP3 capsid proteins, having 100% sequence identity to the VP1, VP2, and / or VP3 capsid proteins. In certain embodiments, the AAV vector contains or consists of one or more sequences identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, or AAV3B, and one or more ITRs by at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.).

[0149] In certain embodiments, recombinant AAV (rAAV) vectors include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, RhlO, Rh74 and their AAV-2i8 variants (e.g., ITR and capsid variants, e.g., amino acid insertions, additions, substitutions and deletions) as described in International Publication No. 2013 / 158879 (International Application US2013 / 037170), International Publication No. 2015 / 013313 (International Application US2014 / 047670), and U.S. Patent Application Publication No. 2013 / 0059732 (U.S. Patent Application No. 13 / 594,773).

[0150] rAAVs, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, as well as mutant, hybrid, and chimeric sequences, can be constructed using recombination techniques known to those skilled in the art, such as containing one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAV ITR sequences. Such AAV vectors typically retain at least one functional flanking ITR sequence necessary for the rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will contain sequences (e.g., functional ITR sequences) necessary for cis-replication and packaging.

[0151] In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, AAV3B, AAV-2i8 capsids, or mutant capsids derived therefrom.

[0152] In some embodiments, recombinant adeno-associated virus (rAAV) comprises a vector genome containing at least one promoter sequence. In some embodiments, the promoter is selected from the group consisting of the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Roussarcoma virus long terminal repeat sequence, Chinese hamster elongation factor 1α (CHEF-1), human EF-1α, ubiquitin, and the human cytomegalovirus major pre-early promoter (hCMV MIE).

[0153] In a further embodiment, a method for producing a recombinant adeno-associated virus (rAAV) vector is provided.

[0154] In one embodiment, a method for generating a recombinant adeno-associated virus (rAAV) vector is provided, which comprises the following steps: (a) A step of providing a host cell comprising recombinant nucleic acid as previously described herein, wherein a first polynucleotide sequence encodes a therapeutic payload, a second nucleotide sequence encodes viral vector rep and cap proteins, and a third nucleotide sequence encodes E4, E2a and VA proteins, (b) A step of incubating host cells under conditions suitable for the preparation of recombinant rAAV vectors, and (c) A step of recovering the viral vector from the cell culture, Optional, (d) A step of formulating the recovered target polypeptide for therapeutic use. Includes.

[0155] In the context of rAAV vector production, host cells are used to replicate the viral genome and package it within the AAV capsid. For example, human embryonic kidney cells (HEK cells) genetically engineered to produce proteins necessary for AAV replication and capsid assembly are widely used to produce rAAV vectors. During rAAV vector production, host cells are typically transfected with multiple plasmids containing the AAV genome, along with therapeutic genes and the rep and cap genes necessary for replicating the viral genome and packaging it into the capsid. The plasmids provide the genetic material necessary to produce rAAV particles. The host cells replicate the AAV genome, package it into AAV particles, and can then be collected and purified for use, for example, in gene therapy. Well-characterized host cells, such as HEK293 cells, can help ensure consistent and reliable production of rAAV particles. Other cells that can be used in the context of rAAV production are known in the art.

[0156] In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is selected from the group consisting of CHO cells, BHK cells, HEK cells, and Sp2 / 0 cells. In preferred embodiments, the host cell is an HEK host cell, particularly an HEK293 host cell.

[0157] In some embodiments, polypeptides and rAAV vectors produced according to the present invention are further processed, for example, by formulation for therapeutic use. Accordingly, pharmaceutical compositions comprising polypeptides produced according to the present invention or rAAV vectors produced according to the present invention are also provided herein. In one embodiment, the pharmaceutical composition comprises either the polypeptide or viral vector provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises either the polypeptide or viral vector provided herein and at least one further therapeutic agent, for example, described below.

[0158] Pharmaceutical compositions (formulations) can be prepared by combining polypeptides or viral vectors with pharmaceutically acceptable carriers or excipients known to those skilled in the art. Exemplary pharmaceutical compositions described herein include lyophilized, aqueous, and frozen formulations.

[0159] Pharmaceutically acceptable carriers are typically non-toxic to the recipient at the doses and concentrations used and include, but are not limited to, buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-crezo Low molecular weight (less than approximately 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0160] Pharmaceutical compositions used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0161] Any polypeptide or viral vector produced in accordance with the present invention may be used in therapeutic methods.

[0162] In one embodiment, an rAAV vector is provided for use as a pharmaceutical. In a further embodiment, an rAAV vector is provided for use in treating a disease caused by loss of function of a gene in a patient. In a particular embodiment, an rAAV vector is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an rAAV vector for use in a method of treating an individual having a loss-of-function genetic disorder, comprising administering an effective amount of the rAAV vector to the individual. "Loss-of-function genetic disorder" refers to a type of genetic disorder in which the production of a functional protein is reduced or absent due to a gene mutation or other genetic defect, resulting in a disease phenotype. Therapies for treating such diseases are also called gene replacement therapies in the art. Examples of such diseases include, but are not limited to, cystic fibrosis, sickle cell anemia, hemophilia, and Tay-Sachs disease. In such one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents) to the individual, as described below, for example.

[0163] In further embodiments, the present invention provides the use of rAAV vectors in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of loss-of-function genetic disorders. In further embodiments, the pharmaceutical is for use in a method of treating loss-of-function genetic disorders, comprising administering an effective amount of the pharmaceutical to an individual having a loss-of-function genetic disorder. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., one of those described below, to the individual.

[0164] In further embodiments, the present invention provides a method for treating loss-of-function genetic disorders. In one embodiment, the method comprises administering an effective amount of rAAV vector to an individual having such loss-of-function genetic disorder. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below.

[0165] The individual according to any of the above embodiments is preferably a human.

[0166] In further embodiments, the present invention provides a pharmaceutical composition comprising one of the rAAV vectors provided herein for use, for example, in any of the therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises one of the rAAV vectors provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the rAAV vectors provided herein and at least one additional therapeutic agent, for example, one of those described below.

[0167] The rAAV vector of the present invention can be administered alone or used in combination therapy. For example, combination therapy may include administering the rAAV vector of the present invention and administering at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents).

[0168] Such combination therapies described above include combination administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical compositions) and individual administration, in which case the rAAV vector of the present invention may be administered before, simultaneously with, and / or after the administration of one or more additional therapeutic agents. In one embodiment, the administration of the rAAV vector and the administration of the additional therapeutic agents occur within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. In one embodiment, the rAAV vector and the additional therapeutic agent are administered to the patient on day one of treatment.

[0169] The rAAV vector (and any additional therapeutic agent) prepared in accordance with the present invention may be administered by any suitable means, including parenteral administration, intrapulmonary administration, intranasal administration, and, if local treatment is desired, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing schedules are considered herein, including but not limited to single doses, multiple doses at various time points, bolus administration, and pulse infusion.

[0170] The rAAV vectors prepared according to the present invention will be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals. The rAAV vector may, but is not necessarily, be formulated with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs will depend on the amount of rAAV vector present in the pharmaceutical composition, the type of disorder or treatment, and the other factors discussed above.

[0171] For the prevention or treatment of disease, the appropriate dosage of the rAAV vector prepared according to the present invention (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of rAAV vector, the severity and course of the disease, whether the rAAV vector is administered for prophylactic or therapeutic purposes, previous treatments, and the discretion of the attending physician. The rAAV vector is administered appropriately to the patient, either as a single dose or over a series of treatments. The progress of this treatment is readily monitored by conventional techniques and assays.

[0172] In another aspect of the present invention, a product is provided containing a material useful for the treatment, prevention and / or diagnosis of the above-mentioned disease. The product comprises a container and a label or accompanying information attached to or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, and infusion bags. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for treating, preventing and / or diagnosing the symptoms and may have a sterile access port (for example, the container may be an infusion bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is an rAAV vector prepared according to the present invention. The label or accompanying information indicates that the composition is used to treat a selected symptom. Furthermore, the product may include (a) a first container containing a composition comprising an rAAV vector prepared according to the present invention; and (b) a second container containing a composition comprising a further cytotoxic agent or other therapeutic agent. A product of this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition thereto, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. This may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0173] The following description illustrates specific embodiments of the present invention.

[0174] 1. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, the recombinant polyadenylation signal sequence having a sequence length of less than 100 nucleotides, and eukaryotic cells transformed with the recombinant nucleic acid comprising the recombinant transcription unit are capable of expressing the polypeptide at the same or higher expression levels compared to eukaryotic cells transformed with a reference nucleic acid comprising the recombinant polyadenylation signal sequence of SEQ ID NO: 2, wherein the nucleotide sequence of the reference nucleic acid is identical to the sequence of the recombinant nucleic acid without regard to the recombinant polyadenylation signal sequence with respect to sequence identity.

[0175] 2. The recombinant transcription unit according to Embodiment 1, wherein the recombinant polyadenylation signal sequence comprises or comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs: 12.

[0176] 3. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, and the recombinant polyadenylation signal sequence comprises or comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0177] 4. A recombinant transcription unit according to any one of Embodiments 1 to 3, wherein the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12.

[0178] 5. Recombinant nucleic acids, (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence Includes, The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%. Recombinant nucleic acids.

[0179] 6. Recombinant nucleic acid according to Embodiment 5, (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence. It further includes, The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence individually have 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% sequence identity with the third recombinant polyadenylation signal sequence. Recombinant nucleic acids.

[0180] 7. The recombinant nucleic acid according to Embodiment 5 or 6, wherein the first recombinant polyadenylation signal sequence, the second recombinant polyadenylation signal sequence, and, if present, the third recombinant polyadenylation signal sequence, have a sequence length of less than 100 nucleotides.

[0181] 8. Recombinant nucleic acids according to embodiments 5 to 7, wherein the first recombinant polyadenylation signal sequence, the second recombinant polyadenylation signal sequence, and the third recombinant polyadenylation signal sequence, if present, are unable to participate in DNA strand exchange for forming a recombinant intermediate.

[0182] 9. Recombinant nucleic acid according to any one of embodiments 5 to 8, wherein recombination events between a nucleic acid containing a first recombinant polyadenylation signal sequence and a nucleic acid containing a second recombinant polyadenylation signal sequence are reduced or prevented.

[0183] 10. Recombinant nucleic acid according to any one of embodiments 5 to 9, wherein recombination events between a nucleic acid containing a first recombinant polyadenylation signal sequence and a nucleic acid containing a third recombinant polyadenylation signal sequence are reduced or prevented, and / or recombination events between a nucleic acid containing a second recombinant polyadenylation signal sequence and a nucleic acid containing a third recombinant polyadenylation signal sequence are reduced or prevented.

[0184] 11. Recombinant nucleic acids according to any one of Embodiments 5 to 10, wherein the first, second, and, if present, the third polypeptide are expressed in eukaryotic cells.

[0185] 12. Recombinant nucleic acid according to any one of Embodiments 5 to 11, wherein the first recombinant transcription unit is the recombinant transcription unit described in any one of Embodiments 1 to 4, the second recombinant transcription unit is the recombinant transcription unit described in any one of Embodiments 1 to 4, and if present, the third recombinant transcription unit is the recombinant transcription unit described in any one of Embodiments 1 to 4.

[0186] 13. Recombinant nucleic acid according to any one of Embodiments 5 to 12, (a) The first recombinant transcription unit further comprises a first promoter operably ligated to a nucleotide sequence encoding a first polypeptide, (b) further comprising a second promoter operably ligated to a nucleotide sequence encoding a second polypeptide, The first promoter and the second promoter have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%. Recombinant nucleic acids.

[0187] 14. Recombinant nucleic acid according to any one of embodiments 6 to 13, (c) If present, further comprising a first promoter operably ligated to a nucleotide sequence encoding a first polypeptide, The first and second promoters have 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or less than 60% sequence identity with the third promoter. Recombinant nucleic acids.

[0188] 15. Recombinant nucleic acids according to Embodiment 13 or 14, (i) The first, second, and third promoters present are active in eukaryotic cells, (ii) The first promoter drives the expression of the first polypeptide, (iii) The second promoter drives the expression of the second polypeptide, (iv) The third promoter drives the expression of the third polypeptide, and / or (v) The first promoter, the second promoter, and the third promoter, if present, drive the expression of the first, second, and third polypeptides, respectively. Recombinant nucleic acids.

[0189] 16. Recombinant nucleic acid according to any one of embodiments 13 to 15, wherein the first promoter, the second promoter, and the third promoter, if present, are individually selected from the group consisting of the hPGK1 promoter, the CMV promoter, and the hEF1α promoter.

[0190] 17. Recombinant nucleic acid according to any one of embodiments 5 to 16, wherein the recombinant nucleic acid comprises at least one vector.

[0191] 18. The recombinant nucleic acid according to any one of Embodiments 5 to 17, wherein the recombinant nucleic acid comprises a first vector containing a first recombinant transcription unit, a second vector containing a second recombinant transcription unit, and, if a third recombinant transcription unit is present, a third vector containing a third recombinant transcription unit.

[0192] 19. Recombinant nucleic acid according to any one of Embodiments 17 or 18, wherein at least one vector comprises a selection marker operably linked to a first, second, or, if present, a third recombinant transcription unit.

[0193] 20. Recombinant nucleic acid according to Embodiment 19, wherein the selectable marker is selected from the group consisting of hygromycin selectable marker, neomycin selectable marker, G418 selectable marker, dihydrofolate reductase (DHFR), thymidine kinase, glutamine synthase, asparagine synthase, tryptophan synthase, histidinol dehydrogenase, and nucleic acids that confer resistance to puromycin, bleomycin, phleomycin, chloramphenicol, zeosin, and mycophenolic acid.

[0194] 21. Recombinant nucleic acids according to Embodiments 17-20, wherein the first, second, and / or third vector, if present, includes a bacterial origin of replication, particularly a pUC19 origin of replication.

[0195] 22. A host cell comprising a recombinant transcription unit according to any one of Embodiments 1 to 4 and / or a recombinant nucleic acid according to any one of Embodiments 5 to 21.

[0196] 23. A host cell according to Embodiment 22, which is a eukaryotic host cell.

[0197] 24. A host cell according to Embodiment 22 or 23, selected from the group consisting of CHO, BHK, HEK, and Sp2 / 0.

[0198] 25. Recombinant viral vector comprising a vector genome, wherein the vector genome is arranged in the order from 5' to 3', (i) 5'ITR sequence, (ii) Promoter sequence, (iii) Sequence encoding polypeptide, (iv) Recombinant polyadenylated signal sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and (v)3'ITR sequence Recombinant viral vectors containing this vector.

[0199] 26. The recombinant viral vector according to Embodiment 25, wherein the recombinant polyadenylation signal sequence is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12.

[0200] 27. Recombinant viral vectors according to Embodiment 25 or 26, wherein the recombinant viral vector is selected from the group consisting of retroviral vectors, adenovirus vectors, helper-dependent adenovirus vectors, hybrid adenovirus vectors, herpes simplex virus vectors, lentivirus vectors, poxvirus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, human cytomegalovirus vectors, lentivirus vectors, adenovirus vectors or adeno-associated virus (AAV) vectors, or recombinant variants derived therefrom.

[0201] 28. The recombinant viral vector according to any one of Embodiments 25 to 27, wherein the recombinant viral vector is a recombinant adeno-associated virus (rAAV) vector.

[0202] 29. The rAAV according to Embodiment 28, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, AAV3B, AAV-2i8 capsid or mutant capsids derived therefrom.

[0203] 30. A method for producing a target polypeptide, wherein the method comprises the following steps: (a) A step of providing a host cell according to any one of embodiments 22 to 24, (b) A step of incubating host cells under conditions suitable for polypeptide expression, (c) A step of recovering the target polypeptide from the cell culture. Methods that include...

[0204] 31. A method for producing a target polypeptide, the method comprising the following steps: (a) Providing a host cell comprising a recombinant nucleic acid according to any one of Embodiments 4 to 21, wherein the target polypeptide is a first polypeptide, and the second and third polypeptides are necessary for the production of the target polypeptide or improve its production; (b) Incubating host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide; (c) A step of recovering the target polypeptide from the cell culture, Optional, (d) A step of formulating the recovered target polypeptide for therapeutic use. Methods that include...

[0205] 32. A method for producing a recombinant adeno-associated virus (rAAV) vector, the method comprising the following steps: (a) Providing a host cell comprising a recombinant nucleic acid according to any one of Embodiments 4 to 21, wherein a first polynucleotide sequence encodes a therapeutic payload, a second nucleotide sequence encodes viral vector rep and cap proteins, and a third nucleotide sequence encodes E4, E2a and VA proteins; (b) A step of incubating host cells under conditions suitable for the preparation of recombinant rAAV vectors, (c) A step of recovering the viral vector from the cell culture, Optional, (d) A step of formulating the recovered target polypeptide for therapeutic use. Methods that include...

[0206] 33. The method according to any one of embodiments 30 to 32, wherein the host cells are selected from the group consisting of CHO cells, BHK cells, HEK cells, and Sp2 / 0 cells.

[0207] 34. The method according to Embodiment 32 or 33, wherein the rAAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, AAV3B, AAV-2i8 vectors, or vector variants derived therefrom.

[0208] 35. Use of a recombinant transcription unit for recombinant production of a target polypeptide, wherein the recombinant transcription unit is as defined in any one of Embodiments 1 to 4.

[0209] 35. Use of recombinant nucleic acid for recombinant production of a polypeptide of interest, wherein the recombinant nucleic acid is as defined in any one of Embodiments 5 to 21.

[0210] 36. The present invention is as described above with reference to the examples and drawings contained herein.

[0211] TIFF2026516648000001.tif243170TIFF2026516648000002.tif83170

[0212] This disclosure includes combinations of the described embodiments and preferred features, unless such combinations are clearly unacceptable or expressly avoided.

[0213] Herein, aspects and embodiments of the present disclosure will be described by reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0214] Throughout this Spec., including the following claims, unless contextually required, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to mean the inclusion of the described integer or process, or group of integers or processes, but not the exclusion of any other integer or process, or group of integers or processes.

[0215] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references unless otherwise explicitly indicated by the context. In this specification, ranges may be expressed as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, another embodiment includes one particular value and / or another particular value. Similarly, the use of the antecedent “about” will be understood to mean that when a value is expressed as an approximation, a particular value forms another embodiment.

[0216] Where a nucleic acid sequence is disclosed herein, its reverse complement is also expressly intended.

[0217] The methods described herein may preferably be carried out in vitro. The term “in vitro” is intended to encompass procedures performed using cells in culture, while the term “in vivo” is intended to encompass procedures performed using / on intact multicellular organisms. [Examples]

[0218] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be implemented based on the general description provided above.

[0219] Example 1 material and method 1.1 Gene Synthesis The desired gene segments and, if necessary, plasmids were synthesized by GenScript Biotech (Rijswijk, Netherlands).

[0220] 1.2 Cell culture and transfection of human fetal kidney cells (HEK293T) HEK293T cells were cultured in DMEM (high glucose, GlutaMAX, pyruvate; Gibco, catalog number 31966) supplemented with 10% (v / v) fetal bovine serum (Gibco, catalog number A5209402) and 50 U / mL penicillin-streptomycin (Gibco, catalog number 15070063), and routinely subculturised using 0.25% trypsin-EDTA (Gibco, catalog number 25200).

[0221] For transient transfection of HEK293T cells, 2,500 cells per well at 20 μL were seeded into 384-well plates 1 day before transfection. Next, a 5 μL transfection mixture consisting of 25 ng of plasmid DNA complexed with 0.05 μL of Lipofectamine 2000 (Invitrogen, catalog number 11668019) in Opti-MEM low serum medium (Gibco, catalog number 31985) was transiently transfected into each well at room temperature for 20 minutes. All reporter plasmids used to evaluate the polyadenylation signal were transfected in equimolar amounts, and the total amount of transfected plasmid for each experimental condition was normalized to 25 ng using a mock plasmid lacking active transcription and open reading frames.

[0222] 1.3 Quantification of NanoLuc luciferase (Nluc) production Total Nluc production was quantified by adding 25 μL of 2X luciferase assay solution (mixing 1 volume of Nano-Glo luciferase assay substrate and 50 volumes of assay buffer) to each well of a 384-well plate using the Nano-Glo luciferase assay system (Promega, catalog number N1110). The assay plate was incubated in the dark at room temperature for 10 minutes, and then luminescence was quantified using a PHERAstar FSX (BMG Labtech) plate reader.

[0223] Example 2 A transient transfection reporter plasmid was constructed (Figure 1) using a DNA sequence encoding a constitutive promoter (Prom.), enhanced green fluorescent protein (EGFP), P2A self-cleaving peptide sequence, NanoLuc luciferase (Nluc), PEST proteolytic signal, and 3' untranslated region (3'UTR) to evaluate the ability of polyadenylation (polyA) signals to support high expression levels by efficient transcriptional termination and polyadenylation.

[0224] Two copies of the standard BGH polyadenylation signal sequence and the short sNRP-1 polyadenylation signal sequence (2×sNRP-1; McFarland et al. (2006)) were inserted immediately downstream of the 3’UTR of the reporter plasmid, respectively (Figure 2A). To evaluate the relative transcription termination efficiency of the recombinant polyadenylation signal sequence and its ability to support high protein expression levels, HEK293T cells were transiently transfected with the corresponding reporter plasmid for 24 hours, and then the total Nluc expression level was assayed. Figure 2B shows the relative Nluc expression levels from the two reporter plasmids, and the results are normalized to the average luminescence value of the cells transfected with the reporter plasmid encoding BGH. Notably, the short 2-fold sNRP-1 polyA coding construct expresses less than 25% compared to the BGH coding construct, highlighting that short polyadenylation signal sequences known in the art are less reliable in supporting high expression levels of the gene of interest.

[0225] Example 3 To establish a set of short recombinant polyadenylation signal sequences that can support high expression levels while having high sequence heterogeneity, enabling their use in multi-gene expression vectors without the risk of recombination, a 95-nucleotide (nt) recombinant polyadenylation signal sequence design was created, consisting of the core elements of the polyadenylation signal sequence derived from the synthetic rabbit β-globin polyadenylation signal sequence defined by Levitt et al. (Levitt et al. (1989)), including the polyadenylation signal (PAS), and two GU / U-rich downstream sequence element (DSE) regions. In addition, two cytosine-adenine (CA) mRNA cleavage sites were introduced 15 - 20 nt downstream of the PAS, and a 26-nt U-rich upstream sequence element (USE) region was introduced (Figure 3).

[0226] Based on the poly-A design described above, an initial set of four recombinant polyadenylation signal sequences was designed (Poly-A-1.1, -2.1, -3.1, and -4.1). Each of the four recombinant polyadenylation signal sequences was then rationally modified by extending the USE region to 46 nt and designed to contain a unique primer annealing site with a Tm of 70–72°C compatible with Gibson assemblies. Furthermore, small nt modifications were introduced, focusing on the variable region between the USE or PSA region and the DSE region, to increase heterogeneity between one or more poly-A sequences or to reduce strong secondary RNA structures within the poly-A sequences. Twelve recombinant polyadenylation signal sequences were selected and introduced into the reporter plasmid described above (Figure 4A) and transiently tested with HEK293T. Figure 4B shows the relative Nluc expression levels from reporter plasmids encoding 12 recombinant polyadenylated signal sequences 24 hours after transfection, with results normalized to the mean luminescence values ​​for all transfection conditions. From the set of recombinant polyadenylated signal sequences tested, three (Poly-A-2.3, -3.3, and -4.3) were selected for further characterization due to their ability to maintain relatively high expression levels and having less than 50% sequence identity compared to one another.

[0227] Example 4 To evaluate the three selected recombinant polyadenylation signal sequences, the rabbit β-globulin polyadenylation signal sequence defined by Levitt et al. (1989) was introduced into a reporter plasmid (Figure 5A) and transiently tested on HEK293T. Figure 5B shows the relative Nluc expression levels of Levitt et al.'s polyA and the three synthetic polyA-coding plasmids 24 hours after transfection, with results normalized to the mean luminescence values ​​of cells transfected with Levitt et al.'s polyA-coding reporter plasmid. In particular, all three synthetic polyA-coding constructs showed Nluc expression levels twice as high as Levitt et al.'s rabbit β-globulin polyadenylation signal sequence.

[0228] Example 5 To evaluate the three selected recombinant polyadenylation signal sequences against known larger polyadenylation signal sequences, hGH poly(A) and SV40 poly(A) were introduced into reporter constructs and tested against plasmids containing the recombinant polyadenylation signal sequences using a BGH-containing reporter plasmid, as described above (Figure 6A). To evaluate the relative transcription termination efficiency and ability to support high protein expression levels of the recombinant polyadenylation signal sequences, HEK293T cells were transiently transfected with the corresponding reporter plasmid for 24 hours and then assayed for total Nluc expression levels. Figure 6B shows the relative Nluc expression levels from the reporter plasmid. Results are normalized to the mean luminescence values ​​of cells transfected with the BGH-coding reporter plasmid. In particular, the recombinant polyadenylation signal sequences support expression levels equivalent to or higher than those of known larger polyadenylation signal sequences.

[0229] Example 6 To evaluate how recombinant polyadenylation signal sequences are affected by upstream 3'UTR sequence configurations, three different novel 3'UTR sequences with less than 50% pairwise sequence identity and less than 30% sequence identity were introduced into reporter plasmids encoding recombinant polyadenylation signal sequences (Figure 7A). As shown in Figure 7B, all constructs were transiently tested on HEK293T. Figure 7B shows the relative Nluc expression levels from the reporter plasmid 24 hours after transfection, and the results are normalized to the mean luminescence values ​​for all transfection conditions. In particular, the recombinant polyadenylation signal sequences show minimal influence from the three different upstream 3'UTR sequences.

[0230] Example 7 To evaluate how recombinant polyadenylation signal sequences are affected by the promoter strength used, and how this affects the resulting expression levels, three constitutive promoters of different strengths—hPGK1, CMV, and hEF1α—were introduced into reporter plasmids encoding their respective recombinant polyadenylation signal sequences (Figure 8A). All constructs were transiently tested on HEK293T, and relative Nluc expression levels from the reporter plasmids were quantified 24 hours after transfection. Figures 8B (hPGK1), 8C (CMV), and 8D (hEF1α) show the relative Nluc expression levels from different reporter plasmids, respectively, with results normalized to the mean luminescence values ​​for all transfection conditions in the corresponding graphs. In particular, recombinant polyadenylation signal sequences, in combination with different constitutive promoters, support robust expression at different levels.

Claims

1. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, the recombinant polyadenylation signal sequence having a sequence length of less than 100 nucleotides, and eukaryotic cells transformed with a recombinant nucleic acid comprising the recombinant transcription unit are capable of expressing the polypeptide at the same or higher expression level as eukaryotic cells transformed with a reference nucleic acid comprising a recombinant polyadenylation signal sequence of Sequence ID No. 2, and the nucleotide sequence of the reference nucleic acid is identical to the sequence of the recombinant nucleic acid with respect to sequence identity, without considering the recombinant polyadenylation signal sequence.

2. The recombinant transcription unit according to claim 1, wherein the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs:

12.

3. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operably linked to a recombinant polyadenylation signal sequence, and the recombinant polyadenylation signal sequence comprises or comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs:

12.

4. The recombinant transcription unit according to any one of claims 1 to 3, wherein the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO:

12.

5. (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide, operably linked to a first recombinant polyadenylation signal sequence, (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide, operably linked to a second recombinant polyadenylation signal sequence Recombinant nucleic acids, including The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%, The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence each have a sequence length of less than 100 nucleotides. Recombinant nucleic acids.

6. (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide, operably linked to a third recombinant polyadenylation signal sequence. It further includes, The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence individually have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40% with the third recombinant polyadenylation signal sequence. The third recombinant polyadenylation signal sequence has a sequence length of less than 100 nucleotides. Recombinant nucleic acid according to claim 5.

7. The recombinant nucleic acid according to claim 5 or 6, wherein the first recombinant polyadenylation signal sequence, the second recombinant polyadenylation signal sequence, and the third recombinant polyadenylation signal sequence, if present, are unable to participate in DNA strand exchange for forming a recombinant intermediate.

8. The recombinant nucleic acid according to any one of claims 5 to 7, wherein the first recombinant transcription unit is the recombinant transcription unit according to any one of claims 1 to 4, the second recombinant transcription unit is the recombinant transcription unit according to any one of claims 1 to 4, and if present, the third recombinant transcription unit is the recombinant transcription unit according to any one of claims 1 to 4.

9. (a) The first recombinant transcription unit further comprises a first promoter operably ligated to the nucleotide sequence encoding the first polypeptide, (b) The second recombinant transcription unit further comprises a second promoter operably ligated to the nucleotide sequence encoding the second polypeptide, The first promoter and the second promoter have sequence identity of 70%, 65%, 60%, 55%, 50%, 45%, or less than 40%, Recombinant nucleic acid according to any one of claims 5 to 8.

10. (c) If present, the first recombinant transcription unit further comprises a first promoter operably ligated to the nucleotide sequence encoding the first polypeptide, The first promoter and the second promoter have sequence identity with the third promoter of 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or less than 60%. Recombinant nucleic acid according to any one of claims 6 to 9.

11. The recombinant nucleic acid according to any one of claims 5 to 10, wherein the recombinant nucleic acid comprises a first vector containing the first recombinant transcription unit, and a second vector containing the second recombinant transcription unit, and if a third recombinant transcription unit is present, a third vector containing the third recombinant transcription unit.

12. A host cell comprising a recombinant transcription unit according to any one of claims 1 to 4 and / or a recombinant nucleic acid according to any one of claims 5 to 11.

13. A recombinant viral vector comprising a vector genome, wherein the vector genome is arranged in the order from 5' to 3', (i) 5' ITR sequence, (ii) Promoter sequence, (iii) Sequence encoding polypeptide, (iv) Recombinant polyadenylated signal sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and (v) 3' ITR sequence Recombinant viral vectors containing this vector.

14. The recombinant viral vector according to claim 13, wherein the recombinant polyadenylation signal sequence is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO:

12.

15. A recombinant viral vector according to claim 13 or 14, selected from the group consisting of retroviral vectors, adenovirus vectors, helper-dependent adenovirus vectors, hybrid adenovirus vectors, herpes simplex virus vectors, lentivirus vectors, poxvirus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, human cytomegalovirus vectors, lentivirus vectors, adenovirus vectors or adeno-associated virus (AAV) vectors, or recombinant variants derived therefrom.

16. A recombinant viral vector according to any one of claims 13 to 15, which is a recombinant adeno-associated virus (rAAV) vector.

17. A method for producing a target polypeptide, wherein the method comprises the following steps: (a) A step of providing the host cell according to claim 12, (b) A step of incubating the host cells under conditions suitable for the expression of the polypeptide, (c) A step of recovering the target polypeptide from the cell culture. Methods that include...

18. A method for producing a target polypeptide, wherein the method comprises the following steps: (a) A step of providing a host cell comprising a recombinant nucleic acid according to any one of claims 5 to 11, wherein the target polypeptide is a first polypeptide, and a second polypeptide and, if present, a third polypeptide are necessary for the production of the target polypeptide or improve the production of the target polypeptide, (b) A step of incubating the host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide, (c) A step of recovering the target polypeptide from the cell culture, Optional, (d) A step of formulating the recovered target polypeptide for therapeutic use. Methods that include...

19. The method according to claim 17 or 18, wherein the host cells are selected from the group consisting of CHO cells, BHK cells, HEK cells, and Sp2 / 0 cells.

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